# US 9,040,278 B2 · Production of glucose from starch using alpha-amylases from Bacillus subtilis

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- Number: US 9,040,278 B2
- Document ID: US9040278B2
- Status: Granted
- Kind: B2
- Application number: 12/478,266
- Filed: 2009-06-04
- Published: 2009-12-10
- Granted: 2015-05-26
- Earliest claimed: 2008-06-06
- Assignees: Danisco US Inc.
- Applicants: Luis G. Cascao-Pereira; Regina Chin; William A. Cuevas; David A. Estell; Sang-Kyu Lee; Michael J. Pepsin; Scott D. Power; Sandra W. Ramer; Carol A. Requadt; Andrew Shaw; Amr R. Toppozada; Louise Wallace
- Inventors: Luis G. Cascao-Pereira; Regina Chin; William A. Cuevas; David A. Estell; Sang-Kyu Lee; Michael J. Pepsin; Scott D. Power; Sandra W. Ramer; Carol A. Requadt; Andrew Shaw; Amr R. Toppozada; Louise Wallace
- Web page: https://patentgrep.com/patent/US9040278B2

## CPC

- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C11D 3/221: {Mono, di- or trisaccharides or derivatives thereof}
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C12N 9/2417: {from microbiological source}
- D06L 1/14: De-sizing
- Y02E 50/10: Biofuels, e.g. bio-diesel
- 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)}
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- D06L: DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

An α-amylase from Bacillus subtilis (AmyE) produces significant amounts of glucose from various carbohydrate substrates, including vegetable starch, maltoheptaose, and maltotriose. Among other things, this advantageous property allows AmyE or variants thereof to be used in a saccharification reaction having a reduced or eliminated requirement for glucoamylase. The reduction or elimination of the glucoamylase requirement significantly improves the efficiency of the production of ethanol or high fructose corn syrup, for example.

## Claims

### Claim 1

A method of using a Bacillus subtilis α-amylase (AmyE) to produce glucose from an oligosaccharide or starch substrate solution, comprising:
(i) contacting AmyE with the oligosaccharide or polysaccharide substrate; and
(ii) converting the substrate solution to a solution comprising glucose, wherein the AmyE consists of the amino acid sequence of SEQ ID NO: 1, wherein said α-amylase (AmyE) is not the α-amylase (AmyE) having the amino acid sequence of SEQ ID NO: 1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the oligosaccharide solution comprises predominantly maltoheptaose (DP7) or higher oligosaccharides.

### Claim 3 (depends on 1)

The method of claim 1, wherein the starch solution is uncooked corn starch.

### Claim 4 (depends on 1)

The method of claim 1, wherein the pH of the substrate solution during said converting is pH 5.6 to pH 5.8.

### Claim 5 (depends on 1)

The method of claim 1, where said converting does not comprise contacting the substrate solution with a glucoamylase.

### Claim 6 (depends on 1)

The method of claim 1, wherein step (i) further comprises contacting the starch substrate with a glucoamylase or pullulanase.

### Claim 7 (depends on 6)

The method of claim 6, wherein the glucoamylase is added to a concentration of less than 0.5 GAU/g ds (glucoamylase activity units per gram of dry solids).

### Claim 8 (depends on 7)

The method of claim 7, wherein the glucoamylase is added to a concentration of less than 0.02 GAU/g ds.

### Claim 9 (depends on 1)

The method of claim 1, wherein the solution comprising glucose contains at least 0.2 g/L glucose.

### Claim 10 (depends on 9)

The method of claim 9, wherein the solution comprising glucose contains at least 0.4 g/L glucose.

### Claim 11 (depends on 10)

The method of claim 10, wherein the solution comprising glucose contains at least 1.4 g/L glucose.

### Claim 12 (depends on 1)

The method of claim 1, wherein the AmyE comprises the amino acid sequence set forth in SEQ ID NO: 1.

### Claim 13 (depends on 1)

The method of claim 1, further comprising (iii) fermenting the solution comprising glucose to produce ethanol.

### Claim 14 (depends on 13)

The method of claim 13, wherein the ethanol concentration is at least 6% v/v ethanol.

### Claim 15 (depends on 14)

The method of claim 14, wherein the ethanol concentration is at least 14% v/v ethanol.

### Claim 16 (depends on 1)

The method of claim 1, further comprising (iii) contacting the solution comprising glucose with a glucose isomerase to produce high fructose corn syrup.