# US 10,865,399 B2 · Glucoamylase variants and polynucleotides encoding same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,399 B2 - Document ID: US10865399B2 - Status: Granted - Kind: B2 - Application number: 16/135,407 - Filed: 2018-09-19 - Published: 2019-01-03 - Granted: 2020-12-15 - Earliest claimed: 2011-10-11 - Assignees: Novozymes A/S - Applicants: Novozymes A/S - Inventors: Esben Peter Friis; Leonardo De Maria; Jesper Vind; Thomas A. Poulsen; Allan Svendsen; Steffen Danielsen; Rolf T. Lenhard; Henrik Friis-Madsen; Lars K. Skov - Web page: https://patentgrep.com/patent/US10865399B2 ## CPC - C12N 9/2428: {Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase} - C12N 9/242: {Fungal source} - C12P 5/002: {cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00)} - C12P 5/02: acyclic {(C12P5/007 takes precedence)} - C12P 7/06: Ethanol, i.e. non-beverage - C12P 7/46: Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid - C12P 7/48: Tricarboxylic acids, e.g. citric acid - C12P 7/54: Acetic acid (vinegar C12J) - C12P 7/56: Lactic acid - C12P 13/20: Aspartic acid; Asparagine - C12P 19/20: produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose - C12P 37/00: Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin - C12Y 302/01001: Alpha-amylase (3.2.1.1) - C12Y 302/01003: Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase - Y02E 50/10: Biofuels, e.g. bio-diesel - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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 - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The present invention relates to glucoamylase variants having improved thermostability. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants. ## Claims ### Claim 1 A glucoamylase variant of Penicillium having glucoamylase activity, the glucoamylase variant comprising: a) a substitution or deletion at two or more positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526, 563, 564, 568, 571 of the polypeptide of SEQ ID NO: 3; and b) an amino acid sequence that has at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 3. ### Claim 2 (depends on 1) The variant of claim 1, further comprising a substitution or deletion in at least one position selected from the group consisting of position 65, 327, 501, and 504 of SEQ ID NO: 3. ### Claim 3 (depends on 1) The variant of claim 1, wherein the variant further comprises or consists of one or more substitutions selected from the group consisting of T65A, Q327F, E501V, Y504T, and Y504*. ### Claim 4 (depends on 1) The variant of claim 1, wherein the variant further comprises at least one of the following substitutions or combinations of substitutions: T65A; Q327F; E501V; Y504T; Y504*; T65A+Q327F; T65A+E501V; T65A+Y504T; T65A+Y504*; Q327F+E501V; Q327F+Y504T; Q327F+Y504*; E501V+Y504T; E501V+Y504*; T65A+Q327F+E501V; T65A+Q327F+Y504T; T65A+E501V+Y504T; Q327F+E501V+Y504T; T65A+Q327F+Y504*; T65A+E501V+Y504*; Q327F+E501V+Y504*; T65A+Q327F+E501V+Y504T; or T65A+Q327F+E501V+Y504*. ### Claim 5 (depends on 1) The variant of claim 1, wherein the variant comprises at least one of the following combinations of substitutions: P11F+T65A+Q327F; R1K+D3W+K5Q+G7V+N8S+T10K+P11S+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F; P11F+D26C+K33C+T65A+Q327F; P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; P11F+T65A+Q327W; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; P11F+T65A+Q327W+E501V+Y504T; P2N+P4S+P11F+K34Y+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F+D445N+V447S; P2N+P4S+P11F+T65A+I172V+Q327F; P2N+P4S+P11F+T65A+Q327F+N502*; P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; P2N+P4S+P11F+T65A+Q327F+S377T; P2N+P4S+P11F+T65A+V325T+Q327W; P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; K5A+P11F+T65A+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; K5A+P11F+T65A+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T. ### Claim 6 (depends on 1) The variant of claim 1, which has reduced sensitivity to protease degradation compared to SEQ ID NO: 3. ### Claim 7 (depends on 1) The variant of claim 1, which has improved thermostability compared to SEQ ID NO: 3. ### Claim 8 (depends on 1) A composition comprising the Penicillium glucoamylase variant of claim 1. ### Claim 9 (depends on 1, 8) The composition of claim 8, comprising an alpha-amylase and Penicillium glucoamylase variant of claim 1. ### Claim 10 (depends on 1) A process of producing a fermentation product from starch-containing material comprising the steps of: (a) liquefying starch-containing material in the presence of an alpha amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermenting organism; wherein step (a) and/or step (b) is carried out using at least the Penicillium glucoamylase variant of claim 1. ### Claim 11 (depends on 10) The process of claim 10, wherein the fermentation product is ethanol and the starch-containing material is corn. ### Claim 12 (depends on 1) A process of producing a fermentation product from starch-containing material, comprising the steps of: (a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and (b) fermenting with a fermenting organism, wherein step (a) is carried out using at least the Penicillium glucoamylase variant of claim 1. ### Claim 13 (depends on 12) The process of claim 12, wherein the fermentation product is ethanol and the starch-containing material is corn. ### Claim 14 (depends on 1) The variant of claim 1, wherein the variant further comprises at least one of the following combinations of substitutions: E501V+Y504T; T65A+K161S; T65A+Q405T; T65A+Q327W; T65A+Q327F; T65A+Q327Y; T65A+Q327F+E501V+Y504T; T65A+S105P+Q327W; T65A+S105P+Q327F; T65A+Q327W+S364P; T65A+Q327F+S364P; T65A+S103N+Q327F; P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; or S255N+Q327F+E501V+Y504T.