# US 9,040,279 B2 · Saccharification enzyme composition and method of saccharification thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,279 B2 - Document ID: US9040279B2 - Status: Granted - Kind: B2 - Application number: 12/478,368 - Filed: 2009-06-04 - Published: 2009-12-10 - Granted: 2015-05-26 - Earliest claimed: 2008-06-06 - Assignees: Danisco US Inc. - Applicants: Suzanne Breneman; Sung Ho Lee; Vivek Sharma; Jayarama K. Shetty - Inventors: Suzanne Breneman; Sung Ho Lee; Vivek Sharma; Jayarama K. Shetty - Web page: https://patentgrep.com/patent/US9040279B2 ## CPC - C12P 7/00: Preparation of oxygen-containing organic compounds - 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)} - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The present disclosure relates to a Bacillus subtilis alpha-amylase (AmyE) or its variants thereof. AmyE or its variants thereof may be used to more efficiently produce fermentable sugars from starch. Also disclosed are a composition comprising a glucoamylase and AmyE or variant thereof and a method of processing starch utilizing the described enzyme composition. ## Claims ### Claim 1 A composition for saccharifying a starch comprising a glucoamylase and an alpha-amylase, wherein the alpha-amylase is a Bacillus subtilis alpha-amylase (AmyE) or an AmyE variant, wherein the alpha-amylase consists of the amino acid sequence of SEQ ID NO: 1 and the AmyE variant consists of the amino acid sequence of SEQ ID NO: 2. ### Claim 2 (depends on 1) The composition of claim 1, wherein the alpha-amylase consisting of the amino acid sequence set forth in SEQ ID NO: 1. ### Claim 3 (depends on 1) The composition of claim 1, wherein the AmyE variant has one or more altered properties compared to the AmyE having an amino acid sequence of SEQ ID NO: 1. ### Claim 4 (depends on 3) The composition of claim 3, wherein the one or more altered properties is: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, stability at lower level of calcium ion (Ca2+), specific activity, or any combination thereof. ### Claim 5 (depends on 1) The composition of claim 1 further comprising a phytase, a pullulanase, a beta-amylase, a fungal alpha-amylase, a protease, a cellulose, a hemicellulase, a lipase, a cutinase, an isoamylase, or any combination thereof. ### Claim 6 (depends on 1) A method of processing a starch comprising admixing the composition of claim 1 for a time sufficient to saccharify the starch. ### Claim 7 (depends on 6) The method of claim 6 further comprising producing high fructose corn syrup. ### Claim 8 (depends on 7) The method of claim 7, wherein high fructose corn syrup production is achieved by admixing a glucose isomerase. ### Claim 9 (depends on 6) The method of claim 6 further comprising fermenting the starch to produce ethanol. ### Claim 10 (depends on 9) The method of claim 9, wherein saccharifying and fermenting are performed simultaneously. ### Claim 11 (depends on 9) The method of claim 9 further comprising recovering the ethanol. ### Claim 12 (depends on 9) The method of claim 9 further comprising distilling the starch to obtain the ethanol, wherein the fermenting and the distilling are carried out simultaneously, separately, or sequentially. ### Claim 13 (depends on 1) A method of saccharifying a starch comprising admixing a composition of claim 1 comprising a glucoamylase and an alpha-amylase with an oligosaccharide or a starch substrate, wherein the alpha-amylase is a Bacillus subtilis alpha-amylase (AmyE) or an AmyE variant having the amino acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 2. ### Claim 14 (depends on 13) The method of claim 13, wherein the alpha-amylase consists of the amino acid sequence set forth in SEQ ID NO: 1. ### Claim 15 (depends on 13) The method of claim 13, wherein the AmyE variant has one or more altered properties compared to the AmyE having an amino acid sequence of SEQ ID NO: 1. ### Claim 16 (depends on 15) The method of claim 15, wherein the one or more altered properties is: substrate specificity, substrate binding, substrate cleavage pattern, thermal stability, pH/activity profile, pH/stability profile, stability towards oxidation, stability at lower level of calcium ion (Ca2+), specific activity, or any combination thereof. ### Claim 17 (depends on 13) The method of claim 13, wherein the glucoamylase is used at an amount no higher than 0.11 glucoamylase units per gram dry solid (GAU/g ds). ### Claim 18 (depends on 13) The method of claim 13 further comprising admixing a phytase, a pullulanase, a beta-amylase, a fungal alpha-amylase, a protease, a cellulose, a hemicellulase, a lipase, a cutinase, an isoamylase, or any combination thereof, with the oligosaccharide or the starch substrate. ### Claim 19 (depends on 13) The method of claim 13 further comprising producing high fructose corn syrup. ### Claim 20 (depends on 19) The method of claim 19, wherein high fructose corn syrup production is achieved by admixing a glucose isomerase. ### Claim 21 (depends on 13) The method of claim 13 further comprising fermenting the starch to produce ethanol. ### Claim 22 (depends on 21) The method of claim 21, wherein saccharifying and fermenting are performed simultaneously. ### Claim 23 (depends on 21) The method of claim 21 further comprising recovering the ethanol. ### Claim 24 (depends on 21) The method of claim 21 further comprising distilling the starch to obtain the ethanol, wherein the fermenting and the distilling are carried out simultaneously, separately, or sequentially.