Carboxyesterase polypeptides for amide coupling

Patent metadata

Field	Value
Publication number	US-11015180-B2
Application number	US-201816218003-A
Country	US
Kind code	B2
Filing date	Dec 12, 2018
Priority date	Dec 13, 2017
Publication date	May 25, 2021
Grant date	May 25, 2021

Abstract

The present invention provides engineered carboxyesterase enzymes having improved properties as compared to a naturally occurring wild-type carboxyesterase enzymes, as well as polynucleotides encoding the engineered carboxyesterase enzymes, host cells capable of expressing the engineered carboxyesterase enzymes, and methods of using the engineered carboxyesterase enzymes in amidation reactions.

First claim

The invention claimed is:

1. An engineered carboxyesterase comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2, wherein said engineered carboxyesterase comprises a substitution in said polypeptide sequence at position 282, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2, and wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase of SEQ ID NO:2.

2. The engineered carboxyesterase of claim 1 , wherein said polypeptide sequence further comprises substitutions at positions selected from: 39, 39/323, 62, 62/117, 63, 64, 65, 66, 68, 69, 70, 71, 71/263, 77, 77/184, 103, 103/147, 104, 104/429, 105, 107, 107/185, 108, 109, 109/117, 110, 111, 113, 114, 115, 117, 118, 118/269, 118/349, 119, 126, 147, 153, 153/215, 164, 164/271, 174, 183, 184, 184/249, 185, 186, 187, 188, 190, 209, 210, 211, 212, 213, 213/271, 213/345, 214, 215, 215/271, 216, 217, 217/231, 224, 224/268/372, 231, 249, 249/284, 263, 268, 269, 270, 270/470, 271, 271/416, 276, 277, 278, 279, 280, 281, 281/374, 283, 283/429, 284, 284/438, 285, 286, 311, 317, 320, 320/323, 320/323/372, 320/372/376, 320/376/377, 321, 323, 324, 345, 349, 372, 372/376, 373, 374, 376, 377, 405, 416, 420, 427, 428, 429, 438, and 470, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

3. The engineered carboxyesterase of claim 1 , wherein said amino acid at position 282 is a polar amino acid.

4. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase comprises an amino acid substitution selected from: X282Q, X282S, and X282T, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 2.

5. The engineered carboxyesterase of claim 1 , wherein said polypeptide sequence is selected from: SEQ ID NOs: 4, 6, 8, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132 and 134.

6. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase comprises improved amidation activity as compared to the wild-type T. fusca carboxyesterase of SEQ ID NO:2.

7. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase on a substrate comprising isobutylamine.

8. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase on the substrate set comprising isobutylamine and ethyl benzoate.

9. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase in producing a product comprising N-isobutyl-benzamide.

10. The engineered carboxyesterase of claim 1 , further comprising at least one substitution selected from: L343V, L372L, L320W/G, L214R, L271Y and L65G, wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2.

11. The engineered carboxyesterase of claim 1 , further comprising at least one substitution selected from: L268A, L63A/R, L189Q/I/E, L214R, L381L, and L69W, wherein said engineered carboxyesterase exhibits greater enzymatic activity than wild-type T. fusca carboxyesterase, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:2.

12. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase is purified.

13. The engineered carboxyesterase of claim 1 , wherein said engineered carboxyesterase is immobilized on a solid support.

14. A composition comprising at least one engineered carboxyesterase of claim 1 .

Assignees

• Codexis Inc

Inventors

• Voladri Rama
• Entwistle David
• Micklitsch Christopher Michael
• Mitchell Vesna
• Dellas Nikki
• Zhang Xiyun
• Sero Antoinette
• Dorr Brent
• Fuerst Douglas E

Classifications

• C12N9/18 Primary

  Carboxylic ester hydrolases {(3.1.1)} · CPC title

• C12Y301/01001

  Carboxylesterase (3.1.1.1) · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title