PENICILLIN-G ACYLASES
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.
1 . An engineered penicillin G acylase capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is at least 85% or more identical to SEQ ID NO: 40 and comprises a substitution as compared to SEQ ID NO: 40 at a position selected from positions 55, 71, 74, and 390.
2 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase further comprises a substitution as compared to SEQ ID NO: 40 at a position selected from positions 38, 185, 279, 348, 352, 444, and 466.
3 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises SEQ ID NO: 154, 160, or 174.
4 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises a sequence that is at least 85% identical to a sequence set forth in Table 17.1, 18.1, 19.1, 20.1, 22.1, 23.1, and/or 23.2.
5 . The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises a sequence set forth in Table 17.1, 18.1, 19.1, 22.1, 23.1, and/or Table 23.2.
6 . The engineered penicillin G acylase of claim 1 , wherein said engineered penicillin G acylase comprises a histidine tag.
8 . An engineered polynucleotide sequence encoding the engineered penicillin G acylase of claim 1 .
9 . The engineered polynucleotide sequence of claim 8 , wherein said sequence comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO: 1, 11, 23, 39, 55, 69, 81, 99, 107, 109, 115, 135, 141, 153, and/or 159.
10 . A vector comprising the polynucleotide sequence of claim 8 .
11 . The vector of claim 10 , further comprising at least one control sequence.
12 . A host cell comprising the vector of claim 10 .
13 . A method for producing acylated insulin, comprising: providing the engineered penicillin G acylase of claim 1 and insulin; exposing said engineered penicillin G acylase and said insulin under conditions such that said engineered penicillin G acylase acrylates said insulin, thereby producing acylated insulin.
14 . The method of claim 13 , wherein said acylation is conducted in the presence of methyl phenylacetate.
15 . The method of claim 13 , wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
16 . The method of claim 14 , wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
17 . The method of claim 13 , wherein said acylation occurs at position A1 of said insulin.
18 . The method of claim 13 , wherein said acylation occurs at position B1 of said insulin.
19 . The method of claim 13 , wherein said acylation occurs at position B29 of said insulin.
20 . The method of claim 13 , wherein said acylation occurs at positions A1, B1, and B29 of said insulin.
21 . The method of claim 13 , 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.
22 . A composition comprising acylated insulin produced according to the method of claim 13 .