# Patent

## Title
Variants of a lysozyme and polynucleotides encoding same

## Abstract

The present invention relates to variant lysozymes. The present invention also relates to polynucleotides encoding the variant lysozymes and to nucleic acid constructs, vectors, and host cells comprising the polynucleotide.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/EP2011/052812 filed Feb. 25, 2011, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 10154756.0 filed Feb. 25, 2011 and U.S. provisional application No. 61/309,065 filed Mar. 1, 2010 the contents of which are fully incorporated herein by reference.


REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to variants of a lysozyme, polynucleotides encoding the variants and methods of producing the variants.
BACKGROUND OF THE INVENTION
Lysozyme (EC 3.2.1.17), also known as muramidase or N-acetylmuramide glycanhydrolase, catalyzes hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins.
Lysozyme is typically produced as a defensive mechanism against bacteria by many organisms as viruses, plants, insects, birds, reptiles and mammals. The enzyme causes the hydrolysis of bacterial cell walls by cleaving the glycosidic bonds of peptidoglycan; an important structural molecule in bacteria. After having their cell walls weakened by lysozyme action, bacterial cells lyse resulting from osmotic pressure. There is an increasing interest in the potential of lysozyme enzymes as antimicrobial agents. For example, lysozyme activity has been shown against pathogens such as Streptococcus pneumoniae, Bacillus anthracis, Enterococcus faecium, Bacillus stearothermophilus, Clostridium botulinum, Clostridium butyricum, Clostridium perfringens, Clostridium sporogenes, Clostridium tyrobutyricum, and Listeria monocytogenes. 
Lysozyme has been classified into five different glycoside hydrolase (GH) families (CAZy, www.cazy.org): hen egg-white lysozyme (GH22), goose egg-white lysozyme (GH23), bacteriophage T4 lysozyme (GH24), Sphingomonas flagellar protein (GH73) and Chalaropsis lysozymes (GH25). The lysozyme family GH25 has been found to be structurally unrelated to the other lysozyme families.
Use of lysozyme has been suggested in animal feed (see for example WO 00/21381 and WO 04/026334), in cheese production (see for example WO 05/080559), food preservation (Hughey and Johnson (1987) Appl Environ Microbiol 53:2165), detergents (see for example U.S. Pat. No. 5,041,236 and EP 0425016), in oral care (see for example U.S. Pat. No. 4,355,022, WO 04/017988 and WO 08/124,764), cosmetology and dermatology, contraception, urology, and gynaecology (see for example WO 08/124,764).
Hen egg-white lysozyme is a commercially available lysozyme product. Lysozymes isolated from microbial sources are also known.
SUMMARY OF THE INVENTION
The present invention relates to lysozyme variants and nucleotide sequence encoding same. In one aspect, the present invention relates to lysozyme variants belonging to glycosyl hydrolase family 25 (GH25) comprising an alteration of an amino acid sequence at one or more positions selected from the group consisting of position number 47, 111, 108, 45, 22, 110, 120, 147, 196, 49, 55, 193, 161, 128, 131, 203, 98, 112, 55, 32, 89, 206 121, 120, 185, 113, 119, 35, 153, 158, 171, 195, 76, 164, 30, 85, 178, 183, 186, 112, 174, 187, 197, 102, 134, 108, 196, 197, 198, 56, 19, 120, 20, 135, and 203, which position corresponds to a position in amino acid sequence SEQ ID NO:3, and wherein the lysozyme variant has antimicrobial and/or lysozyme activity. In one embodiment, the lysozyme variant is a variant of a fungal lysozyme. In another embodiment, the lysozyme variant comprises an amino acid sequence having at least 75% identity to SEQ ID NO:3. In another embodiment, the lysozyme variant is a variant of an Aspergillus fumigatus lysozyme.
The variants of the present invention have altered properties compared to the parental lysozyme, such as altered temperature-dependent activity profile, e.g. improved thermal activity or improved activity at low or moderate temperatures (psychrophilic or mesophilic activity), improved temperature stability, improved pH activity, improved pH stability and/or, increased resistance to protease degradation.
The present invention also relates to antimicrobial compositions and antimicrobial methods comprising the lysozyme variants of the present invention. In embodiments, the present invention also relates to the use of the variants of the present invention in animal feed, cheese production, food preservation, detergents, oral care, cosmetology and dermatology.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is an alignment of a number of mature family 25 lysozyme amino acid sequences as follows:
Aspergillus fumigatus lysozyme (SEQ ID NO:3);
Aspergillus fumigatus lysozyme (SEQ ID NO:4);
Aspergillus fischerianus lysozyme (SEQ ID NO:5);
Aspergillus clavatus lysozyme (SEQ ID NO:6);
Aspergillus oryzae lysozyme (SEQ ID NO:7);
Aspergillus terreus lysozyme (SEQ ID NO:8);
Aspergillus fischerianus lysozyme (SEQ ID NO:9);
Aspergillus fumigatus lysozyme (SEQ ID NO:10);
Aspergillus clavatus lysozyme (SEQ ID NO:11);
Aspergillus terreus lysozyme (SEQ ID NO:12); and
Penicillum marneffei lysozyme (SEQ ID NO:13).
The signal peptide has been predicted using the program Signal P version 3.0. The signal peptide is not in the alignment.
FIG. 2 lists the atomic coordinates of the three-dimensional structure of the Aspergillus fumigatus lysozyme.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to lysozyme variants, especially variants of lysozyme belonging to glycosyl hydrolase family 25 (GH25), comprising an alteration, preferably in the form of a substitution and/or an insertion and/or a deletion at one or more (several) positions, where the numbering of the positions corresponds to the numbering of the positions of SEQ ID NO:3. The variants of the present invention have antimicrobial and/or lysozyme activity.
DEFINITIONS
Antimicrobial activity: The term “antimicrobial activity” is defined herein as is an activity that kills or inhibits the growth of microorganisms such as algae, archea, bacteria, fungi or protozoans. The antimicrobial activity can for example be bactericidal meaning the killing of bacteria, bacteriostatic meaning the prevention of bacterial growth, or prevention of sporulation. For purposes of the present invention, antimicrobial activity is determined according to the lysozyme turbidity activity assay described in the “Materials and Methods” section.
Lysozyme activity: The term “lysozyme activity” is defined herein as a peptidoglycan N-acetylmuramoylhydrolase activity (EC 3.2.1.17) that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. For purposes of the present invention, lysozyme activity is determined according to the lysozyme turbidity activity assay described in the “Materials and Methods” section.
Variant: The term “variant” is defined herein as a polypeptide having antimicrobial activity and/or lysozyme activity comprising an alteration, such as a substitution, insertion, and/or deletion, of one or more (several) amino acid residues at one or more (several) specific positions which positions correspond to the amino acid positions in SEQ ID NO:3. The altered polypeptide (variant) may be obtained through human intervention by modification of the polynucleotide sequence encoding the parental enzyme. The parental enzyme may be encoded by SEQ ID NO:1 or a sequence which is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to one of these sequences and which encode a polypeptide having antimicrobial and/or lysozyme activity. The variant polypeptide sequence is preferably one which is not found in nature.
Wild-Type enzyme: The term “wild-type” lysozyme denotes a lysozyme expressed by a naturally occurring organism, preferably from a naturally occurring microorganism such as algae, archea, bacteria, yeast, filamentous fungus, or protzoans found in nature. The term wild-type may be used interchangeably with the term “naturally occurring”.
Parent enzyme: The term “parent” lysozyme or “parental” lysozyme as used herein means a lysozyme to which a modification, e.g. substitution(s), insertion(s), deletion(s), and/or truncation(s), is made to produce the enzyme variants of the present invention. This term also refers to the polypeptide with which a variant is compared and aligned. The parent may be a naturally occurring (wild-type) polypeptide such as the enzyme of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13 or a polypeptide which is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% identical to one of these sequences. The parent polypeptide may also be a variant of a naturally occurring polypeptide which has been modified or altered in the amino acid sequence. A parent may also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.
Isolated variant or polypeptide: The term “isolated variant” or “isolated polypeptide” as used herein refers to a variant or a polypeptide that is isolated from a source, e.g. the host cell from which it is expressed or the enzyme complex it is normally present in. Preferably, the polypeptide is at least 40% pure, such as, at least 60% pure, at least 80% pure, at least 90% pure or at least 95% pure, as determined by SDS-PAGE.
Substantially pure variant or polypeptide: The term “substantially pure variant” or “substantially pure polypeptide” denotes herein a polypeptide preparation that contains at most 10%, such as, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure variant or polypeptide is at least 92% pure, such as, at least 94% pure, at least 95% pure, at least 96% pure, at least 96% pure, more at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure or even 100% pure by weight of the total polypeptide material present in the preparation. The variants and polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the variant or polypeptide by well-known recombinant methods or by classical purification methods.
Mature polypeptide: The term “mature polypeptide” is defined herein as a polypeptide having antimicrobial and/or lysozyme activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. For the polypeptide defined by SEQ ID NO:2, an example of a mature lysozyme sequence starts at position 18 of SEQ ID NO:2 and ends at position 235 of SEQ ID NO:2. This mature lysozyme sequence is also shown in SEQ ID NO:3. Another example is when the lysozyme is expressed in Aspergillus oryzae, then the mature polypeptide start at position 26 of SEQ ID NO:2 and ends at position 235 of SEQ ID NO:2. Depending on expression system, however, the length of the actual mature polypeptide may vary, such as, e.g. 1 to 10 amino acids in length (longer or shorter) at the N and/or C termini from the predicted mature polypeptide.
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” is defined herein as a nucleotide sequence that encodes a mature polypeptide having antimicrobial and/or lysozyme activity. In one aspect, the mature polypeptide coding sequence is nucleotides 51 to 705 of SEQ ID NO:1. The mature polypeptide coding sequence may vary 3 to 30 nucleotides in length depending on the expression system. When expressed in Aspergillus oryzae the mature polypeptide coding sequence can for example correspond to nucleotides or 75 to 705 of SEQ ID NO:1 of SEQ ID NO:1.
Identity: The parameter “identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences. For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277; http://emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) supra; http://emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein as a predicted polypeptide that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with the Aspergillus fumigatus lysozyme (UniProt Accession No. A4DA29).
Functional fragment of a polypeptide: The term “functional fragment of a polypeptide” is used to describe a polypeptide which is derived from a longer polypeptide, e.g., a mature polypeptide, and which has been truncated either in the N-terminal region or the C-terminal region or in both regions to generate a fragment of the parent polypeptide. To be a functional polypeptide the fragment must maintain at least 20% of the antimicrobial and/or lysozyme activity of the full-length/mature polypeptide, such as, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the antimicrobial and/or lysozyme activity of the full-length/mature polypeptide.
Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In one aspect, the isolated polynucleotide is at least 40% pure, such as, at least 60% pure, at least 80% pure, at least 90% pure or at least 95% pure, as determined by agarose electrophoresis.
Substantially pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered polypeptide production systems. Thus, a substantially pure polynucleotide contains at most 10%, such as, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, or at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′ and 3′ untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, such as, at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, or even at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.
Coding sequence: When used herein, the term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of its polypeptide product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant polynucleotide.
Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or a vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Altered property: The term “altered property” is defined herein as a characteristic associated with a variant that is altered, unless otherwise stated to another reference lysozyme, and which is altered as compared relative to the parent lysozyme. Such altered properties include, but are not limited to, altered temperature-dependent activity profile, altered temperature stability, altered pH activity, altered pH stability and/or, altered resistance to protease degradation.
Improved property: The term “improved property” is defined herein as a characteristic associated with a variant that is improved, unless otherwise stated to another reference lysozyme, and which is improved compared relative to the parent lysozyme. Such improved properties include, but are not limited to improved temperature-dependent activity profile such as improved thermal activity or improved activity at low temperatures, improved temperature stability, improved pH activity, improved pH stability and/or, increased resistance to protease degradation.
Altered temperature-dependent activity profile: The term “altered temperature-dependent activity profile” as used herein describes a variant enzyme displaying an alteration of the temperature-dependent activity profile when compared to the temperature-dependent activity profile of the parent lysozyme. The temperature-dependent activity profile provides a measure of the enzyme's efficiency in preventing microbial growth, eliminating microbial cells and/or performing catalysis of a hydrolysis reaction over a range of temperatures at given conditions such as pH and solvent composition. A lysozyme has a specific temperature range wherein the polypeptide is stable and retains its enzymatic activity, outside this range the lysozyme becomes less active and potentially also less stable. Within the temperature range there generally is a temperature optimum where the lysozyme shows the highest activity.
When the alteration in the temperature-dependent activity profile is towards higher temperatures, a more thermoactive or thermophilic lysozyme is generated. By improving the activity of the lysozyme at higher temperatures, it will be able to function under conditions that require higher temperatures (e.g. from 45° C. to 110° C., preferably from 50° C. to 100° C., more preferably from 60° C. to 90° C., even more preferably from 70° C. to 80° C.), for example a disinfection or sterilization process. Furthermore, the initial rate of a reaction catalyzed by a lysozyme can be accelerated by an increase in temperature that is measured by determining thermal activity of a variant. A more thermoactive variant will lead to an increase in the rate of hydrolysis decreasing the time required and/or decreasing the enzyme concentration required for preventing microbial growth, eliminating microbial cells and/or hydrolysis when used at a temperature above the optimum temperature of the parent lysozyme.
When the alteration in the temperature-dependent activity profile is towards lower or moderate temperatures, a lysozyme with improved activity at low temperatures (e.g. from 0° C. to 20° C., preferably from 2° C. to 18° C., preferably from 5° C. to 15° C., more preferably from 8° C. to 12° C., even most preferably from 10° C. to 15° C.) or moderate temperatures (e.g. from 15° C. to 45° C., preferably from 20° C. to 40° C., preferably from 22° C. to 35° C., most preferably from 25° C. to 30° C.,) is generated. A lysozyme with increased activity at lower or moderate temperatures will prevent microbial growth, eliminate microbial cells and/or catalyze a hydrolysis reaction faster than the parent enzyme at a temperature lower than the temperature optimum of the parent enzyme defined by the temperature-dependent activity profile of the parent. Such an enzyme may for example be advantageous at washing conditions at decreased temperatures such as 5° C. to 40° C., preferably 10° C. to 30° C., more preferably from 15° C. to 25° C., even more preferably from 18° C. to 20° C. It can also be advantageous in the processing of heat-sensitive food such as lactic products, juice products or wine, or for biomediation of soils and waste water during winter in temperate countries. Another example of the utility of a lysozyme with increased activity at low temperatures is for treating cold water fish species e.g salmon and trout; such a water treatment in fish breeding plants.
In one aspect of the present invention the alteration of the temperature-dependent activity profile of a lysozyme variant improves the activity of the variant at low or moderate temperature. Preferably, the activity of the lysozyme variant is compared with the activity of the parent lysozyme at a temperature of 5° C. below the optimum temperature of the parent enzyme, preferably 10° C., more preferably 15° C. 20° C., 25° C., 30° C., 35° C., 40° C., even more preferred 45° C. and most preferred 50° C. below the optimum temperature of the parent enzyme, under which conditions the variant has an activity which is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold and even most preferably at least 20-fold higher than that of the parent enzyme. Preferably, the lysozyme variant at the same time maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, 90%, more preferably at least 95%, even more preferably at least 100% of the activity that parent lysozyme exhibits at its temperature optimum. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the temperature to the desired decreased temperature.
In one aspect of the present invention the alteration of the temperature-dependent activity profile of a lysozyme variant improves the activity of the variant at high temperatures. Preferably, the activity of the lysozyme variant is compared with the activity of the parent lysozyme at a temperature of 5° C. above the optimum temperature of the parent enzyme, preferably 10° C., more preferably 15° C., 20° C., 25° C., 30° C., 35° C. or 40° C., even more preferred 45° C. and most preferred 50° C. above the optimum temperature of the parent enzyme, under which conditions the variant has an activity which is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold and even most preferably at least 20-fold higher than that of the parent enzyme. Preferably, the lysozyme variant at the same time maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, 90%, more preferably at least 95%, even more preferably at least 100% of the activity that parent lysozyme exhibits at its temperature optimum. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the temperature to the desired increased temperature.
Improved temperature stability: The term “temperature stability” or “improved thermostability” is defined herein as a variant enzyme displaying retention of enzymatic activity after a period of incubation at elevated temperature relative to the parent enzyme. Such a variant may or may not display an altered thermal activity profile relative to the parent. For example, the variant may not be active at the elevated temperatures, but is able to maintain its three dimensional structure and then regain activity once it is returned to lower temperatures. Alternatively, the variant may have an improved ability to refold following incubation at elevated temperature relative to the parent enzyme.
In one aspect, the thermal stability of a lysozyme variant is improved such that the variant can survive high temperatures e.g. temperatures from 45° C. to 110° C., preferably from 50° C. to 100° C., more preferably from 60° C. to 90° C., even more preferably from 70° C. to 80° C. Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, more preferably at least 90%, even more preferably at least 95% residual activity after incubation at a given high temperature for 1 hour when compared to the variant which has been maintained at room temperature for the same time. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme which has been treated under the same conditions. Preferably the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the temperature to the desired increased temperature.
Improved pH activity: The term “improved pH activity” is defined herein as a variant enzyme displaying an alteration of the pH-dependent activity profile when compared to the pH activity profile of the parent lysozyme. The pH activity profile provides a measure of the enzyme's efficiency in preventing microbial growth, eliminating microbial cells and/or performing catalysis of a hydrolysis reaction over a pH range at given conditions such as temperature and solvent composition. A lysozyme has a specific pH range wherein the polypeptide is stable and retains its enzymatic activity, outside this range the lysozyme becomes less active and potentially also less stable. Within the pH range there generally is a pH optimum, where the lysozyme shows the highest activity.
A lysozyme variant with improved activity at alkaline pH (e.g. from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5) will be able to function in more alkaline environments such as detergents.
A variant with improved activity at acidic pH (e.g. from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4) will be able to function under more acidic conditions, such as preservative in certain foods or as a eubiotic molecule in feeds.
In one aspect, the pH activity profile is altered such that a lysozyme variant has improved activity at alkaline pH. Preferably, the activity of the lysozyme variant is compared with the activity of the parent lysozyme at a pH at least 0.5 units above the optimum pH of the parent enzyme, preferably 1, 1.5, 2, 2.5 or 3 pH units above the optimum pH of the parent enzyme, most preferably at least 3.5 pH units above the optimum pH of the parent enzyme and most preferably at least 4 pH units above the optimum pH of the parent enzyme, under which conditions the variant has an activity which is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold and even most preferably at least 20-fold higher than that of the parent enzyme. Preferably, the lysozyme variant at the same time maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, or 90%, more preferably at least 95%, even more preferably at least 100% of the activity that parent lysozyme exhibits at its pH optimum. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the pH to the desired increased pH.
In one aspect, the pH activity profile is altered such that a lysozyme variant has improved activity at acidic pH. Preferably, the activity of the lysozyme variant is compared with the activity of the parent lysozyme at a pH at least 0.5 units below the optimum pH of the parent enzyme, preferably 1, 1.5, 2, 2.5 or 3 pH units below the optimum pH of the parent enzyme, most preferably at least 3.5 pH units below the optimum pH of the parent enzyme and most preferably at least 4 pH units below the optimum pH of the parent enzyme, under which conditions the variant has an activity which is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold and even most preferably at least 20-fold higher than that of the parent enzyme. Preferably, the lysozyme variant at the same time maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, or 90%, more preferably at least 95%, even more preferably at least 100% of the activity that parent lysozyme exhibits at its pH optimum. Preferably the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the pH to the desired decreased pH.
Improved pH stability: The term “improved pH stability” is defined herein as a variant enzyme displaying structural stability relative to the parent enzyme after a period of incubation at a pH which is outside the pH range where the enzyme is active (pH activity range). Such a variant may or may not display an altered pH activity profile relative to the parent. For example, the variant may not be active at the increased or decreased pH, but is able to maintain its three dimensional structure and then regain activity once it is returned to the pH activity range. Alternatively, the variant may have an improved ability to refold relative to the parent following incubation at increased or decreased pH.
In one aspect, the pH stability profile is altered such that a lysozyme variant has improved stability at acidic pH (e.g. from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4). Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, more preferably at least 90%, even more preferably at least 95% residual activity after incubation at a given pH for 1 hour when compared to the variant which has been maintained at pH 6.5 for the same time. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme which has been treated under the same conditions. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the pH to the desired decreased pH.
In one aspect, the pH stability profile is altered such that a lysozyme variant has improved stability at alkaline pH (e.g. from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5). Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, more preferably at least 90%, even more preferably at least 95% residual activity after incubation at a given pH for 1 hour when compared to the variant which has been maintained at pH 6.5 for the same time. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme which has been treated under the same conditions. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the pH to the desired increased pH.
Improved protease stability: The term “improved protease stability” is defined herein as a variant enzyme displaying structural stability relative to the parent enzyme after a period of incubation with a protease (e.g. a pepsin or a serine-proteases such as trypsin or chymotrypsin from the digestive system of an animal or human) and/or bile salts. Preferably, the protease stable variant is also stable at acidic pH (e.g. from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4). Such a variant may or may not display an altered activity profile relative to the parent. The variant may for example have an improved ability to refold relative to the parent following incubation with proteases and/or incubation at a decreased pH. With respect to the decreased pH, the variant may not be active during the incubation at the decreased pH, but is able to maintain its three dimensional structure and then regain activity once it is returned to the pH activity range.
In one aspect, the protease stability is improved such that a lysozyme variant has improved stability following incubation with a protease and/or a bile salt. Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, more preferably at least 90%, even more preferably at least 95% residual activity after incubation with a serine-protease and/or bile salt for 1 hour when compared to the variant which has been incubated without protease and/or bile salt for the same time. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme which has been treated under the same conditions. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation that serine-protease, pepsin and/or bile salts are added.
In a further aspect, the protease stability is improved as described above and the pH stability profile is altered such that a lysozyme variant has improved stability at acidic pH (e.g. from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4). Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70% or 80%, more preferably at least 90%, even more preferably at least 95% residual activity after incubation at a given pH with a serine-protease and/or bile salt for 1 hour when compared to the variant which has been maintained at pH 6.5 without protease and/or bile salts for the same time. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme which has been treated under the same conditions. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the “Materials and Methods” section, with the deviation of the pH to the desired decreased pH and serine-protease, pepsin and/or bile salts is added.
Conventions for Designation of Variants
For purposes of the present invention, the amino acid sequence of the lysozyme disclosed in SEQ ID NO:3 is used to determine the corresponding amino acid residue in another lysozyme. The amino acid sequence of another lysozyme is aligned with the amino acid sequence of the lysozyme disclosed in SEQ ID NO:3, and based on the alignment the amino acid position number corresponding to any amino acid residue in the amino acid sequence of the lysozyme disclosed in SEQ ID NO:3 can be determined.
An alignment of polypeptide sequences may be made using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) Trends in Genetics 16:276-277; http://emboss.org), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
In describing the various lysozyme variants of the present invention, the nomenclature described below is adapted for ease of reference. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.
The alteration(s) are an insertion and/or deletion of the amino acid which occupies the position, and/or a substitution of the amino acid which occupies the position with a different amino acid.
Substitutions. For an amino acid substitution, the following nomenclature is used: original amino acid,/position/substituted amino acid. Accordingly, the substitution of threonine with alanine at position 226 is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”), e.g., “G205R+S411F”, representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively. Where an original amino acid may be substituted by an amino acid selected from a group it is designated as “K129R,S,A,I,F,Q” representing the substitution of a lysine (K) at position 129 with an amino acid selected from the group consisting of: arginine (R), serine (S), alanine (A), isoleucine (I), phenylalanine (F) and glutamine (Q). Alternatively, “K129R,S,A,I,F,Q” could be written as K129R or K129S, or K129A, or K129I or K129F or K129Q
Deletions. For an amino acid deletion, the following nomenclature is used: original amino acid/position/asterisk (*). Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”), e.g. G195*+S411*.
Insertions. For an amino acid insertion, the following nomenclature is used: asterisk (*)/position/lower case letter/inserted amino acid, where the lower case letter indicates the addition of an amino acid downstream of the position number. Accordingly, the insertion of a glutamic acid (E) downstream of position 10 is designated “*10aE”. If a second amino acid, e.g. a valine (V), is to be inserted downstream of position 10 after the glutamic acid (E) it is designated “*10aE+*10bV”. Additions to the N-terminal of the polypeptide are designated with a 0 (zero). The addition of a glutamic acid (E) and a valine (V) added to the N-terminal amino acid of a polypeptide is designated as “*0aE+*0bV”. A “downstream” insertion can also be described as the addition of one or more amino acids between the named position and the position immediately following the named position, e.g. an insertion downstream of position 195 results in the addition of one or more amino acids between position 195 and 196, thereby generating new positions *195a, *195b and so forth.
Parent Lysozymes
In the present invention, the parent lysozyme is either a lysozyme belonging to family 25 of glycosyl hydrolases also termed “family 25 lysozymes”, preferably a fungal family 25 lysozyme. Family 25 of glycosyl hydrolases or lysozymes is defined herein as a polypeptide falling into the glycoside hydrolase family 25 according to Henrissat B. (1991) Biochem. J. 280:309-316; and Henrissat B., and Bairoch A. (1996) Biochem. J. 316:695-696.
Examples or parent lysozymes include lysozymes derived from Aspergillus or Penicillium, such as, Aspergillus fumigatus, Aspergillus fischerianus, Aspergillus clavatus, Aspgerillus oryzae, Aspergillus terreus, Penicillum marneffei. Specific examples of lysozymes include the Aspergillus fumigatus lysozyme (SEQ ID NO:3); Aspergillus fumigatus lysozyme (SEQ ID NO:4); Aspergillus fischerianus lysozyme (SEQ ID NO:5); Aspergillus clavatus lysozyme (SEQ ID NO:6); Aspergillus oryzae lysozyme (SEQ ID NO:7); Aspergillus terreus lysozyme (SEQ ID NO:8); Aspergillus fischerianus lysozyme (SEQ ID NO:9); (SEQ ID NO:10); Aspergillus fumigatus lysozyme Aspergillus clavatus lysozyme (SEQ ID NO:11); Aspergillus terreus lysozyme (SEQ ID NO:12); and Penicillum marneffei lysozyme (SEQ ID NO:13). An alignment of these lysozyme amino acid sequences is shown in FIG. 1.
A representative structure of the family 25 lysozymes is provided in the atomic coordinates attached as FIG. 2 for the Aspergillus fumigatus lysozymes of SEQ ID NO:3.
In one embodiment a GH25 lysozyme is defined as a polypeptide comprising the following motifs:
[GA]-X-Y-[HF]-[FY]-X(6,15)-[QED]-[AV]-X(2,5)-[FYW]-X(8,17)-[PKMYASRVLI]-X(2)-[VLI]-D-X-E,
wherein the standard IUPAC one-letter codes for the amino acids are used. The symbol “X” is used for a position where any amino acid is accepted. Ambiguities are indicated by listing the acceptable amino acids for a given position, between square brackets “[ ]”. For example stands [QED] for Gln or Glu or Asp. A gap (“X”) in the pattern is indicated by a numerical range between parentheses. For example corresponds “X(2)” to any amino acid at two contiguous positions (e.g. X-X), and X(2,5) corresponds to any amino acid from two to five contiguous positions (e.g. X-X or X-X-X or X-X-X-X or X-X-X-X-X).
In another embodiment, the parental lysozyme is (a) a polypeptide corresponding to the mature peptide of SEQ ID NO:2; or (b) a polypeptide comprising an amino acid sequence having at least at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% with the mature polypeptide of SEQ ID NO:2; or (c) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide encoded by sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a full-length complementary strand of (i) or (ii); or (d) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the mature polypeptide coding sequence of SEQ ID NO:1.
The parent lysozyme may also comprise an amino acid sequence having a degree of identity to the polypeptide of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13 of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and which amino acid sequence has antimicrobial and/or lysozyme activity (hereinafter “homologous polypeptides”). In one aspect, the homologous polypeptides have an amino acid sequence that differs by twenty amino acids, nineteen amino acids, eighteen amino acids, seventeen amino acids, sixteen amino acids, fifteen amino acids, fourteen amino acids, thirteen amino acids, twelve amino acids, eleven amino acids, ten amino acids, nine amino acids, eight amino acids, seven amino acids, six amino acids, five amino acids, four amino acids, three amino acids, two amino acids, or only one amino acid from the mature polypeptide of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13.
Substantially homologous parent lysozymes may have one or more (several) amino acid alterations such as substitutions, deletions and/or insertions. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the three-dimensional folding or activity of the protein or polypeptide; small deletions, typically of one to about 9 amino acids, one to about 15 amino acids or one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about five to ten residues, 10 to 15 residues, or 20 to 25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tag, or protein A (Nilsson et al. (1985) EMBO J. 4:1075; Nilsson et al. (1991) Methods Enzymol. 198:3. See also, in general, Ford et al. (1991) Protein Expression and Purification 2:95-107.
Although the changes described above preferably are of a minor nature, such changes may also be of a substantive nature such as fusion of larger polypeptides of up to 300 amino acids or more both as amino- or carboxyl-terminal extensions. Examples of fusion polypeptides include the addition of a binding domains and/or linker segments to the lysozymes of the present invention.
Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter specific activity are known in the art and are described, for example, by Neurath and Hill (1979) The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
Essential amino acids in the lysozyme polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells (1989) Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e. antimicrobial and/or lysozyme activity) to identify amino acid residues that are critical to the activity of the molecule. See also Hilton et al. (1996) J. Biol. Chem. 271:4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photo affinity labeling, in conjunction with mutation of putative contact site amino acids. See for example de Vos et al. (1992) Science 255:306-312; Smith et al. (1992) J. Mol. Biol. 224:899-904; Wlodaver et al. (1992) FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from analysis of homologies with polypeptides which are related to a polypeptide according to the invention. The crystal structure of the fungal GH25 from Aspergillus fumigatus (the parent lysozyme) at a resolution of 1.8 Å is included in the present invention. This is the first crystal structure of a eukaryotic representative of the lysozyme GH25 family. The structure was used as input to the Dictionary of Protein Secondary Structure (DSSP) program in order to calculate its secondary structure. The DSSP program was designed by Wolfgang Kabsch and Chris Sander to standardize secondary structure assignment. DSSP is a database of secondary structure assignments for all protein entries in the Protein Data Bank. DSSP is also the program that calculates DSSP entries from PDB entries. DSSP is described in Kabsch W. and Sander C. (1983) “Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features”, Biopolymers Dec, 22(12):2577-637.
The following are the secondary structure elements of Aspergillus fumigatus GH25 lysozyme:














beta-strands
alpha-helices
disulfide bridge




















1
β1
T11-I17
α1
F26-K31
C115-C154


2
β2
F36-E43
α2
F52-K61



3
β3
L65-F71
α3
G79-K88



4
β4
M103-D105
α4
H120-T138



5
β5
M143-T146
α5
A148-T155



6
β6
L167-A170
α6
M209-S217



7
β7
I189-S193





8
β8
D201-N206









The structure of the Aspergillus fumigatus GH25 lysozyme comprises a single domain, with the shape of a flattened ellipsoid with dimensions of 50 Å×39 Å×36 Å. The enzyme has a β/α-barrel fold, which is different from the (β/α)8-barrels found in triosephosphate isomerase (TIM) and many other enzymes (For more details see the paper by Rau A., Hogg T., Marquardt R. and Hilgenfeld R., A new lysozyme fold. Crystal structure of the muramidase from Streptomyces coelicolor at 1.65 A resolution, Journal of Biological Chemistry 2001, volume 276, pp. 31994-9). It is composed of eight β-strands and six α-helices, with the strands forming the staves of the barrel and the helices located around it. As in regular TIM barrels, the first five β-strands and α-helices alternate. However, the fifth α-helix is followed by strands β6 to β8, which are connected by loops lacking any helices. Helix α6 is located at the C terminus of the polypeptide chain, sitting at the bottom (N-terminal end) of the barrel. All β-strands are arranged parallel to one another, except strand β8 which, very unusually, is in an antiparallel orientation with respect to the other strands.
Based on the structure residues, D105 and E107 in SEQ ID NO:3 have been identified as catalytic residues. In a preferred embodiment the variant of the present invention contains aspartic acid position at 105 (using SEQ ID NO:3 numbering) and contains glutamic acid at position 107 (using SEQ ID NO:3 numbering). Furthermore, based on the structure the following residues, D16, Q191, Y145, Y69, D201, G200, G44, G67, G14, and H70 in SEQ ID NO:3, have been identified as closely related to the active site. In a further preferred embodiment the variant of the present invention contains aspartic acid at position 16, glutamine at position 191, tyrosine at position 145, tyrosine at position 69, aspartic acid at position 201, glycine at position 200, glycine at position 44, glycine at position 67, glycine at position 14 and/or histidine at position 70 (using SEQ ID NO:3 numbering).
The parent lysozyme preferably comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13 or an allelic variant thereof, or a fragment thereof having antimicrobial and/or lysozyme activity. In one aspect, the parent lysozyme comprises the amino acid sequence of SEQ ID NO:2. In another aspect, the parent lysozyme comprises the mature polypeptide of SEQ ID NO:2. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:3 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:4 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:5 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:6 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:7 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:8 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:9 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:10 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:11 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:12 or a mature polypeptide thereof. In another aspect, the parent lysozyme comprises the polypeptide of SEQ ID NO:13 or a mature polypeptide thereof. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:3 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:4 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:5 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:6 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:7 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:8 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:9 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:10 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:11 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:12 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect, the parent lysozyme consists of the amino acid sequence of SEQ ID NO:13 or an allelic variant thereof; or a fragment thereof having antimicrobial and/or lysozyme activity. In another aspect the parent lysozyme comprises an amino acid sequence which is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13. A fragment of polypeptide is a polypeptide having one or more (several) amino acids deleted from the amino- and/or carboxyl-terminus of this amino acid sequence and still maintaining antimicrobial and/or lysozyme activity.
The parent lysozymes may in another aspect be encoded by polynucleotides that hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor, N.Y.). The subsequence may encode a polypeptide fragment having antimicrobial and/or lysozyme activity. In one aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO:1.
A subsequence of the mature polypeptide coding sequence of SEQ ID NO:1, or a homolog thereof, is a nucleotide sequence where one or more (several) nucleotides have been deleted from the 5′- and/or 3′-end, where the polypeptide encoded by the subsequence possess antimicrobial and/or lysozyme activity.
The parent enzymes may also be allelic variants of the polypeptides that have antimicrobial and/or lysozyme activity.
The polynucleotide of SEQ ID NO:1, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:13, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding parent lysozymes from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, at least 25, at least 35, or at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, or at least 500 nucleotides in length. Even longer probes may be used, e.g. nucleic acid probes that are at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides in length, at least 1000 nucleotides in length, at least 1100 nucleotides in length, at least 1200 nucleotides in length, at least 1300 nucleotides in length, at least 1400 nucleotides in length, at least 1500 nucleotides in length or at least 1600 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA library prepared from other organisms may be screened for DNA that hybridizes with the probes described above and encodes a parent lysozyme. Genomic or other DNA from other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO:1, or a subsequence thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleotide probe corresponding to the polynucleotide shown in SEQ ID NO:1, its complementary strand, or a subsequence thereof, under low to very high stringency conditions. Molecules to which the probe hybridizes can be detected using, for example, X-ray film or any other detection means known in the art.
In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO:1. In another aspect, the nucleic acid probe is nucleotides 51 to 705 of SEQ ID NO:1, or nucleotides 75 to 705 of SEQ ID NO:1. In another aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO:2, or a subsequence thereof. In another aspect, the nucleic acid probe is SEQ ID NO:1.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).
For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (see Bolton and McCarthy (1962) Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.
For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
In a third aspect, the parent lysozyme is encoded by a polynucleotide comprising or consisting of a nucleotide sequence having a degree of identity to the mature polypeptide coding sequence of SEQ ID NO:1 of preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably 96%, 97%, 98%, or 99%, which encode an active polypeptide. In one aspect, the mature polypeptide coding sequence is nucleotides 82 to 1653 of SEQ ID NO:1, or nucleotides 97 to 1653 of SEQ ID NO:1.
The parent lysozyme may be obtained from microorganisms of any genus. In one aspect, the parent lysozyme is secreted extracellularly.
In one embodiment, the parent lysozyme is a fungal lysozyme. Examples or parent fungal lysozymes include lysozymes derived from Aspergillus or Penicillium, such as, Aspergillus fumigatus, Aspergillus fischerianus, Aspergillus clavatus, Aspgerillus oryzae, Aspergillus terreus, Penicillum marneffei. Specific examples of parent fungal lysozymes include the Aspergillus fumigatus lysozyme (SEQ ID NO:3); Aspergillus fumigatus lysozyme (SEQ ID NO:4); Aspergillus fischerianus lysozyme (SEQ ID NO:5); Aspergillus clavatus lysozyme (SEQ ID NO:6); Aspergillus oryzae lysozyme (SEQ ID NO:7); Aspergillus terreus lysozyme (SEQ ID NO:8); Aspergillus fischerianus lysozyme (SEQ ID NO:9); (SEQ ID NO:10); Aspergillus fumigatus lysozyme Aspergillus clavtus lysozyme (SEQ ID NO:11); Aspergillus terrus lysozyme (SEQ ID NO:12); and Penicillum marneffei lysozyme (SEQ ID NO:13).
In a further aspect, the parent lysozyme may be a bacterial lysozyme. For example, the lysozyme may be a Gram positive bacterial polypeptide such as a Bacillus, preferably from the Bacillus anthracis BA_GH25C (Martinez-Fleites et al. (2009) Carbohydr Res, 344(13):1753-1757). Other parent bacterial lysozymes may be Streptomyces coelicolor cellosyl (Rau et al. (2001) J Biol Chem, 276(34):31994-31999), the bacteriophage lysin PIyB (Porter et al. (2007) J Mol Biol, 366(2):540-550), or Clp-1 lysozyme from a Streptococcus pneumoniae phage (Perez-Dorado et al. (2007) J Biol Chem, 282(34):24990-24999).
Generation of Variants
Variants of a parent lysozyme can be prepared according to any mutagenesis procedure known in the art, such as random and/or site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide molecule of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (Tian et al., Nature 432:1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.
Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide fragments may then be shuffled.
Site-directed mutagenesis is a technique in which one or several mutations are created at a defined site in a polynucleotide molecule encoding the parent lysozyme. The technique can be performed in vitro or in vivo.
Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent lysozyme and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another. For further description of suitable techniques reference is made to: Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) Current protocols in Molecular Biology, John Wiley and Sons (1995); Harwood, C. R., and Cutting, S. M. (eds.) Molecular Biological Methods for Bacillus, John Wiley and Sons (1990); WO 96/34946; Scherer and Davis (1979) Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al. (1990) Nucleic Acids Research 18:7349-4966.
After the ligase reaction the ligation mixture may be used to transform a host cell, for cloning purposes E. coli cells are often used as described in Ausubel, F. M. et al. The transformed E. coli cells can be propagated in liquid media or on solid agar plates, plasmids can be rescued from the transformed cells and used to transform B. subtilis cells. Suitable competent Bacillus cells, such as MB1510, a 168-derivative (e.g. available from BGSC with accession no. 1A1 168 trpC2), may be transformed as described in WO 03/095658. An E. coli plasmid-borne integration cassette for library construction may be used for Bacillus transformation. The method is described in detail in WO 03/095658. Alternatively, an in vitro amplified PCR-SOE-product (Melnikov and Youngman, Nucleic Acid Research 27:1056) may be used.
Site-directed mutagenesis can be accomplished in vivo by methods known in the art (see for example U.S. Patent Application Publication 2004/0171154; Storici et al. (2001) Nature Biotechnology 19: 773-776; Kren et al. (1998) Nat. Med. 4: 285-290; and Calissano and Macino (1996) Fungal Genet. Newslett. 43: 15-16).
Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants of parent lysozymes.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer (1988) Science 241:53-57; Bowie and Sauer (1989) Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g. Lowman et al. (1991) Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al. (1986) Gene 46:145; Ner et al. (1988) DNA 7:127).
Mutagenesis/shuffling methods as described above can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells, e.g. Bacillus as described above. Mutagenized DNA molecules that encode polypeptides with antimicrobial and/or lysozyme activity can be recovered from the host cells and rapidly sequenced using standard methods in the art.
Variants
The isolated variants of the present invention comprise an alteration of an amino acid at one or more (several) positions selected from the group consisting of positions number 47, 111, 108, 45, 22, 110, 120, 147, 196, 49, 55, 193 161, 128, 131, 95, 203, 98, 112, 55, 32, 89, 206 121, 120, 185, 186, 176, 113, 122, 119, 35, 65, 139, 141, 153, 158, 171, 195, 76, 164, 30, 85, 178, 183, 186, 112, 174, 187, 197, 102, 134, 108, 196, 197, 198, 56, 19, 120, 20, 135, and 203, wherein the variant has antimicrobial and/or lysozyme activity. The numbering of the positions is relative to the amino acid sequence of SEQ ID NO:3.
The isolated variants of the present invention comprise an alteration of an amino acid at one or more (several) positions selected from the group consisting of positions number 47, 111, 108, 45, 22, 110, 120, 147, 196, 49, 55, 193, 161, 128, 131, 203, 98, 112, 55, 32, 89, 206 121, 120, 185, 113, 119, 35, 153, 158, 171, 195, 76, 164, 30, 85, 178, 183, 186, 112, 174, 187, 197, 102, 134, 108, 196, 197, 198, 56, 19, 120, 20, 135, and 203 wherein the variant has antimicrobial and/or lysozyme activity. The numbering of the positions is relative to the amino acid sequence of SEQ ID NO:3.
In one embodiment, the variants described above comprise an additional amino acid alteration at one or more positions selected from the group consisting of position number 95, 186, 65, 122, 139, 141 and 176.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 19 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 19 with Asn. For example, the isolated variants of the present invention comprise or consist of an alteration of N19D.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 20 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 20 with His or Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of H20W or H20Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 22 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 22 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of K22G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 30 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 30 with Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of K30Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 32 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 32 with Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of D32S.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 35 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 35 with Arg. For example, the isolated variants of the present invention comprise or consist of an alteration of Q35R.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 45 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 45 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of T45G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 47 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 47 with Phe. For example, the isolated variants of the present invention comprise or consist of an alteration of Y47F.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 49 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 49 with Ala. For example, the isolated variants of the present invention comprise or consist of an alteration of D49A.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 55 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 55 with Ala or Asn. For example, the isolated variants of the present invention comprise or consist of an alteration of H55A or H55N.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 56 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 56 with Trp. For example, the isolated variants of the present invention comprise or consist of an alteration of Y56W.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 65 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 65 with ILe. For example, the isolated variants of the present invention comprise or consist of an alteration of L65I.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 76 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 76 with Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of K76S.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 85 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 85 with Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of K85Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 89 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 89 with Thr. For example, the isolated variants of the present invention comprise or consist of an alteration of N89T.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 95 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 95 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of D95G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 98 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 98 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of R98G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 102 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 102 with Pro. For example, the isolated variants of the present invention comprise or consist of an alteration of G102P. The variant may also further comprise an alteration of Y134V.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 108 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 108 with Phe or Trp. For example, the isolated variants of the present invention comprise or consist of an alteration of Y108F or Y108W.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 110 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 110 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of P110G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 111 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 111 with Phe. For example, the isolated variants of the present invention comprise or consist of an alteration of Y111F.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 112 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 112 with Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of G112S.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 113 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 113 with Pro. For example, the isolated variants of the present invention comprise or consist of an alteration of A113P.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 119 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 119 with Asn. For example, the isolated variants of the present invention comprise or consist of an alteration of S119N.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 120 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 120 with Ala, Gln or Pro. For example, the isolated variants of the present invention comprise or consist of an alteration of H120A, H120Q or H120P.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 121 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 121 with Ala. For example, the isolated variants of the present invention comprise or consist of an alteration of S121A.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 122 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 122 with Ala. For example, the isolated variants of the present invention comprise or consist of an alteration of Q122A.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 128 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 128 with Arg. For example, the isolated variants of the present invention comprise or consist of an alteration of H128R.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 131 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 131 with Cys. For example, the isolated variants of the present invention comprise or consist of an alteration of V131C.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 134 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 134 with Val. For example, the isolated variants of the present invention comprise or consist of an alteration of Y134V. The variant may also further comprise an alteration of G102G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 135 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 135 with Asn. For example, the isolated variants of the present invention comprise or consist of an alteration of H135N.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 139 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 139 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of S139G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 141 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 141 with Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of W141Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 147 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 147 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of T147G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 153 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 153 with Thr. For example, the isolated variants of the present invention comprise or consist of an alteration of R153T.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 158 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 158 with Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of A158S.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 161 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 161 with Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of F161Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 164 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 164 with Thr. For example, the isolated variants of the present invention comprise or consist of an alteration of K164T.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 171 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 171 with Arg. For example, the isolated variants of the present invention comprise or consist of an alteration of A171R.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 176 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 176 with Val. For example, the isolated variants of the present invention comprise or consist of an alteration of P176V.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 178 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 178 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of K178G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 183 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 183 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of D183G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 185 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 185 with Pro. For example, the isolated variants of the present invention comprise or consist of an alteration of K185P.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 186 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 186 with Tyr. For example, the isolated variants of the present invention comprise or consist of an alteration of T186Y.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 193 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 193 with Ala. For example, the isolated variants of the present invention comprise or consist of an alteration of S193A.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 195 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 195 with Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of K195S.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 196 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 196 with Gly. For example, the isolated variants of the present invention comprise or consist of an alteration of Y196G. The variant may further comprise an alteration of K197P.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 197 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 197 with Pro. For example, the isolated variants of the present invention comprise or consist of an alteration of K197P. The variant may further comprise an alteration of Y196G.
In one embodiment, the isolated variant of the present invention comprises or consists of an alteration at position 198 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 198 with Asn or Phe. For example, the isolated variants of the present invention comprise or consist of an alteration of H198N or H198F.
In one embodiment, the isolated variant of the present invention comprise or consists of an alteration at position 203 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 203 with Asn. For example, the isolated variants of the present invention comprise or consist of an alteration of D203N.
In one embodiment, the isolated variants of the present invention comprises or consists of an alteration at position 206 (using SEQ ID NO:3 for numbering). In an embodiment the alteration is a substitution of the amino acid at position 19 with Ala or Ser. For example, the isolated variants of the present invention comprise or consist of an alteration of N206A or N206S.
In one embodiment, the isolated variants of the present invention comprises an amino acid sequence having a degree of amino acid sequence identity of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of a lysozyme belonging to family 25 of glycosyl hydrolases, preferably a fungal family 25 lysozyme.
In another embodiment, the isolated variants of the present invention comprise an amino acid sequence having a degree of amino acid sequence identity of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of a polypeptide corresponding to the mature peptide of SEQ ID NO:2.
In another embodiment, the variants comprise an amino acid sequence having a degree of amino acid sequence identity of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:3.
In another embodiment, the isolated variants of the present invention comprise an amino acid sequence of a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide encoded by sequence of SEQ ID NO:1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) a full-length complementary strand of (i) or (ii).
In another embodiment, the isolated variants of the present invention comprise a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with the mature polypeptide coding sequence of SEQ ID NO:1.
In an embodiment, the isolated variants further comprise aspartic acid at position 105 and glutamic acid at position 107 (using SEQ ID NO:3 for numbering). In another embodiment, the isolated variants further comprise aspartic acid at position 16, glutamine at position 191, tyrosine at position 145, tyrosine at position 69, aspartic acid at position 201, glycine at position 200, glycine at position 44, glycine at position 67, glycine at position 14 and/or histidine at position 70 (using SEQ ID NO:3 numbering).
In an embodiment, the present invention provides lysozyme variants having altered properties relative to the parent lysozyme. In one embodiment, the lysozyme variant has improved pH stability and/or activity at alkaline pH conditions (such as from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5), as compared to the parent lysozyme. Accordingly, the present invention provides a variant of a parent lysozyme, wherein the variant comprises or consists of an alteration at one or more positions selected from the group consisting of position number 102, 134, 108, 196, 197, 198, 56, 19, 120, 20, 135, and/or 203 (using SEQ ID NO:3 for numbering), and wherein the variant has increased stability and/or activity at alkaline pH as compared to the parent lysozyme. The following lysozyme variants are constructed to have improved pH stability and/or activity at an alkaline pH conditions (such as pH from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5) compared to the parent enzyme (using SEQ ID NO:3 for numbering):















G102P;
Y134V;



G102P;
T108W;



T196G;
K197G;



Y196G;
K197P;



Y56W;
N19D;



H120Q;
H198N;



H198F;
H20W;



H20Y; and/or
H135N.









The following combination of alterations is constructed to have improved pH stability and/or activity at alkaline pH conditions (from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5) compared to the parent enzyme (using SEQ ID NO:3 for numbering): G102P and Y134V and/or Y196G and K197P.
In one embodiment, the lysozyme variant has improved activity at alkaline pH conditions (such as, from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5), as compared to the parent lysozyme. An example of a lysozyme variant constructed to have improved activity at an alkaline pH (such as pH from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, even more preferably from 9 to 9.5) compared to the parent enzyme is D203N (using SEQ ID NO:3 for numbering).
In one embodiment, the lysozyme variant has improved pH stability at acidic pH conditions (such as, from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4), as compared to the parent lysozyme. Accordingly, the present invention provides a variant of a patent lysozyme, wherein the variant comprises or consists of an alteration at one or more positions selected from the group consisting of position number 161, 128, 131, 95, 203, 98, 112, 55, 32, 89, and/or 206 (using SEQ ID NO:3 for numbering), and wherein the variant has increased activity at acidic pH conditions as compared to the parent lysozyme. The following lysozyme variants are constructed to have improved pH stability at an acidic pH (such as from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4) compared to the parent enzyme (using SEQ ID NO:3 for numbering):















F161Y;
H128R;



V131C;
D95G;



D203N;
R98G;



G112S;
H55N;



D32S;
N89T;



N206A; and/or
N206S.









In one embodiment, the lysozyme variant has improved stability under conditions of acidic pH (such as, from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4), and in the presence of gastric proteases as compared to the parent lysozyme. In this connection gastric proteases is intended to mean proteases excreted by the organism into the gastrointestinal tract during digestion, such as trypsin, chymotrypsin and pepsin; or externally added proteases, such as proteases added to the feed e.g. the commercially available protease sold under the name of ProAct© (Novozymes A/S), or any mixtures thereof. Accordingly, the present invention provides a variant of a patent lysozyme, wherein the variant comprises or consists of an alteration at one or more positions selected from the group consisting of position number 56, 131 and/or 161 (using SEQ ID NO:3 for numbering), and wherein the variant has increased stability at acidic pH conditions in the presence of one or more gastric proteases compared to the parent lysozyme. The lysozyme variants constructed to have improved stability at an acidic pH (such as from 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, even more preferably from 3.5 to 4) in the presence of one or more gastric proteases (such as trypsin, chymotrypsin, pepsin and any mixtures thereof) compared to the parent enzyme includes variants comprising following substitutions (using SEQ ID NO:3 for numbering):















Y56W;
V131C and/or



F161C.









In one embodiment, the lysozyme variant has improved activity at low temperatures (e.g. from 0° C. to 20° C., preferably from 2° C. to 18° C., preferably from 5° C. to 15° C., more preferably from 8° C. to 12° C., even most preferably from 10° C. to 15° C.) or moderate temperatures (e.g. from 15° C. to 45° C., preferably from 20° C. to 40° C., preferably from 22° C. to 35° C., most preferably from 25° C. to 30° C.,), as compared to the parent lysozyme. Accordingly, the present invention provides a variant of a parent lysozyme, wherein the variant comprises or consists of an alteration at one or more positions selected from the group consisting of position number 47, 111, 108, 45, 22, 110, 120, 147, 196, 49, 55 and/or 193, and wherein the variant has improved activity at a low temperature as compared to the parent lysozyme. The following variants are constructed to have improved activity at low temperatures (e.g. from 0° C. to 20° C., preferably from 2° C. to 18° C., preferably from 5° C. to 15° C., more preferably from 8° C. to 12° C., even most preferably from 10° C. to 15° C.) or moderate temperatures (e.g. from 15° C. to 45° C., preferably from 20° C. to 40° C., preferably from 22° C. to 35° C., most preferably from 25° C. to 30° C.), as compared to the parent lysozyme (using SEQ ID NO:3 for numbering):















Y47F;
Y111F;



Y108F;
T45G;



K22G;
P110G;



H120A;
T147G;



Y196G;
D49A;



H55A; and/or
S193A.









In one embodiment, the lysozyme variant has improved thermostability (such as, a temperature from 45° C. to 110° C., preferably from 50° C. to 100° C., more preferably from 60° C. to 90° C., even more preferably from 70° C. to 80° C.,), as compared to the parent lysozyme. Accordingly, the present invention provides a variant of a parent lysozyme, wherein the variant comprises or consists of an alteration at one or more positions selected from the group consisting of position number 121, 120, 185, 186, 176, 113, 122, 119, 35, 65, 139, 141, 153, 158, 171, 195, 76, 164, 30, 85, 178, 183, 186, 112, and/or 197 and wherein the variant has improved thermostability as compared to the parent lysozyme. The following variants are constructed to have improved thermostability (such as, a temperature from 45° C. to 110° C., preferably from 50° C. to 100° C., more preferably from 60° C. to 90° C., even more preferably from 70° C. to 80° C.), as compared to the parent lysozyme (using SEQ ID NO:3 for numbering):















S121A;
H120P;



T186T;
A113P;



K185P;
P176V;



Q122A;
S119N;



Q35R;
L65I;



S139G;
W141Y;



R153T;
A158S;



A171R;
K195S;



K76S;
K164T;



K30Y;
F85Y;



K178C;
D183G;



Y186Y;
G112S; and/or



K197P.









Preferred variants having improved thermostability compared to the parent lysozyme include variants comprising following substitutions (Using SEQ ID NO: for numbering):















N19D;
H20Y;



K22G;
T45G;



Y47F;
D49G;



H55A;
R98G;



A158S and/or
K164T.









The variants comprising alterations at one or more of the above identified positions have an increased stability in detergent, preferably in liquid detergent as compared to the parent lysozyme.
The lysosyme variants of the present invention have antimicrobial activity and/or lysozyme activity. Lysozyme variants which are not capable of catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins may still have an antimicrobial effect since such inactive lysozyme variants can bind to the surface of the microorganism and potentially inhibit its growth. Such lysozyme variants may also be termed “bacteriostatics”.
It is understood that the embodiment described above can be combined if desired, especially that the indicated positions and specific substitutions may be combined with the sequence identities of the variants.
Polynucleotides
The present invention also relates to isolated polynucleotides that encode variants of a parent lysozyme according to the present invention. In particular polynucleotides that encode a lysozyme variant as described in the variant section above, is encompassed by the present invention. Polynucleotides of the invention will hybridize to a denatured double-stranded DNA probe comprising either the full variant sequence corresponding to positions 1 to 705 of SEQ ID NO:1 or position 51 to 705 of SEQ ID NO:1 or 75 to 705 of SEQ ID NO:1 with proper sequence alterations corresponding to actual amino acid alterations in the variant or any probe comprising a variant subsequence thereof having a length of at least about 100 base pairs under at least medium stringency conditions, but preferably at high stringency conditions. The variant polynucleotides of the present invention may also comprise silent mutations in addition to the mutations giving rise to the amino acid alterations described in the variant section above. Silent mutations are mutations in the three-letter code which do not give rise to a change in the amino acid, e.g. GTT to GAT, which both code for valine.
The polynucleotides encoding the lysozyme variants of the present invention include DNA and RNA. Methods for isolating DNA and RNA are well known in the art. DNA and RNA encoding genes of interest can be cloned in Gene Banks or DNA libraries by means of methods known in the art.
Polynucleotides encoding polypeptides having antimicrobial and/or lysozyme activity of the invention are then identified and isolated by, for example, hybridization or PCR.
Expression Vectors
The present invention also relates to expression vectors, in particular recombinant expression vectors, comprising a nucleic acid construct of the invention. Nucleic acid constructs of the invention comprise an isolated polynucleotide encoding a variant lysozyme of the present invention, preferably operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The control sequences may either be provided by the vector or by the nucleic acid construct inserted into the vector.
The control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Such promoters are well known in the art. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention; such terminators are well known in the art. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention, such leader sequences are well known in the art. The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention. The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
An isolated polynucleotide encoding a variant lysozyme of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide sequence prior to insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art. Furthermore, tags which may aid purification or immobilization of the polypeptide may be added to the polypeptide. Such a tag may for example be a polyhistidine tag (His tag). Preferably, the tag located in the N-terminal or C-terminal of the polypeptide, and may be encoded by the vector. Alternatively, the tag may be located internally in the polypeptide, as long as it does not affect the functionality of the polypeptide.
The recombinant expression vector may be any vector (e.g. a plasmid, phagemid, phage or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
The vectors of the present invention preferably contain one or more selectable markers that permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus lichenifonnis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. 
The vectors of the present invention may contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
More than one copy of a nucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleotide sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see for example Sambrook et al. (1989) supra).
In one embodiment of the present invention the plasmid vector may contain the following elements:

    
    
        i) a signal peptide coding region (e.g. obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA), followed by a polynucleotide sequence encoding the mature lysozyme variant. This sequence may be preceded by and operably linked to:
        ii) a DNA sequence comprising a mRNA stabilising segment (e.g. derived from the Cryllla gene, as shown in WO 99/043835);
        iii) a marker gene (e.g. a chloramphenicol resistance gene); and
        iv) genomic DNA from Bacillus subtilis as 5′ and 3′ flanking segments upstream and downstream of the polynucleotide, respectively, to enable genomic integration by homologous recombination between the flanking segments and the Bacillus genome. The vectors described above may also be useful in the generation and screening of the variants using the previously described mutagenesis procedures.

Host Cells

    
    


The present invention also relates to a recombinant host cell comprising a polynucleotide encoding a variant lysozyme of the invention, which is advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.
The host cell may be a prokaryote such as bacterial cells, an archaea or a eukaryote such as fungal cells, plant cells, insect cells, or mammalian cells.
Useful prokaryotes are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g. Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus halodurans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus. 
The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see for example Chang and Cohen (1979) Molecular General Genetics 168:111-115), using competent cells (see for example Young and Spizizin (1961) Journal of Bacteriology 81:823-829; or Dubnau and Davidoff-Abelson (1971) Journal of Molecular Biology 56:209-221), electroporation (see for example Shigekawa and Dower (1988) Biotechniques 6:742-751), or conjugation (see for example Koehler and Thorne (1987) Journal of Bacteriology 169:5771-5278).
In a preferred embodiment, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al. (1995) Ainsworth and Bisby's Dictionary of The Fungi (8th edition), CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al. (1995) Ainsworth and Bisby's Dictionary of The Fungi (8th edition), CAB International, University Press, Cambridge, UK, page 171) and all mitosporic fungi (Hawksworth et al. (1995) Ainsworth and Bisby's Dictionary of The Fungi (8th edition), CAB International, University Press, Cambridge, UK). In a more preferred embodiment, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport (eds) (1980) Soc. App. Bacteriol. Symposium Series No. 9).
In an even more preferred embodiment, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell. In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al. (1995) Ainsworth and Bisby's Dictionary of The Fungi (8th edition), CAB International, University Press, Cambridge, UK). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In an even more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma. In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238023 and Yelton et al. (1984) Proceedings of the National Academy of Sciences USA 81:1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al. (1989) Gene 78:147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, Abelson and Simon (editors) Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology 194:182-187, Academic Press, Inc., New York; Ito et al. (1983) Journal of Bacteriology 153:163; and Hinnen et al. (1978) Proceedings of the National Academy of Sciences USA 75:1920.
A particular embodiment of the present invention is a recombinant host cell transformed with a polynucleotide encoding a variant lysozyme of the present invention. Preferably, such a host cell does not contain an inherent lysozyme encoding gene, or such a gene has been disrupted. Thereby the recombinant variant lysozyme is the only lysozyme produced by the recombinant host cell of the present invention.
Methods of Production
The present invention also relates to methods of producing a lysozyme variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant from the cultivation medium.
In the production methods of the present invention, the host cells are cultivated in a nutrient medium suitable for production of the lysozyme variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g. in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
One embodiment of the present invention is a method of producing a variant of a parent lysozyme, wherein said variant has antimicrobial and/or lysozyme activity, said method comprising: a) culturing a cell under conditions suitable for expression of the variant, where said cell contains a polynucleotide sequence encoding a variant of a parent lysozyme in which said variant is altered in one or more (several) amino acid position(s) selected from the group consisting of positions: 47, 111, 108, 45, 22, 110, 120, 147, 196, 49, 55, 193 161, 128, 131, 95, 203, 98, 112, 55, 32, 89, 206 121, 120, 185, 186, 176, 113, 122, 119, 35, 65, 139, 141, 153, 158, 171, 195, 76, 164, 30, 85, 178, 183, 186, 112, 174, 187, 197, 102, 134, 108, 196, 197, 198, 56, 19, 120, 20, 135, and 203, and said polynucleotide sequence is prepared by mutagenesis of a parent polynucleotide sequence of SEQ ID NO:1, or a parent polynucleotide sequence having at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% identity to the nucleotide sequence of SEQ ID NO:1; and b) recovering the lysozyme variant from the cultivation medium. In an alternative aspect, the lysozyme variant is not recovered, but rather a host cell of the present invention expressing a variant is used as a source of the variant.
The lysozyme variant may be detected using methods known in the art that are specific for the expressed polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant lysozyme as described herein in the Examples.
The resulting lysozyme variant may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
A lysozyme variant of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g. preparative isoelectric focusing), differential solubility (e.g. ammonium sulfate precipitation), SDS-PAGE, or extraction (see e.g. J.-C. Janson and Lars Ryden (editors) (1989) Protein Purification VCH Publishers, New York) to obtain substantially pure lysozyme variants.
Compositions
The present invention also relates to compositions comprising a variant lysozyme or a polypeptide having antimicrobial and/or lysozyme activity of the present invention and a carrier and/or an exhibient. Preferably, the compositions are enriched in such a variant or polypeptide. The term “enriched” indicates that the antimicrobial and/or lysozyme activity of the composition has been increased, e.g. with an enrichment factor of 1.1 or more. Preferably, the compositions are formulated to provide desirable characteristics such as low color, low odor and acceptable storage stability.
The composition may comprise a variant or polypeptide of the present invention as the major enzymatic component, e.g. a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.
The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid, paste, gel or a dry formulation. For instance, the polypeptide may be formulated in the form of a granulate or a microgranulate. The variant or polypeptide to be included in the composition may be stabilized in accordance with methods known in the art. In a preferred embodiment the variant lysozyme is formulated in a liquid composition.
A preferred embodiment of the present invention is a feed composition comprising a lysozyme variant of the present invention. In particular a variant with improved stability at acidic pH and/or increased protease stability is preferred.
Detergent Compositions
The present invention also encompasses detergent compositions comprising a lysozyme variant of the present invention. In particular a variant with improved stability and/or activity at alkaline pH and/or a variant with improved activity at low or moderate temperature is preferred. The detergent composition may be adapted for specific uses such laundry, in particular household laundry, dish washing or hard surface cleaning.
The detergent composition typically comprises conventional detergent ingredients such as surfactants, builders, bleaches, enzymes and other ingredients.
In a preferred embodiment the detergent composition comprises a lysozyme and a protease.
The detergent composition can be in any form, such as a solid, liquid, paste, gel or any combination thereof. The composition may be in the form of a tablet, bar or pouch, including multi-compartment pouches. The composition can be in the form of a powder, for example a free-flowing powder, such as an agglomerate, spray-dried powder, encapsulate, extrudate, needle, noodle, flake, or any combination thereof.
Enzymes
In one aspect, the present invention provides a detergent additive comprising a lysozyme variant of the present invention. The detergent additive as well as the detergent composition may comprise one or more enzymes such as a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g. a laccase, and/or peroxidase.
In general the properties of the selected enzyme(s) should be compatible with the selected detergent (i.e. pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may for example be a metalloprotease (EC 3.4.17 or EC 3.4.24) or a serine protease (EC 3.4.21), preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins (EC 3.4.21.62), especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.
Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novozymes A/S), Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect OxP®, FN2TM, and FN3TM (Genencor International Inc.).
Protease enzymes may be incorporated into detergent compositions in accordance with the invention at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants of such lipases are included in this connection. The lipase may for example be triacylglycerol lipase (EC3.1.1.3), phospholipase A2 (EC 3.1.1.4), Lysophospholipase (EC 3.1.1.5), Monoglyceride lipase (EC 3.1.1.23), galactolipase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32), Lipoprotein lipase (EC 3.1.1.34). Examples of useful lipases include a Humicola lanuginosa lipase, e.g. as described in EP 258 068 and EP 305 216; a Rhizomucor miehei lipase, e.g. as described in EP 238 023 or from H. insolens as described in WO 96/13580; a Candida lipase, such as a C. antarctica lipase, e.g. the C. antarctica lipase A or B described in EP 214 761; a Pseudomonas lipase, such as one of those described in EP 721 981 (e.g. a lipase obtainable from a Pseudomonas sp. SD705 strain having deposit accession number FERM BP-4772), in PCT/JP96/00426, in PCT/JP96/00454 (e.g. a P. solanacearum lipase), in EP 571 982 or in WO 95/14783 (e.g. a P. mendocina lipase), a P. alcaligenes or P. pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as described in EP 331 376, a P. stutzeri lipase, e.g. as disclosed in GB 1,372,034, or a P. fluorescens lipase; a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al. (1993) Biochemica et Biophysica Acta 1131:253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. A preferred lipase variant is that of Humicola lanuginosa DSM 4109 as described in WO 00/60063. Especially preferred are the variants disclosed in the Example in WO 00/60063 with improved first wash performance., i.e., T231R+N233R; G91A+D96W+E99K+G263Q+L264A+I265T+G266D+T267A+L269N+R209P+T231R+N233R; N33Q+D96S+T231R+N233R+Q249R; E99N+N101S+T231R+N233R+Q249R; E99N+N101S+T231R+N233R+Q249R.
Suitable commercially available lipases include Lipex®, Lipolase® and Lipolase Ultra®, Lipolex®, Lipoclean® (available from Novozymes A/S), M1 LipaseTM and LipomaxTM (available from Genencor Inc.) and Lipase P “Amano” (available from Amano Pharmaceutical Co. Ltd.). Commercially available cutinases include LumafastTM from Genencor Inc.
The lipases are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Cutinases: Potentially useful types of cutinases include (EC 3.1.1.74), e.g. a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (described e.g. in WO 90/09446). Due to the lipolytic activity of cutinases they may be effective against the same stains as lipases. Commercially available cutinases include LumafastTM from Genencor Inc.
The cutinases are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Carbohydrases: Carbohydrases covers glycoside hydrolases (EC 3.2.1.-) and polysaccharide lyases (EC 4.2.2.-). Glycoside hydrolases catalyze the hydrolysis of the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Polysaccharide lyases catalyze the cleavage of polysaccharide chains by a beta elimination mechanism resulting in a double bond of the newly formed reducing end. Carbohydrases include for example amylases, hemicellulases, pectinases and cellulases described in more detail below. Other carbohydrases may be xanthanases or pullulanases.
Suitable xanthanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Sources of xanthanases are for example described in Cadmus et al. (1988) J of Industrial Microbiology and Biotechnology 4:127-133; EP0030393; and Hashimoto et al. (1998) Appl Environ Microbiol. 64:3765-3768.
Suitable pullulanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Sources of pullulanase are for example Dextrozyme® and Promozyme® D2 (Novozymes A/S).
Amylases: Amylases comprise e.g. alpha-amylases (EC 3.2.1.1), beta-amylases (EC 3.2.1.2) and/or glucoamylases (EC 3.2.1.3) of bacterial or fungal origin. Chemically or genetically modified mutants of such amylases are included in this connection. Alpha-amylases are preferred in relation to the present invention. Relevant alpha-amylases include, for example, α-amylases obtainable from Bacillus species, in particular a special strain of B. licheniformis, described in more detail in GB 1296839.
Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.
Further examples of useful amylases are the alpha-amylases derived from Bacillus sp. strains NCIB 12289, NCIB 12512, NCIB 12513 and DSM 9375; the alpha-amylases shown in SEQ ID NO 1 and 2 of WO 95/26397 (hereby incorporated by reference); the AA560 alpha-amylase derived from Bacillus sp. DSM 12649 disclosed as SEQ ID NO:2 in WO 00/60060 (hereby incorporated by reference); and the variants of the AA560 alpha-amylase, including the AA560 variant disclosed in Examples 7 and 8 (hereby incorporated by reference).
Relevant commercially available amylases include Natalase®, Stainzyme®, Duramyl®, Termamyl®, TermamylTM Ultra, Fungamyl® and BAN® (all available from Novozymes A/S, Bagsvaerd, Denmark), and Rapidase® and Maxamyl® P (available from DSM, Holland) and Purastar®, Purastar OxAm and PoweraseTM (available from Danisco A/S).
Other useful amylases are CGTases (cyclodextrin glucanotransferases, EC 2.4.1.19), e.g. those obtainable from species of Bacillus, Thermoanaerobactor or Thermoanaerobacterium. 
The amylases are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Hemicellulases: Suitable hemicellulases include enzymes with xylanolytiactivity, arabinolytic activity, galactolytic activity and/or mannolytic activity. The hemi-cellulases of the present invention may for example be selected from xylanases (EC 3.2.1.8, EC 3.2.1.32, and EC 3.2.1.136), xyloglucanases (EC 3.2.1.4 and EC 3.2.1.151), arabinofuranosidases (EC 3.2.1.55), acetylxylan esterases (EC EC 3.1.1.72), glucuronidases (EC 3.2.1.31, EC 3.2.1.56, 3.2.1.128 and 3.2.1.139), glucanohydrolase (EC 3.2.1.11, EC 3.2.1.83 and EC 3.2.1.73), ferulic acid esterases (EC 3.1.1.73), coumaric acid esterases (EC 3.1.1.73), mannanases (EC 3.2.1.25; EC 3.2.1.78 and EC 3.2.1.101), arabinosidase (EC 3.2.1.88), arabinanases (EC 3.2.1.99), galactanases (EC 3.2.1.89, EC 3.2.1.23 and 3.2.1.164) and lichenases (EC 3.2.1.73). This is, however, not to be considered as an exhausting list.
Mannananase is a preferred hemicellulase in relation to the present invention. Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. In a preferred embodiment the mannanase is derived from a strain of the genus Bacillus, especially Bacillus sp. 1633 disclosed in positions 31-330 of SEQ ID NO:2 or in SEQ ID NO:5 of WO 99/64619 (hereby incorporated by reference) or Bacillus agaradhaerens, for example from the type strain DSM 8721. A suitable commercially available mannanase is Mannaway® produced by Novozymes A/S or PurabriteTM produced by Genencor a Danisco division.
Xylanase is a preferred hemicellulase in relation to the present invention. A suitable commercially available xylanase is Pulpzyme® HC (available from Novozymes A/S).
The hemicellulases are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Pectinases: Suitable pectinolytic enzymes include those described in WO 99/27083, WO 99/27084, WO 00/55309 and WO 02/092741.
Suitable pectate lyases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. In a preferred embodiment the pectate lyase is derived from a strain of the genus Bacillus, especially a strain of Bacillus subtilis, especially Bacillus subtilis DSM14218 disclosed in SEQ ID NO:2 or a variant thereof disclosed in Example 6 of WO 02/092741 (hereby incorporated by reference) or a variant disclosed in WO 03/095638 (hereby incorporated by reference). Alternatively the pectate lyase is derived from a strain of Bacillus licheniformis, especially the pectate lyases disclosed as SEQ ID NO:8 in WO 99/27083 (hereby incorporated by reference) or variants thereof as described in WO 02/06442.
Suitable commercially available pectate lyases are Pectaway® or Pectawash® produced by Novozymes A/S.
The pectinolytic enzymes are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Cellulases: Suitable cellulases include complete cellulases or mono-component endoglucanases of bacterial or fungal origin. Chemically or genetically modified mutants are included. The cellulase may for example be a mono-component or a mixture of mono-component endo-1,4-beta-glucanase often just termed “endoglucanases” (EC 3.2.1.4). Some xyloglucanases may also have endoglucanases activity and are also considered as suitable cellulases in the present invention. Suitable cellulases are disclosed in U.S. Pat. No. 4,435,307, which discloses fungal cellulases produced from Humicola insolens. Especially suitable cellulases are the cellulases having textile care benefits. Examples of such cellulases are cellulases described in European patent application No. 0 495 257.
Suitable mono-component endoglucanases may be obtained from one or more of the following species Exidia glandulosa, Crinipellis scabella, Fomes fomentarius, Spongipellis sp., Rhizophlyctis rosea, Rhizomucor pusillus, Phycomyces nitens, and Chaetostylum fresenii, Diplodia gossypina, Microsphaeropsis sp., Ulospora bilgramii, Aureobasidium sp., Macrophomina phaseolina, Ascobolus stictoides, Saccobolus dilutellus, Peziza, Penicillium verruculosum, Penicillium chrysogenum, and Thermomyces verrucosus, Trichoderma reesei aka Hypocrea jecorina, Diaporthe syngenesia, Colletotrichum lagenarium, Xylaria hypoxylon, Nigrospora sp., Nodulisporum sp., and Poronia punctata, Cylindrocarpon sp., Nectria pinea, Volutella colletotrichoides, Sordaria fimicola, Sordaria macrospora, Thielavia thermophila, Syspastospora boninensis, Cladorrhinum foecundissimum, Chaetomium murorum, Chaetomium virescens, Chaetomium brasiliensis, Chaetomium cunicolorum, Myceliophthora thermophila, Gliocladium catenulatum, Scytalidium thermophila, Acremonium sp Fusarium solani, Fusarium anguioides, Fusarium poae, Fusarium oxysporum ssp. lycopersici, Fusarium oxysporum ssp. passiflora, Humicola nigrescens, Humicola grisea, Fusarium oxysporum, Thielavia terrestris or Humicola insolens. One preferred endoglucanase is disclosed in WO 96/29397 as SEQ ID NO:9 (hereby incorporated by reference) or an enzyme with at least 70% identity thereto and variants thereof as disclosed in Example 1 of WO 98/12307. Another preferred endoglucanase is disclosed in WO 91/017243 (SEQ ID NO:2) or endoglucanases variants as disclosed in WO 94/007998.
Endoglucanases with an anti-redeposition effect may be obtained from fungal endoglucanases lacking a carbohydrate-binding module (CBM) from a number of bacterial sources. Some sources are Humicola insolens, Bacillus sp. deposited as DSM 12648, Bacillus sp. KSMS237 deposited as FERM P-16067, Panibacillus polymyxa, and Panibacillus pabuli. Specific anti-redeposition endoglucanase are disclosed in WO 91/17244 (FIG. 14) (hereby incorporated by reference), WO 04/053039 (SEQ ID NO:2) (hereby incorporated by reference), JP 2000210081 (position 1 to 824 of SEQ ID NO:1) (hereby incorporated by reference).
Xyloglucanases with an anti-redeposition effect may be obtained from a number of bacterial sources. Some sources are Bacillus licheniformis, Bacillus agaradhaerens (WO 99/02663), Panibacillus polymyxa, and Panibacillus pabuli (WO01/62903). Suitable variants of xyloglucasnes are also described in PCT/EP2009/056875. A commercially available xyloglucanase is Whitezyme® (Novozymes A/S).
Commercially available cellulases include Celluclas® produced from Trichoderma reesei, Celluzyme® produced from Humicola insolens. Commercially available endoglucanases are Carezyme®, Renozyme®, Endolase® and Celluclean® (Novozymes A/S), and KAC-500(B)TM (Kao Corporation) and ClazinaseTM, PuradaxTM EG L and Puradax HA (Danisco A/S).
Cellulases are normally incorporated in the detergent composition at a level of from 0.000001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.00001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.0001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.001% to 0.2% of enzyme protein by weight of the composition.
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful oxidases are laccases (EC 1.10.3.2). Examples of useful peroxidases include catalases (EC 1.11.1.6) and peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
Commercially available peroxidases include GuardzymeTM (Novozymes A/S).
Arylesterases: Suitable arylesterases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful arylesterase are for example obtained from M. Smegmatis as described in WO 05/056782.
Surfactants
Typically, the detergent composition comprises (by weight of the composition) one or more surfactants in the range of 0% to 50%, preferably from 2% to 40%, more preferably from 5% to 35%, more preferably from 7% to 30%, most preferably from 10% to 25%, even most preferably from 15% to 20%. Preferred surfactants are anionic surfactants, non-ionic surfactants, cationic surfactants, zwitterionic surfactants, amphoteric surfactants, and mixtures thereof. Preferably, the major part of the surfactant is anionic. Suitable anionic surfactants are well known in the art and may comprise fatty acid carboxylates (soap), branced-chain, linear-chain and random chain alkyl sulfates or fatty alcohol sulfates or primary alcohol sulfates or alkyl benzenesulfonates such as LAS and LAB or phenylalknesulfonates or alkenyl sulfonates or alkenyl benzenesulfonates or alkyl ethoxysulfates or fatty alcohol ether sulfates or alpha-olefin sulfonate or dodecenyl/tetradecnylsuccinic acid. The anionic surfactants may be alkoxylated. The detergent composition may also comprise from 1 wt % to 10 wt % of non-ionic surfactant, preferably from 2 wt % to 8 wt %, more preferably from 3 wt % to 7 wt %, even more preferably less than 5 wt % of non-ionic surfactant. Suitable non-ionic surfactants are well known in the art and may comprise alcohol ethoxylates, and/or alkyl ethoxylaes, and/or alkylphenol ethoxylates, and/or glucamides such as fatty acid N-glucosyl N-methyl amides, and/or alkyl polyglucosides and/or mono- or diethanolamides or fatty acid amides. The detergent composition may also comprise from 0 wt % to 10 wt % of cationic surfactant, preferably from 0.1 wt % to 8 wt %, more preferably from 0.5 wt % to 7 wt %, even more preferably less than 5 wt % of cationic surfactant. Suitable cationic surfactants are well known in the art and may comprise alkyl quaternary ammonium compounds, and/or alkyl pyridinium compounds and/or alkyl quaternary phosphonium compounds and/or alkyl ternary sulphonium compounds. The composition preferably comprises surfactant in an amount to provide from 100 ppm to 5,000 ppm surfactant in the wash liquor during the laundering process. The composition upon contact with water typically forms a wash liquor comprising from 0.5 g/l to 10 g/l detergent composition. Many suitable surface active compounds are available and fully described in the literature, for example, in “Surface-Active Agents and Detergents”, Volumes 1 and 11, by Schwartz, Perry and Berch.
Builders
The main role of a builder is to sequester divalent metal ions (such as calcium and magnesium ions) from the wash solution that would otherwise interact negatively with the surfactant system. Builders are also effective at removing metal ions and inorganic soils from the fabric surface, leading to improved removal of particulate and beverage stains. Builders are also a source of alkalinity and buffer the pH of the wash water to a level of 9.5 to 11. The buffering capacity is also termed “reserve alkalinity”, and should preferably be greater than 4.
The detergent compositions of the present invention may comprise one or more detergent builders or builder systems. Many suitable builder systems are described in the literature, for example in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Builder may comprise from 0% to 60%, preferably from 5% to 45%, more preferably from 10% to 40%, most preferably from 15% to 35%, even more preferably from 20% to 30% builder by weight of the subject composition. Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates (e.g. tripolyphosphate STPP), alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders (e.g. zeolite) and polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. Ethanole amines (MEA, DEA, and TEA may also contribute to the buffering capacity in liquid detergents.
Bleaches
The detergent compositions of the present invention may comprise one or more bleaching agents. In particular powdered detergents may comprise one or more bleaching agents. Suitable bleaching agents include other photobleaches, pre-formed peracids, sources of hydrogen peroxide, bleach activators, hydrogen peroxide, bleach catalysts and mixtures thereof. In general, when a bleaching agent is used, the compositions of the present invention may comprise from about 0.1% to about 50% or even from about 0.1% to about 25% bleaching agent by weight of the subject cleaning composition. Examples of suitable bleaching agents include:
(1) other photobleaches, for example Vitamin K3;
(2) preformed peracids: suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone, and mixtures thereof. Suitable percarboxylic acids include hydrophobic and hydrophilic peracids having the formula R—(C═O)O—O-M wherein R is an alkyl group, optionally branched, having, when the peracid is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when the peracid is hydrophilic, less than 6 carbon atoms or even less than 4 carbon atoms; and M is a counterion, for example, sodium, potassium or hydrogen;

(3) sources of hydrogen peroxide, for example, inorganic perhydrate salts, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulphate, perphosphate, persilicate salts and mixtures thereof. In one aspect of the invention the inorganic perhydrate salts are selected from the group consisting of sodium salts of perborate, percarbonate and mixtures thereof. When employed, inorganic perhydrate salts are typically present in amounts of from 0.05 to 40 wt %, or 1 to 30 wt % of the overall composition and are typically incorporated into such compositions as a crystalline solid that may be coated. Suitable coatings include inorganic salts such as alkali metal silicate, carbonate or borate salts or mixtures thereof, or organic materials such as water-soluble or dispersible polymers, waxes, oils or fatty soaps. Useful bleaching compositions are described in U.S. Pat. Nos. 5,576,282, and 6,306,812;

(4) bleach activators having R—(C═O)-L wherein R is an alkyl group, optionally branched, having, when the bleach activator is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when the bleach activator is hydrophilic, less than 6 carbon atoms or even less than 4 carbon atoms; and L is leaving group. Examples of suitable leaving groups are benzoic acid and derivatives thereof—especially benzene sulphonate. Suitable bleach activators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED) and nonanoyloxybenzene sulphonate (NOBS). Suitable bleach activators are also disclosed in WO 98/17767. While any suitable bleach activator may be employed, in one aspect of the invention the subject cleaning composition may comprise NOBS, TAED or mixtures thereof; and

(5) bleach catalysts that are capable of accepting an oxygen atom from peroxyacid and transferring the oxygen atom to an oxidizable substrate are described in WO2008/007319 (hereby incorporated by reference). Suitable bleach catalysts include, but are not limited to: iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulphonyl imines; N-phosphonyl imines; N-acyl imines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof. The bleach catalyst will typically be comprised in the detergent composition at a level of from 0.0005% to 0.2%, from 0.001% to 0.1%, or even from 0.005% to 0.05% by weight.

When present, the peracid and/or bleach activator is generally present in the composition in an amount of from about 0.1 to about 60 wt %, from about 0.5 to about 40 wt % or even from about 0.6 to about 10 wt % based on the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracid or precursor thereof.
The amounts of hydrogen peroxide source and peracid or bleach activator may be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.
Adjunct Materials
Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.
Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the composition.
Fluorescent whitening agent: The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of:

    4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2′-disulphonate,
    4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2,2′-disulphonate,
    4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,
    4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate,
    4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2′-disulphonate and,
    2-(stilbyl-4′′-naptho-1.,2′:4,5)-1,2,3-trizole-2′′-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2′-bis-(phenyl-styryl) disulphonate.


Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India.
Other fluorescers suitable for use in the invention include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.
Fabric hueing agents: The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions, thus altering the tint of said fabric through absorption of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO 05/03274, WO 05/03275, WO 05/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch.
Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 09/087,523 (hereby incorporated by reference). Furthermore random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 07/138,054, WO 06/108856 and WO 06/113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 03/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, structurants for liquid detergents and/or structure elasticizing agents.
In one aspect the detergent is a compact fluid laundry detergent composition comprising: a) at least about 10%, preferably from 20 to 80% by weight of the composition, of surfactant selected from anionic surfactants, non ionic surfactants, soap and mixtures thereof; b) from about 1% to about 30%, preferably from 5 to 30%, by weight of the composition, of water; c) from about 1% to about 15%, preferably from 3 to 10% by weight of the composition, of non-aminofunctional solvent; and d) from about 5% to about 20%, by weight of the composition, of a performance additive selected from chelants, soil release polymers, enzymes and mixtures thereof; wherein the compact fluid laundry detergent composition comprises at least one of:
(i) the surfactant has a weight ratio of the anionic surfactant to the nonionic surfactant from about 1.5:1 to about 5:1, the surfactant comprises from about 15% to about 40%, by weight of the composition, of anionic surfactant and comprises from about 5% to about 40%, by weight of the composition, of the soap; (ii) from about 0.1% to about 10%, by weight of the composition, of a suds boosting agent selected from suds boosting polymers, cationic surfactants, zwitterionic surfactants, amine oxide surfactants, amphoteric surfactants, and mixtures thereof; and (iii) both (i) and (ii). All the ingredients are described in WO 07/130,562 hereby incorporated by reference in its entirety further polymers useful in detergent formulations are described in WO 07/149,806, which is hereby incorporated by reference in its entirety.
In another aspect the detergent is a compact granular (powdered) detergent comprising a) at least about 10%, preferably from 15 to 60% by weight of the composition, of surfactant selected from anionic surfactants, non ionic surfactants, soap and mixtures thereof; b) from about 10 to 80% by weight of the composition, of a builder, preferably from 20% to 60% where the builder may be a mixture of builders selected from i) phosphate builder, preferably less than 20%, more preferably less than 10% even more preferably less than 5% of the total builder is a phosphate builder; ii) a zeolite builder, preferably less than 20%, more preferably less than 10% even more preferably less than 5% of the total builder is a zeiolite builder; iii) citrate, preferably 0 to 5% of the total builder is a citrate builder; iv) polycarboxylate, preferably 0 to 5% of the total builder is a polycarboxylate builder v) carbonate, preferably 0 to 30% of the total builder is a carbonate builder and vi) sodium silicates, preferably 0 to 20% of the total builder is a sodium silicate builder; c) from about 0% to 25% by weight of the composition, of fillers such as sulphate salts, preferably from 1% to 15%, more preferably from 2% to 10%, more preferably from 3% to 5% by weight of the composition, of fillers; and d) from about 0.1% to 20% by weight of the composition, of enzymes, preferably from 1% to 15%, more preferably from 2% to 10% by weight of the composition, of enzymes.
Uses
A lysozyme variant, or a composition thereof, of the present invention may be used in several applications to degrade a material comprising a peptidoglycan or a chitodextrin by treating the material with the polypeptide or composition thereof (see for example Proctor and Cunningham, (1988) Critical Reviews in Food Science and Nutrition 26:359-395; Carini et al. (1985) Microbiol. Alimen. Nutr. 3:299-320; Hughey and Johnson (1987) Appl. Environ. Microbiol. 53:2165-2170; Cunningham et al. (1991) World's Poultry Science Journal 47:141-163).
Examples of preferred uses of the lysozyme variants or compositions thereof of the present invention are given below. The dosage of the lysozyme variant and other conditions under which the lysozyme variants is used may be determined on the basis of methods known in the art.
The variant lysozymes of the present invention may be used as antimicrobial agents. One aspect of the present invention is a method for reducing microbial contamination, comprising treating a microbial contaminated surface with a lysozyme variant of the present invention.
To assess whether a lysozyme variant of the present invention is capable of acting as an antimicrobial agent it can be tested in a turbidity assay. In this assay it is tested whether the lysozyme is capable of degrading microbial cells e.g. a dried substrate of Exiguobacterium undae cells (isolated from a smelly sock) or Clostridium perfringens cells dissolved in buffer or detergent, and thereby reducing the optical density (OD) at for example 540 nm, when compared to a microbial suspension only treated with buffer.
The variant lysozyme is preferably incorporated into and/or used together with detergent compositions as described above.
When washing is performed repeatedly at temperatures below 60° C. there is an increased risk of malodour in the washing machine (laundry as well as dishwashing) and on the textiles washed in the machine. This malodour is likely to be caused by microbial organisms such as bacteria, fungi, algae or other unicellular organisms growing in the washing machine. The present invention provides a method of reducing microbial contamination on a surface, such as a textile garment or hard surface such as metal, plastic or rubber parts in a washing machine, bathroom tiles, floors, table tops, drains, sinks and washbasin, by treating the microbial contaminated surface with a lysozyme variant or lyzysyme variant composition of the present invention. Such a treatment is also expected to reduce the malodour on textiles and hard surfaces containing microbial contamination.
The reduction of microbial contamination can be assessed in several ways, for example by letting a panel assess whether the smell has been decreased, alternatively a sample may be taken from the surface and cultivated to assess whether the microbial count has been reduced as a result of the treatment compared to a treatment without lysozyme.
Furthermore, the invention relates to a process for laundering of fabrics comprising treating fabrics with a washing solution containing a detergent composition and a lysozyme variant or a lysozyme variant composition of the invention. The laundering treatment can for example be carried out in a machine washing process or in a manual washing process. The washing solution can for example be an aqueous washing solution containing the detergent composition and with a pH between 3 and 12, preferably between pH 7 and 12, more preferably between pH 8 and 10.
The fabrics subjected to the methods of the present invention may be conventional washable laundry, for example household laundry. Preferably, the major part of the laundry is garments and fabrics, including knits, wovens, denims, yarns, and towelling, made from cotton, cotton blends or natural or manmade cellulosics (e.g. originating from wood pulp) or blends thereof. Examples of blends are blends of cotton or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fibers, lyocell).
The lysozyme variant of the invention may also be used in animal feed. In an embodiment, the present invention provides a method for preparing an animal feed composition comprising adding a lysozyme variant of the present invention to one or more animal feed ingredients. Preferably, the animal feed does not contain antibiotics.
The lysozyme variants of the present invention may for example be used to stabilize the healthy microflora of animals, in particular livestock such as, but not limited to, cattle, dear, goats, pigs, poultry, rabbits and sheep, but also in fish by suppressing growth/intestinal colonization of pathogens such as Clostridium perfringens, Escherichia coli, Salmonella, such as Salmonella typhimurium and Salmonella Mbandaka, and Camplylobacter sp. In a preferred embodiment, the lysozyme variant replaces antibiotics in animal diets. Especially variants with increased stability at acidic pH and improved stability towards proteases are suitable for feed applications since they can survive the passage through the digestive tract. In a preferred embodiment a lysozyme variant is applied to chicken and has anti-microbal activity against Clostridium perfringens. In a further embodiment a lysozyme variant of the present invention is used as a feed additive, where it may provide a positive effect on the microbial balance of the chicken digestive tract and in this way improve animal performance.
Lysozyme variants may also be used in animal feed as feed enhancing enzymes that improve feed digestibility to increase the efficiency of its utilization according to WO 00/21381 and WO 04/026334.
The lysozyme variants may be used for disinfection and for preventing or removing biofilm on a surface according to U.S. Pat. No. 6,777,223.
The lysozyme variants may also be used to selectively inhibit the uncontrolled growth of Clostridium tyrobutyricum during the maturation of cheeses, in particular those made from pressed and cooked curds, e.g. Swiss Cheese, Parmesan, Edam, Gouda, Cheddar, and many others.
The lysozyme variants may also be used in oral care. For example, lysozyme can be used alone or in combination with other enzymes or even antimicrobial peptides in toothpaste or other oral care products. The polypeptides may be introduced into the oral cavity or applied to an article that is to be introduced into the oral cavity. See for example WO 08/124,764.
The lysozyme variants may also be used in topical treatment of dystrophic and inflammatory lesions of the skin and soft tissues. See for example Palmieri and Boraldi (1977) Arch. Sci. Med. (Torino) 134:481-485.
The lysozyme variants may also be used in skin care. For example, the polypeptide is applied to the skin of a patient suffering from a skin infection, such as acne. The lysozyme variants may also be used in a wound dressing, which is applied to wounded skin, for example, to aid in healing of the wound. See for example U.S. Application No. 20080254079.
The lysozyme variants may also be used in lipstick, lip balm, lip gel, or lip gloss. For example, such products can be used for treatment of a localized lip infection, for example, a cold sore. See for example U.S. Application No. 20080254079.
The lysozyme variants may also be used in the treatment of bronchopulmonary diseases.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Materials and Methods
Turbidity Assay
The activity of Lysozyme was determined by measuring the decrease (drop) in absorbance/optical density of a solution of resuspended dried cells of Exiguobacterium undae measured in a spectrophotometer at 540 nm.
Preparation of Dried Cells of Exiguobacterium Undae (The Substrate)
A culture of E. undae (DSM14481) was grown in 100 mL LB medium (Fluka 51208, 25 g/L) in a 500 mL shake-flask at 30° C., 250 rpm overnight. The overnight culture was then centrifuged at 20° C., 5000 g for 10 minutes, and the pellet was washed two times in sterile milliQ water, and resuspended in Milli-Q water. The washed cells were centrifuged for 1 minute at 13000 rpm and as much as possible of the supernatant was decanted. The washed cells were dried in a vacuum centrifuge for 1 hour before the weight of the cells was determined. The dried cells were stored at −20° C. Before use the cells were resuspended in Universal buffer at pH 6 to a concentration of 0.5 mg cells/mL and the optical density (OD) at 540 nm was measured. The cell suspension was then adjusted so the cell concentration equaled an OD540=1.0. The adjusted cell suspension was then stored cold before use. Resuspended cells should be used within 4 hours.
Measurement of Lysozyme Antimicrobial Activity in the Turbidity Assay
The lysozyme sample to be measured was diluted to a concentration of 100-200 mg enzyme protein/L in Universal buffer (see below) at pH 6, and kept on ice until use. In a 96 well microtiterplate (Nunc) 200 μL of the substrate was added to each well, and the plate was incubated at 37° C. for 5 minutes in a PowerWaveX spectrophotometer (Bio-Tek Instruments INC). Following the incubation the absorbance of each well was measured at 540 nm (start value). To start the activity measurement 20 μL of the diluted lysozyme samples was added to the 200 μL substrate in each well and kinetic measurement of absorbance at 540 nm was initiated for minimum 30 minutes up to 24 hours at 37° C. The measured absorbance at 540 nm was monitored for each well and over time a drop in absorbance was seen if the lysozyme had antimicrobial activity. The larger the drop in absorbance was the larger was the lysozyme antimicrobial activity.
Preparation of Universal Buffer Stock
Stock:
















100 mM Succinic acid
118.09 g/mol
5.90 g



100 mM HEPES
 238.3 g/mol
11.9 g



100 mM CHES
207.29 g/mol
10.4 g



100 mM CAPS
221.31 g/mol
11.1 g



150 mM NaCl
 58.44 g/mol
4.38 g









Mix in 500 mL water. Adjust pH with HCl or NaOH to pH 6. Dilute buffer 10 times before use.
In order to compare results from the turbidity assay the samples to be compared should preferably be tested in the same experimental run using the same buffer and substrate batch.
Variations in the Lysozyme Antimicrobial Activity in the Turbidity Assay
The assay described above, is the preferred standard microbial activity test for the present invention. However, for lysozyme variants which are generated to achieve an improved property, the assay may be adapted.
To test improved thermal activity the incubation temperature can be increased to the desired temperature, e.g. from 45° C. to 110° C. If the temperature exceeds 50° C., the sample must be incubated in a external heat-source instead of directly in the spectrophotometer measurements are then performed by measuring a start value and an end value.
To test improved activity at low or moderate temperatures the incubation temperature can be changed to the desired temperature, e.g. from 0° C. to 40° C.
To test improved temperature stability the lysozyme sample may be pre-incubated at a desired temperature, e.g. 45° C. to 110° C. for 30 min to 24 hours, and then cooled down to the temperature of the assay described above before measuring the activity.
To test improved pH activity the pH may be increased to for example pH 7.5 to 12 or decreased pH for example from 2 to 5.5.
To test improved pH stability the lysozyme sample may be pre-incubated at a desired pH for 30 min to 24 h, and then returned to the pH of the assay described above before measuring the activity.
To test increased resistance to protease degradation the lysozyme sample may be pre-incubated with pepsin at 0.5 to 2 mg/ml, preferably from 1 to 1.5 mg/ml or with a serine-proteases such as 10 mg/L Savinase at 25-40° C. for 30 min to 24 h prior to conducting the assay described above.
The temperature and pH optimum of a lysozyme may also be assessed using the turbidity assay. For temperature optimum assessment the assay is run in a range of temperatures, for example from 5° C. to 80° C., while the pH is maintained at 6. For pH optimum the pH of the assay is varied over a range, for example pH 2 to 12 while the temperature is maintained at 37° C.
Example 1
Generation of Variants
Using the methods described above following variants of A fumigates lyzozyle (SEQ ID NO: 2) was prepared















N19D,
H20W,



H20Y,
K22G,



K30Y,
Q35R,



T45G,
Y47F,



D49G,
H55A,



Y56W,
L65I,



K76S,
F85Y,



N89S,
D95G,



R98G,
G102P + Y134V,



Y108F,
Y111F,



H120Q,
V131C,



H136N,
S139G,



W141Y,
T147G,



R153T,
A158S,



F161Y,
K164T,



A171R,
K178G,



D183G,
T186Y + W187Y,



S193A,
K195S,



Y196G + K197P,
H198F,



D203N,
N206A,



N206S,
E107A,



D105A.









Example 2
Stability at Low pH in Gastric Juice
Measurement of Lysozyme Stability to pH 2 and Pepsin Incubation
To measure lysozyme stability under simulated stomach conditions we incubated wt lysozyme as well as selected variants in artificial gastric juice (0.01 M HCl, 0.1 M NaCl, 1 mg/ml pepsin). Lysozyme samples were diluted to a concentration of 50 mg enzyme protein/L in milliQ water. In a 96 well microtiterplate (Nunc) 162 μL of artificial gastric juice was added to each well together with 18 μL lysozyme sample at t=0 min. Samples were incubated for 1 hour at 37° C., 350 rpm in an Eppendorf termomixer. Each lysozyme was tested in triplicate. To stop the stomach incubation at t=60 min 20 μL citric acid Na2PO4 buffer solution pH 6.8 (Prepared by mixing 22.75 ml 0.1 M citric acid with 77.25 ml 0.2 M Na2PO4) was added to each well. Microtitre wells without lysozyme were used as negative controls and wells with citric acid Na2PO4 buffer solution pH 6.8 added at t=0 min before addition of lysozyme was used as positive controls.
To measure residual lysozyme activity 20 μL E. undae suspension was added to each well and OD 540 nm was measured each minute for 1 hour at 37° C. in a Sunrise spectrophotometer (Tecan). OD 540 nm measurements at t=60 min were used for calculations. Lysozyme residual activity in percent was calculated as Lysozyme residual activity=((OD (540 nm) negative control−OD (540 nm) gastric test)/OD (540 nm) positive control)*100%
Results
Lysozyme wt and selected variants were tested for stability towards incubation in artificial gastric juice. Residual activities of the tested variants are shown in table 1. 3 out of 7 variants had improved stability compared to the wt.







TABLE 1







Residual activity of lysozyme variant incubated in artificial gastric juice.










Variant
Residual activity %













Y56W
98



V131C
81



F161C
51



Wildtype
40



D95G
38



R98G
15



D203N
3



N206A
0









Example 3
Thermostability of Variants
Measurement of Lysozyme Thermostability
Purified samples lysozyme and variants were suspended to a concentration of 10 ppm in 40× diluted buffer originally consisting of 11.8 g succinic acid; 23.8 g HEPES; 20.8 g CHES; 22.2 g CAPS and 8.76 g NaCl pr. L. Buffer pH was adjusted to pH 6 with HCl or NaOH prior to use. Lysozyme thermostability was tested by incubation of purified lysozyme and variants thereof in a Veriti thermal Cycler (Applied Biosystems) for 10 min at 90° C. Simultaneously, control samples were incubated on ice. Immediately after incubation samples were put on ice, substrate added (substrate was prepared as described above). Residual activity was essentially assessed as described above except that activity was measured for 20 minutes.
Results Thermostability
Lysozyme wt and selected variants were tested for thermostability. Residual activities of the tested variants relative to WT are shown in table 2. 10 variants had relative residual activities greater than 5% and may therefore be considered as having enhanced thermostability in comparison to WT.







TABLE 2







Residual activity of lysozyme variants relative to WT after heating










Variant




Mutations
Relative residual activity %













N19D
15



H20Y
37



K22G
26



T45G
21



Y47F
18



D49G
14



H55A
20



R98G
9



G102P + Y134V
−5



Y111F
−1



H120Q
−6



H136N
2



S139G
−10



R153T
4



A158S
11



K164T
10



A171R
−11



K178G
−5



S193A
−3



H198F
−9



D203N
−13



N206A
−4



E107A
3

## Claims

1. An isolated lysozyme variant of a parent lysozyme, comprising an amino acid alteration at one or more positions selected from the group consisting of
19, 20, 22, 30, 32, 35, 45, 47, 49, 55, 56, 76, 89, 98, 102, 108, 110, 111, 112, 113, 119, 120, 121, 131, 134, 135, 147, 153, 158, 161, 164, 171, 178, 183, 185, 186, 187, 193, 195, 196, 198, 203, and 206,
wherein SEQ ID NO: 3 is used for numbering, the lysozyme variant is at least 90% identical to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3, and the lysozyme variant has antimicrobial activity.

2. The lysozyme variant of claim 1, which is at least 95% identical to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3.

3. The lysozyme variant of claim 2, which comprises a substitution at position 18.

4. The lysozyme variant of claim 2, which comprises a substitution at position 19.

5. The lysozyme variant of claim 2, which comprises a substitution at position 20.

6. The lysozyme variant of claim 2, which comprises a substitution at position 22.

7. The lysozyme variant of claim 2, which comprises a substitution at position 45.

8. The lysozyme variant of claim 2, which comprises a substitution at position 49.

9. The lysozyme variant of claim 2, which comprises a substitution at position 55.

10. The lysozyme variant of claim 2, which comprises a substitution at position 56.

11. The lysozyme variant of claim 10, which comprises Y56W.

12. The lysozyme variant of claim 2, which comprises a substitution at position 131.

13. The lysozyme variant of claim 12, which comprises V131C.

14. The lysozyme variant of claim 2, which comprises a substitution at position 161.

15. A detergent composition comprising the lysozyme variant of claim 2 and a surfactant.

16. A feed composition comprising the lysozyme variant of claim 2 and a feed component.

17. A method for reducing microbial contamination, comprising treating a microbial contaminated surface with a lysozyme variant of claim 2.

18. An isolated polynucleotide encoding the lysozyme variant of claim 2.

19. A recombinant host cell comprising an expression vector comprising the polynucleotide of claim 18.

20. A method of producing a lysozyme variant, comprising:
(a) culturing a host cell of claim 19 under conditions suitable for expression of the variant; and
(b) recovering the lysozyme variant from the cultivation medium.

21. An isolated lysozyme variant of a parent lysozyme, comprising an amino acid alteration at one or more positions selected from the group consisting of
85, 128, and 174,

wherein SEQ ID NO: 3 is used for numbering, the lysozyme variant is at least 90% identical to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3, and the lysozyme variant has antimicrobial activity.

22. The lysozyme variant of claim 21, which is at least 95% identical to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3.

23. A detergent composition comprising the lysozyme variant of claim 22 and a surfactant.

24. A feed composition comprising the lysozyme variant of claim 22 and a feed component.

25. A method for reducing microbial contamination, comprising treating a microbial contaminated surface with a lysozyme variant of claim 22.

26. An isolated polynucleotide encoding the lysozyme variant of claim 22.

27. A recombinant host cell comprising an expression vector comprising the polynucleotide of claim 25.

28. A method of producing a lysozyme variant, comprising:
(a) culturing a host cell of claim 27 under conditions suitable for expression of the variant; and
(b) recovering the lysozyme variant from the cultivation medium.