Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-11-05
2004-10-12
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C510S276000, C510S300000
Reexamination Certificate
active
06803222
ABSTRACT:
TECHNICAL FIELD
The present invention relates to alkaline proteases having high specific activity and strong oxidant resistance. As the alkaline proteases of the present invention have an excellent detergency, these enzymes may be added to a detergent.
BACKGROUND ART
Proteases have conventionally been used in a variety of fields such as various detergents (including laundry detergents), cosmetics, bath agents, food modifiers, and pharmaceuticals (such as digestion aids and anti-inflammatory agents). Of these uses, proteases for detergents are industrially produced in the largest amount and have a great market value. Accordingly, a number of proteases are now available on the market.
In most cases, stains on clothes contain not only proteins but also plural components such as lipids and solid particles. Therefore, there is a demand for detergents having a sufficient detergency to remove complex stains. To address this demand, the present inventors applied for a patent (WO99/18218), which provided alkaline proteases having a molecular weight of about 43,000 that are capable of retaining caseinolytic activity even in the presence of a high concentration of fatty acids. The alkaline protease provided in WO99/18218 also exhibited excellent detergency even when the stain is composed of not a simple protein component but plural components, for example, protein and lipid.
Alkaline proteases having improved specific activity, oxidant resistance and detergency that are usable for detergents of wide-ranging compositions remain in demand.
DISCLOSURE OF THE INVENTION
The present inventors searched for improved alkaline proteases to address the aforementioned demand mainly from enzyme variants. The above-described alkaline proteases possess significant differences in enzymological properties from serine proteases typified by subtilisin. Accordingly, the modified site of subtilisin did not provide useful information. As a result of a further investigation, the present inventors have found that in order to obtain novel alkaline proteases having improved specific activity, stability against an oxidant and detergency while maintaining the properties of the above-described alkaline proteases, specific amino acid residues must be present at a predetermined position of their amino acid sequence.
In one aspect of the present invention, is an alkaline protease wherein an amino acid residue at (a) position 84, (b) position 104, (c) position 256 or (d) position 369 of SEQ ID NO:1, or at a position corresponding thereto, has been deleted or specifically mutated.
In the case of position 84 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with an arginine, residue.
In the case of position 104 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a proline residue.
In the case of position 256 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with an alanine, serine, glutamine, valine, leucine, asparagine, glutamic acid or aspartic acid residue.
In the case of position 369 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with asparagine residue.
In another aspect of the present invention, is an alkaline protease wherein an amino acid residue at (e) position 66 or 264, (f) position 57, each of 101 to 106, 136, 193 or 342, (g) position 46 or 205, (h) position 54, 119, 138, 148 or 195, (i) position 247, (j) position 124, (k) position 107 or (l) position 257 of SEQ ID NO:1, or at a position corresponding thereto, has been deleted or specifically mutated.
In the case of Position 66 or 264 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a glutamine, aspartic acid, serine, glutamic acid, alanine, threonine, leucine, methionine, cysteine, valine, glycine or isoleucine residue.
In the case of position 57, 101 to 106, 136, 193, or 342 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a lysine, serine, glutamine, phenylalanine, valine, arginine, tyrosine, leucine, isoleucine, threonine, methionine, cysteine, tryptophan, aspartic acid, glutamic acid, histidine, proline or alanine residue.
In the case of position 46 or 205 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a tyrosine, tryptophan, alanine, asparagine, glutamic acid, threonine, valine, leucine, isoleucine, histidine, serine, lysine, glutamine, methionine or cysteine residue.
In the case of position 54, 119, 138, 148, or 195 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a tryptophan, phenylalanine, alanine, asparagine, glutamic acid, threonine, valine, histidine, serine, lysine, glutamine, methionine, glycine, aspartic acid, proline, arginine or cysteine residue.
In the case of position 247 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a tryptophan, phenylalanine, alanine, asparagine, glutamic acid, threonine, valine, leucine, isoleucine, histidine, serine, glutamine, methionine or cysteine residue.
In the case of position 124 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with an alanine or lysine residue.
In the case of position 107 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a lysine, arginine, alanine or serine residue.
In the case of position 257 of SEQ ID NO:1, or a position corresponding thereto, the preferred mutation is to replace the original amino acid present in the sequence with a valine or isoleucine residue.
In a further aspect of the present invention, is a gene encoding the alkaline protease, a recombinant vector containing the gene and a transformant containing the vector.
In a still further aspect of the present invention, is a detergent composition containing the alkaline protease of the present invention.
REFERENCES:
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Tobe et al, 1997 EMBL Acc# .E03808 Alignment with SID NO: 1.*
Christianson et al, 1998 from WO9856927. Alignment with NO: 1.*
Hitomi et al, 1999 from WO9918218. Alignment with SEQ ID NO: 1.*
R. J. Siezen, et al., Protein Engineering, vol. 4, No. 7, pp. 719-737, XP-0022008733, “Homology Modelling and Protein Engineering Strategy of Subtilases, The Family of Subtilisin-like Serine Proteinases”, 1991.
R. J. Siezn, et al., Protein Science, vol. 6, No. 3, pp. 501-523, XP-000990399, “Subtilases: The Superfamily of Subtilisin-like Serine Proteases”, Mar. 1997.
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Araki Hiroyuki
Hatada Yuji
Kageyama Yasushi
Ogawa Akinori
Okuda Mitsuyoshi
Kao Corporation
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
Swope Sheridan
LandOfFree
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