Polypeptides having alpha-amylase activity and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-2017240873-A1
Application number	US-201715587576-A
Country	US
Kind code	A1
Filing date	May 5, 2017
Priority date	Sep 30, 2011
Publication date	Aug 24, 2017
Grant date	—

Abstract

The present invention relates to isolated polypeptides having alpha-amylase activity, catalytic domains, carbohydrate binding domains and polynucleotides encoding the polypeptides, catalytic domains or carbohydrate binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or carbohydrate binding domains.

First claim

What is claimed is:

1 . A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having alpha-amylase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide in an expression host, and wherein the polypeptide has at least 85% sequence identity to amino acids 21 to 619 of SEQ ID NO:4.

2 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide has at least 90% sequence identity to amino acids 21 to 619 of SEQ ID NO:4.

3 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide has at least 95% sequence identity to amino acids 21 to 619 of SEQ ID NO:4.

4 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide has at least 97% sequence identity to amino acids 21 to 619 of SEQ ID NO:4.

5 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide has at least 99% sequence identity to amino acids 21 to 619 of SEQ ID NO:4.

6 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide comprises or consists of SEQ ID NO: 4, or amino acids 21 to 619 of SEQ ID NO: 4.

7 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide comprises a catalytic domain selected from the group consisting of:
(a) a catalytic domain having at least 95% sequence identity to amino acids 21 to 493 of SEQ ID NO: 4; (b) a variant of amino acids 21 to 493 of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more positions; and (c) a fragment of the catalytic domain of (a), or (b) that has alpha-amylase activity.

8 . The nucleic acid construct or expression vector of claim 1 , wherein the polypeptide comprises a carbohydrate binding domain, wherein the binding domain is selected from the group consisting of:
(a) a carbohydrate binding domain having at least 95% sequence identity to amino acids 511 to 619 of SEQ ID NO: 4; (b) a carbohydrate binding domain encoded by a polynucleotide that hybridizes under very high stringency conditions with (i) nucleotides 2025 to 2351 of SEQ ID NO: 3, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii), wherein very high stringency conditions are defined for probes of at least 100 nucleotides in length as prehybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.; (c) a carbohydrate binding domain encoded by a polynucleotide having at least 90% sequence identity to nucleotides 2025 to 2351 of SEQ ID NO: 3 or the cDNA sequence thereof; (d) a variant of amino acids 511 to 619 of SEQ ID NO: 4 comprising a substitution, deletion, and/or insertion at one or more positions; and (e) a fragment of (a), (b), (c), or (d) that has carbohydrate binding activity.

9 . A host cell transformed with the nucleic acid construct or expression vector of claim 1 .

10 . The host cell of claim 9 , wherein the host cell is a yeast host cell.

11 . The host cell of claim 10 , wherein the yeast host cell is selected from the group consisting of a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , and Yarrowia cell.

12 . The host cell of claim 11 , wherein the Kluyveromyces cell is a Kluyveromyces lactis cell.

13 . The host cell of claim 11 , wherein the Saccharomyces cell is selected from the group consisting of Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis , and Saccharomyces oviformis.

14 . The host cell of claim 11 , wherein the Yarrowia cell is a Yarrowia lipolytica cell.

15 . A method of producing a polypeptide having alpha-amylase activity, comprising:
(a) cultivating a host cell transformed with the nucleic acid construct or expression vector of claim 1 under conditions conducive for production of the polypeptide; and optionally (b) recovering the polypeptide.

Assignees

• Novozymes As

Inventors

• Li Ming
• Duan Junxin
• Tsutsumi Noriko
• Coward-Kelly Guillermo
• Lundkvist Henrik

Classifications

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• A21D8/042

  with enzymes · CPC title

• C12N9/242 Primary

  Fungal source · CPC title

• C12P7/14

  Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms · CPC title