Kao Corporation

Mitsuyoshi Okuda

Tsuyoshi Sato

Katsuhisa Saeki

Nobuyuki Sumitomo

Tohru Kobayashi

Masafumi Nomura

Yasushi Kageyama

Yasushi Takimura

Hiromi Kubota

Shitsuw Shikata

Jun Hitomi

Mikio Takaiwa

Yoshifumi Izawa

Keiji Endo

Masatoshi Tohata

Hiroyuki Araki

Yuji Hatada

Akinori Ogawa

Kazuhiro Saito

Shingo Koyama

Yasunao Wada

Ayako Shimazu

Yumi Nishimura

Providing an alkaline protease exhibiting an improved solubility in a liquid detergent. A mutant alkaline protease consisting of the amino acid sequence represented by SEQ ID No: 2 or an amino acid sequence having 80% or more sequence identity therewith, in which at least one amino acid residue selected from the group consisting of the amino acid residues at predetermined positions of the amino acid sequence represented by SEQ ID No: 2 or corresponding positions thereto are substituted.

Improving the solubility of an alkaline protease in a liquid detergent by amino acid substitution

The present invention provides a mutant alkaline cellulase having enhanced anti-redeposition ability. The present invention provides a mutant alkaline cellulase having an amino acid sequence in which at least amino acid residue selected from the amino acid residues at the positions corresponding to positions 58, 45, 52, 56, 60, 64, 66, 71, 103, 119, 122, 123, 124, 125, 127, 130, 140, 161, 164, 175, 176, 178, 179, 181, 193, 194, 195, 196, 197, 199, 202, 203, 217, 225, 227, 228, 251, 267, 272, 276, 277, 280, 282, 297, 310, 312, 318, 324, 345, 354, 356, 357, 360, 363, 368, 418, 419, 420, 421, 422, 453, 454, 455, 457, 458, 459, 494, 495, 496, 500, 501, 502, 503, 504, 550, 551, 552, 604, 605, 606, 607, 608, 640, 641, 642, 644, 645, 646, 683, 684, 685, 690, 691, 692, 693, 694, 739, 740, and 741 of the amino acid sequence of an alkaline cellulase set forth in SEQ ID NO: 2 or of the amino acid sequence of an alkaline cellulase having at least 70% identity with the amino acid sequence of SEQ ID NO: 2, is substituted with other amino acid residue.

Mutated alkaline cellulase

The invention is directed to an alkaline protease that has the amino acid sequence of SEQ ID NO: 2 or of an amino acid sequence having an identity of 90% or more thereto, except that one or more amino acid residues at positions selected from (a) position 6, (b) position 15, (c) position 16, (d) position 65, (e) position 66, (f) position 82, (g) position 83, (h) position 204, (i) position 319, and (j) position 337 of SEQ ID NO: 2, or at positions corresponding thereto, are substituted with certain other amino acid residues. The invention is also directed to detergent compositions and culture medium that contain the protease.

Alkaline protease variants

It is an object of the present invention to provide alkaline proteases having industrially sufficient protein productivity and a significant detergency. In the alkaline proteases, the amino acid residues at (a) position 9, (b) position 49, (c) position 194, (d) position 212, (e) position 237, (f) position 245, (g) position 281, (h) position 313, (i) position 379 and (j) position 427 in SEQ ID NO: 2 are selected from the following amino acid residues; Position (a); glutamine, Position (b); glutamine, Position (c); lysine or arginine, Position (d); arginine, asparagine or glutamine, Position (e); asparagines, Position (f); asparagines. Position (g); arginine, Position (h); asparagines, Position (i); lysine, arginine, glutamic acid or aspartic acid, and Position (j); arginine.

Alkaline protease

Provided in the present invention is an alkaline protease wherein an amino acid residue at (a) position 65, (b) position 101, (c) position 163, (d) position 170, (e) position 171, (f) position 273, (g) position 320, (h) position 359 or (i) position 387 of SEQ. ID NO:1 or at a position corresponding thereto has been selected from the following amino acid residues: position (a): proline, position (b): asparagine, position (c): histidine, aspartic acid, phenylalanine, lysine, asparagine, serine, isoleucine, leucine, glutamine, threonine and valine, position (d): valine and leucine, position (e): alanine, glutamic acid, glycine and threonine, position (f): isoleucine, glycine and threonine, position (g): phenylalanine, valine, threonine, leucine, isoleucine and glycine, position (h): serine, leucine, valine, isoleucine and glutamine, position (i): alanine, lysine, glutamine, glutamic acid, arginine and histidine. The present invention makes it possible to efficiently produce and provide alkaline proteases having activity even in the presence of a highly concentrated fatty acid, and exhibiting excellent detergency for the removal of a complex stain containing protein, sebum and the like, and therefore being useful as an enzyme to be incorporated in a detergent.

The present invention provides an alkaline protease exhibiting high detergency and productivity, which is obtained by further enhancing the detergency of an alkaline protease effective against complex dirt and enhancing the specific activity.

An alkaline protease having an amino acid with one or more amino acid residues selected from among those located at (a) position 15, (b) position 16, (c) position 166, (d) position 167, (e) position 187, (f) position 346, and (g) position 405 of the amino acid sequence of SEQ No; 1, or at positions corresponding to these positions, are the following amino acid residues, respectively: (a): histidine, (b): threonine or glutamine, (c): glycine, (d): valine, (e): serine, (f): arginine, and (g); and a gene encoding the alkaline protease.

An alkaline protease having an amino acid with one or more amino acid residues selected from among those located at (a) position 63, (b) position 89, (c) position 120, (d) positions 63 and 187, (e) position 226, (f) position 296, (g) position 304 of the amino acid sequence of SEQ ID NO: 1, or at positions corresponding to these positions, are the following amino acid residues, respectively: (a): serine, (b): histidine, (c): arginine, (d): serine,(e): tyrosine, (f): valine, and (g): serine; and a gene encoding the alkaline protease.

The present invention relates to alkaline proteases having high specific activity and strong oxidant resistance. The present invention also relates to alkaline proteases having excellent detergency that are to be added to a detergent.

Alkaline proteases

An alkaline protease wherein an amino acid residue at (a) position 65, (b) position 101, (c) position 163, (d) position 17-0, (e) position 171, (f) position 273, (g) position 320, (h) position 359 or (i) position 387 of SEQ. ID NO: 2 or at a position corresponding thereto has been selected from the following amino acid residues: position (a): proline, position (b): asparagine, position (e): histidine, aspartic acid, phenylalanine, lysine, asparagine, seine, isoleucine, leucine, glutamine, threonine and valine, position (d): valine and leucine, position (e): alanine, glutamic acid, glycine and threonine, position (K): isoleucine, glycine and threonine, position (g): phenylalanine, valine, threonine, leucine, isoleucine and glycine, position (h): seine, leucine, valine, isoleucine and glutamine, position (i): alanine, lysine, glutamine, glutamic acid, arginine and histidine. A method to an alkaline protease having activity even in the presence of a highly concentrated fatty acid, and exhibiting excellent detergency for the removal of a complex stain containing protein, sebum and the like, and therefore useful as an ingredient in a detergent.

An alkaline protease having the following properties; a gene encoding the same; a microorganism producing the same; and washing compositions containing the same; (i) acting over a broad pH value range of 4 to 13 and achieving, at pH 6 to 12, 80% or more the activity at the optimum pH value; (ii) when treated at 40&#xb0; C. for 30 minutes, being stable over a pH value range of 6 to 11; (iii) having an isoelectric point of about 8.9 to 9.1; and (iv) having casein digesting activity that is not inhibited by oleic acid. The alkaline protease of the present invention is highly stable to various surface active agents and fatty acids, and exhibits high stability to oxidizing agents, and is therefore useful as an enzyme to be used in detergents for automatic dishwashers and laundry detergents, both containing bleaching components.

The present invention relates to genes expressing an alkaline protease having a prepro sequence, vectors containing the same, and transformants containing said vector. The present invention also relates to a method for producing an alkaline protease.

Nucleic acid sequences encoding modified prepro peptides of an alkaline protease

The present invention relates to an alkaline protease having a prepro sequence, wherein the prepro sequence is a mutant sequence of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or higher homology to the amino acid sequence of SEQ ID NO: 1, in which amino acid residues at (a) position 52, (b) position 75, and (c) position 142 of SEQ ID NO: 1, or amino acid residues at positions corresponding to these positions are substituted by the following amino acid residues: According to the present invention, an alkaline protease increased in production can be produced. In particular, there can be efficiently produced an alkaline protease having an activity even in the presence of a fatty acid of high concentration and exhibiting excellent detergency against complex soils containing proteins and sebum.

The present invention relates to alkaline proteases having high specific activity and strong oxidant resistance. The present invention also relates to alkaline proteases having excellent detergency that is to be added to a detergent.

An alkaline protease having the following properties; a gene encoding the same; a microorganism producing the same; and washing compositions containing the same; (i) acting over a broad pH value range of 4 to 13 and achieving, at pH 6 to 12, 80% or more the activity at the optimum pH value; (ii) when treated at 40&deg; C. for 30 minutes, being stable over a pH value range of 6 to 11; (iii) having an isoelectric point of about 8.9 to 9.1; and (iv) having casein digesting activity that is not inhibited by oleic acid. The alkaline protease of the present invention is highly stable to various surface active agents and fatty acids, and exhibits high stability to oxidizing agents, and is therefore useful as an enzyme to be used in detergents for automatic dishwashers and laundry detergents, both containing bleaching components.

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Haga-gun, Japan

Mitsuyoshi Okuda