CN-121975751-A - 3-Sterone-delta 1-dehydrogenase mutant with improved heat resistance and application thereof

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a 3-sterone-delta 1-dehydrogenase mutant with improved heat resistance and application thereof. The invention carries out structural analysis and site-directed mutagenesis design on 3-sterone-delta 1-dehydrogenase AuKsdD gene from Arthrobacter urate, obtains a mutant with higher catalytic activity and obviously enhanced heat resistance compared with wild AuKsdD through directional mutagenesis on key sites E244, K267, T301 and A335, can keep high catalytic efficiency for a long time under high temperature condition, and is suitable for industrialized synthesis of steroid medicine raw materials.

Inventors

• WANG MIN
• SHEN YANBING
• CHEN CHEN
• SHI CHANG
• SU ZHENHUA
• XIA MENGLEI

Assignees

• 天津科技大学

Dates

Publication Date

20260505

Application Date

20260409

Claims (9)

1. 1. A3-sterone-delta 1-dehydrogenase mutant is characterized in that the mutant is obtained by mutating 244 th glutamic acid to leucine, 267 th lysine to arginine, 301 th threonine to glutamic acid and 335 rd alanine to proline on the basis of a wild type AuKsdD shown in SEQ ID NO. 1.
2. 2. The 3-sterone-delta 1-dehydrogenase mutant according to claim 1, wherein the mutant is AuKsdD-M4 mutant and has an amino acid sequence shown in SEQ ID NO. 3.
3. 3. A gene encoding the 3-sterone- Δ1-dehydrogenase mutant according to claim 1.
4. 4. The coding gene according to claim 3, wherein the coding gene is shown in SEQ ID NO. 4.
5. 5. A recombinant vector or recombinant strain comprising a gene encoding the 3-sterone- Δ1-dehydrogenase mutant of claim 1.
6. 6. Use of the recombinant vector or recombinant strain of claim 5 for the production of the 3-sterone- Δ1-dehydrogenase mutant of claim 1.
7. 7. The use of the recombinant vector or recombinant strain according to claim 5 in the dehydrogenation of steroid C1, 2.
8. 8. Use of the 3-sterone- Δ1-dehydrogenase mutant according to claim 1 for the dehydrogenation of steroid C1, 2.
9. 9. Use according to claim 7 or 8, comprising the synthesis of prednisolone by catalytic hydrogenation of cortisone, the synthesis of androsta-4-ene-3, 17-dione by catalytic synthesis of androsta-1, 4-diene-3, 17-dione, or the synthesis of 11 alpha-hydroxy-16, 17 alpha-epoxyprogesterone 11 alpha-hydroxy-16, 17 alpha-epoxypregna-1, 4-diene-3, 20-dione.

Description

3-Sterone-delta 1-dehydrogenase mutant with improved heat resistance and application thereof Technical Field The invention belongs to the technical field of genetic engineering, and particularly relates to a 3-sterone-delta 1-dehydrogenase mutant with improved heat resistance and application thereof. Background Steroid drugs occupy extremely important positions in the pharmaceutical field due to their remarkable anti-inflammatory, antiallergic, anti-infective and immunoregulatory effects, and are the second most common clinical drugs next to antibiotics. The key step of the synthesis process of prednisolone is dehydrogenation reaction of C1 and 2 positions on the ring A of the steroid mother nucleus, and the dehydrogenation reaction is catalyzed by 3-sterone-delta 1-dehydrogenase (KsdD), so that the prednisolone is a core link for realizing high-efficiency biosynthesis of steroid medicines. However, natural KsdD has the common problems of poor heat resistance, low specific enzyme activity, insufficient organic solvent tolerance and the like, so that the natural KsdD is easy to inactivate under the conditions of high temperature, high substrate concentration or long-time reaction, and the industrialized green production of steroid medicaments is severely restricted. Especially in continuous catalytic and amplification reaction systems, the thermal deactivation of enzymes has become a major bottleneck limiting production efficiency and economy. Therefore, molecular transformation is carried out on KsdD heat resistance, so that the heat resistance and the catalytic performance of the novel molecular transformation are improved, and the novel molecular transformation is an urgent need for realizing high-efficiency, energy-saving and low-cost biosynthesis of steroid medicines. The invention aims at the problem, and site-directed mutation is carried out on KsdD key residues through structure-oriented protein engineering, so that mutants with high catalytic activity and excellent heat resistance are obtained, and a new solution is provided for industrial production of steroid medicaments. Disclosure of Invention The invention aims to solve the problems of poor heat resistance, low catalytic efficiency and the like of wild 3-sterone-delta 1-dehydrogenase (KsdD), provide KsdD mutants and genetically engineered bacteria thereof with obvious improvement in heat resistance and enzyme activity, and realize the high-efficiency application of the KsdD mutants in steroid C1 and 2 dehydrogenation reactions. In order to solve the technical problems, the invention carries out structural analysis and site-directed mutagenesis design on the 3-sterone-delta 1-dehydrogenase (AuKsdD) gene from Arthrobacter urate, and obtains a plurality of KsdD mutants with excellent performance by carrying out directed mutagenesis on key sites E244, K267, T301 and A335. Experiments prove that the mutant has higher catalytic activity and obviously enhanced heat resistance compared with the wild AuKsdD, can keep high catalytic efficiency for a long time under high temperature conditions, and is suitable for industrialized synthesis of steroid medicine raw materials. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: According to one of the technical schemes provided by the invention, the 3-sterone-delta 1-dehydrogenase mutant is obtained by mutating 244 th glutamic acid (E) into leucine (L), 267 th lysine (K) into arginine (R), 301 th threonine (T) into glutamic acid (E) and 335 rd alanine (A) into proline (P) on the basis of wild type AuKsdD shown in SEQ ID NO. 1; Furthermore, the mutant is AuKsdD-M4 mutant, which is obtained by simultaneously generating E244L, K267R, T301E, A335P mutation on the basis of wild type AuKsdD shown in SEQ ID NO. 1, and the amino acid sequence of the AuKsdD-M4 mutant is shown in SEQ ID NO. 3. The invention also provides a coding gene of the mutant in one of the technical schemes; Further, the coding gene is shown as SEQ ID NO. 4. The second technical scheme provided by the invention is a recombinant vector containing the coding gene of the 3-sterone-delta 1-dehydrogenase mutant in one technical scheme; further, expression vectors employed by the recombinant vector include, but are not limited to, pET series plasmids, such as pET28a plasmid, and the like. The third technical scheme provided by the invention is a recombinant strain containing the coding gene of the 3-sterone-delta 1-dehydrogenase mutant in one technical scheme; further, host bacteria employed by the recombinant strain include, but are not limited to, E.coli, preferably E.coli BL21 (DE 3). The invention provides a fourth technical scheme, which is the application of the recombinant vector in the third technical scheme or the recombinant bacterium in the fourth technical scheme, particularly the application in the 3-sterone-delta 1-dehydrogenase mutant in one of the production technical schemes, more particu