Method of using α-amylase from Aspergillus clavatus for saccharification

Patent metadata

Field	Value
Publication number	US-9365871-B2
Application number	US-201414561085-A
Country	US
Kind code	B2
Filing date	Dec 4, 2014
Priority date	May 11, 2012
Publication date	Jun 14, 2016
Grant date	Jun 14, 2016

Abstract

A fungal α-amylase is provided from Aspergillus clavatus (AcAmy1). AcAmy1 has an optimal pH of 4.5 and is operable at 30-75° C., allowing the enzyme to be used in combination with a glucoamylase in a saccharification reaction. This obviates the necessity of running a saccharification reaction as a batch process, where the pH and temperature must be readjusted for optimal use of the α-amylase or glucoamylase. AcAmy1 also catalyzes the saccharification of starch substrates to an oligosaccharide composition significantly enriched in DP2 and (DP1+DP2) compared to the products of saccharification catalyzed by an α-amylase from Aspergillus kawachii . This facilitates the utilization of the oligosaccharide composition by a fermenting organism in a simultaneous saccharification and fermentation process, for example.

First claim

What is claimed is:

1. A method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein said method comprises:
(i) contacting said solution comprising starch with an isolated AcAmy1 or variant thereof having α-amylase activity comprising an amino acid sequence with at least 90% amino acid sequence identity to (a) residues 20-636 of SEQ ID NO:1 or (b) residues 20-497 of SEQ ID NO:1;
(ii) saccharifying said solution comprising starch to produce said composition comprising glucose; wherein said isolated AcAmy1 or variant thereof catalyzes the saccharification of the starch solution to glucose; and
(iii) fermenting the glucose composition to produce an End of Fermentation (EOF) product.

2. The method of claim 1 , wherein said fermentation is a simultaneous saccharification and fermentation (SSF) reaction.

3. The method of claim 1 , wherein said fermentation is conducted for 48-70 hours at pH 2-8 and in a temperature range of 25° C.-70° C.

4. The method of claim 1 , wherein the EOF product comprises ethanol.

5. The method of claim 4 , wherein the EOF product comprises 8%-18% (v/v) ethanol.

6. The method of claim 1 , wherein said method further comprises contacting a mash and/or a wort with the AcAmy1 or variant thereof.

7. The method of claim 6 , wherein said method further comprises:
(a) preparing a mash;
(b) filtering the mash to obtain a wort; and
(c) fermenting the wort to obtain a fermented beverage,
wherein AcAmy1 or variant thereof are added to:
(i) the mash of step (a) and/or
(ii) the wort of step (b) and/or
(iii) the wort of step (c).

8. The method of claim 1 , wherein the EOF product comprises a metabolite.

9. The method of claim 8 , wherein the metabolite is citric acid, lactic acid, succinic acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, potassium gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty acid, butanol, an amino acid, lysine, itaconic acid, 1,3-propanediol, or isoprene.

10. The method of claim 1 , further comprising adding glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not AcAmy1, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, beta-glucosidase, or a combination thereof, to said starch solution.

11. The method of claim 10 , wherein said glucoamylase is added to 0.1-2 glucoamylase units (GAU)/g ds.

12. The method of claim 1 , wherein said isolated AcAmy1 or a variant thereof is expressed and secreted by a host cell.

13. The method of claim 12 , wherein said composition comprising starch is contacted with said host cell.

14. The method of claim 12 , wherein the host cell further expresses and secretes a glucoamylase.

15. The method of claim 12 , wherein the host cell is capable of fermenting the glucose composition.

Assignees

• Danisco Us Inc

Inventors

• Ge Jing
• Hua Ling
• Scheffers Martijn Silvan
• Tang Zhongmei
• Van Brussel-Zwijnen Marco
• Vroemen Casper
• Zhang Bo
• Clarkson Kathleen A
• Huitink Jacquelyn A
• Teunissen Paula Johanna Maria

Classifications

• C12P19/14 Primary

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12C7/04

  Preparation or treatment of the mash · CPC title

• C12P2201/00

  Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis · CPC title

• C12P7/14 Primary

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

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title