(en)The present invention relates to novel subtilases from wild-type strains of Bacillus , especially the Bacillus strains ZI344, EP655, P203, EP63, ZI120, ZI130, ZI1342 and ZI140, and to methods of construction and production of these proteases. Further, the present invention relates to use of the claimed subtilases in detergents, such as a laundry detergent or an automatic dishwashing detergent.

1.ApplicationNumber: US-94928010-A
1.PublishNumber: US-7951573-B2
2.Date Publish: 20110531
3.Inventor: NIELSEN PREBEN
BORCHERT MARTIN

4.Inventor Harmonized: NIELSEN PREBEN(DK)
BORCHERT MARTIN(DK)

5.Country: US
6.Claims:
(en)The present invention relates to novel subtilases from wild-type strains of Bacillus , especially the Bacillus strains ZI344, EP655, P203, EP63, ZI120, ZI130, ZI1342 and ZI140, and to methods of construction and production of these proteases. Further, the present invention relates to use of the claimed subtilases in detergents, such as a laundry detergent or an automatic dishwashing detergent.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/363,309 filed on Jan. 30, 2009, which is a divisional of U.S. application Ser. No. 11/504,743 filed on Aug. 15, 2006, now U.S. Pat. No. 7,642,080, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 2005 01155 and PA 2005 01366 filed Aug. 16, 2005 and Sep. 30, 2005, respectively, and U.S. provisional application Nos. 60/709,403 and 60/722,517 filed Aug. 18, 2005 and Sep. 30, 2005, respectively, the contents of which are fully incorporated herein by reference.


SEQUENCES
This application contains the following sequences:
SEQ ID NO: 1—DNA encoding subtilase from Bacillus sp. strain Zi344. Nucleic acids 337 to 1143 encodes the mature subtilase.
SEQ ID NO: 2—Amino acid sequence of subtilase from Bacillus sp. strain Zi344. The mature subtilase is amino acids 113 to 381.
SEQ ID NO: 3—DNA encoding subtilase from Bacillus sp. strain EP655. Nucleic acids 343 to 1149 encodes the mature subtilase.
SEQ ID NO: 4—Amino acid sequence of subtilase from Bacillus sp. strain EP655. The mature subtilase is amino acids 115 to 383.
SEQ ID NO: 5—DNA encoding subtilase from Bacillus sp. strain p203. Nucleic acids 343 to 1149 encodes the mature subtilase.
SEQ ID NO: 6—Amino acid sequence of subtilase from Bacillus sp. strain p203. The mature subtilase is amino acids 115 to 383.
SEQ ID NO: 7 to SEQ ID NO: 27 are artificial primers.
SEQ ID NO: 28—Partial DNA sequence encoding subtilase from Bacillus sp. strain EP63.
SEQ ID NO: 29—Partial amino acid sequence of subtilase from Bacillus sp. strain EP63.
SEQ ID NO: 30—Partial DNA sequence encoding subtilase from Bacillus sp. strain ZI120.
SEQ ID NO: 31—Partial amino acid sequence of subtilase from Bacillus sp. strain ZI120.
SEQ ID NO: 32—Partial DNA sequence encoding subtilase from Bacillus sp. strain ZI130.
SEQ ID NO: 33—Partial amino acid sequence of subtilase from Bacillus sp. strain ZI130.
SEQ ID NO: 34—Partial DNA sequence encoding subtilase from Bacillus sp. strain ZI132.
SEQ ID NO: 35—Partial amino acid sequence of subtilase from Bacillus sp. strain ZI132.
SEQ ID NO: 36—Partial DNA sequence encoding subtilase from Bacillus sp. strain ZI340.
SEQ ID NO: 37—Partial amino acid sequence of subtilase from Bacillus sp. strain ZI340.
The amino acid sequences of SEQ ID NOs: 29, 31, 33, 35 and 37 are mature subtilases where the C-terminals are truncated.
Deposited Microorganisms
The wild type strain referred to as p203 was deposited on 23 Jun. 2005 under the Budapest treaty at the Deutsche Sammlung von Mikroorganismen and Zellkulturen under the deposit number DSM 17419. The deposit contains the subtilase gene referred to as p203A herein, which is identical with SEQ ID NO: 5.
FIELD OF THE INVENTION
The present invention relates to novel subtilases from wild-type strains of Bacillus and to methods of construction and production of these proteases. Further, the present invention relates to use of the claimed subtilases in detergents, such as a laundry detergent or an automatic dishwashing detergent.
BACKGROUND OF THE INVENTION
Enzymes have been used within the detergent industry as part of washing formulations for more than 30 years. Proteases are from a commercial perspective the most relevant enzyme in such formulations, but other enzymes including lipases, amylases, cellulases, hemicellulases or mixtures of enzymes are also often used.
The search for proteases with appropriate properties include both discovery of naturally occurring proteases, i.e., so called wild-type proteases but also alteration of well-known proteases by e.g., genetic manipulation of the nucleic acid sequence encoding said proteases.
One family of proteases, which is often used in detergents, is the subtilases. This family has been further grouped into 6 different sub-groups (Siezen and, 1997, Protein Science 6: 501-523). One of these sub-groups, the Subtilisin family was further divided into the subgroups of “true subtilisins (I-S1)”, “high alkaline proteases (I-S2)” and “intracellular proteases”. Siezen and Leunissen identified also some proteases of the subtilisin family, but not belonging to any of the subgroups. The true subtilisins include proteases such as subtilisin BPN′ (BASBPN), subtilisin Carlsberg (ALCALASE®, NOVOZYMES NS) (BLSCAR), mesentericopeptidase (BMSAMP) and subtilisin DY (BSSDY). The high alkaline proteases include proteases such as subtilisin 309 (SAVINASE®, NOVOZYMES A/S) (BLSAVI) subtilisin PB92 (BAALKP), subtilisin BL or BLAP (BLSUBL), subtilisin 147 (ESPERASE®, NOVOZYMES A/S), subtilisin Sendai (BSAPRS) and alkaline elastase YaB. Outside this grouping of the subtilisin family a further subtilisin subgroup was recently identified on the basis of the 3-D structure of its members, the TY145 like subtilisins. The TY145 like subtilisins include proteases such as TY145 (a subtilase from Bacillus sp. TY145, NCIMB 40339 described in WO 92/17577) (BSTY145), subtilisin TA41 (BSTA41), and subtilisin TA39 (BSTA39).
The PD138 type of protease was first described physico-chemically in WO 93/18140 to Novo Nordisk A/S disclosing one strain producing this type of protease. In WO 93/18140, PD138 type of protease was described based on immunological cross reaction with a polyclonal rabbit antibody directed towards the purified protease. The primary structure of the protease was not disclosed. Later the Bacillus species producing this protease was taxonomically classified as Bacillus gibsonii (Nielsen et al., 1995). The type strain of Bacillus gibsonii is identical with the strain described in WO 93/18140. WO 2003/054184 and WO 2003/054185 disclose alkaline subtilases from strains of Bacillus gibsonii.
BRIEF DESCRIPTION OF THE INVENTION
The inventors have isolated novel proteases belonging to the PD138 like proteases subgroup of the subtilisin family that possess advantageous properties, such as improved performance in detergent at low temperature.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 , Phylogenetic tree showing the relationship of the mature subtilase peptide sequences were constructed upon alignment with default settings in the ClustalV function of program MegAlign™ version 5.05 in DNAStar™ program package.
FIG. 2 . The alignment of the sequences from the PCR screening from FIG. 1 .



DEFINITIONS
Prior to discussing this invention in further detail, the following terms and conventions will first be defined.
The term “subtilases” refer to a sub-group of serine proteases according to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue.
The subtilases may be divided into 6 sub-divisions, i.e., the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
The Subtilisin family (EC 3.4.21.62) may be further divided into 3 sub-groups, i.e., I-S1 (“true” subtilisins), I-S2 (highly alkaline proteases) and intracellular subtilisins. Definitions or grouping of enzymes may vary or change, however, in the context of the present invention the above division of subtilases into sub-division or sub-groups shall be understood as those described by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al., 1997, Protein Science 6: 501-523.
The term “parent” is in the context of the present invention to be understood as a protein, which is modified to create a protein variant. The parent protein may be a naturally occurring (wild-type) polypeptide or it may be a variant thereof prepared by any suitable means. For instance, the parent protein may be a variant of a naturally occurring protein which has been modified by substitution, chemical modification, deletion or truncation of one or more amino acid residues, or by addition or insertion of one or more amino acid residues to the amino acid sequence, of a naturally-occurring polypeptide. Thus the term “parent subtilase” refers to a subtilase which is modified to create a subtilase variant.
“Homology” or “homologous to” is in the context of the present invention to be understood in its conventional meaning and the “homology” between two amino acid sequences should be determined by use of the “Similarity” defined by the GAP program from the University of Wisconsin Genetics Computer Group (UWGCG) package using default settings for alignment parameters, comparison matrix, gap and gap extension penalties. Default values for GAP penalties, i.e., GAP creation penalty of 3.0 and GAP extension penalty of 0.1 (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711). The method is also described in S. B. Needleman and C. D. Wunsch, Journal of Molecular Biology, 48, 443-445 (1970). Identities can be extracted from the same calculation. The homology between two amino acid sequences can also be determined by “identity” or “similarity” using the GAP routine of the UWGCG package version 9.1 with default setting for alignment parameters, comparison matrix, gap and gap extension penalties can also be applied using the following parameters: gap creation penalty=8 and gap extension penalty=8 and all other parameters kept at their default values. The output from the routine is besides the amino acid alignment the calculation of the “Percent Identity” and the “Similarity” between the two sequences. The numbers calculated using UWGCG package version 9.1 is slightly different from the version 8.
The term “position” is in the context of the present invention to be understood as the number of an amino acid in a peptide or polypeptide when counting from the N-terminal end of said peptide/polypeptide. The position numbers used in the present invention refer to different subtilases depending on which subgroup the subtilase belongs to.
DETAILED DESCRIPTION OF THE INVENTION
Selection of Strains Producing Novel Subtilisins
In the search for Bacillus strains producing novel subtilases we selected a number of strains, which based on 16S rDNA similarity was related to Bacillus gibsonii . The Bacillus strains P203, EP655, ZI344, EP63, ZI120, ZI130, ZI132 and ZI140 were fermented in a standard Bacillus fermentation medium (BP-X added 0.1 M NaHCO3 to adjust pH to 9).
The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity test can be performed either by the well known ouchterlony double immuno diffusion procedure or by tandem crossed immunoelectro-phoresis according to N. H Axelsen, Handbook of immunoprecipitation-in-gel Techniques. Blackwell Scientific Publications (1983) chapters 5 &14. The terms “antigenic identity” and “partial antigenic identity” are described in the same book chapters 5, 19 and 20.
Culture fluids were analyzed for protease activity using Alcalase™ as standard. Fluids with 10 CPU/L or more activity was included in the immunological analysis. The analysis included two different polyclonal rabbit antibodies; AB41 was antibody raised against the PD138 protease (WO 93/18140). The other antibody was AB65 raised against PD490 protease (Not published). The analysis gave two groups of proteases with a partial reaction against the AB41. One of these groups also has a partial reaction against AB65, whereas the other group reacted identical with AB65. A third group including PD138 gave identical reaction with AB41 and partial reaction with AB65.
PCR Screening
A part of the genes encoding the proteases which exhibited novel immunochemical properties as described above was amplified with a standard PCR reaction with PCR primers designed from available sequences, see Example 1.
The nucleotide sequences were analyzed with DNA STAR™, and based on nucleotide sequence diversity with PD138 as benchmark the novel groups identified with antibodies were confirmed. A phylogenetic tree based on the sequences from the PCR screening is presented in FIG. 1 . A ClustalV alignment of the sequences from the PCR screening is shown in FIG. 2 .
Cloning and Expression of Full Length Subtilase of the Invention
Inverse PCR
Inverse PCR was performed with specific DNA primers designed to complement the DNA sequence obtained from PCR product of the partial protease gene and chromosomal DNA extracted from the appropriate bacterial strain. Inverse PCR was made on the strains P203, EP655 and ZI344, whereas the strains EP63, ZI120, ZI130, ZI1342 and ZI140 were not further investigated. The inverse PCR products were nucleotide sequenced to obtain the region encoding the N and C terminal parts of the genes.
Production of Full Length Subtilase
The subtilase genes were amplified with specific primers with restriction sites in the 5′ end of primers that allow gene fusion with the Savinase signal peptide of plasmid pDG268NeoMCS-PramyQ/Prcrylll/crylllAstab/Sav (U.S. Pat. No. 5,955,310). Protease positive colonies were selected and the coding sequence of the expressed enzyme from the expression construct was confirmed by DNA sequence analysis.
Subtilases of the Invention
The subtilases of the present invention include subtilases from the Bacillus strains ZI344, EP655, P203, EP63, ZI120, ZI130, ZI1342 and ZI140 as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37, respectively. WO 2003/054184 disclose an alkaline protease from Bacillus gibsonii , DSM 14393 which has app. 85.9% amino acid sequence identity with ZI344 and app. 87% amino acid sequence identity with EP655 and P203. Further, the alkaline protease from Bacillus gibsonii , DSM 14393 has 88.2% identity with the partial sequence of the subtilases from ZI120 and ZI130 (SEQ ID NOs: 31 and 33); and 88.1%, 86.8% and 83.8% identity with the partial sequence of the subtilases from EP63, ZI132 and ZI340 (SEQ ID NO: 29, 35 and 37) respectively.
The protease from Bacillus gibsonii , DSM 14393 is encoded by a nucleic acid sequence which is app. 75.5% identical with SEQ ID NO: 1 and app. 80.2% identical with SEQ ID NO's: 3 and 5. The nucleic acid sequence encoding the protease from Bacillus gibsonii , DSM 14393 is 72.2%, 75.7%, 75.7%, 76.2% and 75.5% identical with the nucleic acid sequence encoding the mature part of the partial sequence of the subtilases from ZI340 (SEQ ID NO: 36), ZI120 (SEQ ID NO: 30), ZI130 (SEQ ID NO: 32), EP63 (SEQ ID NO: 28) and ZI132 (SEQ ID NO: 34) respectively.
Thus, the subtilase of the present invention is at least 90% identical with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37. Preferably, said subtilase is at least 91% identical with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37, more preferably said subtilase is at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 or SEQ ID NO: 37.
Correspondingly, the subtilases according to the present invention are encoded by an isolated nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36. Preferably, said nucleic acid sequence is at least 81% identical with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, more preferably said nucleic acid sequence is at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36.
Further the isolated nucleic acid sequence encoding a subtilase of the invention hybridizes with a complementary strand of the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 under low stringency conditions, at least under medium stringency conditions, at least under medium/high stringency conditions, at least under high stringency conditions, at least under very high stringency conditions as described below.
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 concentration of 10 ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32 P-dCTP-labeled (specific activity >1×10 9 cpm/μg) probe for 12 hours at ca. 45° C. For various stringency conditions the filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS and at least 55° C. (low stringency), more preferably at least 60° C. (medium stringency), still more preferably at least 65° C. (medium/high stringency), even more preferably at least 70° C. (high stringency), and even more preferably at least 75° C. (very high stringency).
Variants
Combined Modifications
The present invention also encompasses any of the above mentioned subtilase variants in combination with any other modification to the amino acid sequence thereof. Especially combinations with other modifications known in the art to provide improved properties to the enzyme are envisaged.
Such combinations comprise the positions: 222 (improves oxidation stability), 218 (improves thermal stability), substitutions in the Ca 2+ -binding sites stabilizing the enzyme, e.g., position 76, and many other apparent from the prior art. In further embodiments a subtilase variant described herein may advantageously be combined with one or more modification(s) in any of the positions: 27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 120, 123, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (BPN' numbering). The novel subtilases differ from the primary structure of BPN′ by deletion at the following positions 36, 57 and 158 to 162. The novel subtilase are 6 amino acids shorter than BPN′.
Methods for Expression and Isolation of Proteins
To express an enzyme of the present invention the above mentioned host cells transformed or transfected with a vector comprising a nucleic acid sequence encoding an enzyme of the present invention are typically cultured in a suitable nutrient medium under conditions permitting the production of the desired molecules, after which these are recovered from the cells, or the culture broth.
The medium used to culture the host cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). The media may be prepared using procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
If the enzymes of the present invention are secreted into the nutrient medium, they may be recovered directly from the medium. If they are not secreted, they may be recovered from cell lysates. The enzymes of the present invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like, dependent on the enzyme in question.
The enzymes of the invention may be detected using methods known in the art that are specific for these proteins. These detection methods include use of specific antibodies, formation of a product, or disappearance of a substrate. For example, an enzyme assay may be used to determine the activity of the molecule. Procedures for determining various kinds of activity are known in the art.
The enzymes 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 (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J-C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
When an expression vector comprising a DNA sequence encoding an enzyme of the present invention is transformed/transfected into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme. An advantage of using a heterologous host cell is that it is possible to make a highly purified enzyme composition, characterized in being free from homologous impurities, which are often present when a protein or peptide is expressed in a homologous host cell. In this context homologous impurities mean any impurity (e.g., other polypeptides than the enzyme of the invention) which originates from the homologous cell where the enzyme of the invention is originally obtained from.
Detergent Applications
The enzyme 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, especially for automatic dish washing (ADW).
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, 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 Relase®, Alcalase®, Savinase®, Primase®, Everlase®, Esperase®, Ovozyme®, Coronase®, Polarzyme® and Kannase® (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™, FN2™, FN3™, FN4™ and Purafect Prime™ (Genencor International, Inc.), BLAP X and BLAP S (Henkel).
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 Lipolase™ and Lipolase Ultra™ (Novozymes A/S).
Amylases: Suitable amylases (α and/or β) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, α-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 used amylases are Duramyl®, Termamyl®, Stainzyme®, Fungamyl® and BAN® (Novozymes A/S), Rapidase™, Purastar™ and Purastar OxAm™ (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™, Renozyme® and Carezyme™ (Novozymes A/S), 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).
Hemicellulases: Suitable hemicellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable hemicellulases include mannanase, lichenase, xylanase, arabinase, galactanase acetyl xylan esterase, glucorunidase, ferulic acid esterase, coumaric acid esterase and arabinofuranosidase as described in WO 95/35362. Suitable mannanases are described in WO 99/64619.
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 030% 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 carboxymethyl-cellulose, 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 H 2 O 2 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 redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
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 which is hereby incorporated as reference.
Typical powder detergent compositions for automated dishwashing include:











1)






Nonionic surfactant
0.4-2.5%


Sodium metasilicate
0-20%


Sodium disilicate
3-20%


Sodium triphosphate
20-40%


Sodium carbonate
0-20%


Sodium perborate
2-9%


Tetraacetyl ethylene diamine (TAED)
1-4%


Sodium sulphate
5-33%


Enzymes
0.0001-0.1%

































2)




Nonionic surfactant (e.g., alcohol ethoxylate)
1-2%

Sodium disilicate
2-30%

Sodium carbonate
10-50%

Sodium phosphonate
0-5%

Trisodium citrate dehydrate
9-30%

Nitrilotrisodium acetate (NTA)
0-20%

Sodium perborate monohydrate
5-10%

Tetraacetyl ethylene diamine (TAED)
1-2%

Polyacrylate polymer (e.g., maleic acid/acrylic acid
6-25%

copolymer)


Enzymes
0.0001-0.1%

Perfume
0.1-0.5%

Water
5-10







































3)






Nonionic surfactant
0.5-2.0%


Sodium disilicate
25-40%


Sodium citrate
30-55%


Sodium carbonate
0-29%


Sodium bicarbonate
0-20%


Sodium perborate monohydrate
0-15%


Tetraacetyl ethylene diamine (TAED)
0-6%


Maleic acid/acrylic acid copolymer
0-5%


Clay
1-3%


Polyamino acids
0-20%


Sodium polyacrylate
0-8%


Enzymes
0.0001-0.1%







































4)






Nonionic surfactant
1-2%


Zeolite MAP
15-42%


Sodium disilicate
30-34%


Sodium citrate
0-12%


Sodium carbonate
0-20%


Sodium perborate monohydrate
7-15%


Tetraacetyl ethylene diamine (TAED)
0-3%


Polymer
0-4%


Maleic acid/acrylic acid copolymer
0-5%


Organic phosphonate
0-4%


Clay
1-2%


Enzymes
0.0001-0.1%


Sodium sulphate
Balance





























5) Nonionic surfactant 1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate 12-20% Monopersulphate (2 KHSO 5 •KHSO 4 •K 2 SO 4 ) 15-21% Bleach stabilizer 0.1-2% Maleic acid/acrylic acid copolymer 0-6% Diethylene triamine pentaacetate, 0-2.5% pentasodium salt Enzymes 0.0001-0.1% Sodium sulphate, water Balance
Powder and liquid dishwashing compositions with cleaning surfactant system typically include the following ingredients:

6) Nonionic surfactant 0-1.5% Octadecyl dimethylamine N-oxide dihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyl dimethylamine 0-4% N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5% (hydroxylethyl)amine N-oxide anhydrous and hexadecyl bis (hydroxyethyl)amine N-oxide anhydrous C 13 -C 15 alkyl ethoxysulfate with an average degree of 0-10% ethoxylation of 3 C 12 -C 15 alkyl ethoxysulfate with an average degree of 0-5% ethoxylation of 3 C 13 -C 15 ethoxylated alcohol with an average degree of 0-5% ethoxylation of 12 A blend of C 12 -C 15 ethoxylated alcohols with an 0-6.5% average degree of ethoxylation of 9 A blend of C 13 -C 15 ethoxylated alcohols with an 0-4% average degree of ethoxylation of 30 Sodium disilicate 0-33% Sodium tripolyphosphate 0-46% Sodium citrate 0-28% Citric acid 0-29% Sodium carbonate 0-20% Sodium perborate monohydrate 0-11.5% Tetraacetyl ethylene diamine (TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5% Sodium sulphate 0-12.5% Enzymes 0.0001-0.1%
Non-aqueous liquid ADW compositions typically include the following ingredients:









7)




Liquid nonionic surfactant e.g., alcohol ethoxylates
2.0-10.0%

Alkali metal silicate
3.0-15.0%

Alkali metal phosphate
20.0-40.0%

Liquid carrier selected from higher
25.0-45.0%

glycols, polyglycols, polyoxides, glycolethers


Stabilizer (e.g., a partial ester of phosphoric acid
0.5-7.0%

and a C 16 -C 18 alkanol)


Foam suppressor (e.g., silicone)
0-1.5%

Enzymes
0.0001-0.1%
























8) Liquid nonionic surfactant e.g., alcohol ethoxylates 2.0-10.0% Sodium silicate 3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate 0.0-1.5% Stabilizing system (e.g., mixtures of finely divided 0.5-7.0% silicone and low molecular weight dialkyl polyglycol ethers) Low molecule weight polyacrylate polymer 5.0-15.0% Clay gel thickener (e.g., bentonite) 0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes 0.0001-0.1% Liquid carrier selected from higher lycols, poly- Balance glycols, polyoxides and glycol ethers
Thixotropic liquid ADW compositions typically include the following ingredients:

9) C 12 -C 14 fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0% Sodium citrate 0-12% Sodium tripolyphosphate 0-15% Sodium carbonate 0-8% Aluminium tristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylate thickener 1.32-2.5% Sodium polyacrylate 2.4-6.0% Boric acid 0-4.0% Sodium formate 0-0.45% Calcium formate 0-0.2% Sodium n-decydiphenyl oxide disulphonate 0-4.0% Monoethanol amine (MEA) 0-1.86% Sodium hydroxide (50%) 1.9-9.3% 1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1% Suds suppressor, dye, perfumes, water Balance
Liquid automatic dishwashing compositions typically include the following ingredients:

10) Alcohol ethoxylate 0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20% Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate 18-33% Sodium citrate dihydrate 18-33% Sodium stearate 0-2.5% Sodium perborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED) 0-8% Maleic acid/acrylic acid copolymer 4-8% Enzymes 0.0001-0.1%
Liquid ADW compositions containing protected bleach particles typically include the following ingredients:

11) Sodium silicate 5-10% Tetrapotassium pyrophosphate 15-25% Sodium triphosphate 0-2% Potassium carbonate 4-8% Protected bleach particles, e.g., chlorine 5-10% Polymeric thickener 0.7-1.5% Potassium hydroxide 0-2% Enzymes 0.0001-0.1% Water Balance
12) Automatic dishwashing compositions as described in 1), 2), 3), 4), 6) and 10), wherein perborate is replaced by percarbonate.

13) Automatic dishwashing compositions as described in 1)-6) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in “Efficient manganese catalysts for low-temperature bleaching”, Nature 369: 637-639 (1994).

Materials and Methods

Method for Producing a Subtilase Variant

The present invention provides a method of producing an isolated enzyme according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the enzyme, is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
When an expression vector comprising a DNA sequence encoding the enzyme is transformed into a heterologous host cell it is possible to enable heterologous recombinant production of the enzyme of the invention. Thereby it is possible to make a highly purified subtilase composition, characterized in being free from homologous impurities.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed subtilase may conveniently be secreted into the culture medium and may be recovered there-from by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
Example 1
Selection of Strains and Screening with Antibodies
In the search for Bacillus strains producing novel subtilases of the PD138 group we selected a number of strains, which based on 16S rDNA similarity was related to Bacillus gibsonii . A number of such Bacillus strains were fermented in a standard Bacillus fermentation medium (BP-X added 0.1 M NaHCO 3 to adjust pH to 9).
The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity test can be performed either by the well known ouchterlony double immuno diffusion procedure or by tandem crossed immunoelectro-phoresis according to N. H Axelsen, Handbook of immunoprecipitation-in-gel Techniques. Blackwell Scientific Publications (1983) chapters 5 &14.
Culture fluids were analyzed for protease activity using Alcalase™ as standard. Fluids with 10 CPU/L or more activity was included in the immunological analysis.
The analysis included two different antibodies; AB41 is a polyclonal rabbit antibody raised against the PD138 protease (WO 93/18140). The other antibody is AB65 raised against a bacterial subtilisin isolated from wild type Bacillus sp. PD490 (not published). The analysis revealed two novel groups of proteases with a partial reaction against the AB41. One of these groups also had a partial reaction against AB65 (EP655, ZI120, EP63, ZI130 and ZI132), whereas the other group reacted identical with AB65 (ZI344 and ZI430). A third group including the PD138 protease reacted identical with AB41 and partially identical with AB65.





TABLE 1



Different proteases and their reaction with two different antibodies.






Antibody








Protease
AB41
AB65




PD138
Identical
Partial


EP655
Partial
Partial


ZI120
Partial
Partial


EP63
Partial
Partial


ZI130
Partial
Partial


ZI132
Partial
Partial


ZI344
Partial
Identical


ZI340
Partial
Identical
































A part of the subtilase gene was amplified with a standard PCR reaction with PCR primers:
PD138A0 (SEQ ID NO: 7)/PD138A2 (SEQ ID NO: 9) gave a PCR product of about 900 nt;
PD138A1 (SEQ ID NO: 8)/PD138A2 (SEQ ID NO: 9) gave a PCR product of about 450 nt;
ZI344F (SEQ ID NO: 10)/PD138A2 (SEQ ID NO: 9) gave a PCR product of about 800 nt.
GAGGAGGCNGAGTTNGARGC (SEQ ID NO: 7), the symbols for degenerations are: N for inosine and R for an equal mixture of A and G.
AGTTAGCAGATATAAATAATTCAA (SEQ ID NO: 8),
GTGGAGTAGCCATAGATGTACCA (SEQ ID NO: 9),
TGCAAACGAGGTTGAACAGG (SEQ ID NO: 10).
The PCR reaction that included 50 U/ml of Ampli-Taq™ DNA polymerase (Perkin Elmer) 10× Amplitaq buffer (final concentration of MgCl 2 is 1.5 mM) 0.2 mM of each of the dNTPs (dATP, dCTP, dTTP and dGTP), 0.2 pmol/microliter of the primers and 1 microliter DNA template.
Template DNA was recovered from the various Bacillus strains using HighPure™ PCR template preparation kit (Boehringer Mannheim art. 1796828) as recommended by the manufacturer for DNA recovery from bacteria. The quality of the isolated template was evaluated by agarose gel electrophoresis. If a high molecular weight band was present the quality was accepted. PCR was run in the following protocol: 94° C., 2 minutes 40 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 68° C. for 1 minute] completed with 68° C. for 10 minutes. PCR products were analyzed on a 1% agarose gel in TAE buffer stained with Ethidium bromide to confirm a single band of app. 1050 nucleotides. The PCR product was recovered by using Qiagen™ PCR purification kit as recommended by the manufacturer. The nucleotide sequences were determined by sequencing on an ABI PRISM™ DNA sequencer (Perkin Elmer).
The nucleotide sequences were analyzed with DNA STAR™, and based on nucleotide sequence diversity with PD138 as benchmark the novel groups identified with antibodies were confirmed. A phylogenetic tree based on the sequences from the PCR screening is presented in FIG. 1 . A ClustalV alignment of the sequences from the PCR screening is shown in FIG. 2 .
Example 2
Production of Full Length Subtilases
Inverse PCR
Three digestions of the chromosomal DNA of the strains EP655, P203 and ZI344 were made using the restriction enzymes Mlu1, EcoR1 and Sac1. Upon digestion the DNA was separated from the restriction enzymes using Qiaquick™ PCR purification kit (art. 28106, Qiagen, Germany). The digestions were religated and subjected to a PCR reaction using primers (PCR primers SEQ ID NOs: 11-16) designed to recognise the sequence of the PCR product already obtained. The following PCR protocols were applied: 94° C. 2 min 30 cycles of [94° C. for 15 s, 52° C. for 30 s, 72° C. for 2 min] 72° C. 20 min. In the PCR the amount of primer, DNA polymerase and buffer were the same as in Example 1. Alternatively a protocol with 94° C. 2 min 30 cycles of [94° C. for 15 s, 52° C. for 30 s, 68° C. for 3 min] 68° C. 20 min. and replacing Amplitaq® and Amplitaq® buffer with Long-template Taq Polymerase™ (Boehringer Mannheim) with the buffer supplied with the polymerase. The PCR reactions were analyzed on 0.8% agarose gels stained with ethidium bromide. All PCR fragments were recovered and the nucleotide sequence was determined by using specific oligo primers different from those used in the PCR reaction (Sequencing primers SEQ ID NOs: 17-22).
The following primers were used for obtaining the inverse PCR and sequencing:
Inverse PCR Primers
P203A-PCR-R (SEQ ID NO: 11) ACACGAGTAATACCCCAAGG
P203A-PCR-F (SEQ ID NO: 12) GCTAATGCAATGGCAGTAGG
ZI344-PCR-R (SEQ ID NO: 13) ACTCTTTGAATGCCCCAAGG
ZI344-PCR-F (SEQ ID NO: 14) AGGTGTACTTGTTGTGGCAG
EP655-PCR-R (SEQ ID NO: 15) AGTAATACCCCAAGGCACCG
EP655-PCR-F (SEQ ID NO: 16) GCGGCTTCAGGTAATAACGG
Sequencing Primers
P203A-seq-R (SEQ ID NO: 17) CAACTCAACTGATAATACGG
P203A-seq-F (SEQ ID NO: 18) TTCTCTCAATATGGTGCAGG
EP655-seq-R (SEQ ID NO: 19) AATGCATCAACATCTTCAGG
EP655-seq-F (SEQ ID NO: 20) GGATATCCTGCACGTTATGC
ZI344-seq-R (SEQ ID NO: 21) AGTGCTTCTACATCCTCAGG
ZI344-seq-F (SEQ ID NO: 22) AACGTTGGCTACCCTGCACG
Production of the Full Length Subtilase
To produce the subtilases of strains P203, EP655 and ZI344 the protease gene was amplified from chromosomal DNA of the wild type strains. For P203 chromosomal DNA of the strain DSM 17419 can be used. The protease gene was amplified as a app. 1200 nt (nucleotide) PCR product. For P203 primers P203A-Sac1/P203A-BamH1 for Zi344 primers ZI344-Sac1/ZI344-Mlu1 and for EP655 primers P203A-Sac1/EP655-MLu1 were used. Template DNA was chromosomal DNA of the respective wild type Bacillus strains.
Primers:
P203A-Sac1: TTATGGAGCTCCTAAAAATGAGGAGGCGACC (SEQ ID NO: 23)
P203A-BamH1: TGTATGGATCCAAATAGAGACGAAACCGCCC (SEQ ID NO: 24)
EP655-MLu1: GATTAACGCGTCTGCTCTTATCGACTAGCGG (SEQ ID NO: 25)
ZI344-Sac1: TTATGGAGCTCGATCAATACAAGGAGGCGAC (SEQ ID NO: 26)
ZI344-Mlu1: GATTAACGCGTGTTCTTTTATCGTGTAGCTG (SEQ ID NO: 27)
EP655-Sac1: use P203A-Sac1.
The PCR products were recovered using Qiaquick™ spin columns as recommended (Qiagen, Germany). The quality of the isolated template was evaluated by agarose gel electrophoresis. PCR was run in the following protocol: 94° C., 2 minutes 40 cycles of [94° C. for 30 seconds, 52° C. for 30 seconds, 68° C. for 1 minute] completed with 68° C. for 10 minutes. PCR products were analyzed on a 1% agarose gel in TAE buffer stained with Ethidium bromide to confirm a single band of the correct size. The PCR products were digested with restriction enzymes Sac1 and Mlu1 and purified on GFX™ PCR and Gel Band Purification Kit (Amerham Biosciences).
The digested and purified PCR fragment was ligated to the Sac I and Mlu I digested plasmid pDG268NeoMCS-PramyQ/Prcrylll/crylllAstab/Sav (U.S. Pat. No. 5,955,310). The ligation mixture was used for transformation into E. coli TOP10F′ (Invitrogen BV, The Netherlands) and several colonies were selected for miniprep (QIAprep® spin, QIAGEN GmbH, Germany). The purified plasmids were checked for insert before transformation into a strain of Bacillus subtilis derived from B. subtilis DN 1885 with disrupted apr, npr and pel genes (Diderichsen et al., 1990, J. Bacteriol. 172: 4315-4321). The disruption was performed essentially as described in “ Bacillus subtilis and other Gram-Positive Bacteria,” American Society for Microbiology, p. 618, eds. A. L. Sonenshein, J. A. Hoch and Richard Losick (1993). Transformed cells were plated on 1% skim milk LB-PG agar plates, supplemented with 6 micrograms/ml chloramphenicol. The plated cells were incubated over night at 37° C. and protease containing colonies were identified by a surrounding clearing zone. Protease positive colonies were selected and the coding sequence of the expressed enzyme from the expression construct was confirmed by DNA sequence analysis.
Example 3
Purification and Characterisation
Purification
This procedure relates to purification of a 2 liter scale fermentation for the production of the subtilases of the invention in a Bacillus host cell.
Approximately 1.6 liters of fermentation broth are centrifuged at 5000 rpm for 35 minutes in 1 liter beakers. The supernatants are adjusted to pH 6.5 using 10% acetic acid and filtered on Seitz Supra® S100 filter plates.
The filtrates are concentrated to approximately 400 ml using an Amicon® CH2A UF unit equipped with an Amicon® S1Y10 UF cartridge. The UF concentrate is centrifuged and filtered prior to absorption at room temperature on a Bacitracin affinity column at pH 7. The protease is eluted from the Bacitracin column at room temperature using 25% 2-propanol and 1 M sodium chloride in a buffer solution with 0.01 dimethylglutaric acid, 0.1 M boric acid and 0.002 M calcium chloride adjusted to pH 7.
The fractions with protease activity from the Bacitracin purification step are combined and applied to a 750 ml Sephadex® G25 column (5 cm dia.) equilibrated with a buffer containing 0.01 dimethylglutaric acid, 0.2 M boric acid and 0.002 m calcium chloride adjusted to pH 6.5.
Fractions with proteolytic activity from the Sephadex® G25 column are combined and applied to a 150 ml CM Sepharose® CL 6B cation exchange column (5 cm dia.) equilibrated with a buffer containing 0.01 M dimethylglutaric acid, 0.2 M boric acid, and 0.002 M calcium chloride adjusted to pH 6.5.
The protease is eluted using a linear gradient of 0-0.1 M sodium chloride in 2 liters of the same buffer.
In a final purification step subtilase containing fractions from the CM Sepharose® column are combined and concentrated in an Amicon® ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.).
Example 4
Stability of Subtilases
The stability of the subtilases of the invention can be evaluated in a standard Western European dishwashing tablet detergent without other enzymes than the experimentally added subtilases. The stability of the subtilases can be determined as the residual proteolytic activity after incubation of the subtilase in a detergent.
The formulation of a standard Western European Tablet detergent is defined as:
Component Percentage Non-ionic surfactants 0-10% Foam regulators 1-10% Bleach (per-carbonate or per-borate) 5-15% Bleach activators (e.g., TAED) 1-5% Builders (e.g., carbonate, phosphate, tri-phosphate, Zeolite) 50-75% Polymers 0-15% Perfume, dye etc. <1% Water and fillers (e.g., sodium sulphate) Balance
Assay for Proteolytic Activity

The proteolytic activity is determined with casein as substrate. One Casein Protease Unit (CPU) is defined as the amount of protease liberating about 1 micro-M of primary amino groups (determined by comparison with a serine standard) per minute under standard conditions, i.e., incubation for about 30 minutes at about 25° C. at pH 9.5.
The proteolytic activity may also be determined by measuring the specific hydrolysis of succinyl-Ala-Ala-Pro-Leu-p-nitroanilide by said protease. The substrate is initially dissolved in for example, DMSO (Dimethyl Sulfoxide) and then diluted about 50 fold in about 0.035 M borate buffer, about pH 9.45. All protease samples may be diluted about 5-10 fold by the same borate buffer. Equal volumes of the substrate solution and sample are mixed in a well of an ELISA reader plate and read at about 405 nm at 25° C. All sample activities and concentrations are normalized to the standard protease solution activity and concentration, respectively.
A typical Western European tablet detergent for automated dishwashing is dissolved (5.5 g/L) in 9° dH water at ambient temperature maximum 30 minutes prior to start of analyses. Samples of subtilases are diluted to a concentration of 2-4 CPU/ml in Britten Robinson buffer (Britten Robinson buffer is: 40 mM Phosphate, 40 mM Acetate and 40 mM Borate) pH 9.5. For the analyses every sample is divided and tested under two conditions: For the control the subtilase is diluted 1:9 in Britten Robinson buffer pH 9.5 to a final volume of 1 ml. This sample is analyzed immediately after dilution. For the detergent stability the subtilase sample is diluted 1:9 in detergent solution (detergent concentration in the stability test is 5 g/L) these samples are incubated at 55° C. for 30 minutes prior to analysis by addition of casein substrate.
The assay is started by addition of 2 volumes of casein substrate (casein substrate is 2 g of casein (Merck, Hammerstein grade) in 100 ml of Britten Robinson buffer pH 9.5, pH is re-adjusted to 9.5 when the casein is in solution). Samples are kept isothermic at 25° C. for 30 minutes. The reaction is stopped by addition of 5 ml TCA solution (TCA solution is 89.46 g of Tri-chloric acid, 149.48 g of Sodium acetate-tri-hydrate and 94.5 ml of glacial acetic acid in 2.5 L of deionised water). The samples are incubated at ambient temperature for at least 20 minutes and filtered through Whatman® paper filter no. 42.
400 microliters of filtrate is mixed with 3 ml OPA reagent (OPA reagent is composed of: 3.812 g of borax, 0.08% EtOH, 0.2% DTT and 80 mg of o-phthal-dialdehyd in 100 ml water). Absorption at 340 nm is measured and CPU is calculated from the concentration of free amines on a standard of a solution of 0.01% L-serine (Merck art. 7769).
Example 5
Microtiter Egg Assay (MEA)
In this assay the digestion of denatured egg proteins by proteases in the presence of detergent can be followed in a 96-well microtiter plate. Heating of egg proteins produces visual changes and changes in physicochemical properties. The clear translucent material is transformed to a milky substance. This is partly due to sulfhydryl-disulfide interchange reactions of denatured proteins. For example, heating unmasks the sulfhydryl group of ovalbumin, and the unmasked groups form disulfide linkages. The digestion of the denatured egg proteins by proteases converts the milky egg solution to a more clear solution dependent on the ability of the enzymes to degrade egg proteins.
Procedure
a) Prepare an egg solution by dissolving 200 mg egg powder (Sanovo International AS) in 93.7 mL, where the water hardness in adjusted to 16° dH. Denature the egg solution by increasing the temperature to 85° C. over an 8 minutes time period.
b) Dilute the subtilase enzyme to 320 nM in succinic acid buffer: 10 mM succinic acid+2 mM CaCl 2 +0.02% non-ionic detergent (such as Brij35) adjusted to pH 6.5;
c) Prepare the detergent solution just before use by mixing 5 g detergent & 937.5 mL water (16° dH (Ca 2+ /Mg 2+ 4:1)). The dishwash detergent could be a typical Western Europe 2 in1 (use 8° dH) or 3 in1 tablet (use 16° dH) or an automatic dishwash powder product (use 8° dH). If the detergent already contains proteases, the detergent solution should be inactivated in a microwave oven at 85° C. for 5 minutes
d) Add to each well in a 96 well microtiter plate: 10 microliters of 320 nM enzyme solution (final concentration 20 nM)+150 microliters detergent solution (final concentration 5 g/L, 16° d)+egg solution (320 micrograms egg protein/well).
Measure OD 410 nm immediately (time 0 minutes) on a spectrophotometer. Incubate exactly 20 minutes at 55° C. and then measure OD 410 nm again. Calculate ΔOD and compare the variants with the performance of a reference subtilase, such as Savinase® or Alcalase® from Novozymes A/S. The performance of the reference is set to ΔOD=100%.
By use of the above mentioned procedure the digestion of denatured egg proteins by the subtilase enzymes of the invention was compared with that of Savinase®. The results are presented in Table 1 as performance % of Savinase performance:









TABLE 1



Savinase
Alcalase
EP655
ZI344
P203



100
10
211
230
212