17β-hydroxysteroid dehydrogenase mutants and application thereof

Patent metadata

Field	Value
Publication number	US-11098287-B2
Application number	US-202017094982-A
Country	US
Kind code	B2
Filing date	Nov 11, 2020
Priority date	Sep 10, 2020
Publication date	Aug 24, 2021
Grant date	Aug 24, 2021

Abstract

The disclosure discloses 17β-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17β-hydroxysteroid dehydrogenase (1.66 U/mg).

First claim

What is claimed is:

1. A 17β-hydroxysteroid dehydrogenase mutant, wherein the 17β-hydroxysteroid dehydrogenase mutant comprises an amino acid sequence having all of SEQ ID NO: 2 except for:
a mutation of valine at position 107 to alanine;
a mutation of threonine at position 155 to asparagine;
a mutation of histidine at position 164 to tyrosine; or
a mutation of threonine at position 155 to asparagine, and a mutation of histidine at position 164 to tyrosine.

2. A gene, wherein the gene encodes the 17β-hydroxysteroid dehydrogenase mutant according to claim 1 .

3. A recombinant plasmid, wherein the recombinant plasmid carries the gene according to claim 2 .

4. A host cell, wherein the host cell carries the gene according to claim 2 .

5. The host cell according to claim 4 , wherein the host cell is Escherichia coli.

6. A method for producing boldenone, comprising: adding the host cell according to claim 4 to a transformation system containing 1,4-androstenedione to be transformed to obtain a transformation solution containing boldenone; and separating the transformation solution containing boldenone to obtain boldenone.

7. The method for producing boldenone according to claim 6 , wherein the transformation temperature is 35 to 40° C. and the pH is 7.0 to 8.0.

8. The method for producing boldenone according to claim 6 , wherein the transformation system further contains methylated-β-cyclodextrin.

9. A host cell, wherein the host cell comprises the recombinant plasmid according to claim 3 .

Assignees

• Univ Jiangnan

Inventors

• Rao Zhiming
• Shao Minglong
• Wang Yetong
• Wu Yuling
• Yang Taowei
• Zhang Xian
• Xu Meijuan

Classifications

• C12P33/06

  Hydroxylating · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y101/01051

  3 (or 17)-Beta-hydroxysteroid dehydrogenase (1.1.1.51) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title