# US 12,595,471 B2 · Amylase variants > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,595,471 B2 - Document ID: US12595471B2 - Status: Granted - Kind: B2 - Application number: 17/636,972 - Filed: 2020-08-21 - Published: 2022-08-25 - Granted: 2026-04-07 - Earliest claimed: 2019-08-22 - Assignees: BASF SE - Applicants: BASF SE - Inventors: Stefan Jenewein; Zachary David Miles; Priya Anand - Web page: https://patentgrep.com/patent/US12595471B2 ## CPC - C11D 3/38681: {Chemically modified or immobilised enzymes} - C12N 9/2414: {Alpha-amylase (3.2.1.1.)} - A21D 8/042: {with enzymes} - C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase} - C12Y 302/01001: Alpha-amylase (3.2.1.1) - Y02E 50/10: Biofuels, e.g. bio-diesel - 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) - A21D: TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS (preservation of flour or dough before baking A23B40/00; preservation of bakery products A23B45/00) - C12Y: ENZYMES - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract Described herein are genetically engineered enzymes having amylase enzyme activity, compositions comprising the enzymes, and methods of making and using the enzymes. The genetically engineered amylase enzymes are useful in many different applications such as laundry detergents, dish washing detergents, and cleaning products for homes, industry, vehicle care, baking, animal feed, pulp and paper processing, starch processing, brewing, and ethanol production. ## Claims ### Claim 1 An isolated, synthetic, or recombinant polypeptide having alpha-amylase activity comprising an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 44. ### Claim 2 (depends on 1) The polypeptide according to claim 1 having alpha-amylase activity consisting of an A and B domain and a C domain wherein the amino acid sequence of the A and B domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 42 and the amino acid sequence of the C domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 44. ### Claim 3 (depends on 1) The polypeptide according to claim 1, wherein the A and B domain has at least 92%, sequence identity to the A and B domain having the amino acid sequence of SEQ ID NO: 42. ### Claim 4 (depends on 1) The polypeptide according to claim 1, wherein the C domain has at least 92% sequence identity to the C domain having the amino acid sequence of SEQ ID NO: 44. ### Claim 5 (depends on 1) The polypeptide according to claim 1 comprising a substitution, deletion, and/or insertion at one or more positions. ### Claim 6 (depends on 1) The polypeptide according to claim 1 comprising the sequence TQXDYLDHPDVIGWTREGDXXHXXSGLAXLMSDGPXGXKWMX VGKNNAGEXWXDITGNQTNTVTINXDGXGQFXVXXGSXSIYXQX(SEQ ID NO: 56), wherein X can be any amino acid. ### Claim 7 (depends on 1) The polypeptide according to claim 1, wherein the polypeptide has one or more amino acid residues selected from the group consisting of 402R, H; 419S, G, D; 420V, I; 422, A, V; 423N, D, G, K; 428T, A; 435G, R, E; 437S, A; 441N, E, D; 444K, E; 450V, I; 452Y, H, R; 466K, R; 469W, S; 473H, Q, R; 475S, N; 476G, E; 479V, A; 483V, I; and 485R, Q, K according to the numbering of SEQ ID NO: 39. ### Claim 8 (depends on 1) The polypeptide according to claim 1 comprising a substitution at one or more positions selected from the group consisting of 430 and 454 according to the numbering of SEQ ID NO: 39. ### Claim 9 (depends on 1) The polypeptide according to claim 1 comprising an amino acid residue at one or more of the amino acid positions selected from the group consisting of 401, 403, 405, 411, 413, 415, 424, 426, 428, 432, 455, 477, 479 and 481 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 39. ### Claim 10 (depends on 1) The polypeptide according to claim 1 comprising an amino acid residue at one or more of the amino acid positions selected from the group consisting of 309, 313, 347, 348, 350, 351, 354, 355, 358, 359, 388, 389, 392 and 396 (according to the numbering of SEQ ID NO: 39) as present in SEQ ID NO: 40. ### Claim 11 (depends on 1) The polypeptide according to claim 1 comprising a deletion of one or more amino acids corresponding to positions 181, 182, 183 and 184 corresponding to the numbering of SEQ ID NO: 39. ### Claim 12 (depends on 1) The polypeptide according to claim 1 comprising a substitution at one or more positions selected from the group consisting of 9, 130, 195, 206, 244, 202, 179, 181, 186, and 190 according to the numbering of SEQ ID NO: 39. ### Claim 13 (depends on 1) The polypeptide according to claim 1 comprising: an amino acid sequence having at least 90% sequence identity to SEQ ID NO:54. ### Claim 14 (depends on 1) The polypeptide according to claim 1, wherein the amylase has an increase in expression, activity, thermostability, stability, performance in laundry, specific activity, substrate specificity, pH-dependent activity, pH-dependent stability, oxidative stability, Ca2+ dependency, or any combination thereof compared to the amylase having the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40. ### Claim 15 (depends on 1) A composition comprising the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to claim 1. ### Claim 16 (depends on 15) The composition of claim 15, further comprising at least one second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a mannanase, a pectinase, xylanase, and a nuclease. ### Claim 17 (depends on 1) A method of making the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to claim 1, comprising: providing a nucleic acid sequence encoding the polypeptide, transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the polypeptide, and optionally purifying the polypeptide. ### Claim 18 (depends on 1) A method of preparing a dough or a baked product prepared from the dough, the method comprising adding the isolated, synthetic, or recombinant polypeptide having alpha-amylase activity according to claim 1 to the dough and baking it.