Nucleic acid construct with gene encoding an arabinofuranosidase polypeptide and methods of using same

Patent metadata

Field	Value
Publication number	US-9428741-B2
Application number	US-201514680942-A
Country	US
Kind code	B2
Filing date	Apr 7, 2015
Priority date	Apr 26, 2005
Publication date	Aug 30, 2016
Grant date	Aug 30, 2016

Abstract

The present invention relates to isolated polypeptides having alpha-L-arabinofuranosidase activity and isolated nucleic acid sequences encoding the polypeptides, such that the nucleic acid is operably linked to a heterologous control sequence for expression. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides set forth in SEQ ID NO: 2 and fragments of SEQ ID NO: 2.

First claim

The invention claimed is:

1. A nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide having arabinofuranosidase activity, wherein the nucleic acid sequence is operably linked to one or more heterologous control sequences capable of directing the expression of the polypeptide in a suitable expression host, and wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide with at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2; and
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with the full complement of the nucleic acid sequence of nucleotides 55-1677 of SEQ ID NO: 1; wherein the high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide for high stringency, following standard Southern blotting procedures, followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

2. The nucleic acid construct of claim 1 , wherein the polypeptide has at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

3. The nucleic acid construct of claim 1 , wherein the polypeptide has at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2.

4. The nucleic acid construct of claim 1 , wherein the polypeptide comprises the mature polypeptide of SEQ ID NO: 2.

5. The nucleic acid construct of claim 1 , wherein the polypeptide is a fragment of the mature polypeptide of SEQ ID NO: 2 and has arabinofuranosidase (EC 3.2.1.55) activity, wherein the fragment has one or more amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2 and has at least 430 amino acids of the mature polypeptide of SEQ ID NO: 2.

6. The nucleic acid construct of claim 1 , wherein the polypeptide is native to a strain of Humicola.

7. The nucleic acid construct of claim 1 , wherein the polypeptide is native to a strain of Humicola insolens.

8. The nucleic acid construct of claim 1 , wherein the polypeptide comprises amino acids 19 to 558 of SEQ ID NO: 2 and exhibits arabinofuranosidase activity.

9. A recombinant expression vector comprising the nucleic acid construct of claim 1 .

10. A recombinant host cell transformed with the nucleic acid construct of claim 1 .

11. A method for producing a polypeptide having arabinofuranosidase activity, comprising cultivating the recombinant host cell of claim 10 under conditions conducive for production of the polypeptide, and recovering the polypeptide.

12. A recombinant host cell transformed with a nucleic acid sequence encoding a polypeptide having arabinofuranosidase activity, wherein the nucleic acid sequence is operably linked to one or more heterologous control sequences and wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide with at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2; and
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with the full complement of the nucleic acid sequence of nucleotides 55-1677 of SEQ ID NO: 1;
wherein the high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mL sheared and denatured salmon sperm DNA, and 50% formamide for high stringency, following standard Southern blotting procedures, followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

13. A method for producing a polypeptide having arabinofuranosidase activity, comprising cultivating the recombinant host cell of claim 12 under conditions conducive for production of the polypeptide, and recovering the polypeptide.

Assignees

• Novozymes As

Inventors

• Soerensen Hanne Risbjerg
• Joergensen Christel Thea
• Christensen Lars Hylling
• Joergensen Christian Isak
• Hansen Carsten Hoerslev
• Kofod Lene Venke

Classifications

• A21D8/042

  with enzymes · CPC title

• C12Y302/01055

  Alpha-N-arabinofuranosidase (3.2.1.55) · CPC title

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• A23L29/06

  Enzymes · CPC title

• A23K20/168

  Steroids · CPC title