Honda Motor Co., Ltd.

Kazusa Dna Research Institute

Migiwa Suda

Yoshitsugu Hirose

Yasuhiro Kondo

Asuka Yamaguchi

Jiro Okuma

Daisuke Shibata

Masaru Sato

Tomohiko Kato

Takahiro Gunji

Jiro Ohkuma

A thermostable cellobiohydrolase, having a cellulose-binding motif domain including (A1) a polypeptide including an amino acid sequence represented by SEQ ID NO: 1, (B1) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and having a cellulose-binding function, or (C1) a polypeptide including an amino acid sequence having 70% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and having a cellulose-binding function, and also having a cellobiohydrolase catalytic domain, wherein the thermostable cellobiohydrolase exhibits hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 95&#xb0; C. and pH 5.5.

Thermostable cellobiohydrolase

A thermostable cellobiohydrolase, having a cellobiohydrolase catalytic domain including: (A) a polypeptide including the amino acid sequence represented by SEQ ID NO: 1, (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 65&#xb0; C. and pH 6, or (C) a polypeptide including an amino acid sequence having 80% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 65&#xb0; C. and pH 6.

A thermostable cellobiohydrolase, having a cellobiohydrolase catalytic domain including: (A) a polypeptide including the amino acid sequence represented by SEQ ID NO: 1 or 2, (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 75&#xb0; C. and pH 5.5, or (C) a polypeptide including an amino acid sequence having 75% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 75&#xb0; C. and pH 5.5.

A thermostable glycoside hydrolase includes a polypeptide including the amino acid sequence represented by SEQ ID NO: 1, and at least one moiety selected from the group consisting of a cellulose-binding module, a linker region, a signal peptide and a tag. A glycoside hydrolase mixture includes a hermostable glycoside hydrolase and at least one other glycoside hydrolase, in which the thermostable glycoside hydrolase is encoded by a polynucleotide including the nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence encoding at least one moiety selected from the group consisting of a cellulose-binding module, a linker region, a signal peptide and a tag. The thermostable glycoside hydrolase is produced by a method including generating the thermostable glycoside hydrolase in a transformant. The transformant is introduced in an expression vector.

Thermostable glycoside hydrolase

A thermostable cellobiohydrolase, having a cellobiohydrolase catalytic domain including: (A) a polypeptide including the amino acid sequence represented by SEQ ID NO: 1 or 2, (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 75&#xb0; C. and pH 5, or (C) a polypeptide including an amino acid sequence having 60% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of phosphoric acid swollen Avicel at least under conditions of 75&#xb0; C. and pH 5.

The thermostable cellobiohydrolase of the present invention is a polypeptide which has cellobiohydrolase activity at least under conditions of a temperature of 75&#xb0; C. and a pH of 5.5, and which includes a polypeptide including an amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, a polypeptide including an amino acid sequence in which one or several amino acids are deleted, substituted, or added in an amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7, or a polypeptide including an amino acid sequence having 80% or greater but less than 100% sequence identity with an amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7.

A method for selectively obtaining a natural variant of an enzyme having activity includes (1) a step of detecting an ORF sequence of a protein having enzyme activity from a genome database including base sequences of metagenomic DNA of environmental microbiota; (2) a step of obtaining at least one PCR clone including the ORF sequence having a full length, a partial sequence of the ORF sequence, or a base sequence encoding an amino acid sequence which is formed by deletion, substitution, or addition of at least one amino acid residue in an amino acid sequence encoded by the ORF sequence, by performing PCR cloning on at least one metagenomic DNA of the environmental microbiota by using a primer designed based on the ORF sequence; (3) a step of determining a base sequence and an amino acid sequence which is encoded by the base sequence for each PCR clone obtained in the step (2); and (4) a step of selecting a natural variant of an enzyme having activity by measuring enzyme activity of proteins encoded by each PCR clone obtained in the step (2).

Method for obtaining natural variant of enzyme and super thermostable cellobiohydrolase

A hyperthermostable endoglucanase, having an endoglucanase catalytic domain including: (A) a polypeptide including the amino acid sequence represented by SEQ ID NO: 1 or 2, (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of carboxymethyl cellulose at least under conditions of 90&#xb0; C. and pH 5.5, or (C) a polypeptide including an amino acid sequence having 80% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and having hydrolysis activity against a substrate of carboxymethyl cellulose at least under conditions of 90&#xb0; C. and pH 5.5.

Hyperthermostable endoglucanase

A hyperthermostable endoglucanase, having an endoglucanase catalytic domain including: (A) a polypeptide including an amino acid sequence represented by SEQ ID NO: 1; (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and having hydrolysis activity against a substrate of carboxymethyl cellulose at least under conditions of a temperature of 110&#xb0; C. and a pH of 5.5; or (C) a polypeptide including an amino acid sequence having at least 55% sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and having hydrolysis activity against a substrate of carboxymethyl cellulose at least under conditions of a temperature of 110&#xb0; C. and a pH of 5.5.

A thermostable &#x3b2;-glucosidase including a &#x3b2;-glucosidase catalytic domain, the &#x3b2;-glucosidase catalytic domain including:

Thermostable &#x3b2;-glucosidase

A thermostable xylanase having a xylanase catalytic domain including: (A) a polypeptide including the amino acid sequence represented by SEQ ID NO: 1, (B) a polypeptide including an amino acid sequence in which at least one amino acid has been deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 1, and having xylanase activity at least under conditions of 85&#xb0; C. and pH 6.0, or (C) a polypeptide including an amino acid sequence having 80% or greater sequence identity with the amino acid sequence represented by SEQ ID NO: 1, and having xylanase activity at least under conditions of 85&#xb0; C. and pH 6.0.

Thermostable xylanase belonging to GH family 10

An isolated recombinant thermostable &#x3b2;-glucosidase includes a polypeptide including the amino acid sequence of SEQ ID NO: 1 and at least one region selected from the group consisting of a cellulose-binding module, linker domain, a signal peptide and a tag.

A hyperthermostable endoglucanase including an endoglucanase catalytic domain, the endoglucanase catalytic domain including:

Hyperthermostable endoglucanase belonging to GH family 12

A thermostable &#x3b2;-xylosidase including a &#x3b2;-xylosidase catalytic domain, the &#x3b2;-xylosidase catalytic domain including:

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Wako, Japan

Migiwa Suda