# US 2021/0277434 A1 · Glucoamylases and methods of use, thereof

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- Number: US 2021/0277434 A1
- Document ID: US20210277434A1
- Status: Published application
- Kind: A1
- Application number: 17/257,641
- Filed: 2019-07-02
- Published: 2021-09-09
- Earliest claimed: 2018-07-04
- Assignees: Danisco US Inc.
- Applicants: Danisco US Inc
- Inventors: Zhongmei TANG; Qihui WU; Xingxiang XI; Zhenghong ZHANG
- Web page: https://patentgrep.com/patent/US20210277434A1

## CPC

- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

Described are methods of saccharifying starch-containing materials using a glucoamylase, the methods of producing fermentation products and the fermentation products produced by the method thereof.

## Claims

### Claim 1

A method for saccharification of a starch substrate, comprising contacting the substrate with a glucoamylase having at least two, at least three, or at least four times more activity on an α-1,6 bond-containing substrate compared to the glucoamylase from Aspergillus niger under equivalent conditions, wherein saccharifying with the glucoamylase produces a glucose syrup having a higher level of glucose compared to saccharifying the same starch substrate with the glucoamylase from Aspergillus niger under equivalent conditions.

### Claim 2

A method for increasing the amount of glucose in a syrup produced by saccharifying a starch substrate, comprising contacting the substrate with a glucoamylase having at least two, at least three, or at least four times more activity on an α-1,6 bond-containing substrate compared to the glucoamylase from Aspergillus niger under equivalent conditions, wherein the saccharifying with the glucoamylase produces a glucose syrup having a higher level of glucose compared to saccharifying the same starch substrate with the glucoamylase from Aspergillus niger under equivalent conditions.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein the α-1,6 bond-containing substrate is amylopectin, panose or isomaltose.

### Claim 4

The method of any of the preceding claims, wherein the glucoamylase has at least 20% more activity at pH 4.5 on soluble starch substrate compared to the glucoamylase from Aspergillus niger under equivalent conditions.

### Claim 5

The method of any of the preceding claims, wherein the glucose syrup comprises at least 4%, at least 10%, or at least 25% more glucose compared to a syrup produced by saccharifying with the glucoamylase from Aspergillus niger at a temperature between 60 and 69° C.

### Claim 6

The method of any of the preceding claims, wherein the glucose syrup comprises at least a 4% reduction, at least a 10% reduction, or at least a 20% reduction in DP3+ compared to a glucose syrup prepared by contacting the same starch substrate with the glucoamylase from Aspergillus niger under equivalent conditions.

### Claim 7

The method of any of the preceding claims, wherein the glucose syrup comprises at least 90% glucose, at least 91% glucose, at least 92% glucose, at least 93% glucose, at least 94% glucose, at least 95% glucose, at least 96% glucose, at least 97% glucose, at least 98% glucose or at least 99% glucose.

### Claim 8

The method of any of the preceding claims, wherein saccharifying the starch substrate is performed at a temperature above 60° C., above 65° C., above 70° C., above 75° C., or above 80° C.

### Claim 9

The method of any of the preceding claims, wherein saccharifying the starch substrate is performed at a pH below 4.5, below 4.0, or below 3.5.

### Claim 10

The method of any of the preceding claims, performed within a simultaneous saccharification and fermentation process.

### Claim 11

The method of any of the preceding claims, wherein the glucoamylase has at least 50% residual activity at 80° C. after 10 minutes at pH 5.0.

### Claim 12

The method of any of the preceding claims, wherein the glucoamylase has at least 20% more activity at pH 3 compared to the glucoamylase from Aspergillus niger under equivalent conditions.

### Claim 13

The method of any of the preceding claims, wherein the glucoamylase is from Penicillium glabrum, Symbiotaphrina kochii, Penicillium brasilianum or a variant, thereof.

### Claim 14

The method of any of the preceding claims, wherein the glucoamylase is selected from the groups consisting of:
d) a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;
e) a polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or
f) a polypeptide having at least 80% identity to a catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

### Claim 15

A recombinant construct comprising a nucleotide sequence encoding a glucoamylase, wherein said coding nucleotide sequence is operably linked to at least one regulatory sequence functional in a production host and is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 or a nucleotide sequence with at least 80% sequence identity thereto, wherein said regulatory sequence is heterologous to the coding nucleotide sequence, or said regulatory sequence and coding sequence are not arranged as found together in nature.