IP Library Patent Application 14496956
Patent Application
App. No. 14/496,956

POLYPEPTIDE HAVING ENDOGLUCANASE ACTIVITY

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Patent No.
US None
App. No.
14/496,956
Abstract

The present invention relates to a family 5 glycoside hydrolase variant having endoglucanase activity, polynucleotides encoding the family 5 glycoside hydrolase variant, vectors, host cells comprising the polynucleotides, and methods for using the family 5 glycoside hydrolase variant.

Claims (57)

1 . A family 5 glycoside hydrolase variant, the variant encoded by a mutated version of a wild-type parental polynucleotide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete sequence identity to a (cDNA) nucleotide sequence of SEQ ID NO: 1 and comprising at least one of the following nucleotide residue changes:

the nucleotides at positions 157 to 159 are TCT;

the nucleotides at positions 175 to 177 are AAG;

the nucleotides at positions 175 to 177 are CAT;

the nucleotides at positions 181 to 183 are CCT;

the nucleotides at positions 190 to 192 are AAT;

the nucleotides at positions 208 to 210 are ATG;

the nucleotides at positions 259 to 261 are CAT;

the nucleotides at positions 259 to 261 are ATG;

the nucleotides at positions 274 to 276 are GCT;

the nucleotides at positions 547 to 549 are TTG;

the nucleotides at positions 550 to 552 are GAG;

the nucleotides at positions 574 to 576 are AAT;

the nucleotides at positions 589 to 591 are GCT;

the nucleotides at positions 595 to 597 are ATG;

the nucleotides at positions 598 to 600 are GTT; and

the nucleotides at positions 898 to 900 are GCG;

wherein the variant has at least one of the following activities: endoglucanase activity, a beta-mannanase activity, an exo-1,3-glucanase activity, an endo-1,6-glucanase activity, a xylanase activity, and an endoglycoceramidase activity; and wherein the activity of the variant is greater than a polypeptide encoded by the wild-type parental polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

2 . The family 5 glycoside hydrolase variant of claim 1 , wherein family 5 glycoside hydrolase variant has endoglucanase activity.

3 . An expression cassette, a vector or a cloning vehicle comprising the mutated version of a wild-type parental polynucleotide sequence SEQ ID NO: 1 of claim 1 ,

wherein optionally the cloning vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome,

and optionally the viral vector comprises an adenovirus vector, a retroviral vector or an adeno-associated viral vector, and optionally the cloning vehicle comprises a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

4 . A transformed cell comprising a nucleic acid comprising the mutated version of a wild-type parental polynucleotide sequence SEQ ID NO: 1 of claim 1 ,

wherein optionally the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

5 . A transformed cell comprising the expression vector of claim 3 .

6 . The transformed cell of claim 5 , wherein the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

7 . The transformed cell of claim 6 , wherein the bacterial cell is selected from Zymomonas mobilis, Escherichia coli and Klebsiella oxytoca.

8 . The transformed cell of claim 6 , wherein the yeast cell is selected from Saccharomyces cerevisiae, Saccharomyces uvarum, Kluyveromyces fragilis, Kluyveromyces lactis, Candida pseudotropicalis , and Pachysolen tannophilus.

9 . The transformed cell of claim 6 , wherein the fungal cell is selected from the genus Aspergillus, Penicillium, Rhizopus, Chrysosporium, Myceliophthora, Trichoderma, Humicola, Acremonium or Fusarium.

10 . The transformed cell of claim 9 , wherein the fungal cell is of the species Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Penicillium chrysogenum, Myceliophthora thermophila , or Rhizopus oryzae.

11 . The transformed cell of claim 6 , wherein the yeast cell is selected from the genus Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Klockera, Schwanniomyces or Yarrowia.

12 . The transformed cell of claim 11 , wherein the yeast cell is of the species S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus or K. fragilis.

13 . A mature family 5 glycoside hydrolase variant, the variant having a mutated version of a wild-type parental polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete sequence identity to an amino acid sequence of SEQ ID NO: 2 and comprising at least one of the following amino acid residue changes:

proline is substituted with serine at position 53;

proline is substituted with histidine at position 59;

proline is substituted with lysine at position 59;

threonine is substituted with proline at position 61;

lysine is substituted with asparagine at position 64;

glycine is substituted with methionine at position 70;

threonine is substituted with histidine at position 87;

threonine is substituted with methionine at position 87;

glycine is substituted with alanine at position 92;

serine is substituted with leucine at position 183;

threonine is substituted with glutamic acid at position 184;

lysine is substituted with asparagine at position 192;

glutamine is substituted with alanine at position 197;

lysine is substituted with methionine at position 199;

serine is substituted with valine at position 200; and

lysine is substituted with alanine at position 300;

wherein the variant has at least one of the following activities: endoglucanase activity, a beta-mannanase activity, an exo-1,3-glucanase activity, an endo-1,6-glucanase activity, a xylanase activity, and an endoglycoceramidase activity; and wherein the activity of the variant is greater than the wild-type parental polypeptide having an amino acid sequence of SEQ ID NO: 2.

14 . The variant of claim 13 , wherein the variant has endoglucanase activity.

15 . A method for hydrolyzing cellulose comprising contacting biomass with the polypeptide of claim 13 .

16 . The method of claim 15 , wherein the biomass is subjected to a pretreatment process prior to being contacted with the polypeptide.

17 . The method of claim 16 , wherein the pretreatment process comprises the step of heating the biomass to at least 50° Celsius.

18 . The method of claim 16 , wherein the pretreatment process comprises the step of contacting the biomass with an aqueous solution.

19 . The method of claim 18 , wherein the aqueous solution has a pH of less than 7.

20 . The method of claim 18 , wherein the aqueous solution has a pH of more than 7.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 18, 2015
From: POLAND, JOHN; STEGE, JUSTIN T.
To: BP CORPORATION NORTH AMERICA INC.
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