# Patent

## Title
Polypeptides of strain Bacillus sp. P203

## Abstract

A new strain Bacillus sp. P203 (Bacillus plakortiensis) is disclosed. Isolated mature functional polypeptide which is obtainable from the bacterium strain Bacillus sp. P203 deposited under accession number DSM 17419 are disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK2006/000447 filed Aug. 15, 2006 which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2005 01156 filed Aug. 16, 2005 and U.S. provisional application No. 60/709,264 filed Aug. 18, 2005, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to functional polypeptides encoded by polynucleotides comprised in the genome of strains of a novel Bacillus species Bacillus plankortiensis. The reference strain of this species is Bacillus sp. P203 deposited under DSM 17419. The invention relates further to the polynucleotides and constructs of such polynucleotides encoding such polypeptides or facilitating their expression as well as to method for preparing the polypeptide. Still further the invention relates to compositions comprising the polypeptides and polynucleotides and to uses of the polypeptide. Still further the invention relates to a bacterium Bacillus sp. P203 as deposited under accession number DSM 17419.
BACKGROUND OF THE INVENTION
Alkaliphilic Bacillus strains are in focus of industrial biotechnology. Alkaliphilic strains and especially enzymes of those species have great potential in biotechnical applications where enzymatic activity (or even maximum activity) at high pH values is required for biotechnical processes (Horikoshi: Alkaliphiles: Some Applications of Their Products for Biotechnology; Microbiology and Molecular Biology Reviews, Vol. 63, No. 4, 1999). Examples for alkaliphilic Bacillus strains as source for novel catalysts are B. clausii, B. pseudofirmus, B. clarkii, B. gibsonii. In the pursuit of novel enzymes it is also known to screen for such new enzymes by subjecting potential candidates to specific enzyme assays. This approach is limited to the availability of enzyme assays and does not allow the identification of functional enzymes or polypeptides for which the activity is still unknown.
Further, whole genome sequencing is a known method to obtain the information on all genes from a given microorganism e.g. as described in Fleischmann et al.; Whole genome sequences and assembly of Haemophilus influenzae Rd; Nature 269: 496-512; (1995).
Most enzymes for industrial use are enzymes which are secreted to the medium by a microorganism. However, only a few percent of a microorganisms' genome encodes secreted proteins. For example only approx. 4% of the Bacillus subtilis genome or its closest relatives encode secreted proteins (Van Dijl et al.: Protein transport pathways in Bacillus subtilis: a genome-based road map; in “Bacillus subtilis and its closest relatives—From genes to cells; p. 337-355; A. L. Sonenshein (ed.); ASM Press 2002).
One disadvantage of genome sequencing is that the vast majority of the obtained sequences encode non secreted proteins.
An additional disadvantage of genomic sequencing particular to eukaryotes such as fungi is that the genome size is many times larger than a bacterial genome making gene discovery by this method more costly and time consuming.

    
    
        The random sequencing of cDNAs (Expressed sequence tags or ESTs) is another approach that allows for discovery of secreted proteins. In general, EST approaches suffer two drawbacks with regard to secreted protein identification; 1) Depending on the induction conditions used for the cDNA library sequenced, very few, typically between 0.5%-15% or even 1 and 5% of the cDNAs encode secreted proteins. 2) The clones all come from a cDNA pool derived from mRNAs that are present in the organism in proportion to the induction level of each particular gene.
    
    


Also known is signal trapping which is a method to identify genes including nucleotides encoding a signal peptide using a translational fusion to an extra cellular reporter gene lacking its own signal (WO 01/77315).
SUMMARY OF THE INVENTION
The present inventors have found a strain of a novel Bacillus species Bacillus plankortiensis. The reference/type strain of this species is Bacillus sp. P203 deposited under DSM 17419. The strain grows at pH (7-11) and at low temperatures (4-30° C.). This strain is interesting because the phylogenetic distance between the public known strains and strain DSM17419 is significant and because the growth conditions are similar to conditions for several applications for industrial enzymes.
The genome of a microorganism contains thousands of different genes; some encoding polypeptides some coding for RNAs. Only a limited number of the genes in the genome of a microorganism encode functional polypeptides which are secreted by the microorganism to the surrounding medium serving an external purpose for the microorganism. Such polypeptides are interesting for industry from the point of view that such polypeptides may be produced in considerable amounts in continuous processes without destroying the cells producing the polypeptides.
It is an object of the present invention to identify and provide polypeptides secreted from strain Bacillus sp. P203 deposited under accession number DSM 17419 which have functional purpose for the strain Bacillus sp. P203 because such polypeptides may not only be used for industrial purposes but they may also be produced in industrially relevant processes and amounts.
In a first aspect the invention provides functional polypeptides encoded by polynucleotides comprised in the genome of strains of Bacillus species Bacillus plankortiensis deposited under accession number DSM 17419, and an isolated mature functional polypeptide which is at least 70% identical to and exhibits the same function of a corresponding secreted polypeptide obtainable from the bacterium strain Bacillus sp. P203.
In further aspects the invention provides a polynucleotide encoding the polypeptide of the invention; a nucleotide construct comprising the polynucleotide encoding the polypeptide, operably linked to one or more control sequences that direct the production of the polypeptide in a host cell; a recombinant expression vector comprising the nucleotide construct of the invention and to a recombinant host cell comprising the nucleotide construct of the invention.
In still further aspects the invention provides a method of preparing a polypeptide of the invention comprising:

    (a) cultivating a strain comprising a nucleotide sequence encoding a polypeptide of the invention which strain is capable of expressing and secreting the polypeptide and
    (b) recovering the polypeptide.


In a further aspect the invention provides a composition comprising a polypeptide of the invention and a method for preparing such a composition comprising admixing the polypeptide of the invention with an excipient.
In a further aspect the invention provides a composition comprising a polynucleotide of the invention and a method for preparing such a composition comprising admixing the polynucleotide of the invention with an excipient.
In further aspects the invention provides use of the polypeptide of the invention or a composition comprising said polypeptide in various applications.
In a final aspect the invention provides an electronic storage medium comprising information of the amino acid sequence of polypeptides of the invention or the nucleotide sequences of the polynucleotide of the invention.
Sequence Listing
The present application contains information in the form of a sequence listing, which is appended to the application and also submitted on a data carrier accompanying this application. The contents of the data carrier are fully incorporated herein by reference.
SEQ ID NO: 1 encodes serine protease comprised in SEQ ID NO: 2; SEQ ID NO: 3 encodes the carbonic anhydrase comprised in SEQ ID NO: 4; SEQ ID NO: 5 encodes the carbonic anhydrase comprised in SEQ ID NO:6; SEQ ID NO: 7 encodes xylanase comprised in SEQ ID NO: 8; SEQ ID NO: 9 encodes rhamnogalacturonan lyase comprised in SEQ ID NO: 10; SEQ ID NO: 11 encodes galactanase comprised in SEQ ID NO: 12.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows growth characteristics of Bacillus sp. P203.
FIG. 2 shows stability of carbonic anhydrase from Bacillus sp. P203.
FIG. 3 shows inhibition of carbonic anhydrase with acetazolamide.
FIG. 4 shows inhibition of carbonic anhydrase with metal ions, azide and other reagents.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “identity” as used herein, is to be understood as the homology 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 was determined by using AlignX in the program of Vector NTI ver. 7.1 (Informax inc., 7600. Wisconsin Avenue, Suite #1100, Bethesda, Md. 20814, USA). Amino acid alignment was created using the Clustal W algorithm (Nucleic Acid Research, 22 (22): 4673-4680, 1994). The following additional parameters are used: Gap opening penalty of 10, Gap extension penalty of 0.05, Gap separation penalty range of 8. Pairwise alignment parameters were Ktuple=1, gap penalty=3, gap length opening penalty=10, gap extension penalty=0.1, window size=5 and diagonals=5. The degree of identity between two nucleotide sequences is determined using the same algorithm and software package as described above for example with the following settings: Gap penalty of 10, and gap length penalty of 10. Pairwise alignment parameters is Ktuple=3, gap penalty=3 and windows=20.
The term “functional polypeptide” as used herein in the context of the present invention means a polypeptide which can be expressed and secreted by a cell and which constitutes an operational unit capable of operating in accordance with the function it is designed to fulfil by the cell. Optionally, co-factors may be required for the polypeptide to adopt the intended function. One example of functional polypeptides is catalytically active polypeptides or enzymes which help the cell catalyzing reactions in the environment surrounding the cell. Another example could be polypeptides which serve as signal substance. Further examples are polypeptides which function as sensors (receptors) for environmental parameters (chemicals in the environment surrounding the cell) or polypeptides, which are active against other organisms (antimicrobial (poly)peptides) or polypeptides, which contributes to the structural integrity of the cell.
The term “mature region” as used herein about portion of an amino acid sequences or polypeptide means the portion or region or domain or section of the amino acid sequences or polypeptide which is the mature functional polypeptide.
The term “region of nucleotide sequence encoding a mature polypeptide” as used herein means the region of a nucleotide sequence counting from the triplet encoding the first amino acid of a mature polypeptide to the last triplet encoding the last amino acid of a mature polypeptide.
The term “secreted polypeptide” as used herein is to be understood as a polypeptide which after expression in a cell is either transported to and released to the surrounding extracellular medium or is associated/embedded in the cellular membrane so that at least a part of the polypeptide is exposed to the surrounding extracellular medium.
Polypeptides of the Invention
The present invention relates to polypeptides similar to those secreted polypeptides obtainable from strain Bacillus sp. P203 deposited under accession number DSM 17419. In particular the invention provides an isolated mature functional polypeptide which is at least 70%, preferably 80% or more, such as 90%, 95%, 96%, 97%, 98% or 99% identical to and exhibits the same function of a corresponding secreted polypeptide obtainable from the strain Bacillus sp. P203 deposited under accession number DSM 17419.
Polypeptides of the invention are, in particular, secreted by strain Bacillus sp. P203 deposited under accession number DSM 17419 with the purpose of serving a function for that particular cell.
Among the thousands of potential genes in the genome of strain Bacillus sp. P203 deposited under accession number DSM 17419 the polynucleotides of this genome encoded 6 secreted functional mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, which were determined to be functional, that is translated into functional polypeptides by the chosen host cell.
Further in a particular embodiment the genes encoding the said mature polypeptides can all be expressed and their corresponding mature polypeptides can be secreted when culturing an E. coli host transformed with polynucleotides comprising those regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide. By comparing homology or identity of the sequences of the 6 polypeptide sequences to known sequences the particular function of the polypeptides were annotated. All 6 secreted functional polypeptides were determined to be enzymes.
In particular the isolated polypeptide is selected from the group consisting of:

    (a) a polypeptide having an amino acid sequence which has at least 70% identity with an amino acid sequence selected from the group consisting of the mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and
    (b) a polypeptide which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence selected from the group consisting of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide,
        (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide;

wherein the polypeptide exhibits the function of the corresponding mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.

    
    


In one particular embodiment the polypeptide of the invention is selected among the enzymes secreted by strain Bacillus sp. P203 deposited under accession number 17419 and isolated by the present inventors, i.e. the group of enzymes consisting of xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase.
The invention also provides an isolated enzyme selected from the group consisting of:

    (a) an enzyme comprising an amino acid sequence which has at least 70% identity with the amino acid sequence of a mature enzyme selected from the group consisting of xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase secreted from the strain of strain. Bacillus sp. P203 deposited under accession number 17419 and
    (b) an enzyme which is encoded by a nucleotide sequence which hybridize under high stringency conditions with a polynucleotide probe selected from the group consisting of
    
        (i) the complementary strand to a nucleotide sequence comprised in the strain of strain Bacillus sp. P203 deposited under accession number 17419 encoding a mature enzyme selected from the group consisting of xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase secreted from that strain;
        (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences comprised in the strain of strain Bacillus sp. P203 deposited under accession number DSM 17419 encoding a mature enzyme selected from the group consisting of xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase secreted from that strain and

wherein the enzyme have a function selected from xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase.

    
    


The polypeptide of the invention is an isolated polypeptide, preferably the preparation of the polypeptide of the invention contains at the most 90% by weight of other polypeptide material with which it may be natively associated (lower percentages of other polypeptide material are preferred, e.g. at the most 80% by weight, at the most 60% by weight, at the most 50% by weight, at the most 40% at the most 30% by weight, at the most 20% by weight, at the most 10% by weight, at the most 9% by weight, at the most 8% by weight, at the most 6% by weight, at the most 5% by weight, at the most 4% at the most 3% by weight, at the most 2% by weight, at the most 1% by weight and at the most 0.5% by weight). Thus, it is preferred that the isolated polypeptide of the invention is at least 92% pure, i.e. that the polypeptide of the invention constitutes at least 92% by weight of the total polypeptide material present in the preparation, and higher percentages are preferred such as at least 94% pure, at least 95% pure, at least 96% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, and at the most 99.5% pure. In particular, it is preferred that the polypeptide of the invention is in “essentially pure form, i.e. that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide of the invention by means of well-known recombinant methods.
The polypeptide of the invention of the invention may be synthetically made, naturally occurring or a combination thereof. In a particular embodiment the polypeptide of the invention may be obtained from a microorganism such as a prokaryotic cell, an archaeal cell or a eukaryotic cell. The cell may further have been modified by genetic engineering
In a particular embodiment, the polypeptide of the invention is an enzyme exhibiting optimum enzyme activity at a temperature within the range from about 10° C. to about 80° C., particularly in the range from about 20° C. to about 60° C.
In a particular embodiment the polypeptide of the invention is an enzyme, which is functionally stabile at a temperature of up to 100° C., in particular up to 80° C., more particularly up to 60° C.
In a particular embodiment the polypeptide of the invention is an enzyme exhibiting at least 20%, in particular at least 40%, such as at least 50%, in particular at least 60%, such as at least 70%, more particularly at least 80%, such as at least 90%, most particularly at least 95%, such as about or at least 100% of the enzyme activity of an enzyme selected from mature enzymes comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
In particular the isolated mature functional polypeptide is at least 70. % identical to and exhibits the same function of a corresponding secreted polypeptide obtainable from the bacterium strain Bacillus sp. P203 deposited under accession number DSM 17419 and specially the polypeptide of the invention comprises, contains or consists of an amino acid sequence which has at least 70% identity with a polypeptide sequence selected from the group consisting of mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12. The percent identity is particularly at least 95%, e.g. at least 96%, such as at least 97%, and even more particularly at least 98%, such as at least 99% or even 100% identity.
In another particular embodiment the percent identity is at least 50%; particularly at least 60%, particularly at least 65%, particularly at least 70%, particularly at least 75%, particularly at least 80%, and even more particularly at least 85% identity.
In a particular embodiment, the amino acid sequence of the polypeptide of the invention differs by at the most ten amino acids (e.g. by ten amino acids), in particular by at the most five amino acids (e.g. by five amino acids), such as by at the most four amino acids (e.g. by four amino acids), e.g. by at the most three amino acids (e.g. by three amino acids), in particular by at the most two amino acids (e.g. by two amino acids), such as by one amino acid from the mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO 10 and SEQ ID NO: 12.
The polypeptide of the invention may be a wild-type polypeptide isolated from a natural source such as the strain of strain Bacillus sp. P203, deposit number DSM 17419, or another wild type strain, however the present invention also encompass artificial variants, where a polypeptide of the invention has been mutated for example by adding, substituting and/or deleting one or more amino acids from said polypeptide while retaining the function of the polypeptide and/or other properties. Hence, the polypeptide of the invention may be an artificial variant, wherein at least one substitution, deletion and/or insertion of an amino acid has been made to an amino acid sequence comprising or consisting of the mature polypeptide comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
The polypeptides of the invention also include functional fragments of the amino acid sequences described herein and nucleic acids encoding functional fragments of the amino acid sequences described herein, including fragments of the mature enzymes secreted from the strain, of strain Bacillus sp. P203, deposited under accession number DSM 17419, as described herein, including fragment of an enzyme selected from the group consisting of xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase secreted from the strain of strain Bacillus sp. P203 deposited under accession number DSM 17419.
Artificial variants may be constructed by standard techniques known in the art usually followed by screening and/or characterization. Standard techniques includes classical mutagenesis, e.g. by UV irradiation of the cells or treatment of cells with chemical mutagens as described by Gerhardt et al. (1994); in vivo gene shuffling as described in WO 97/07205; in vitro shuffling as described by Stemmer, (1994) or WO 95/17413, random mutagenesis as described by Eisenstadt E. et al., (1994); PCR techniques as described by Poulsen et al. (1991); family shuffling as described by J. E. Ness, et al, Nature Biotechnology, vol. 17, pp. 893-896 (1999); site-directed mutagenesis as described by Sambrook et al. (1989), Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y. A general description of nucleotide substitution can be found in e.g. Ford et al., 1991, Protein Expression and Purification 2, p. 95-107.
Such standard genetic engineering methods may also be used prepare a diversified library of variant nucleotide sequences from the genes encoding one or more parent enzymes of the invention, expressing the enzyme variants in a suitable host cell and selecting a preferred variant(s). A diversified library can be established by a range of techniques known to the art (Reetz M T; Jaeger K E, in Biocatalysis—from Discovery to Application edited by Fessner W D, Vol. 200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p. 389-391, 1994; Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000, 1997; or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp 5511-5515, 1998).
In a particular embodiment of the invention, amino acid changes (in the artificial variant as well as in wild-type enzyme) are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
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, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions which do not generally alter and or impair the function of a protein are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, 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 as well as these in reverse.
In a particular embodiment the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may be performed, which improve the thermal stability of the enzyme, which alter the substrate specificity, which changes the pH optimum, and the like.
Particularly, the number of such substitutions, deletions and/or insertions in the polypeptide of the invention, particularly in those polypeptides selected from the group consisting of mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ. ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 to produce an artificial variant is at the most 10, such as at the most 9, e.g. at the most 8, more preferably at the most 7, e.g. at the most 6, such as at the most 5, most preferably at the most 4, e.g. at the most 3, such as at the most 2, in particular at the most 1.
In a particular embodiment the artificial variant is a variant, which has an altered, preferably reduced, immunogenicity, especially allergenicity, in animals including man as compared to a parent enzyme. The term “immunogenicity in this context is to be understood as the artificial variant capability of invoking a an altered, in particular reduced, immunological response when administered to an animal, including intravenous, cutaneous, subcutaneous, oral and intratracheal administration. The term “immunological response in this context means that the administration of the artificial variant causes an alteration in the immunoglobulin levels in the animal body, such as in IgE, IgG and IgM or an alteration in the cytokine level in the animal body. Methods for mapping immunogenic/antigenic epitopes of a protein, preparing variants with altered immunogenicity and methods for measuring an immunological response is well known to the art and are described e.g. in WO 92/10755, WO 00/26230, WO 00/26354 and WO 01/31989. The term “allergenicity in this context is to be understood as the artificial variant ability of invoking an altered, in particular reduced, production of IgE in an animal as well as the ability to bind IgE from said animal. Particularly allergenicity arising from intratracheal administration of the polypeptide variant to the animal is particularly of interest (also known as respiratory allergenicity).
In a further embodiment, the polypeptide of the invention is a polypeptide which is encoded by nucleotide sequences which hybridize under at least high stringency conditions, particularly under very high stringency conditions with a polynucleotide probe selected from the group consisting of

    (i) the complementary strand to a nucleotide sequence selected from the group of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide,
    (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from regions of SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide
    (iii) a fragment of (i) or (ii) encoding a secreted polypeptide having the function of the corresponding mature polypeptide comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12

(J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).



In particular, the polypeptide of the invention is encoded by a polynucleotide comprising a nucleotide sequence selected from the group of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide or a sequences differing there from by virtue of the degeneracy of the genetic code. More particularly, the polypeptide of the invention is encoded by a polynucleotide consisting of a nucleotide sequence selected from the group of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide or a sequence differing there from by virtue of the degeneracy of the genetic code.
The nucleotide sequences of regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide or a subsequence thereof, as well as the amino acid sequences of the mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 or a fragment thereof, may be used to design a polynucleotide probe to identify and clone DNA encoding enzymes of the invention 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 15, preferably at least 25, more preferably at least 35 nucleotides in length, such as at least 70 nucleotides in length. It is; however, preferred that the polynucleotide probe is at least 100 nucleotides in length. For example, the polynucleotide probe may be at least 200 nucleotides in length, at least 300 nucleotides in length, at least 400 nucleotides in length or at least 500 nucleotides in length. Even longer probes may be used, e.g., polynucleotide probes which are at least 600 nucleotides in length, at least 700 nucleotides in length, at least 800 nucleotides in length, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labelled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin).
Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA, which hybridizes with the probes described above and which encodes enzymes of the invention. Genomic or other DNA from such 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 materials. In order to identify a clone or DNA which has the required homology and/or identity or is homologous and/or identical with of nucleotides selected from regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide, the carrier material with the immobilized DNA is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labelled polynucleotide probe which again hybridizes to a nucleotide sequence selected from regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide under high to very high stringency conditions. Molecules to which the polynucleotide probe hybridizes under these conditions may be detected using X-ray film or by any other method known in the art. Whenever the term “polynucleotide probe is used in the present context, it is to be understood that such a probe contains at least 15 nucleotides.
In an interesting embodiment, the polynucleotide probe is the complementary strand of a nucleotide sequence selected from regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide.
In another interesting embodiment, the polynucleotide probe is the complementary strand of a nucleotide sequence which encodes an enzyme selected from the group of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11. In a further interesting embodiment, the polynucleotide probe is the complementary strand of a mature polypeptide coding region of a nucleotide sequence selected from regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide.
For long probes of at least 100 nucleotides in length, high to very high stringency conditions are defined as pre-hybridization and hybridization at 42° C. in 5×SSPE, 1.0% SDS, 5×Denhardt's solution, 100 microgram/ml sheared and denatured salmon sperm DNA, following standard Southern blotting procedures. Preferably, the long probes of at least 100 nucleotides do not contain more than 1000 nucleotides. For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 60° C. (high stringency), in particular washed three times each for 15 minutes using 0.1×SSC, 0.1% SDS at 68° C. (very high stringency).
Although not particularly preferred, it is contemplated that shorter probes, e.g. probes which are from about 15 to 99 nucleotides in length, such as from about 15 to about 70 nucleotides in length, may be also be used. For such short probes, stringency conditions are defined as pre-hybridization, hybridization, and washing post-hybridization at 5° C. to 10° C. below the calculated Tm using the calculation according to 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 short probes which are about 15 nucleotides to 99 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.
SEQ ID NO: 2, Serine Protease
In a particular embodiment the polypeptide of the invention is an serine protease comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with an serine protease obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature serine protease comprised in SEQ ID NO: 2. The serine protease of the present invention is further described in Example 4.
SEQ ID NO: 4, Carbonic Anhydrase
In a particular embodiment the polypeptide of the invention is an carbonic anhydrase comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a carbonic anhydrase obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature carbonic anhydrase comprised in SEQ ID NO: 4. The carbonic anhydrase of the present invention is further described in Example 8.
SEQ ID NO: 6, Carbonic Anhydrase
In a particular embodiment the polypeptide of the invention is an carbonic anhydrase comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a carbonic anhydrase obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature carbonic anhydrase comprised in SEQ ID NO: 6.
SEQ ID NO: 8, Xylanase
In a particular embodiment the polypeptide of the invention is an xylanase comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a xylanase obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature xylanase comprised in SEQ ID NO: 8. The xylanase of the present invention is further described in Example 6.
SEQ ID NO: 10, Rhamnogalacturonan Lyase
In a particular embodiment the polypeptide of the invention is a rhamnogalacturonan lyase comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a rhamnogalacturonan lyase obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature rhamnogalacturonan lyase comprised in SEQ ID NO: 8. The rhamnogalacturonan lyase of the present invention is further described in Example 5.
SEQ ID NO: 12, Galactanase
In a particular embodiment the polypeptide of the invention is a galactanase comprising or consisting of an amino acid sequence which has at least 70%, such as at least 80% or 85%, particularly at least 95%, more particularly at least 96%, more particularly at least 97%, more particularly at least 98%, more particularly at least 99% or most particularly 100% identity with a galactanase obtainable from strain Bacillus sp. P203, in particular that strain of strain Bacillus sp. P203 deposited under accession number DSM 17419, more particularly the mature galactanase comprised in SEQ ID NO: 8. The galactanase of the present invention is further described in Example 7.
Polynucleotides
The present invention also relates to polynucleotides, particularly isolated polynucleotides, comprising or consisting of a nucleotide sequence encoding a polypeptide of the invention. In a particular embodiment, the nucleotide sequence is set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 including nucleotide sequences differing there from by virtue of the degeneracy of the genetic code. In a further embodiment the polynucleotide of the invention is a modified nucleotide sequence which comprises or consists of a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide and which comprises at least one modification/mutation compared with the parent nucleotide sequence comprised in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.
The techniques used to isolate and/or clone a nucleotide sequence encoding an enzyme are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleotide sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al, 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.
The nucleotide sequence may be obtained by standard cloning procedures used in genetic engineering to relocate the nucleotide sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired fragment comprising the nucleotide sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleotide sequence will be replicated. The nucleotide sequence may be of genomic, cDNA, RNA, semi-synthetic, synthetic origin, or any combinations thereof.
In particular the polynucleotide comprises, preferably consists of, a nucleotide sequence which has at least 50% identity with a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide. Particularly, the nucleotide sequence has at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide. Particularly, the nucleotide sequence comprises a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide. In an even more particular embodiment, the nucleotide sequence consists of a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide.
In particular the polynucleotide comprises, preferably consists of, a nucleotide sequence encoding a mature enzyme selected from xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase and which has at least 50% identity, particularly at least 65% identity, more particularly at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with a nucleotide sequence encoding a mature enzyme selected from xylanase, serine protease, carbonic anhydrase, rhamnogalacturonan lyase and galactanase secreted from the strain of strain Bacillus sp. P203 deposited under accession number 17419.
SEQ ID NO: 1
In a particular embodiment the polynucleotide of the invention encodes a serine protease and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 1 encoding a mature serine protease.
SEQ ID NO: 3
In a particular embodiment the polynucleotide of the invention encodes a carbonic anhydrase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 3 encoding a mature carbonic anhydrase.
SEQ ID NO: 5
In a particular embodiment the polynucleotide of the invention encodes a carbonic anhydrase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 5 encoding a mature carbonic anhydrase.
SEQ ID NO: 7
In a particular embodiment the polynucleotide of the invention encodes a xylanase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 7 encoding a mature xylanase.
SEQ ID NO: 9
In a particular embodiment the polynucleotide of the invention encodes a rhamnogalacturonan lyase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 9 encoding a mature rhamnogalacturonan lyase,
SEQ ID NO: 11
In a particular embodiment the polynucleotide of the invention encodes a galactanase and comprises or consists of an nucleotide sequence which has at least 70% identity, more particularly at least 80% identity, more particularly at least 90% identity, more particularly at least 95% identity, more particularly at least 96% identity, more particularly at least 97% identity, more particularly at least 98% identity, more particularly at least 99% identity or most particularly 100% identity with the nucleotide sequence of SEQ ID NO: 11 encoding a mature galactanase.
Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of a polypeptide which comprises an amino acid sequence that has at least one substitution, deletion and/or insertion as compared to an amino acid sequence selected from mature polypeptide comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID. NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12.
It will be apparent to those skilled in the art that such modifications can be made to preserve the function of the enzyme i.e. made outside regions critical to the function of the enzyme. Amino acid residues which are essential to the function are therefore preferably not subject to modification, such as substitution. Amino acid residues essential to the function may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). Sites of substrate-enzyme interaction can be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).
Moreover, a nucleotide sequence encoding an enzyme of the invention may be modified by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the enzyme encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme.
The introduction of a mutation into the nucleotide sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure, which utilizes a super coiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with DpnI, which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art may also be used. For a general description of nucleotide substitution, one may consult with e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
The present invention also relates to a polynucleotide comprising, preferably consisting of, a nucleotide sequence which encodes a polypeptide of the invention and which hybridizes under high stringency conditions, preferably under very high stringency conditions with a polynucleotide probe selected from the group consisting of:

    (i) the complementary strand to a nucleotide sequence selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11

encoding a mature polypeptide,

    (ii) the complementary strand to the cDNA sequence contained in a nucleotide sequences selected from the regions of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding a mature polypeptide and,
    (iii) a fragment of (i) or (ii) encoding a secreted mature polypeptide having the function of the corresponding mature polypeptides comprised in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12

As will be understood, details and particulars concerning hybridization of the nucleotide sequences will be the same or analogous to the hybridization aspects discussed in the section titled “polypeptides of the invention herein.



The present invention also encompasses a storage medium suitable for use in an electronic, preferably digital, device comprising information of the amino acid sequence of polypeptides of the invention or the nucleotide sequences of the polynucleotide of the invention, in particular any of the polypeptide or polynucleotide sequences of the invention in an electronic or digital form, such as binary code or other digital code. The storage medium may suitably be a magnetic or optical disk and the electronic device a computing device and the information may in particular be stored on the storage medium in a digital form.
Recombinant Expression Vectors.
The present invention also relates to recombinant expression vectors comprising the nucleic acid construct of the invention. The various nucleotide and control sequences described above may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, the nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. 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 recombinant expression vector may be any vector (e.g., a plasmid 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 licheniformis, 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 preferably 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.
For integration into the host cell genome, the vector may rely on the nucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleotides, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).
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, e.g., Sambrook et al., 1989, supra).
Recombinant Host Cells.
The present invention also relates to recombinant a host cell comprising the nucleic acid construct of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleotide 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 unicellular microorganism, e.g., a prokaryote or a non-unicellular microorganism, e.g., a eukaryote.
Useful unicellular cells 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 lautus, Bacillus lentus, Bacillus lichenifonnis, 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, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., 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, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).
The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
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., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). 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, P. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
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, supra). 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 obligately 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 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 238 023 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, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 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.
The Donor Strain
The invention also provides a strain Bacillus sp. P203, as deposited under accession number DSM 17419, and compositions comprising this microorganism.
The invention also provides strains of the genus Bacillus consisting of a novel species (Bacillus plakortiensis). The novel species comprise alkali- and halo-tolerant Bacillus strains which grow from pH 7-11 and from 0-12% NaCl concentration in the media at temperatures ranging from 4-30° C. Optimal conditions were from 4-20 C, at pH 7.5 and in 10% NaCl. Strains of the novel species can be further distinguished from related strains by the following parameters: The strains could utilize glycerol, glucose, D-fructose, D-mannose, D-mannitol, arbutin, aesculin, salicine, gelatine, citrate, glyceryl tributyrate, D-maltose, D-trehalose, skimmed milk, AZCL-casein, AZCL-arabinan and AZCL-galactan but not starch, Tween 20, AZCL-xyloglucan, AZCL-pullulan, AZCL-arabinoxylan, AZCL-dextrain, AZCL-cellulose, AZCL-beta-glucan, AZCL-xylan, red amylose, red pullulan and red starch. Hydrolysis of ONPG was observed. Indol production (using Kováçs reagent) was negative, Voges Proskauer reaction (acetoin production) and test for catalase and oxidase activity was positive.
Strain P203 had 98% nucleotide identity from 16S rDNA sequence to type strain Bacillus gibsonii (deposit number DSM8722). Growth characteristics of strain P203 are shown in FIG. 1. Bacillus plakortiensis is more related to Bacillus gibsonii than to any other known Bacillus species, based on comparative 16S rDNA analysis. However, DNA homology between Bacillus plakortiensis, strain P203, and the closest known Bacillus species B. gibsonii type strain is (25.8-29.5%) DNA-DNA hybridization and G+C DNA content, determined by reverse phase HPLC at the DSMZ, Braunschweig, Germany. DNA was isolated using a French press (Thermo SpectronicTM) and purified by chromatography on hydroxyapatite as described by (Cashion, et al., 1977). DNA-DNA hybridization was carried out as described by Ley, J. D., Cattoir, H. & Reynaerts, A. (1970): The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133-42, with the modifications described by Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983): Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184-192., using a model Cary 100 Bio. UV/VIS-spectrophotometer equipped with a Peltier-thermostated 6×6 multi cell changer and a temperature controller with in situ temperature probe (Varian).
Methods for Preparing Enzyme Polypeptides
The present invention also relates to methods for producing an enzyme of the invention comprising (a) cultivating a strain comprising a nucleotide sequence encoding an enzyme of the invention which strain is capable of expressing and secreting the enzyme and (b) recovering the enzyme. In a particular embodiment the strain is a wild type strain such as the strain Bacillus sp. P203 DSM 17419, while in another embodiment the strain is a recombinant host cell as described, supra.
In these methods of the invention, the cells are cultivated in a nutrient medium suitable for production of the enzyme using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, 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). As the enzyme is secreted into the nutrient medium, the enzyme can be recovered directly from the medium.
The resulting enzyme may be recovered by methods known in the art. For example, the enzyme may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The polypeptides 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., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
The methods of the invention also include the TAST method of WO 01/77315 A1 on a sample of the strain Bacillus sp. P203 deposited under accession number 17419, i.e. by fusing genes (e.g. from a gene library) from the genome of the strain Bacillus sp. P203 deposited under accession number DSM 17419 with a gene encoding a signalless reporter, such as a beta-lactamase, via a transposon tag, growing host cell clones comprising the genes of the strain Bacillus sp. P203 DSM 17419 fused with a gene encoding a signalless reporter, such as a beta-lactamase, via a transposon tag in a medium revealing the presence of the reporter, such as an ampicillin containing medium, detecting clones secreting the reporter and isolating gene and polypeptide of the strain Bacillus sp. P203 deposited under accession number DSM 17419 comprised in that clone.
When growing host cell clones comprising the genes of the strain Bacillus sp. P203 deposited under, accession number 17419 fused with a gene encoding a signalless reporter, such as a beta-lactamase, via a transposon tag in a medium revealing the presence of the reporter, such as an ampicillin containing medium, only those clones expressing and secreting the reporter (e.g. beta-lactamase) will be detected (e.g. survive). However the reporter will only be secreted if the gene to which the reporter gene is fused has an intact promotor and ribosome binding site (i.e. a gene which is expressed by the cell to produce a polypeptide in real life), which can be recognized in the host strain, and if the reporter is translated so that the synthesized polypeptide is transported across the cytoplasma membrane and folded correctly. Hence, when inserting the fused gene into a selected host cell, those clones, for which a reporter presence is detected (e.g. ampicillin resistance), will contain a gene from the strain Bacillus sp. P203 deposited under accession number DSM 17419, which encodes a functional secreted polypeptide.
Transgenic Plants
The present invention also relates to a transgenic plant, plant part, or plant cell that has been transformed with a nucleotide sequence encoding an enzyme of the invention so as to express and produce the enzyme. In one embodiment the plant could be used as host for production of enzyme in recoverable quantities. The enzyme may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant enzyme may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor. In particular the plant or plant parts expressing the enzyme may be used as an improved starting material for production of fuel-alcohols or bio-ethanol.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. 
Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.
Also included within the scope of the present invention are the progeny of such plants, plant parts and plant cells.
The transgenic plant or plant cell expressing an enzyme of the invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding an enzyme of the invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.
Conveniently, the expression construct is a nucleic acid construct which comprises a nucleotide sequence encoding an enzyme of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).
The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding an enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.
For constitutive expression, the 35S-CaMV promoter may be used (Franck et al., 1980, Cell 21: 285-294). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito at al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad at al., 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitre and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya at al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588).
A promoter enhancer element may also be used to achieve higher expression of the enzyme of the invention in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding an enzyme of the present invention. For instance, Xu et al., 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression.
The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).
Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38). However it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.
Following transformation, the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well known in the art.
The present invention also relates to methods for producing an enzyme of the invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleotide sequence encoding an enzyme of the invention under conditions conducive for production of the enzyme and (b) recovering the enzyme.
Compositions Comprising Polypeptides and Methods for their Preparation
The invention provide a composition comprising a polypeptide of the invention and preferably an excipient and a method for preparing such a composition comprising admixing the polypeptide of the invention with an excipient. In particular the composition comprises at least two different polypeptides of the invention, preferably at least 3, more preferable at least 4, more preferable at least 5, more preferable at least 6. Most the composition comprises all polypeptides secreted when fermenting a sample of strain Bacillus sp. P203 deposited under accession number DSM 17419 or a mutant thereof wherein one or more genes has been deleted or added.
In a particular embodiment the polypeptide of the invention is the major (polypeptide) component of the composition, e.g., a mono-component composition. The excipient in this context is to be understood as any auxilliary agent or compound used to formulate the composition and includes solvent, carriers, stabilizers and the like.
The composition may further comprise one or more additional enzymes, 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 compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a solid composition. For instance, the enzyme composition may be formulated using methods known to the art of formulating polypeptides and/or pharmaceutical products, e.g. into coated or uncoated granules or micro-granules. The polypeptide of the invention may thus be provided in the form of a granule, preferably a non-dusting granule, a liquid, in particular a stabilized liquid, a slurry or a protected polypeptide. For certain applications, immobilization of the polypeptide on a solid matrix may be preferred.
The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art e.g. by stabilizing the polypeptide in the composition by adding and antioxidant or reducing agent to limit oxidation of the polypeptide or it may be stabilized by adding polymers such as PVP, PVA, PEG or other suitable polymers known to be beneficial to the stability of polypeptides in solid or liquid compositions
In a further embodiment the composition of the invention is a detergent composition which, in addition to the polypeptide of the invention, comprises a surfactant and optionally compounds selected from the group consisting of builders such as zeolites, bleaching agents such as percarbonate, bleach enhancers such as TAED or NOBS, suds suppressors, fragrants, etc.
In a further embodiment the composition of the invention is a feed composition that in addition to the polypeptide of the invention comprises a cereal or grain product.
In a further embodiment the composition of the invention is a food composition such as a baker's flour composition, a brewed product, a fruit juice, an oil or lard product comprising the polypeptide of the invention.
In a further embodiment the composition of the invention is a pulping composition, which in addition to the polypeptide of the invention, comprises pulp.
Use of Polypeptides or Compositions Comprising them
In still further aspects the invention provides use of the polypeptides or polynucleotides of the invention or a composition comprising said polypeptides or polynucleotides in various applications, particularly (technical) processes such as processes performed in industry or household, herein under for commercial research purposes. Hence the invention encompasses a process comprising employing a polypeptide of the invention or a polynucleotide of the invention in a (technical) industrial, research or household process.
In one embodiment the polypeptide or the composition of the invention is used for cleaning a cellulosic fabric.
In another embodiment the polypeptide or the composition of the invention is used to prepare a food or feed additive.
In yet another embodiment the polypeptide or the composition of the invention is used for treatment of lignolitic materials and pulp.
In particular carbonic anhydrase may be used for carbon fixation from CO2 emission streams, e.g. in membrane reactors for separating CO2 from gas streams using carbonic anhydrase for applications in e.g. electrical generation flue gas stacks, greenhouse gases, but also in unconventional applications such as pilot cockpits and astronaut's space suits to keep breathing air free of toxic CO2 levels.
In another embodiment carbonic anhydrases may be useful to promote water purification or mineralization from aqueous media by producing bicarbonate that can complex with ions dissolved in the aqueous media, especially divalent ions such as calcium, and aid in flocculation or precipitation of these ions from aqueous media. Such precipitation can in some applications be used to reduce water-hardness, and in other applications can have an added benefit of increasing the weight, volume, or opacity of certain products, such as in the paper-making process, or in remediation of soils and rocks.
Further carbonic anhydrase may also used as targets for the drug development such as carbonic anhydrase inhibitors (e.g. sulphonamide derivatives).
Detergent Disclosure
The polypeptide of the invention may be added to and thus become a component of a detergent composition:
The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
In a specific aspect, the invention provides a detergent additive comprising the polypeptide of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
In general the properties of the chosen 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 be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, 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®, FN2®, and FN3® (Genencor International Inc.).
Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (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.
Preferred commercially available lipase enzymes include LipolaseTM, Lipolase UltraTM and Lipex (Novozymes A/S).
Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g. a special strain of B. licheniformis, described in more detail in GB 1,296,839.
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.
Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (Novozymes A/S), RapidaseTM and PurastarTM (from Genencor International Inc.).
Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
Commercially available cellulases include Celluzyme®, and Carezyme® (Novozymes), Clazinase®, and Puradax HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).
Peroxidases/Oxidases: Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include 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 Guardzyme® (Novozymes A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
The detergent composition comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants are typically present at a level of from 0.1% to 60% by weight.
When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap.
When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine (“glucamides).
The detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system which may comprise a H2O2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil re-deposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
It is at present contemplated that in the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor.
The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 that is hereby incorporated as reference.
Deposited Microorganisms
The following microorganism were deposited by the applicant according to the Budapest Treaty on the International Recognition of the deposits of Microorganisms for the Purpose of Patent Procedures at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany:
23 Jun. 2005; strain Bacillus sp. P203; DSM accession number DSM 17419.
EXAMPLES
Example 1
Identifying Functional Polypeptides Secreted by Strain Bacillus sp. P203, DSM 17419
A. Genomic Library Construction
Chromosomal DNA from strain Bacillus sp. P203 (DSM 17419) was prepared by using standard molecular biology techniques (Ausuble et al. 1995 “Current protocols in molecular biology Publ: John Wiley and sons). The prepared DNA was partially cleaved with Mbo I and separated in a sucrose gradient by ultracentrifugation. Fragments of 3 to 10 kilobases were extracted, precipitated and resuspended in a suitable buffer.
A genomic library was made by using the Stratagene ZAP ExpressTM predigested Vector kit and Stratagene ZAP ExpressTM predigested Gigapack® cloning kit (BamHI predigested) (Stratagene Inc., USA) following the instructions/recommendations from the vendor. The resulting lambdaZAP library comprised 1.25×108 pfu of which 40,000 were collected for mass excision. The resulting 10,000 E. coli colonies were pooled and plasmids were prepared by using the Qiagen Spin Mini prep kit (Qiagen, Germany). The eluate of approx. 1 ml containing the plasmid DNA was precipitated in a centrifuge with 1 volume part of Na-acetate pH 5 and 2 volume parts 96% ethanol at 20000 rpm at 4° C., washed with 70% (v/v) ethanol, dried at room temperature and resuspended in 200 microliter TE buffer. The DNA concentration of the plasmid pool DNA of the strain Bacillus sp. P203 genomic library was 0.7 microgram/microliter.
B. Transposon Construction and Preparation
The rationale behind the methology of Transposon Assisted Signal Trapping (TAST) as described in WO 01/77315 A1 is to fuse all genes within a selected genome with a gene encoding a signalless beta-lactamase via a transposon tag. Hence when growing host cell clones comprising the genes of a genome fused with a gene encoding a signalless beta-lactamase via a transposon tag in an ampicillin containing medium only those clones expressing and secreting a beta-lactamase will survive. However the beta-lactamase will only be secreted if the gene to which the beta-lactamase gene is fused has an intact promotor and ribosome binding site (i.e. a gene which is expressed by the cell to produce a polypeptide in real life), which can be recognized in the host strain, and if the beta-lactamase is translated so that the synthesized polypeptide is transported across the cytoplasma membrane and folded correctly. Hence, when inserting the fused gene into a selected host cell, those clones, which are ampicillin resistant contains a gene which encodes a functional secreted polypeptide.
Usually, when employing the TAST methodology it is even not necessary to express the entire gene. When tagging the genes with a transposon, expression of the N-terminal part of the genes as protein fusion shows that the genes contain intact transcription, translation and secretion sequences. Hence expression of the N-terminal part of the genes as protein fusion is usually regarded as sufficient for assuring expression and secretion of the entire gene.
Thus it can be concluded that the genes obtained by the TAST method actually do encode secreted functional polypeptides.
Construction of a SigA4 Transposon Containing the Beta-Lactamase Reporter Gene:
Following the instructions of WO 01/77315 A1, the construction of a transposon containing a signal-less beta-lactamase gene was carried out using standard molecular biology techniques. The signal-less beta-lactamase gene was initially PCR amplified from the vector pUC19) using a proofreading polymerase (Pfu Turbo, Stratagene, USA). The resulting PCR fragment contained the restriction sites NotI and EcoRI in order to aid cloning. The plasmid pEntranceposon(Cam′) containing the Entranceposon and the antibiotic resistance markers CAT (encoding chloramphencol resistance in the transposon) was obtained from Finnzymes, OY (Espoo Finland). The plasmid was digested with the restriction enzymes NotI and EcoRI, gel purified and ligated with the signal-less beta-lactamase containing fragment. The ligation was transformed into electro-competent DH10B cells and the E. coli clone containing the recombinant plasmid with the signal-less beta-lactamase was identified by restriction analysis and named SigA2.
For transposon preparation, a smaller derivative of SigA2 was constructed, which lacked the b/a gene encoding beta-lactamase: Two oligonucleotide primers SigA2NotU-P 5′-TCG CGA TCC GTT TTC GCA TTT ATC GTG AAA CGC T-3′ (SEQ ID NO: 15) and SigA2NotD-P 5′-CCG CAA ACG CTG GTG AAA GTA AAA GAT GCT GAA-3′ (SEQ ID NO: 16), which bind to the start and stop of the bla gene of SigA2 directing outwards were used PCR amplify SigA2 without the bla gene. An amplificate of approx. 3.6 kb generated in the this PCR reaction was relegated and transformed in to a suitable E. coli strain. A plasmid of 3.6 kb was isolated from a transformant which was able to grow on LB chloramphenicol but not on LB ampicillin. This plasmid maintained both BglII sites and lacks the active bla gene and was called pSig4 (see the patent application PA 2004000010).
60 microliter of pSigA4 plasmid DNA preparation with a concentration of 0.3 microgram/microliter was digested with BglII and separated on an agarose gel. The SigA2 transposon DNA band of 2 kb was eluted and purified by using the “GFXTMPCR, DNA and Gel Band Purification Kit” (Amersham Pharmacia Biotech Inc, USA) according to the instructions of the vender and eluted in 200 microliter EB buffer.
C. Transposon Tagging
The transposon prepared from pSigA4 carries a 5′-truncated bla-gene encoding a beta-lactamase from which the secretion signal has been removed. The beta-lactamase conveys ampicillin resistance on E. coli only when the protein is secreted to the periplasm, whereas cytoplasmic, expression of beta-lactamase does not confer ampicillin resistance. Without a signal sequence, the beta-lactamase enzyme will not be transported to the periplasm and therefore the clone will not grow on media containing ampicillin. The signal-less beta-lactamase gene was contained within the transposon in such a way that there was a continuous open reading frame between the transposon border and the beta-lactamase coding region. In this way the modified transposon, when it transposes into a gene encoding a protein that is secreted, could cause an in-frame fusion with the target gene. This resulted in a fusion gene product that is secreted to the periplasm of E. coli and conveys resistance to the ampicillin. If the transposon integrated even in-frame into a gene encoding a non-secreted protein, the respective host will not become ampicillin resistance.
For the in vitro transposon tagging of the strain Bacillus sp. P203 genomic library 250 nanogram of SigA2 transposon were mixed with 2.5 microgramm of the DNA concentration of the plasmid pool DNA of the strain Bacillus sp. P203 genomic library, 2.5 microliter of Finnzymes MuA Transposase (0.22 microgram/microliter) and 10 microliter of 5× buffer from Finnzymes OY, Espoo, Finland) in a total volume of 50 microliter and incubated at 30° C. for 3 h and followed by heat inactivation at 75° C. for 10 min. The DNA was precipitated by addition of 5 microliter 3M Na-acetate pH 5 and 110 microliter 96% ethanol, incubated at −20° C. for 20 h and centrifuged for 30 min at 20000 rpm. The pellet was washed with 70% ethanol, dried and resuspended in 10 microliter TE buffer.
D. Transformation and Selection
Electro-competent E. coli DH10B cells were transformed by electroporation in a Biorad Gene Pulse device (50 uF, 25 mAmp, 1.8 kV with 5 microliter of the transposon tagged plasmid pool, mixed with 1 ml SOC medium, pre-incubated for 1 h at 37° C. and plated on LB with 25 microliter/mililiter ampicillin, 50 microliter/mililiter kanamycin, 10 microliter/mililiter chloramphenicol and incubated for 2-3 days at 30° C. Out of the resistant transformants 1152 colonies were selected. Templates for DNA sequencing from the signal trapped library were prepared by rolling circle amplification using the Templyphi 500 kit (Amersham Biosciences) according to the manufacturer's instructions.
E. Plasmid Preparation and Sequencing
Approximately 500 transposon tagged plasmids were sequenced with the A2up primer AGCGTTTGCGGCCGCGATCC (SEQ ID NO: 13) which read upstream into the into the transposon tagged gene, and, in a second reaction, with B primer TTATTCGGTCGAAAAGGATCC (SEQ ID NO: 14) which read downstream into the transposon tagged gene.
F. Sequence Assembly and Annotation
The obtained sequences were assembled into contigs by using the program PhredPhrap (Brent Ewing, LaDeana Hillier, Michael C. Wendl, and Phil Green, Base-calling of automated sequencer traces using phred I. Accuracy assessment (1998) Genome Research 8:175-185; Brent Ewing and Phil Green, Base-calling of automated sequencer traces using phred II. Error probabilities (1998) Genome Research 8:186-194). The obtained contigs were subsequently compared to sequences available in standard public DNA and protein sequences databases by using the program BLASTX 2.0a19MP-WashU [14 Jul. 1998] [Build linux-x86 18:51:44 30 Jul. 1998] (Gish, Warren (1994-1997). Unpublished; Gish, Warren and David J. States (1993). Identification of protein coding regions by database similarity search. Nat. Genet. 3:266-72).
The obtained sequences were functional genes which encoded intact and functional polypeptides, because they were obtained as ampicillin resistant clones as explained supra.
Example 2
Determining Function by Homology
The function of the polypeptides SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 were annotated by sequences comparison with genes or polypeptides of known function. The polypeptides of the invention were compared to a list of closest related sequences from public and in-house databases of contig's. The contigs were subsequently compared to sequences available in standard public DNA and protein sequences databases by using the program BLASTX 2.0a19MP-WashU [14 Jul. 1998]. A careful analysis of sequence alignments of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 to their closest related sequences with known function from other databases made it possible to predict the function of these polypeptides on the basis of the degree of amino acid identity. Even when the overall amino acid identity was below 40%, which usually makes it difficult to make a good prediction, we were able to predict the function of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 by carefully analysing and interpreting the amino acid residues in the catalytic sites or in important regions of the polypeptide sequences. If the amino acids of the catalytic site of a known sequences were also present in the polypeptide of the invention, combined with a sufficient overall amino acid identity, it was concluded that the polypeptide from Bacillus sp. P203 (DSM 17419) had the same function as the known sequence.
Example 3
Preparing Polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12
To prepare the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, the genes comprised in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11 encoding these polypeptides are expressed by fusing the DNA encoding the open reading frame to DNA a promoter, ribosome-binding site and terminator suitable for genes expression in an appropriate host strain, for example Escherichia coli, Bacillus subtilis, Bacillus licheniformis or Bacillus clausii or a derivative of strain Bacillus sp. P203. The promoter can either be an inducible promotor or a constitutive promoter. Any signal sequences can be exchanged with a suitable signal peptide of another bacterium. The expression construct can either be part of a plasmid or of a linear DNA. It can be integrated into the chromosome of the host strain by recombination or it can be present in the host cell on a plasmid. Then the transformed cells carrying the gene of interest are grown in a suitable medium in the desired volume. If an inducible promoter is used, the gene expression is started by adding the inducer. Otherwise a no inducer is needed and the cells will be grown until a suitable amount of protein from the gene of interest is produced. Then the culture is harvested and the proteins are recovered by standard methods.
Example 4
Protease
The culture fluid or a cell lysate of a host strain synthesising and secreting a serine protease in a suitable buffer may be assayed for that activity. A suitable volume of such a sample is spotted on agarose plates which contain the insoluble chromogenic substrate AZCL-casein (MegazymeTM) and a suitable buffer at suitable pH. The plate is incubated for an appropriate time, e.g. one day, at an appropriate temperature, e.g. 55° C. The activity is visible as blue halos around the spots. As an alternative to AZCL-casein non-labelled casein can be used. On non-labelled casein spotted on agarose plates, clearing zones form in the presence of a serine protease.
Example 5
Rhamnogalacturonan Lyase
Rhamnogalacturonan is a major polysaccharide found in the hairy regions of pectin. Rhamnogalacturonan lyase (PL4 and PL11) and rhamnogalacturonase (GH28) are enzymes that can degrade the rhamnogalacturonan backbone of pectin but have no specificity towards homogalacturonan. A suitable volume of the culture fluid or a cell lysate of a host strain synthesising and secreting a rhamnogalacturonan lyase in a suitable buffer at an appropriate temperature, e.g. 30° C. is used for measuring the activity. A suitable volume of such a sample is spotted on agarose plates which contain the insoluble chromogenic substrate AZCL-rhamnogalacturonan I (Megazyme). The plate is incubated for an appropriate time, e.g. one day at an appropriate temperature, e.g. 30° C. The activity is visible as blue halos around the spots. As an alternative AZCL-galactan or AZCL-debranched arabinan or non-labelled potato pectic galactan or rhamnogalacturonan is added to agar plates or as substrate solution to the enzyme assay. Enzyme activity can be detected as blue halos around the spots clearing zones or by methods published by Mutter, M. et al., Characterization of Recombinant Rhamnogalacturonan alpha-L-Rhamnopyranosyl-(1, 4)-alpha-D-Galactopyranosyluronide Lyase from Aspergillus aculeatus, Plant Physiol. (1998) 117: 141-152 and by McKie, V. et al., A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose, Biochem. J. (2001) 355, 167-177
Example 6
Xylanase
Xylanases are endo-acting enzymes that cleave beta-1,4-xylose polymers. For the measurement of endo-1,4-beta-D-xylanase in enzyme preparations the Xylanase Assay Kit (Megazyme) may be used. Alternatively, a suitable volume of the culture fluid or a cell lysate of a host strain synthesising and secreting a xylanase in a suitable buffer at an appropriate temperature, e.g. 30° C. is used for measuring the activity. A suitable volume of such a sample is spotted on agarose plates which contain the insoluble chromogenic substrate AZCL-xylan or AZCL-arabinoxylan (Megazyme). The plate is incubated for an appropriate time, e.g. one day at an appropriate temperature, e.g. 30° C. The activity is visible as blue halos around the spots.
Example 7
Galactanase
Proteins of this type are hemicellulases that can hydrolyze short xylo-oligosaccharides, e.g. arabinan. The culture fluid or a cell lysate of a host strain synthesizing or secreting a galactanase in a suitable buffer may be assayed for that activity. A suitable volume of the culture fluid or a cell lysate of a host strain synthesizing and secreting a galactanase in a suitable buffer at an appropriate temperature, e.g. 30° C. is used for measuring the activity. A suitable volume of such a sample is spotted on agarose plates which contain the insoluble chromogenic substrate AZCL-arabinan (Megazyme). The plate is incubated for an appropriate time, e.g. one day at an appropriate temperature, e.g. 30° C. The activity is visible as blue halos around the spots.
Example 8
Expression of Carbonic Anhydrase from Strain Bacillus sp. P203 in B. Subtilis 
The signal peptide from the alpha-amylase from B. licheniformis (AmyL) was fused by PCR in frame to the gene encoding the carbonic anhydrase (SEQ ID NO: 3). The DNA coding for the resulting coding sequence was integrated by homologous recombination on the Bacillus subtilis host cell genome. The gene construct was expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter including stabilizing sequence. The gene coding for Chloramphenicol acetyl-transferase was used as maker. (Described e.g in Diderichsen et al., A useful cloning vector for Bacillus subtilis. Plasmid, 30, p. 312, 1993).
Chloramphenicol resistant transformants were analyzed by DNA-sequencing to verify the correct DNA sequence of the construct. One such clone was selected.
Fermentations of the carbonic anhydrase (SEQ ID NO: 4) expression clone was performed on a rotary shaking table in 1 L baffled Erlenmeyer flasks each containing 400 ml LB media supplemented with 34 mg/l chloramphenicol. The clone was fermented for 3 days at 25-30° C. The carbonic anhydrase activity was determined according to Wilbur, K. M. and Anderson, N. G., Electrometric and colorimetric determination of carbonic anhydrase, J Biol Chem, 1948, 176, p. 147-154. Alternatively, the carbonic anhydrase activity can be measured as esterase activity with para-nitrophenolacetate as substrate according to Chirica, L. C. et al., Cloning, expression and some properties of alpha-carbonic anhydrase from Helicobacter pylori, Biochim. Biophys. Acta, 2001, 1544, p. 55-63. A standard assay contained 900 microliter of PNPA, 10 microliter of 300 mM Tris pH 7.5 and 90 microliter of carbonic anhydrase protein solution. After a pre-incubation for 5 min, the enzyme was added to the PNPA buffer mixture and the increase in absorbance (due to the hydrolysis of the substrate and release of p-nitrophenol) at 348 nm was monitored photometrically in a quartz cuvette over a period of 10 min. As a positive control for both activity assays, a solution of bovine carbonic anhydrase (Calbiochem, 1 mg per mL) may be used.
Example 9
Expression of Carbonic Anhydrase from Strain Bacillus sp. P203 in E. coli 
Alternatively to example 8, the carbonic anhydrase can be expressed in E. coli. Plasmid pEG131377-9 was generated by ligation of the pGEM-T vector (Promega) with the carbonic anhydrase PCR product (SEQ NO. 3) amplified from Bacillus sp. P203 genomic DNA (extracted with Qiagen Genomic DNA Kit, 19060) using gene specific primers BOCAfwd (5′ TAGTCCGTACATATGAGAAAAACAA 3′) (SEQ ID NO: 17) and BOCArev (5′ TTCAAAGCTTATAGTGAAATCCAACT 3′) (SEQ ID NO: 18). The plasmid was used as template for generation of plasmid pETBlueCA. The plasmid pETBlueCA for expression of the carbonic anhydrase in E. coli was constructed by PCR amplification with the gene specific primers pQECAfwd (5′ ATCCGCATGCTGAGAAAAACAAA 3′) (SEQ ID NO: 19) and pQECArev (5′ AATTAAGCTTAATAGTGAAATCCAACT 3′) (SEQ ID NO: 20) from plasmid template pEG131377-9 followed by a ligation of the PCR product with the linear pETBlue-1 AccepTorTM vector system (Novagen) according to the manufacturer's instructions. Plasmids were trans-formed into chemical competent E. coli NovaBlue SinglesTM cells (Novagen) for blue-white screening. Inserts of three plasmids were verified by DNA sequencing using primers pETBlueUP, pETBlueDOWN (Novagen, primers flanking the insert region) and primer CAmitSEQrev (5′ CACTTGGTGAATGGAAATG 3′) (SEQ ID NO: 21). The plasmid pETBlueCA clone no. 8 (pETBlueCAB) was found correct and was transformed into chemical competent E. coli Tuner(DE3)pLacI cells for over-expression of carbonic anhydrase from Bacillus sp. P203 under control of the lac promoter (IPTG inducible).
Example 10
Purification and Characterization of Carbonic Anhydrase (SEQ ID NO: 4) from strain Bacillus sp. P203
Purification
Culture broth was centrifuged (10000 g, 15 minutes) and the supernatants were carefully decanted from the precipitates. The pooled culture supernatant was concentrated to 1/10 of the volume by crossflow filtration (MW cutoff 10,000 Da) and purified on a carbonic anhydrase specific chromatography column. For purification of recombinant carbonic anhydrase from Bacillus plankortiensis, a highly selective affinity column was prepared. In principle, the affinity column was prepared as described before (Khalifah, R. G. et al., Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B, Biochem. 1977, 16, p. 2241-7) with the following modifications: The sulfonamide ligand 4-aminomethylbenzenesulfonamide (0.2 g, Sigma) was dissolved in 12 mL of coupling buffer (0.1 M NaHCO3, pH 8, 0.5 M NaCl). 1 g of activated CH Sepharose 4BTM (Amersham Biosciences) was dissolved in approx. 20 ml 1 mM HCl and thoroughly washed with 250 mL 1 mM HCl on a sintered glass filter. The gel was eluted from the filter with 6 mL of 1 mM HCl and given to the dissolved ligand in a 50 mL Falcon tube. The mixture was gently rotated end-over-end for 1 h at room temperature. To remove excess ligand, the gel was washed with 250 mL coupling buffer. Blocking of any remaining active groups was performed by washing the gel with 0.1 M Tris-HCl, pH 8 and incubating in that buffer for 1 hour. Three cycles of a) 250 mL 0.1 M Tris, pH 8, 0.5 M NaCl and b) 0.1 M sodium acetate, pH 4, 0.5 M NaCl were used to wash the gel after blocking. The gel was transferred in a beaker and washed extensively with loading buffer D (0.25 M Na2SO4, 25 mM Tris pH 8.3) prior to use.
5 mL of the affinity media was given to either 50 mL concentrated culture broth of example no. 8 or to the cell free raw protein extract of example no. 9. The suspension was smoothly agitated at room temperature for one hour and then poured into a column letting gravity packing the column. The column was washed intensively with 10 volumes of loading volume with buffer D until no protein further was detectable. Elution of carbonic anhydrase was started by closing the outlet and addition of 10 ml elution buffer E (0.4 M KSCN, 25 mM Tris pH 8.3). After 10 min of incubation with moderately shaking of the column, 500 microliter fractions were collected. Fractions containing carbonic anhydrase activity were pooled after the first active fraction (usually fractions 4-10). Pooled fractions were re-buffered intensively in a Centricon device (Millipore, at 4000×g) with a MW 10,000 Da cutoff using 10 volumes of carbonic anhydrase storage buffer F (100 mM Tris-HCl pH 8.0, 50 micromolar zinc sulphate). Aliquots of purified carbonic anhydrase were stored at −20° C. until further use. The purified recombinant carbonic anhydrase showed a single protein band on SDS polyacrylamid gels.
Characterization
The carbonic anhydrase activity was determined according to Wilbur. K. M. and Anderson, N. G., Electrometric and colorimetric determination of carbonic anhydrase, J Biol Chem, 1948, 176, p. 147-154. The purified recombinant carbonic anhydrase was tested for temperature stability at 4-60° C. for 1 and 17 h. The protein was found to be stable at 4° C. in buffer F for a week with minimal loss of activity (97+/−4% of original activity) and at room temperature (20° C., 95+/−4% of original activity). No loss of activity (99+/−4% of original activity) was detected after storage of the protein at −20° C. for one week in buffer F. The stability of carbonic anhydrase rapidly decreased at temperatures over 37° C. (see FIG. 2). Storage of recombinant carbonic anhydrase in buffer F supplemented with 1 mM dithiothreitol (DTT) was found to stabilize the enzyme.
Inhibition studies of recombinant carbonic anhydrase were performed with azide ions and specific carbonic anhydrase inhibitor acetazolamide (Sigma). The IC50 values were 500 micromolar for azide and 0.09 micromolar for acetazolamide, complete inhibition was observed in the presence of 7.5 micromolar acetazolamide (see FIG. 3).
Inhibition of carbonic anhydrase by two and three valent metal ions, azide and other reagents commonly used in protein science (see FIG. 4):
The following inhibitors were used:







TABLE 1





Inhibitors


















 0:
Control



 1:
CaCl2



 2:
MnCl2



 3:
EDTA



 4:
NaWO4



 5*:
CrCl3



 6:
Guanidin-HCl



 7:
beta-mercaptoethanol



 8:
SDS



 9*:
FeCl3



10:
Urea



11:
MgCl2



12:
DTT



13:
Na2SeO3



14:
ZnSO4



15:
(NH4)Fe(SO4)2



16:
CoCl2



17*:
Triton X 100



18:
CuSO4



19:
NiSO4



20:
NaN3










Enzyme was pre-incubated with 1 mM of the substances for 1 h on ice in a total volume of 50 micro liter before testing activity with 10 micro liter of the mixture with the Wilbur and Anderson, 1948 method. The control with no additives (water) was set to 100% activity. The detergent Triton X 100 and ions Fe3+ and Cr2+ (marked with asterixes in table 1) altered the assay when in given concentration of 1 mM in the total assay volume. Instead activity with 25 micro molar concentration of the chemicals are given.
Example 11
Expression of Galactanase
The signal peptide from the alpha-amylase from B. licheniformis (AmyL) was fused by PCR in frame to the gene encoding the galactanase (SEQ ID NO: 12). The DNA coding for the resulting coding sequence was integrated by homologous recombination on the Bacillus subtilis host cell genome. The gene construct was expressed under the control of a triple promoter system (as described in WO 99/43835), consisting of the promoters from Bacillus licheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIA promoter including stabilizing sequence. The gene coding for Chloramphenicol acetyl-transferase was used as maker. (Described e.g in Diderichsen et al., A useful cloning vector for Bacillus subtilis. Plasmid, 30, p. 312, 1993).
Chloramphenicol resistant transformants were analyzed by DNA sequencing to verify the correct DNA sequence of the construct. One such clone was selected. Fermentations of the galactanase (SEQ ID NO: 12) expression clone was performed on a rotary shaking table in 500 ml baffled Erlenmeyer flasks each containing 100 ml LB media supplemented with 34 mg/l chloramphenicol. The clone was fermented for 3 days at 30° C. The activity was determined with OPNG as substrate.
Example 12
Verification of Galactanase Activity from Strain Bacillus sp. P203
Assay:
50 microliters of the cell free supernatant from example no. 7 were given to 750 microliters of a 4.5 mM solution of ortho-nitrohenylgalactopyranoside (ONPG), in 50 mM phosphate buffer pH 7.6. The mixture was incubated at 40° C. for 15 min and the reaction was stopped by addition of 300 microliters 1 M sodium carbonate. The solutions were centrifuged for 1 min at 13,000 rpm prior to spectrometric measurements and diluted with water if necessary. The absorbation of o-nitrophenolate is measured optically at 420 nm.

## Claims

1. An isolated polypeptide having carbonic anhydrase activity, selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence which has at least 90% sequence identity to SEQ ID NO: 4;
(b) a fragment of SEQ ID NO: 4 having carbonic anhydrase activity.

2. The polypeptide of claim 1, having at least 95% sequence identity to SEQ ID NO: 4.

3. The polypeptide of claim 1, having at least 96% sequence identity to SEQ ID NO: 4.

4. The polypeptide of claim 1, having at least 97% sequence identity to SEQ ID NO: 4.

5. The polypeptide of claim 1, having at least 98% sequence identity to SEQ ID NO: 4.

6. The polypeptide of claim 1, having at least 99% sequence identity to SEQ ID NO: 4.

7. The polypeptide of claim 1, which comprises SEQ ID NO: 4.

8. The polypeptide of claim 1, which consists of SEQ ID NO: 4.

9. A composition comprising the polypeptide of claim 1.

10. The composition of claim 9, further comprising one or more additional enzymes.

11. The composition of claim 9, further comprising a cereal or grain product.

12. The composition of claim 9, further comprising a polysaccharide or a mixture of polysaccharides.

13. A detergent composition comprising a polypeptide of claim 1 and a surfactant.