Immobilized transaminases and process for making and using immobilized transaminase

Patent metadata

Field	Value
Publication number	US-9523107-B2
Application number	US-201414767949-A
Country	US
Kind code	B2
Filing date	Feb 24, 2014
Priority date	Feb 28, 2013
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

The invention is directed to immobilized transaminases and methods of making and using them.

First claim

What is claimed is:

1. A resin comprising an immobilized transaminase polypeptide, wherein the transaminase polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the resin comprises methacrylate, and wherein the transaminase polypeptide is attached to the resin.

2. The resin of claim 1 , wherein the transaminase is attached to the resin by hydrophilic interactions.

3. The resin of claim 1 , wherein the resin comprises DIAION HP2MG.

4. The resin of claim 3 , wherein the transaminase is stable in a solvent system that comprises at least 90% organic solvent.

5. The resin of claim 3 , wherein the transaminase is stable in a solvent system that comprises at least 95% organic solvent.

6. The resin of claim 2 , wherein the resin comprises methacrylate with no functional groups.

7. The resin of claim 2 , wherein the resin comprises methacrylate with no aromatic groups.

8. A resin comprising an immobilized transaminase polypeptide, wherein the transaminase polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the resin comprises DIAION HP2MG, and wherein the transaminase polypeptide is physically attached to the resin via hydrophilic interactions.

9. A process for making (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine comprising the steps of:
1) dissolving 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one in an organic solvent; and
2) contacting the 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8-H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one with the resin comprising the immobilized transaminase of claim 1 and an amino group donor to thereby make (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazi-n-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.

10. The process of claim 9 wherein the immobilized transaminase is physically attached to the resin, and the resin is DIAION HP2MG.

11. A process of making a resin comprising an immobilized transaminase comprising the steps of:
1) incubating a solution of a transaminase polypeptide with a resin to form a resin comprising an immobilized transaminase polypeptide, wherein the transaminase polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the resin comprises methacrylate, and wherein the transaminase polypeptide is attached to the resin; and
2) filtering and rinsing the immobilized transaminase.

Assignees

• Merck Sharp & Dohme

Inventors

• Truppo Matthew D
• Journet Michel
• Strotman Hallena
• Mcmullen Jonathan P
• Grosser Shane T

Classifications

• C12N11/08

  the carrier being a synthetic polymer · CPC title

• C12P13/001

  Amines; Imines · CPC title

• C12N9/1096

  transferring nitrogenous groups (2.6) · CPC title

• C12P17/182 Primary

  Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system (alloxazine or isoalloxazine, e.g. riboflavine C12P25/00) · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title