Source: http://www.google.com/patents/US7816115?ie=ISO-8859-1
Timestamp: 2014-03-12 03:15:01
Document Index: 107759567

Matched Legal Cases: ['application No. 60', 'art. 1796828', 'art. 1796828', 'art. 1796828', 'art. 28106', 'art. 7769']

Patent US7816115 - Subtilases - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe present invention relates to novel JP170 like subtilases from wild-type bacteria, hybrids thereof 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 or an automatic dishwashing...http://www.google.com/patents/US7816115?utm_source=gb-gplus-sharePatent US7816115 - SubtilasesAdvanced Patent SearchPublication numberUS7816115 B2Publication typeGrantApplication numberUS 11/575,551PCT numberPCT/DK2005/000597Publication dateOct 19, 2010Filing dateSep 21, 2005Priority dateSep 21, 2004Also published asEP1794296A1, EP1794296B1, EP2261329A2, EP2261329A3, US7910349, US8232087, US8318470, US20080187958, US20110003730, US20110130319, US20120101019, US20130045524, WO2006032278A1, WO2006032278A9Publication number11575551, 575551, PCT/2005/597, PCT/DK/2005/000597, PCT/DK/2005/00597, PCT/DK/5/000597, PCT/DK/5/00597, PCT/DK2005/000597, PCT/DK2005/00597, PCT/DK2005000597, PCT/DK200500597, PCT/DK5/000597, PCT/DK5/00597, PCT/DK5000597, PCT/DK500597, US 7816115 B2, US 7816115B2, US-B2-7816115, US7816115 B2, US7816115B2InventorsPreben Nielsen, Poul Erik Pedersen, Helle OuttrupOriginal AssigneeNovozymes A/SExport CitationBiBTeX, EndNote, RefManPatent Citations (16), Non-Patent Citations (4), Referenced by (2), Classifications (19), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetSubtilasesUS 7816115 B2Abstract The present invention relates to novel JP170 like subtilases from wild-type bacteria, hybrids thereof 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 or an automatic dishwashing detergent.
1. An isolated subtilase which has an amino acid sequence which is at least 97% identical with the sequence of SEQ ID NO:6.
2. The subtilase of claim 1 which has an amino acid sequence which is at least 98% identical with the sequence of SEQ ID NO:6.
3. The subtilase of claim 1 which has an amino acid sequence which is at least 99% identical with the sequence of SEQ ID NO:6.
4. The subtilase of claim 1 which comprises the sequence of SEQ ID NO 6.
5. The subtilase of claim 1 which consists of the sequence of SEQ ID NO:6.
6. A core subtilase which has an amino acid sequence which is 97% identical with the sequence of amino acids from position 49 through position 391 of SEQ ID NO:6.
7. A hybrid subtilase comprising the core amino acid sequence of claim 6.
8. A detergent composition comprising the subtilase of claim 1 and a surfactant.
9. An isolated nucleic acid sequence encoding the subtilase of claim 1.
10. The nucleic acid sequence of claim 9 as shown in SEQ ID NO:5.
11. A nucleic acid construct comprising the nucleic acid sequence of claim 9 operably liked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
12. A recombinant expression vector comprising the nucleic acid construct of claim 11, a promoter, and transcriptional and translation stop signals.
13. The vector of claim 12, further comprising a selectable marker.
14. A recombinant host cell comprising the nucleic acid construct of claim 11.
15. A method for producing a subtilase, comprising
a) cultivating the recombinant host cell of claim 14; and
b) recovering the subtilase.
CROSS-REFERENCE TO RELATED APPLICATIONS This application is a 35 U.S.C. 371 national application of PCT/DK2005/000597 filed Sep. 21, 2005, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2004 01429 filed Sep. 21, 2004 and U.S. provisional application No. 60/611,720 filed Sep. 21, 2004, the contents of which are fully incorporated herein by reference.
SEQUENCE LISTING AND DEPOSITED MICROOGANISMS Sequence Listing The present invention comprises a sequence listing.
Deposit of Biological Material The following biological material has been deposited under the terms of the Budapest Treaty with Deutsche Sammlung von Mikroorganismen und Zellkulturen and given the following accession numbers:
Deposit Accession Number Date of Deposit JP170/PD456 hybrid DSM16714 15 Sep. 2004 JP170/JT169 hybrid DSM16715 15 Sep. 2004 JT169 DSM16719 15 Sep. 2004 PD456 DSM16722 15 Sep. 2004 JP170/JP75 hybrid DSM16712 15 Sep. 2004 JP170/JP40 hybrid DSM16713 15 Sep. 2004 JP170/AMRK133 hybrid DSM16716 15 Sep. 2004 The deposits contain subtilase genes and hybrid subtilase genes as described in example 1-3.
FIELD OF THE INVENTION The present invention relates to novel JP170 like subtilases from wild-type bacteria, hybrids thereof 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 R. J. and Leunissen J. A. M., 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 A/S) (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).
BRIEF DESCRIPTION OF THE INVENTION The inventors have isolated novel proteases belonging to the JP170 like proteases subgroup of the subtilisin family that possess advantageous properties, such as improved detergent stability.
BRIEF DESCRIPTION OF DRAWINGS FIG. 1, Phylogenetic tree showing the relationship of the mature subtilase peptide sequences were constructed upon alignment with default settings in the ClustalW function of program MegAlign� version 5.05 in DNAStar� program package.
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., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 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. Protein Science 6 (1997) 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.
The term �hybrid� is in the context of this invention to be understood as a protein that has been modified by replacing one or more segments of the gene encoding the parent protein with corresponding segments derived from genes encoding another protein.
The term �core� in the context of this invention is to be understood as a segment that comprises a substantial part of the subtilase gene including the part encoding the active site and a substantial part of the rest of the subtilase molecule, to provide unique traits to a hybrid.
The term �modification(s)� or �modified� is in the context of the present invention to be understood as to include chemical modification of a protein as well as genetic manipulation of the DNA encoding a protein. The modification(s) may be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest. Thus the term �modified protein�, e.g. �modified subtilase�, is to be understood as a protein which contains modification(s) compared to a parent protein, e.g. subtilase.
�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 Construction of Degenerated Primers Degenerated primers were constructed from an alignment of genes of already known proteases such as Ya, KAO KSM-43 and JP170. The primers were degenerated in order to allow screening for protease gene fragments different from Ya, KAO KSM-43 and JP170.
Based on the results of the screening a number of enzymes were selected for further investigation. The selected enzymes are shown in FIG. 1, and they both represent new enzyme molecules and representatives of the prior art. The enzymes selected for further investigation are JP40, JP75, JT169, AMRK133 and PD456, which can be seen as forming a separate group in FIG. 1. Also hybrid subtilases produced as described below can be seen in FIG. 1.
Based on these results the inventors decided to move on with a dual approach; expression of the PCR product by in frame fusions to N and C terminal parts of the known protease of Bacillus halmapalus strain JP170 and inverse PCR to get the full sequences of selected enzymes.
By SOE PCR(SOE: Splicing by Overlapping Extension) hybrid gene products comprising 5 segments were generated as described in Example 2. The hybrid subtilase genes are used for production of a mature protease enzyme of about 433 amino-acids and a molecular weight of approximately 45 kd. The first segment is the nucleotide sequence encoding the pro sequence of JP170 protease (that is not a part of the mature protease) and 40 amino acids of the N terminal of the mature JP170 protease. This is followed by a fusion primer segment encoding 8 amino acids (this segment may contain sequence variation due to the degeneration of the primer SF16A767F). The third segment is encoding the approximately 343 amino acid long core. This segment includes the sequence encoding the active site of the protease. This is followed by a fusion primer segment encoding 7 amino acids (this segment may contain variation due to the degeneration of the primer SF16A1802R). The fifth segment is encoding the 35 amino acids of the C terminal of the JP170 protease.
SOE PCR products based on core segments from the following strains were generated: JP40 (SEQ ID NO:5), PD456 (SEQ ID NO:7), JP75 (SEQ ID NO:9), JT169 (SEQ ID NO:11), AMRK133 (SEQ ID NO:13) (the SEQ ID number of the gene sequence encoding the mature hybrid protease is indicated in brackets).
The N terminal end of the core segment is located in one of positions 1-10, 10-20, 20-30, 30-40, 40-50, 50-60 or 60-70 of the subtilase of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. The C terminal end of the core segment is located in one of positions 70-80, 80-90, 90-100, 100-150, 150-200, 200-250, 250-300, 300-320, 320-340, 340-360, 360-380, 380-400, 400-420 of the subtilase of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. In a preferred embodiment the core of the subtilase of the invention comprises the amino acids in position 49-392 of the hybrids JP40 (SEQ ID NO:6), PD456 (SEQ ID NO:8), JP75 (SEQ ID NO:10), JT169 (SEQ ID NO:12), AMRK133 (SEQ ID NO:14).
The core sequence preferably has 94% identity with the amino acids in position 49-392 of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. More preferably the core sequence has 95% identity, 96% identity, 97% identity, 98% identity or 99% identity with SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
The corresponding nucleotides encoding the core segment can be seen in any of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. In a preferred embodiment the core of the subtilase of the invention is encoded by the nucleotides in position 145-1177 of the hybrids JP40 (SEQ ID NO:5), PD456 (SEQ ID NO:7), JP75 (SEQ ID NO:9), JT169 (SEQ ID NO:11), AMRK133 (SEQ ID NO:13).
The N and C terminals of the hybrids of the present invention could equally well be selected from other subtilases, such as BLSCAR, BMSAMP, BASBPN or BSSDY of I-S1, BLSAVI, BAALKP, BLSUBL or subtilisin 147 of I-S2, a members of the TY145 like subtilases, or another member of the JPI 70 like subtilases.
Inverse PCR was performed with specific DNA primers designed to complement the DNA sequence of the core PCR product and chromosomal DNA extracted from the appropriate bacterial strain. Inverse PCR was made on the strains JT169 and PD456. The inverse PCR products were nucleotide sequenced to obtain the region encoding the N and C terminal parts of the gene.
The subtilase is in the context of the present invention to be understood as including the members of the novel subgroup of FIG. 1: JP40, PD456, JP75, JT169, AMRK133 and homologous or hybrids thereof. According to the identity matrix of FIG. 2 the amino acid sequence identity of the closest related prior art subtilase is 94.7%.
Thus, the subtilase of the present invention is at least 95% identical with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14. In particular said subtilase may be at least 96%, at least 97%, at least 98% or at least 99% identical with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14.
The subtilase of the present invention is encoded by an isolated nucleic acid sequence, which nucleic acid sequence has at least 85% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13. Preferably, said nucleic acid sequence has at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.
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:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13 preferably 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.
27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 120, 123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (BPN′ numbering).
Specifically, the following BLSAVI, BLSUBL, BSKSMK, and BMLKP modifications are considered appropriate for combination:
K27R, *36D, S56P, N76D, S87N, G97N, S101G, S103A, V104A, V104I, V104N, V104Y, H120D, N123S, G159D, Y167, R170, Q206E, N218S, M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A.
Furthermore variants comprising any of the modifications S101G+V104N, S87N+S101 G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I or N76D+V104A, or other combinations of the modifications K27R, N76D, S101G, S103A, V104N, V104Y, V104I, V104A, N123S, G159D, A232V, Q236H, Q245R, N248D, N252K, T274A in combination with any one or more of the modification(s) mentioned above exhibit improved properties.
S101G+S103A+V104I+G159D+A232V+Q236H+ Q245R+N248D+N252K.
Preferred commercially available protease enzymes include Alcalase�, Savinase�, Primase�, Duralase�, Esperase�, and Kannase� (Novozymes A/S), Maxatase�, Maxacal�, Maxapem�, Properase�, Purafect�, Purafect OxP�, FN2�, and FN3� (Genencor International Inc.).
Preferred commercially available lipase enzymes include Lipolase� and Lipolase Ultra� (Novozymes A/S).
Commercially available amylases are Duramyl�, Termamyl�, Fungamyl� and BAN� (Novozymes A/S), Rapidase� and Purastar� (from Genencor International Inc.).
Commercially available cellulases include Celluzyme�, Renozyme� and Carezyme� (Novozymes A/S), Clazinase�, and Puradax HA� (Genencor International Inc.), and KAC-500(B)� (Kao Corporation).
Commercially available peroxidases include Guardzyme� (Novozymes A/S).
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�).
0.4-2.5%
0.0001-0.1% 2)
Nonionic surfactant (e.g. alcohol ethoxylate)
1-2% Sodium disilicate
10-50% Sodium phosphonate
0-5% Trisodium citrate dehydrate
Nitrilotrisodium acetate (NTA)
1-2% Polyacrylate polymer
(e.g. maleic acid/acrylic acid copolymer)
0.0001-0.1% Perfume
0.1-0.5% Water
5-10 3)
0.0001-0.1% 4)
0.0001-0.1% Sodium sulphate
Monopersulphate (2 KHSO5�KHSO4�K2SO4)
0.1-2% Maleic acid/acrylic acid copolymer
Diethylene triamine pentaacetate,
0.0001-0.1% Sodium sulphate, water
Powder and liquid dishwashing compositions with cleaning surfactant system typically include the following ingredients:
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-
70:30 wt. C18/C16 blend of octadecyl bis
0-5% (hydroxyethyl)amine N-oxide anhydrous and
hexadecyl bis (hydroxyethyl)amine N-oxide
C13-C15 alkyl ethoxysulfate with an average degree of
C12-C15 alkyl ethoxysulfate with an average degree of
0-5% ethoxylation of 3
C13-C15 ethoxylated alcohol with an average degree
0-5% of ethoxylation of 12
A blend of C12-C15 ethoxylated alcohols with an
0-6.5%
average degree of ethoxylation of 9
A blend of C13-C15 ethoxylated alcohols with an
0-4% average degree of ethoxylation of 30
0-46%
0-4% Maleic acid/acrylic acid copolymer
0-7.5%
0-12.5%
0.0001-0.1% Non-aqueous liquid automatic dishwashing compositions typically include the following ingredients:
Liquid nonionic surfactant (e.g. alcohol ethoxylates)
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 and
a C16-C18 alkanol)
Foam suppressor (e.g. silicone)
0.0001-0.1% 8)
7.0-20.0%
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,
polyglycols, polyoxides and glycol ethers
Thixotropic liquid automatic dishwashing compositions typically include the following ingredients:
Block co-polymer surfactant
1.5-15.0% Sodium citrate
0-12% Sodium tripolyphosphate
0-15% Sodium carbonate
0-8% Aluminium tristearate
0-1.7%
Polyacrylate thickener
1.32-2.5% Sodium polyacrylate
2.4-6.0% Boric acid
0-4.0%
Sodium n-decydiphenyl oxide disulphonate
Monoethanol amine (MEA)
0-1.86%
1.9-9.3% 1,2-Propanediol
0-9.4%
0.0001-0.1% Suds suppressor, dye, perfumes, water
Liquid automatic dishwashing compositions typically include the following ingredients:
Fatty acid ester sulphonate
Sodium disilicate monohydrate
18-33% Sodium citrate dihydrate
18-33% Sodium stearate
0-8% Maleic acid/acrylic acid copolymer
4-8% Enzymes
0.0001-0.1% Liquid automatic dishwashing 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, 1994, pp. 637-639.
Example 1 PCR Screening The core part of protease gene was amplified in a PCR reaction that included 50 U/ml of Ampli-taq� DNA polymerase (Perkin Elmer) 10� Amplitaq buffer (final concentration of MgCl2 is 1.5 mM) 0.2 mM of each of the dNTPs (dATP, dCTP, dTTP and dGTP) 0.2 μmol/μl of the primers SF16A767F (CNATGCATGAAGCNTTCCGCGG, SEQ ID NO:15) (�N� is degeneration introduced by insertion of inosine)) and SF16A1802R(CNACGTTGTTNCNGCCATCCC, SEQ ID NO:16) and 1 μl template DNA. 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 analysed 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). PCR products of PD456, JT169, JP40, JP75 and AMRK133 were determined. The nucleotide sequences were translated to amino acid sequences, and the diversity analysed by comparative peptide sequence analysis. As can be seen in FIG. 1 the diversity by far exceeded that of the prior art.
Example 2 Production of Subtilase Hybrids Expression of Hybrid Proteases, PCR Amplification
1) The N terminal part of JP170 protease gene. This PCR product is obtained by PCR using the primers
PEP192 5′-CCGCGGAATGCTTCATGCATCG-3′ (SEQ ID NO:27) and
PEP200 5′-GTTCATCGATCTTCTACTATTGGGGCGMC-3′ (SEQ ID NO:28) and 1 μl template DNA. 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 analysed on a 1% agarose gel in TAE buffer stained with Ethidium bromide to confirm a single band of app. 700 nucleotides.
2) The C terminal part of JP170 protease gene. This PCR product is obtained by PCR using the primers
PEP193 5′-GGGATGGCAGAAACAACGTGG-3′ (SEQ ID NO:29) and
PEP201 5′-TTAAACGCGTTTAATGTACAATCGCTAAAGAAAAG-3′ (SEQ ID NO:30) and 1 μl template DNA. 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 analysed on a 1% agarose gel in TAE buffer stained with Ethidium bromide to confirm a single band of app. 370 nucleotides.
3) The core PCR product described in Example 1.
In the SOE PCR reaction the three PCR products are mixed and a fused product is amplified in a standard PCR protocol using the primers PEP200 and PEP201 and 1 μl template DNA. Template DNA is a mixture of the three PCR products described above (1-3). These PCR products may be 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 analysed on a 1% agarose gel in TAE buffer stained with Ethidium bromide to confirm a single band of app. 1850 nucleotides.
The digested and purified PCR fragment was ligated to the Cla I and Mlu I digested plasmid pDG268NeoMCS-PramyQ/PrcryIII/cryIIIAstab/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 μg/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 Production of Full Length Subtilases Inverse PCR
Three digestions of the chromosomal DNA of the strains PD456 and JT169 were made using the restriction enzymes xho1, BamH1 and Pst1. 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 NO:17-20) 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. Using same PCR amount of primer polymerase and buffer as above. 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 polymerease. The PCR reactions were analysed 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 NO:21-26). In some cases the first primer did not give sufficient nucleotide sequence information to characterise the entire open reading frame of the protease gene. In these cases new primers were applied either by using the sequence information obtained with the initial inverse PCR sequencing primer, or by going back to the initial PCR fragment and defining a new primer sequence.
The following primers were used for obtaining the inverse PCR and sequencing:
PD456 PCR Forward: AGGATTCCCGAACGGAAACCAAGG (SEQ ID NO: 17) PD456 PCR Reverse: TCCGTTTCCAAGTACAGACCCGG (SEQ ID NO: 18) JT169 PCR Forward: TCTCTCTAGTATGGTCTGATGCTCC (SEQ ID NO: 19) JT169 PCR Reverse: TCCGTTTCCAAGAACAGATCCGGC (SEQ ID NO: 20) JT169 Forward Sequencing ATCTGCTTCTATAACACTGG (SEQ ID NO: 21) JT169 Reverse Sequencing1 TGCGTTCCGTGACCATTTGG (SEQ ID NO: 22) JT169 Reverse Sequencing2 TTTGCGTCAAGTGCCACAGC (SEQ ID NO: 23) JT169 Reverse Sequencing3 TCAACAATTTCATCTATGCC (SEQ ID NO: 24) PD456 Forward Sequencing GCCGTGTAACAATGGACAAG (SEQ ID NO: 25) PD456 Reverse Sequencing1 TCCCTTTATTGGTTGCTCCG (SEQ ID NO: 26) The gene sequences encoding the mature part of the protease gene of strains JT169 and PD456 are shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively. Sequencing Primers
To produce the subtilases of strain JT169 and PD456 the protease gene was amplified from chromosomal DNA of the wild type strains or from the clones deposited as DSM16719 and DSM16722 using the primers:
JT169 Expression Forward
AGTTCATCGATCGGGGGAGCTAGCAGCTTCGA
JT169 Expression Reverse
TGATTAACGCGTTTAGTTCACAATCGCCAATG
PD456 Expression Forward
AGTTCATCGATCGGGGGGGCTAGCAACTTTGA
PD456 Expression Reverse
These PCR products were digested with restriction enzymes Cla1 and Mlu1 (except AA351 that was digested with (Nar1 and Mlu1) and ligated into pDG268neo, and expressed as described in Example 2.
Example 4 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 Amicono 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 Sephadexe 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 Sephadexe 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.
In a final purification step subtilase containing fractions from the CM Sepharosee column are combined and concentrated in an Amicon� ultrafiltration cell equipped with a GR81PP membrane (from the Danish Sugar Factories Inc.).
Example 5 Stability of Subtilases The stability of the produced subtilases was evaluated in a standard Western European dishwashing tablet detergent without other enzymes than the experimentally added subtilases. The stability of the subtilases is determined as the residual proteolytic activity after incubation of the subtilase in a detergent.
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
400 μl 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-dialdehyde 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).
CPU/L
Everlase16L
Ovozyme
BLAP-S
Enzymatic proteolysis of cloned full length proteases of the invention in the typical Western European tablet detergent:
PD456-1
PD456-2
PD456-3
PD456-4
JT169-1
JT169-2
JT169-3
JT169-4
Enzymatic proteolysis of cloned hybrid proteases of the invention in the typical Western European tablet detergent. The reference is JP170:
CPU/I
JP75-1
JP75-2
JP40-1
JP40-2
As can be seen from the results the subtilases and subtilase hybrids of the invention exhibit a greatly improved proteolytic activity after incubation in a detergent as compared to the prior art subtilase JP170. Therefore, the subtilases and subtilase hybrids of the invention exhibits improved stability in a detergent as compared to the prior art.
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