# Patent

## Title
Alkaline Bacillus amylase

## Abstract

This invention relates to an amylase derived from Bacillus horikoshii. The amylase has a preference for soluble substrates, shows substantial exo-amylase activity and has a pH optimum of about 9.8.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation application of U.S.  application Ser. No. 11/721,353 filed Jun. 11, 2007, now U.S. Pat. No. 7,659,101, which is a 35 U.S.C. 371 national application of PCT/DK2005/000794 filed on Dec. 15, 2005, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2004 01936 filed on Dec. 15, 2004 and U.S. provisional application No. 60/636,212 filed on Dec. 15, 2004, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to amylases having a pH optimum around 9.8.
BACKGROUND OF THE INVENTION
For a number of years alpha-amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing and baking.
Examples of commercial alpha-amylase products are Termamyl®, BAN® and Fungamyl®, all available from Novozymes NS, Denmark. These and similar products from other commercial sources have an acidic to a neutral pH optimum, typically in the range of from pH 5 to pH 7.5, and they do not display optimal activity in detergent solutions at alkaline pH.
WO 95/26397 discloses an alpha-amylase from a Bacillus strain, which has optimum activity at pH 8. WO 96/23873 describes variants of Bacillus amylases with improved performance under washing conditions.
U.S. Pat. No. 5,147,796 describes an alkaline pullulanase having alpha-amylase activity. FIG. 2b of the document shows optimum amylase activity at pH 8-8.5.
M. Takagi et al., J. Ferment. Bioeng, vol 81, No. 6, 557-559 (1996) describe an alkaliphilic alpha-amylase-pullulanase from Bacillus sp. The enzyme has optimum amylase activity at pH 9, but the activity drops rapidly at higher pH, and the activity at pH 10 is lower than at pH 7.
WO 01/14532 and WO 99/50399 disclose alpha-amylases from Bacillus strains, which have optimum activity at pH 9.5.
The amylase of the present invention has a highly alkaline profile with an optimum activity at pH 9.8
SUMMARY OF THE INVENTION
The invention provides an alpha-amylase having one or more of the following characteristics:

    
    
        a) An amino acid sequence which has at least 60% identity with amino acids 1 to 1105 of Seq Id No: 2.
        b) An amino acid sequence comprising Seq Id No: 2.
        c) An amino acid sequence that is identical to Seq Id No: 2.
        d) An amino acid sequence comprised by Seq Id No: 2
    
    


In a further aspect the invention relates to an alpha-amylase having one or more characteristics selected from the group consisting of:

    
    
        a) a theoretical pI of about 4.4; or
        b) a pH-optimum of about 9.8; or
        c) a temperature optimum of about 50° C. at pH 9; or
        d) at least 40% residual activity after 30 minutes at 50° C. and pH 10; or
        e) exhibits substantial exo-amylase activity
        f) obtainable from Bacillus horikoshii. 
    
    


In a further aspect the invention relates to an alpha-amylase encoded by a polynucleotide which hybridizes under medium stringency conditions, more preferably high stringency conditions, with the nucleic acid sequence of Seq Id No:1 or its complementary strand.
In a still further aspect the invention relates to a DNA sequence encoding the alpha-amylase of the invention. I a particular embodiment said DNA sequence comprises or is comprised by Seq. ID No.: 1.
Further aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector.
The invention also provides a method for producing the alpha-amylase and for the use of said alpha-amylase for production of malto-oligosaccharides with a DP of less than 10, and as a detergent enzyme.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a pH activity profile of the alpha-amylase of the invention.
FIG. 2 shows a temperature activity profile of the alpha-amylase of the invention.
FIG. 3 shows the stability of the alpha-amylase of the invention after incubation at various temperatures.
FIG. 4 shows the stability of the alpha-amylase of the invention after incubation at various pH.
FIG. 5 shows the wash performance of the alpha-amylase in a commercial detergent.
FIG. 6 shows the wash performance of the alpha-amylase at pH 9 in Carbonate buffer.



DETAILED DESCRIPTION OF THE INVENTION
Sequence Listing and Deposited Microorganisms
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. In addition, the present application refers to deposited microorganisms. The contents of the data carrier and the deposited microorganisms are fully incorporated herein by reference.
The alpha-amylase of the invention may be derived from Bacillus sp. strain NCIB 12512. This strain was deposited on 29 Jul. 1987 under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at the National Collections of Industrial and Marine Bacteria Limited (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom.
The deposit was made by Novo Nordisk NS and was later assigned to Novozymes NS.
Production of Alpha-Amylase
The alpha-amylase of the invention can be produced by cultivating a suitable amylase-producing strain of Bacillus or the transformed host cell of the invention in a suitable nutrient medium, and recovering the alpha-amylase from the culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
The alpha-amylase secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
Properties of Alpha-Amylase
A preferred alpha-amylase is derived from Bacillus horikoshii. It can be produced as described in the examples. The full length amino acid sequence of the amylase and the DNA encoding it are shown in Seq. ID No. 2 and Seq. ID No. 1 respectively.
In the present context, “derived from” is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question.
The following characteristics were found for the amylase of the invention (purified alpha-amylase):

    A molecular weight of approximately 130 kDa as determined by SDS-PAGE using a Novex, 4-20% gradient gel. The theoretical MW of the polypeptide chain is 123 kDa, using the pc program GPMAW (Lighthouse data).
    A theoretical pI of 4.4 was determined by the pc program Vector NTI 8, 1994-2002 Infomax, Inc.
    A pH-activity curve is shown in FIG. 1, taking the activity at pH 9.8 as 100%. It was determined using the reducing sugar assay (PAHBAH) using 50 mM Britten-Robinson buffer adjusted to various pH-values. FIG. 1 shows that the optimum activity is about pH 9.8.
    A temperature-activity curve was measured using the PAHBAH assay at various temperatures with 50 mM Britten-Robinson buffer adjusted to pH 9.0. The results are shown in FIG. 2. It is seen from FIG. 2 that the amylase of the invention has optimum activity at about 50° C.
    Temperature stability of the amylase was measured at pH 10. A stability profile of the amylase of the invention (FIG. 3) shows superior stability of the amylase of the invention compared to a commercial amylase.
    pH stability of the amylase was measured at 37° C. A stability profile of the amylase of the invention (FIG. 4) shows superior stability of the amylase of the invention compared to a commercial amylase.

Sequence Homology and Alignment



For purposes of the present invention, alignments of sequences and calculation of homology and identity scores may be done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is −12 for proteins and −16 for DNA, while the penalty for additional residues in a gap is −2 for proteins and −4 for DNA. Alignment may be made with the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology, 183:63-98).
Multiple alignments of protein sequences may be made using “ClustalW” (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignments of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence.
The amino acid sequence of the present invention exhibits a degree of identity preferably of at least 60%, or at least 65%, or at least 70%, or at least 75%, preferably at least 80%, or at least 85%, more preferably at least 90%, especially at least 95%, even more especially at least 96%, or at least 97%, or at least 98%, or at least 99% with the amino acid sequence shown in positions 1-1105 of SEQ ID NO: 2.
The DNA construct of the present invention comprises a DNA sequence exhibiting a degree of identity preferably of at least 60%, preferably at least 70%, more preferably at least 80%, especially at least 90% or at least 95%, even more especially at least 97% or at least 99% with the nucleic acid sequence shown in positions 109-3423 of SEQ ID NO: 1.
Hybridization
Suitable experimental conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5×SSC (sodium chloride/sodium citrate, Sambrook, et al., 1989) for 10 min, and prehybridization of the filter in a solution of 5×SSC, 5× Denhardt's solution (Sambrook, et al., 1989), 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA (Sambrook, et al., 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity>1×109 cpm/μg) probe for 12 hours at ca. 45° C. The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at least 55° C. (low stringency), preferably at least 60° C. (medium stringency), more preferably at least 65° C. (medium/high stringency), more preferably at least 70° C. (high stringency), even more preferably at least 75° C. (very high stringency).
Molecules which hybridize to the oligonucleotide probe under these conditions are detected by exposure to x-ray film.
Recombinant Expression Vector
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids 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, a 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) which can be conveniently subjected to recombinant DNA procedures and can bring about 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 which 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 which 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 which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.
The vectors of the present invention preferably contain an element(s) that permits 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 polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration 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 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 nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity 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. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
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 pAMR1 permitting replication in Bacillus. 
More than one copy of a polynucleotide 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 polynucleotide 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 polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, 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.: Molecular Cloning: A Laboratory Manunal, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Host Cells
The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of the alpha-amylase of the invention. The cell may be transformed with the DNA construct of the invention encoding the amylase, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell. Examples of bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram positive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. clausii, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gram negative bacteria such as Escherichia coli. The transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., 1989: Molecular Cloning: A Laboratory Mannual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
Industrial Applications—Please Provide Information About Relevant Applications
Owing to their activity at alkaline pH values, the alpha-amylases of the invention are well suited for use in a variety of industrial processes, e.g. laundry and hard surface cleaning detergent compositions, the production of sweeteners and ethanol from starch. Thus, it may be used in conventional starch-converting processes, such as liquefaction and saccharification processes described in U.S. Pat. No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.
Pulp and Paper Production
The alkaline alpha-amylase of the invention may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch. The alpha-amylase of the invention is especially useful in a process for producing a papermaking pulp from starch-coated printed-paper. The process may be performed as described in WO 95/14807, comprising the following steps:

    a) disintegrating the paper to produce a pulp,
    b) treating with a starch-degrading enzyme before, during or after step a), and
    c) separating ink particles from the pulp after steps a) and b).


The alpha-amylases of the invention may also be very useful in modifying starch where enzymatically modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline alpha-amylases of the invention it becomes possible to modify the starch in the presence of the filler thus allowing for a simpler integrated process.
Desizing
The alpha-amylase of the invention may also be very useful in textile desizing. In the textile processing industry, alpha-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size, which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred because it does not involve any harmful effect on the fibre material. In order to reduce processing cost and increase mill throughput, the desizing processing is sometimes combined with the scouring and bleaching steps. In such cases, non-enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size does lead to some fibre damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the alpha-amylases of the invention as they have an improved performance in alkaline solutions. The alpha-amylases may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile.
Beer Making
The alpha-amylases of the invention may also be very useful in a beer-making process; the alpha-amylases will typically be added during the mashing process.
Production of Malto-Oligosaccharides
The alpha-amylase of the invention exhibits substantial exo-alpha-amylase (see break down pattern of amylase and waxy corn starch, Example 4). This allows for the use of the enzyme of the present invention for production of malto-oligosaccharides of a specific degree of polymerization (DP), a process that otherwise is tedious and expensive. Malto-oligosaccharides with a DP around 10 or less have potential applications in e.g. the food industry. These malto-oligosaccharides have a less sweet taste compared to sucrose's, and a partial replacement of sucrose with e.g. maltotetraose (DP=4) reduces the sweetness of the foods without affecting their inherent taste and flavour. At the same time they have a high moisture retention power, and can thus serve to prevent retrogradation of starch ingredient and makes retain a suitable moisture in foods. A further aspect of application of malto-oligosaccharides is that they show a less Maillard reaction, and thus less coloration. Malto-oligosaccharides show higher viscosity than that of sucrose, and are useful in improving the texture of foods. A further aspect of the application of malto-oligosaccharides is that they may be used to control the freezing points of frozen foods, as they depresses the freezing point of water more moderately than sucrose or high-fructose syrup.
Detergent Compositions
The alpha-amylase 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 enzyme 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, a pectate lyase, 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.
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., 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 nonyl-phenols 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 pre-parations 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 present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.
Materials and Methods
50 mM Britton-Robinson Buffer
50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH.
Detergent:
Commercial European color detergent; alpha-amylases present in the detergent were inactivated by microwaves before adding the alpha-amylase of the present invention.
EXAMPLES
Example 1
Cloning and Expression of the Alpha-Amylase of the Invention
Bacterial Strains and Growth Conditions
TH1 is a Bacillus subtilis strain (amy-, spo-, apr-, npr-), that has been modified by insertion of a construct, from the strain DN3 (Noone et al. 2000, J Bacteriol 182 (6) 1592-1599) by transformation and selection for erytromycin. The changed genotype is: ykdA::pDN3 (PykdA-lacZ Pspac-ykdA) Ermr. TH1 contains the following features: the full ykdA promoter is fused to the LacZ reporter gene. In addition the ykdA gene is placed under control of the IPTG-inducible Pspac promoter, so the ykdA gene no longer has it's naturally regulation. The strain can be used as host for expression clones and libraries and transformants expressing and secreting protein can be selected on plates containing X-gal and IPTG. TH1 can be maintained and propagated in Luria-Bertani (LB) or supplemented with agar (1.5% wt/vol) as appropriate, addition of 6 μg/ml erythromycin and grown at 37° C. with aeration.
DNA Manipulations
Bacillus subtilis transformations were performed as described previously (Anagnostopolous, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746). All routine molecular biological procedures were performed according to the protocols described by Sambrook et al. (1989).
Modification of Vector
The shuttle vector for Bacillus and E. coli pDG268neo (Widner B; Thomas M; Sternberg D; Lammon D; Behr R; Sloma A (2000): Development of marker-free strains of Bacillus subtilis capable of secreting high levels of industrial enzymes. Journal of Industrial Microbiology and Biotechnology, Vol. 25 (4) pp. 204-212) was modified to allow for cloning of partial digested Sau3A or Tsp509I genomic DNA. The vector was modified by inserting a BamHI and EcoRI site between the Sac1 and Not1 sites (fragment between was deleted and a linker was inserted). In this way genomic fragments can be cloned and genes contained in these fragments can be transcribed from either the strong promoter on the vector or by their own promoter.
Construction of a Library in E. coli 
Chromosomal DNA from Bacillus horikoshii was isolated by QIAmp Tissue Kit (Qiagen, Hilden, Germany). The genomic DNA was partial digested by Sau3A by standard methods. DNA fragments from 3-5 kb were gel purified and ligated into BamHI digested and dephosphorylated vector (the modified pDG268neo (=pDG268BE)). 1 micro L of the ligation was transformed by electroporation into competent E. coli cells (Electromax DH10B Cells, Invitrogen) according to standard protocols. The transformed cells were plated on plates containing solid LB media containing ampicillin as selection marker. The plates were incubated for 16 h at 37 C. 20.000 transformants obtained this way were pooled and plasmid DNA was prepared from the pooled cells by using a midiprep Qiagene kit (Qiagene). This plasmid pool represents the library.
Transformation of Library into Bacillus subtilis TH1
The obtained plasmid pool DNA was used to transform Bacillus TH1 competent cells. Bacillus transformations were plated on Petri dishes with LB-media supplemented with agar (1.5% wt/vol) and X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) was added to the media at a concentration of 64 micro g/mL, and IPTG (isopropyl-b-D-thiogalactopyranoside) was added to a final concentration of 0.8 mM. Antibiotics were added at the following concentrations: chloramphenicol, 6 μg/ml; and erythromycin, 3 μg/ml. Transformants were incubated at 37° C. overnight.
Selection and Analysis of Transformants Secreting Recombinant Protein
Blue colonies that occurred after 16 h of incubation or in the following 24 h were selected and restreaked on new plates to obtain pure single blue colonies. For expression studies transformants were grown in liquid PS-1 or TY media at 30° C. and at 250 rpm. Cells were spun down and the supernatant analyzed for secreted recombinant protein on SDS-polyacrylamide gels.
Results
A genomic library of Bacillus horikoshii was made in the vector pDGneo268BE. A plasmid pool was made from the E. coli library. The plasmid pool DNA was transformed into Bacillus strain TH1 (TH1 allows for blue/white selection of secreting recombinant clones).16-20000 Bacillus transformants were obtained on agar plates containing X-gal and IPTG, which allows for blue/white selection of secreting Bacillus transformants. Nineteen intense blue colonies appeared among the 16-20.000 colonies. These blue colonies were fermented in liquid media and the supernatants analyzed on SDS-gels. 5 of the 19 blue colonies gave an intense protein band on a SDS gel. A recombinant protein band of around 120 kD from one of the clones was purified and characterized by N-terminal amino acid sequencing. The DNA insert in the corresponding clone was sequenced to identify the gene giving rise to the recombinant protein band. The resulting gene sequence is the sequence of the alpha amylase of the invention listed as the Seq. ID No.:1 and the encoded protein is listed as the Seq. ID No: 2.
Example 2
Purification Method for the Alpha-Amylase of the Invention
The culture supernatant was centrifuged and cell debris was discarded.
Fermentation supernatant was then sterile filtered using Seitz-EKS filter with pore size 0.22 microns which was purchased from Pall Seitz Schenk Filter system GmbH, Bettringer Strasse 42 D-73550 Germany. Filtration was carried out using pressure filter.
The sterile filtered supernatant was then adjusted to 1 M ammonium sulphate by adding solid ammonium sulphate.
Hydrophobic chromatography on Matrix Phenyl Toyopearl 650M Toshohaas was purchased from Bie and Berntsen Roedovre, Denmark.
Fifty milliliter column was packed with the matrix and equilibrated with 1M ammonium sulphate adjusted to pH 8. Sterile filtered fermentation supernatant was then applied on the column and washed with the 1 Molar ammonium sulphate solution till all unbound material was washed out.
Bound Proteins were then eluted by lowering the salt concentration using 50 mM Borate pH 8 solution without any salt. Fractions 10 ml each were collected and analyzed by SDS-PAGE.
Fractions containing protein with 120 kDa molecular weight were then pooled and dialyzed against Buffer containing 50 mM Borate, 5 mM Dimethyl glutaric acid pH 6.
Anion exchange chromatography on Fast flow Q sepharose matrix was purchased form Amersham Pharmacia and 50 ml column was packed. The column was then equilibrated with the buffer containing 50 mM Boarte, 5 mM Dimetrhyl glutaric acid pH6. The dialyzed pool containing the 120 kDa protein was then applied on the column and washed with the buffer till all unbound material was washed out. The bound protein was then eluted with linear salt gradient using Buffer B pH6 which contained 50 mM Borate, 5 mM Dimethyl glutaric acid and 1 M salt.
Fractions were then analyzed by SDS-PAGE and pooled on the basis of Molecular weight which contained the desired protein.
Example 3
Characterization of the Alpha-Amylase of the Present Invention
Reducing Sugar Assay (PAHBAH)
The enzyme reaction was initiated by adding 50 μl enzyme sample to 100 micro L 0.15% amylose (Merck, 4561), 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH), 0.01% Triton-X-100 in PCR tubes. This solution was incubated for 15 min at different temp. in a Peltier Thermal Cycler (PCT-200, MJ research) and stopped with 75 micro L 1.5% (w/v) p-hydroxybenzoic acid hydrazide (Sigma, H-9882), 5% (w/v) K-Na-tartrate (Merck, 1.08087), 2% (w/v) NaOH and incubated at 95° C. for 10 min. The temperature was lowered to 20° C. and 150 micro L was transferred to a 96 well micro titter plate. The absorbance was then measured at 410 nm with a microplate reader (Spectra Max 190, Molecular Devices).
pH Profile
The pH profile was measured at 37° C., at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 using the Reducing sugar assay described above.
The pH profile is shown in FIG. 1, and it is seen that the alpha-amylase of the invention has an optimum activity at about 9.8.
Temperature Profile
The temperature profile was measured at pH 9 at 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C. using the Reducing sugar assay described above.
The temperature profile is shown in FIG. 2, and it is seen that the alpha-amylase of the invention has an optimum activity at about 50° C.
Temperature Stability
150 micro L enzyme sample (diluted with 50 mM Britton-Robinson buffer: 50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to 10 with NaOH, with 1 mM EDTA or 1 mM CaCl2) in an Eppendorf tube was incubated for 30 min a in Eppendorf Thermomixer at different temperatures and 300 rpm shaking. The incubation was stopped by transferring the eppendorf tupe to an ice bath and adding 75 micro L solution to 75 micro L 0.5 M HEPES pH 7.20 micro L of the above stopped solution was transferred into a microtiter plate and 200 micro L 3.7 mM 4,6-ethylidene-G7PNP, 3.3 kU/L alpha-glucosidase, 52.2 mM HEPES, 73 mM NaCl, 10.5 mM MgCl2, 0.06 mM CaCl2, pH 7 (AMYL kit, Roche Diagnostics GmbH). The absorbance at 405 nm was measured with 11 seconds intervals with a microplate reader (Spectra Max 190, Molecular Devices) and the activity was determined from the slope generated after 5 minutes measurement.
The temperature stability is depicted in FIG. 3, where the stability of the amylase of the present invention is compared with the stability of the commercially available amylase BAN®, available from Novozymes NS. FIG. 3 shows a superior stability of the amylase of the present invention as compared to the commercially available amylase.
pH Stability
150 micro L enzyme sample (diluted with 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, pH adjusted to the value of interest with NaOH, with and without 1 mM CaCl2)) in an Eppendorf tube was incubated for 30 minutes in a Eppendorf Thermomixer at 37° C. and 300 rpm shaking. The incubation was stopped by transferring 75 micro L solution to 75 micro L 0.5 M HEPES pH 7. 20 micro L of the above stopped solution was transferred into a microtiter plate and 200 μl 3.7 mM 4,6-ethylidene-G7PNP, 3.3 kU/L alpha-glucosidase, 52.2 mM HEPES, 73 mM NaCl, 10.5 mM MgCl2, 0.06 mM CaCl2, pH 7(AMYL kit, Roche Diagnostics GmbH). The absorbance at 405 nm was measured with 11 seconds intervals with a micro plate reader (Spectra Max 190, Molecular Devices) and the activity was determined from the slope generated after 5 minutes measurement.
The pH stability is depicted in FIG. 4, where the stability of the amylase of the present invention is compared with the stability of the commercially available amylase BAN®, available from Novozymes NS. FIG. 4 shows a superior stability of the amylase of the present invention as compared to the commercially available amylase.
pI Determination
The pI of the polypeptide chain is determined theoretically by the pc program Vector NTI 8, 1994-2002 Infomax, Inc. pI was determined to 4.4.
Example 4
Amylose/Starch Degradation Pattern
20 micro L sample were added to 1 mL 1% amylose (Merck, 4561) or 1% waxy corn starch (Cerestar), 10 mM sodium borate buffer, 0.1 mM CaCl2, pH 9 and incubated for various times at 37° C. The reaction was stopped by adding 20 micro L 1 M HCl and incubated at 95° C. for 10 minutes. The samples were filtered through a 0.22 micro meter filter and the carbohydrate composition was determined by IMPC HPLC.
The data below is expressed in % area in a top in the HPLC chromatogram, compared to total area of all oligosaccharides in the 1035 minutes chromatogram.







TABLE 1







Degradation pattern of 1% amylose.


1% amylose degradation pattern













0 min
17 min
45 min
120 min
1035 min



%
%
%
%
%


















DP6
<1
3
5
8
<1



DP5
<1
0
1
1
<1



DP4
<1
2
6
15
48



DP3
<1
<1
<1
1
5



DP2
<1
1
2
6
26



DP1
<1
1
1
3
17

















TABLE 2







Degradation pattern of 1% waxy corn starch.


1% waxy corn starch













0 min
17 min
45 min
120 min
1035 min



%
%
%
%
%


















DP6
<1
8
11
8
2



DP5
<1
<1
1
2
1



DP4
<1
3
7
18
30



DP3
<1
<1
<1
<1
5



DP2
<1
1
3
8
19



DP1
<1
1
1
4
14










During amylose/amylopectin degradation no DP products larger than DP6 is seen. Initially the domination degradation product is DP6 and after prolonged incubation, this DP6 is hydrolyzed to DP4 and DP2. Thus, this amylase exhibits substantially exo alpha amylase activity.
Example 5
Wash Performance of the Amylase of the Invention
Wash in Detergent Solution.
Washing performance is evaluated by washing starch-soiled CS-28 swatches (obtainable from CFT, Centre for Test Material, Netherlands) in a beaker under constant shaking.
4 g/L of a commercially available detergent (Mega Perls Color from Henkel GmbH) is added to water. Water hardness is adjusted to 15° dH by addition of Ca:Mg in the ratio 4:1 prior to the addition of the detergent. pH is not adjusted upon addition of detergent.
The soiled swatches are washed for 20 minutes at 40° C. Upon drying the reflectance is measured.
As can be seen from FIG. 5 the alpha-amylase of the invention clearly is able to remove starch from the soiled swatches.
Wash in Buffer Solution.
Washing performance is evaluated by washing starch-soiled CS-28 swatches (obtainable from CFT, Centre for Test Material, Netherlands) in a beaker under constant shaking.
5 mM Carbonate buffer is used to adjust pH to 9.
The soiled swatches are washed for 20 minutes at 40° C. Upon drying the reflectance is measured.
As can be seen from FIG. 6 the alpha-amylase of the invention clearly is able to remove starch from the soiled swatches.

## Claims

1. An isolated alpha-amylase comprising an amino acid sequence which has at least 90% identity with amino acids 1 to 1105 of SEQ ID NO: 2.

2. The alpha-amylase of claim 1 having one or more characteristics selected from the group consisting of:
a) a theoretical pl of about 4.4;
b) a pH-optimum of about 9.8;
c) a temperature optimum of about 50° C. at pH 9;
d) at least 40% residual activity after 30 minutes at 50° C. and pH 10; or
e) obtained from Bacillus horikoshii.

3. An isolated alpha-amylase encoded by a nucleic acid sequence which hybridizes under high stringency conditions with the complement of the nucleic acid sequence of SEQ ID NO; 1, wherein the high
stringency conditions are defined as prehybridization and hybridization in 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 pg/ml of denatured sonicated salmon sperm DNA at 45° C., followed by washing two times for 30 minutes using 2×SSC, 0.2% SDS at 65° C.

4. An isolated DNA sequence encoding the alpha-amylase of claim 1.

5. A recombinant expression vector comprising the DNA sequence according to claim 4.

6. A detergent composition comprising the alpha-amylase of claim 1.

7. A cell transformed with a DNA sequence in accordance with claim 4.

8. A cell transformed with a recombinant expression vector of claim 5.

9. A cell in accordance with claim 7, wherein the cell is a bacterial, fungal or yeast cell.

10. A method of producing an alpha-amylase comprising:
cultivating a cell transformed with a DNA sequence encoding an alpha-amylase comprising an amino acid sequence which has at least 90% identity with amino acids 1 to 1105 of SEQ ID NO: 2 under conditions permitting the production of the alpha-amylase.

11. The method of claim 10 further comprising recovering the alpha-amylase of the culture.

12. The method in accordance with claim 10, wherein said transformed cell selected from the group consisting of bacteria and fungi, and recovering the alpha-amylase from the culture medium.

13. The method in accordance to claim 12, wherein the transformed cell is yeast.