Method for the enzymatic conversion of a phenol substrate into a corresponding catechol product

Patent metadata

Field	Value
Publication number	US-11274283-B2
Application number	US-202017029629-A
Country	US
Kind code	B2
Filing date	Sep 23, 2020
Priority date	Dec 17, 2014
Publication date	Mar 15, 2022
Grant date	Mar 15, 2022

Abstract

A method for the enzymatic conversion of a phenol substrate into a corresponding catechol product comprises the step of incubating the phenol substrate with a Ralstonia solanacearum tyrosinase enzyme, or a functional derivative thereof, in a reaction mixture, for a period of time sufficient to allow the enzyme convert at least some of the phenol substrate into the catechol product.

First claim

What is claimed is:

1. A method for the enzymatic conversion of 4-halophenol into 4-halocatechol, the method comprising the step of incubating the 4-halophenol with a Ralstonia solanacearum tyrosinase enzyme or a functional derivative thereof, in a reaction mixture for a period of time sufficient to allow the Ralstonia solanacearum tyrosinase enzyme to convert at least 90% of the 4-halophenol into 4-halocatechol, in which the reaction mixture comprises ascorbic acid or its sodium salt, wherein the functional derivative is an engineered variant of Ralstonia solanacearum tyrosinase of SEQ ID NO: 1 selected from the group consisting of Y119F, V153A, D317Y and L330V (RV145); T183I, F185Y, N322S, and T359M (RVC10); and N322S (C10_N322S).

2. The method according to claim 1 in which the reaction mixture comprises at least 10 mM of the 4-halophenol.

3. The method according to claim 1 in which the reaction mixture comprises at least 20 mM ascorbic acid.

4. The method according to claim 1 in which the functional derivative of the Ralstonia solanacearum tyrosinase enzyme is capable of the 98% conversion of 10 mM 4-halophenol 4-halocatechol.

5. The method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as an extract from a bacteria that expresses the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof.

6. The method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a bacteria that expresses the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof.

7. The method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a purified enzyme.

8. The method according to claim 1 in which the functional derivative of Ralstonia solanacearum tyrosinase enzyme is RV145.

9. The method according to claim 1 in which the functional derivative of Ralstonia solanacearum tyrosinase enzyme is RVC10.

10. The method according to claim 1 in which the functional derivative of Ralstonia solanacearum tyrosinase enzyme is C10_N322S.

11. The method according to claim 1 in which the Ralstonia solanacearum tyrosinase enzyme, or functional derivative thereof, is provided as a crude mixture in the form of a cell extract.

12. The method according to claim 1 in which the 4-halocatechol is separated from impurities present in the reaction mixture by chilling the reaction mixture after enzymatic conversion.

13. The method according to claim 1 in which the 4-halocatechol is separated from impurities present in the reaction mixture by refrigerating or freezing the reaction mixture after enzymatic conversion.

Assignees

• Univ College Dublin Nat Univ Ireland Dublin

Inventors

• O'Connor Kevin
• Molloy Susan
• Davis Reeta
• Shaw Wesley

Classifications

• C12N9/0071 Primary

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12Y114/18001

  Tyrosinase (1.14.18.1) · CPC title

• C12P7/22

  aromatic · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• C07C39/11

  Alkylated hydroxy benzenes containing also acyclically bound hydroxy groups, e.g. saligenol · CPC title