Novel cellulase complex, and glycosidase hydrolases thereof, and methods of using thereof

Patent metadata

Field	Value
Publication number	US-2018371443-A1
Application number	US-201815985216-A
Country	US
Kind code	A1
Filing date	May 21, 2018
Priority date	Nov 19, 2015
Publication date	Dec 27, 2018
Grant date	—

Abstract

The present invention provides for a purified or isolated cellulase complex comprising two or more glycosidase hydrolase, or enzymatically active fragment thereof, selected from the group consisting of a GH9 polypeptide, a GH48 polypeptide, a GH10 polypeptide, and a GH6 polypeptide, and optionally a GH10_2 polypeptide and/or an AA10 polypeptide.

First claim

What is claimed is:

1 . A composition comprising a purified or isolated cellulase complex comprising two or more glycosidase hydrolase, or enzymatically active fragment thereof, selected from the group consisting of a GH9 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:1, a GH48 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:2, a GH10 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:3, and a GH6 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:4; and optionally the composition or the cellulase complex comprising a GH10_2 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:5, and/or an AA10 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:6.

2 . A composition comprising the composition of claim 1 , and an ionic liquid (IL).

3 . The composition of claim 1 , wherein the purified or isolated cellulase complex comprising the GH9 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:1, or enzymatically active fragment thereof, wherein the amino acid sequence comprises one or more of the following amino acid sequences: SGKLP (SEQ ID NO:13), WRGDS (SEQ ID NO:14), DLTGGW (SEQ ID NO:15), DAGDHVKF (SEQ ID NO:16), WAVYEY (SEQ ID NO:17), DHAWWGPA (SEQ ID NO:18), EVMQM (SEQ ID NO:19), AVWLYLAT (SEQ ID NO:20), WDDVH (SEQ ID NO:21), GLAWLD (SEQ ID NO:22), WGSLRYA (SEQ ID NO:23), FLAFVYSDW (SEQ ID NO:24), RPHHRTAH (SEQ ID NO:25), and SWADSQ (SEQ ID NO:26).

4 . The composition of claim 1 , wherein the purified or isolated cellulase complex comprising the GH48 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:2, or enzymatically active fragment thereof, wherein the amino acid sequence comprises one or more of the following amino acid sequences: PANGYF (SEQ ID NO:27), GIPYHS (SEQ ID NO:28), EAPDYGH (SEQ ID NO:29), TTSEAFSY (SEQ ID NO:30), TGDWSK (SEQ ID NO:31), PATYA (SEQ ID NO:32), DVDNWYG (SEQ ID NO:33), NTFQRG (SEQ ID NO:34), ESVWE (SEQ ID NO:35), QWRYT (SEQ ID NO:36), DADARAIQ (SEQ ID NO:37), KMGDYLRY (SEQ ID NO:38), FDKYF (SEQ ID NO:39), SAHYLLSWY (SEQ ID NO:40), GYQNP (SEQ ID NO:41), GGATNS (SEQ ID NO:42), TFYGM (SEQ ID NO:43), PVYRDP (SEQ ID NO:44), WFGFQAWS (SEQ ID NO:45), GQPDTW (SEQ ID NO:46), YTGNPN (SEQ ID NO:47), and YHRFWAQ (SEQ ID NO:48).

5 . The composition of claim 1 , wherein the purified or isolated cellulase complex comprising the GH10_2 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:5, or enzymatically active fragment thereof, wherein the amino acid sequence comprises one or more of the following amino acid sequences: ADAGLA (SEQ ID NO:49), KFLGNVI (SEQ ID NO:50), YWNQVTPEN (SEQ ID NO:51), TKWGSVE (SEQ ID NO:52), SNGFPFKFHTLVWGSQ (SEQ ID NO:53), PGWISGLS (SEQ ID NO:54), WIQAAGQRYP (SEQ ID NO:55), aDFVDVVNEPLHAKPSYRNAIGGDG (SEQ ID NO:56), TGWDWVIWSF (SEQ ID NO:57), KLLINEYG (SEQ ID NO:58), DPNAA (SEQ ID NO:59), QYVQIINLLKSRGLIDGIGIQ (SEQ ID NO:60), VSVST (SEQ ID NO:61), TGLPIYVSELD (SEQ ID NO:62), TQLARYQ (SEQ ID NO:63), TLWGYIEGQTW (SEQ ID NO:64), ERPALQWLRTYL (SEQ ID NO:65).

6 . The composition of claim 1 , wherein the purified or isolated cellulase complex comprising the AA10 polypeptide comprising an amino acid sequence at least 70% identical to SEQ ID NO:6, or enzymatically active fragment thereof, wherein the amino acid sequence comprises one or more of the following amino acid sequences:

(SEQ ID NO: 66)

MNRRLIARLSGMLAMVLIAA,

(SEQ ID NO: 67)

LAYVPKPEPAEAHGGMVFPATRTYACYVDGKVHGNGGDLNMINPACLDAL

AISGNYQFWNWFGNLISNAGGRHREIIPDGKLCGPTASFDGMNQARTDWW

TTRLQPGATITVRVNAWAPHPGTWYLYVTRDGWDPTQPLKWSDLEPTPFS

QVTNPPINSSGPDGAEYSWQVQLPNKQGRHIIYMIWQRSDSPEAFYNCS

D,

(SEQ ID NO: 68)

YFGSGPIAYEFGDPREGGT.

7 . A recombinant or isolated or purified nucleic acid encoding the cellulase complex, or the GH9 polypeptide, the GH48 polypeptide, the GH10 polypeptide, and/or the GH9 polypeptide of claim 1 .

8 . A vector comprising the recombinant or isolated or purified nucleic acid of claim 7 .

9 . A host cell comprising the nucleic acid of claim 3 or the vector of claim 8 .

10 . A method for producing a cellulase complex of the present invention comprising: providing a host cell of claim 9 , culturing the host cell in a culture medium under conditions whereby the cellulase complex is produced, optionally isolating the cellulase complex from the host cell and/or the culture medium, and optionally contacting the cellulase complex and a cellulose, whereby the cellulose is hydrolyzed by the cellulase complex.

11 . The method of claim 10 , wherein the providing step comprises: introducing an expression vector capable of expressing the cellulase complex in the host cell into the host cell, and optionally constructing the expression vector encoding a promoter operatively linked to a nucleic acid encoding the cellulase complex, wherein the constructing step precedes the introducing step.

12 . A method of hydrolyzing a cellulose, comprising: (a) providing a solution comprising an IL, a cellulose, and the composition of claim 1 , and (b) incubating the solution, such that the cellulose is hydrolyzed by the cellulase complex.

Assignees

• Univ California
• Nat Tech & Eng Solutions Sandia Llc

Inventors

• Singer Steven W
• Kolinko Sebastian D
• Denzel Evelyn
• Hiras Jennifer
• Gladden John M

Classifications

• C12Y302/01025

  Beta-mannosidase (3.2.1.25), i.e. mannanase · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N9/2482

  Endo-1,4-beta-xylanase (3.2.1.8) · CPC title

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12N9/2491

  Beta-mannosidase (3.2.1.25), i.e. mannanase · CPC title