Method for producing glucosidase, enzyme composition, and method for hydrolyzing biomass

摘要

A method for producing a mutant glucosidase includes introducing DNA encoding a secretion signal sequence and DNA encoding Asn-X-Ser or Asn-X-Thr into DNA encoding a glucosidase derived from a thermophile, and introducing the resulting DNA into a eukaryotic microorganism and expressing it as a secretory protein. An enzyme composition contains the mutant glucosidase.

主权项

1. A method of producing a mutant glucosidase derived from a thermophile having a sugar chain bound to an amino acid side chain of a peptide sequence of the mutant glucosidase by covalent binding and having glucosidase activity, wherein the mutant glucosidase demonstrates enhanced ability to increase saccharification of a lignocellulose substrate which has been subjected to pre-treatement when added to cellulase as compared to the corresponding unglycosylated wild-type glucosidase of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, which comprises:
(i) preparing DNA encoding the mutant glucosidase derived from a thermophile by introducing a DNA sequence encoding Asn-X-Ser or Asn-X-Thr (wherein, X is any amino acid except proline) into DNA encoding a glucosidase derived from a thermophile originally devoid of a glycosylation sequence and adding a DNA sequence encoding a secretion signal sequence to the DNA encoding the mutant glucosidase, wherein the glucosidase derived from the thermophile is a protein comprising: (a) any of the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, or (b) an amino acid sequence having 95% or more identity with any of the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20, and β-glucosidase activity, the DNA sequence encoding Asn-X-Ser or Asn-X-Thr is substituted for amino acids of the glucosidase derived from a thermophile corresponding to amino acids of H60-L61-Y62 in the amino acid sequence shown in SEQ ID NO: 4, (ii) introducing the DNA encoding the mutant glucosidase to which the DNA sequence encoding the secretion signal sequence has been added into an eukaryotic microorganism so that a mutant glucosidase encoded by the DNA of the mutant glucosidase is expressed as a secretory protein, and (iii) isolating and purifying the mutant glucosidase thus expressed as a secretory protein.