PENICILLIN-G ACYLASES
The present disclosure relates to engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.
1 . An engineered penicillin G acylase capable of removing the A1/B1/B29 tri-phenyl acetate protecting groups from insulin to produce free insulin, wherein said penicillin G acylase is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 4, 6, 8, 10, and/or 12.
2 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase further comprises at least one additional substitution as provided in Table 5.1, Table 6.2, and/or Table 6.3.
3 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises SEQ ID NO: 6, 8, 10, or 12.
4 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase is encoded by a polynucleotide sequence selected from SEQ ID NOS: 5, 7, 9, and 11.
5 . A vector comprising the polynucleotide sequence of claim 4 .
6 . A host cell comprising the vector of claim 5 .
7 . A method for producing free insulin, comprising: i) providing the engineered penicillin G acylase of claim 1 , and insulin comprising A1/B1/B29 tri-phenyl acetate protecting groups; and ii) exposing said engineered penicillin G acylase to said insulin comprising A1/B1/B29tri-phenyl acetate protecting groups, under conditions such that said engineered penicillin G acylase removes the A1/B1/B29 tri-phenyl acetate protecting groups and free insulin is produced.
8 . The method of claim 7 , wherein said engineered penicillin G acylase produces more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more free insulin.
9 . The method of claim 7 , wherein said penicillin G acylase comprises SEQ ID NO:4, 6, 8, 10, or 12.
10 . A composition comprising free insulin produced according to the method of claim 7 .