Enzyme composition and uses thereof

Patent metadata

Field	Value
Publication number	US-2016201041-A1
Application number	US-201414915484-A
Country	US
Kind code	A1
Filing date	Aug 27, 2014
Priority date	Aug 30, 2013
Publication date	Jul 14, 2016
Grant date	—

Abstract

The invention relates to enzyme compositions comprising a glucoamylase, an alpha-amylase, and optionally a cellulolytic composition and/or a protease. The invention also relates to the use thereof in processes of producing sugars and/or fermentation products from starch-containing material by saccharifying and/or fermenting starch-containing material at a temperature below the initial gelatinization temperature.

First claim

1 . An enzyme composition comprising glucoamylase and alpha-amylase, and optionally protease.

2 . An enzyme composition of claim 1 comprising a
i) glucoamylase;
ii) alpha-amylase;
iii) cellulolytic enzyme composition;
iv) optionally protease.

3 . The enzyme composition of claim 2 , wherein the cellulolytic enzyme composition is derived from a strain of Trichoderma , such as Trichoderma reesei ; a strain of Humicola , such as Humicola insolens ; a strain of Chrysosporium , such as Chrysosporium lucknowense ; or a strain of Penicillium , such as Penicillium decumbens.

4 . The enzyme composition of claim 1 , wherein the glucoamylase is derived from a strain of Trametes , such as Trametes cingulata ; or Pachykytospora , such as Pachykytospora papyracea ; or Leucopaxillus , such as Leucopaxillus giganteus ; a strain of Aspergillus , preferably Aspergillus niger, Aspergillus awamori , or Aspergillus oryzae ; or a strain of Trichoderma , preferably Trichoderma reesei ; or a strain of Talaromyces , preferably Talaromyces emersonii ; a strain of Penicillium , such as a strain of Penicillium oxalicum ; a strain of Gloeophyllum , such as a strain of Gloeophyllum sepiarium or a strain of Gloeophyllum trabeum ; or a strain of Pycnoporus , preferably Pycnoporus sanguineus.

5 . The enzyme composition of claim 1 , wherein the glucoamylase, such as Gloeophyllum sepiarium glucoamylase, is selected from the group consisting of:
(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 4 herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 4 herein.

6 . The enzyme composition of claim 1 , wherein the glucoamylase, such as Gloeophyllum trabeum glucoamylase, is selected from the group consisting of:
(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 18 herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 18 herein.

7 . The enzyme composition of claim 6 , wherein the glucoamylase is Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein, preferably having one or more of the following substitutions: S95P, A121P, especially S95P+A21P.

8 . The enzyme composition of claim 1 , comprising Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 herein, preferably having one or more of the following substitutions: S95P, A121P, especially S95P+A21P, and an alpha-amylase derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

9 . The enzyme composition of claim 1 , wherein the glucoamylase, such as Pycnoporus sanguineus glucoamylase, is selected from the group consisting of:
(i) a glucoamylase comprising the mature polypeptide of SEQ ID NO: 17 herein; (ii) a glucoamylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 17 herein.

10 . The enzyme composition of claim 1 , wherein the enzyme composition comprises the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein, and an alpha-amylase derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably the one disclosed as SEQ ID NO: 13 herein, preferably one having one or more of the following substitutions: G128D, D143N, especially G128D+D143N.

11 . The enzyme composition of claim 1 wherein the alpha-amylase is derived from a strain of the genus Rhizomucor , preferably a strain the Rhizomucor pusillus , or the genus Meripilus , preferably a strain of Meripilus giganteus.

12 . The enzyme composition of claim 1 , wherein the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), such as the one shown in SEQ ID NO: 13 herein, or an alpha-amylase selected from the group consisting of:
an alpha-amylase comprising the mature polypeptide of SEQ ID NO: 13 herein; (ii) an alpha-amylase comprising an amino acid sequence having at least 60%, at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the mature polypeptide of SEQ ID NO: 13 herein.

13 . The enzyme composition of claim 12 , wherein the alpha-amylase is a variant of the alpha-amylase shown in SEQ ID NO: 13 herein having at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C (using SEQ ID NO: 13 for numbering).

14 . The enzyme composition of claim 1 , wherein the alpha-amylase is derived from a Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably disclosed as SEQ ID NO: 13 herein, preferably having one or more of the following substitutions: G128D, D143N, preferably G128D+D143N (using SEQ ID NO: 13 for numbering).

15 . The enzyme composition of claim 1 , further comprising an enzyme selected from the group of trehalase and pectinase, such as pectin lyase or polygalacturonase.

16 . A process of producing a fermentation product from starch containing material, comprising:
(i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermenting using a fermentation organism; wherein saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase and alpha-amylase; and optionally protease.

17 . The process of claim 16 , wherein an enzyme composition of claim 1 are presence during saccharification and/or fermentation

18 . The process of claim 16 , wherein the glucoamylase is the Gloeophyllum trabeum glucoamylase shown in SEQ ID NO: 18 having one of the following substitutions: S95P+A121P and the alpha-amylase is Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), preferably one having the following substitutions G128D+D143N (using SEQ ID NO: 13 for numbering).

19 . The process of claim 16 , wherein the glucoamylase is the Pycnoporus sanguineus glucoamylase shown in SEQ ID NO: 17 herein, and the alpha-amylase is the Rhizomucor pusillus with an Aspergillus niger glucoamylase linker and starch

Assignees

• Novozymes As

Inventors

• Jump Joseph
• Kreel Nathaniel
• Saunders Jeremy
• Akerman Michael John

Classifications

• C12N9/2434

  acting on beta-1,4-glucosidic bonds · CPC title

• C12Y302/01003

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12N9/2428

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12N9/50

  Proteinases {, e.g. Endopeptidases (3.4.21-3.4.25)} · CPC title

• C12N9/2417

  from microbiological source · CPC title