Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Carbonyl reductase mutant and application thereof in preparation of steroid hormone-testosterone

A mutant, reductase technology, applied in the field of bioengineering, to achieve the effects of low cost, high activity and mild catalytic reaction conditions

Active Publication Date: 2021-10-22
FUZHOU UNIV
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant reports on the synthesis of testosterone by using carbonyl reductase to asymmetrically reduce androst-4-ene-3,17-dione

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Carbonyl reductase mutant and application thereof in preparation of steroid hormone-testosterone
  • Carbonyl reductase mutant and application thereof in preparation of steroid hormone-testosterone
  • Carbonyl reductase mutant and application thereof in preparation of steroid hormone-testosterone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of engineering strains co-expressing carbonyl reductase mutant (PmCRm) and formate dehydrogenase (BstFDHm)

[0025] (1) PCR amplification of carbonyl reductase mutant gene pmcr_m and purify

[0026] The whole build process is as follows figure 2 shown.

[0027] The carbonyl reductase mutant PmCRm is obtained by mutating the 136th leucine into serine in the amino acid sequence shown in SEQ ID No.1 (WP_060395036.1).

[0028] According to the amino acid sequence (SEQ ID No.2) of the carbonyl reductase mutant PmCRm, with ( E. coli ) BL21 (DE3) was obtained by codon optimization for the target host pmcr_m Gene (SEQ ID No.3), and entrusted Shanghai Jierui Bioengineering Co., Ltd. to synthesize it. Two primers were designed according to the sequence shown in SEQ ID No.3:

[0029] F1 (SEQ ID No. 4):

[0030] 5'-ATC GGATCC ATGTCAGCATCTAAAAC-3' (the underline is the BamH I restriction site);

[0031] R1 (SEQ ID No. 5):

[0032] 5'-CAG CTCGA...

Embodiment 2

[0070] Example 2 Preparation of co-expression engineering strain quiescent cells

[0071] to pick (E. coli) BL21(DE3)-pET30a- pmcr_m-bstfdh_m A single colony of the engineering strain was added to 20 mL of LB liquid medium supplemented with 50 mg / L kanamycin, and cultured at 37°C and 200 rpm for 8-12 h as the seed solution. According to the inoculation amount of 2vol%, transfer to a 1 L shake flask containing 300 mL of LB medium supplemented with 50 mg / L kanamycin, and continue to culture until OD 600 0.6-0.7, then add lactose at a final concentration of 8% (m / v), place at 25°C and continue to shake for 12 h to induce the expression of the target gene. Then the above culture was centrifuged at 7000 rpm at 16°C for 3 min, the supernatant was discarded, the wet cells were collected, weighed and stored at -20°C for future use.

Embodiment 3

[0072] Example 3 The method of co-expressing engineering bacteria resting cells to convert androst-4-ene-3,17-dione into testosterone

[0073] According to the method of Example 2, resting cells of co-expression engineering strains were prepared and used as biocatalysts.

[0074] Determination of the product by high performance liquid chromatography: Elite high performance liquid chromatography, chromatographic column: Chiralpak AD-H Column (4.6 mm × 250 mm, 5 µm), mobile phase: n-hexane-isopropanol (v / v = 90 / 10), detection wavelength: 254 nm, column temperature: 30°C, flow rate: 1.4 mL / min. Under the detection conditions, the peak eluting times of androst-4-ene-3,17-dione and testosterone were 10.39 min and 11.47 min, respectively.

[0075] (1) Take the wet bacteria and suspend them in 100 mL phosphate buffer (100 mM, pH=7.5) to make the wet bacteria concentration 200 g / L, add the substrate androst-4-ene-3,17-dione 2.88 g, Tween 80 4 mL, co-substrate sodium formate 1.02 g,...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Melting pointaaaaaaaaaa
Specific rotationaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of steroid hormone-testosterone. The method comprises the following steps: co-expressing a carbonyl reductase mutant PmCRm and formate dehydrogenase BstFDH_m in Escherichia coli, and catalyzing androstane-4-ene-3,17-dione to synthesize testosterone by taking resting cells of co-expressed engineering bacteria as a biocatalyst. The biocatalyst has high catalytic activity, regioselectivity and stereoselectivity, 28.8 g / L of androstane-4-ene-3,17-dione can be completely converted within 10 h to generate the target product testosterone, and no by-product is generated. The substrate feeding amount, the conversion rate and the space-time conversion rate of the catalyst all reach the highest level of testosterone prepared by a domestic biological method; after separation and purification, the product recovery rate is up to 95% or above, which indicates that the biocatalyst is an efficient catalyst for green synthesis of testosterone.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a carbonyl reductase mutant and its application in the preparation of steroid hormone-testosterone. Background technique [0002] Testosterone (Testosterone, TS) is an androgen steroid drug, also known as testosterone, testosterone or testosterone, its chemical name is 17 β-hydroxy-androst-4-en-3-one, and its chemical structure is as follows: figure 1 shown. In medicine, testosterone can not only be used as an androgen drug to treat male primary or secondary hypogonadism and maintain secondary sexual characteristics and sexual function, but also can be used as a key precursor for a series of high-efficiency steroid drugs (such as Synthesis of methyltestosterone, nandrolone propionate and testosterone enanthate, etc.). [0003] In the traditional method, the preparation of testosterone is through multi-step chemical reaction, various raw materials (indanone, i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/18C12R1/19
CPCC12N9/0006C12N15/70C12P33/18C12Y101/01184Y02A50/30
Inventor 林娟苏冰梅赵鸿儒许鑫琦许炼
Owner FUZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com