# US 10,865,402 B2 · Penicillin-G acylases > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,402 B2 - Document ID: US10865402B2 - Status: Granted - Kind: B2 - Application number: 16/927,129 - Filed: 2020-07-13 - Published: 2020-10-29 - Granted: 2020-12-15 - Earliest claimed: 2017-01-05 - Assignees: Codexis, Inc. - Applicants: Codexis, Inc. - Inventors: Oscar Alvizo; David Elgart; Robert Kevin Orr; James Nicholas Riggins; Anna Fryszkowska; Katrina W. Lexa; Xiang Yi; Da Duan; Courtney Dianne Moffett; Nikki Dellas; Vesna Mitchell - Web page: https://patentgrep.com/patent/US10865402B2 ## CPC - C12N 9/84: Penicillin amidase {(3.5.1.11)} - C07K 14/62: Insulins - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C12Y 305/01011: Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase - C07K 2319/21: containing a His-tag - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - 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 ## Abstract The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes. ## Claims ### Claim 1 An engineered polynucleotide encoding an engineered penicillin G acylase capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is 90% or more identical to SEQ ID NO: 100, and wherein the residue at position X67 as compared to SEQ ID NO: 100 is an alanine. ### Claim 2 (depends on 1) The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises a sequence that is at least 90% or more identical to at least one sequence set forth in Table 5.1, 5.2, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 20.1, 22.1, 23.1, and/or 23.2. ### Claim 3 (depends on 1) The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises a sequence set forth in Table 5.1, 5.2, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 7.1, 8.1, 9.1, 11.1, 12.1, 13.1, 14.1, 15.1, 16.1, 17.1, 18.1, 19.1, 22.1, 23.1, and/or Table 23.2. ### Claim 4 (depends on 1) The engineered polynucleotide encoding the engineered penicillin G acylase of claim 1, wherein said engineered penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NOS:100 and 110. ### Claim 5 (depends on 1) The engineered polynucleotide sequence of claim 1, wherein said sequence comprises a polynucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOS: 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or 159. ### Claim 6 (depends on 1) A vector comprising the polynucleotide sequence of claim 1. ### Claim 7 (depends on 6) The vector of claim 6, further comprising at least one control sequence. ### Claim 8 (depends on 7) A host cell comprising the vector of claim 7. ### Claim 9 (depends on 1) A method for producing acylated insulin, comprising: providing the engineered penicillin G acylase encoded by the polynucleotide of claim 1 and insulin; exposing said engineered penicillin G acylase and said insulin under conditions such that said engineered penicillin G acylase acylates said insulin, thereby producing acylated insulin. ### Claim 10 (depends on 9) The method of claim 9, wherein said acylation is conducted in the presence of methyl phenylacetate. ### Claim 11 (depends on 9) The method of claim 9, wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin. ### Claim 12 (depends on 10) The method of claim 10, wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin. ### Claim 13 (depends on 9) The method of claim 9, wherein said acylation occurs at position A1 of said insulin. ### Claim 14 (depends on 9) The method of claim 9, wherein said acylation occurs at position B1 of said insulin. ### Claim 15 (depends on 9) The method of claim 9, wherein said acylation occurs at position B29 of said insulin. ### Claim 16 (depends on 9) The method of claim 9, wherein said engineered penicillin G acylase produces more than 90% more acylated insulin as compared to the production of acylated insulin by the polypeptide of SEQ ID NO: 2, 4, 12, 24, 40, 56, 70, 82, 100, 108, 110, 116, 136, 142, 154 and/or 160.