17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, coding gene and engineering bacteria

Abstract

The invention discloses a 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme, a coding gene and engineering bacteria. The 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is abbreviated as 17 beta-HSD3<G186R / Y195W>, and an amino acid sequence of the 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is as shown in SEQ ID NO.1. 17 beta-hydroxysteroid hydroxylase 3 is subjected to molecular modification by adopting a rational design technology, the 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme with improved affinity with a substrate is obtained through methods of homologous modeling, molecular docking, binding energy calculation and the like, the enzyme activity is improved by 1.66 times as compared with the original enzyme activity, and by utilizing the strategy, the catalytic activity of the 17 beta-hydroxysteroid hydroxylase 3 can be obviously improved. The 17 beta-hydroxysteroid hydroxylase 3 mutant enzyme is coded, so that the yield of testosterone is up to 3.95 g / L, and technical support and data reference are provided for large-scale biological preparation of the testosterone which is a male hormone drug.

Description

technical field [0001] The invention belongs to the technical fields of enzyme engineering and bioengineering, and specifically relates to a 17β-hydroxysteroid hydroxylase 3 mutant enzyme, a coding gene, a plasmid containing the coding gene, and an engineering bacterium. [0002] technical background [0003] Steroids are widely used as anti-inflammatory, diuretic, anabolic, contraceptive, androgenic, progestogenic and anticancer drugs, so they are an invaluable resource in the pharmaceutical industry with a global market of over $100 billion per year, Testosterone (TS) is one of the very important male hormones. Testosterone is a key sex hormone that regulates male fertility, muscle mass and fat distribution. It is clinically used to treat male hypogonadism, maintain male secondary sexual characteristics and sexual function. At the same time, testosterone is also a very useful steroid precursor , commonly used in the synthesis of high-performance steroid drugs such as 5α-di...

AI Technical Summary

Problems solved by technology

However, the 17β-hydroxysteroid hydroxylase derived from microorganisms has the following problems: ① The 17β-hydroxysteroid hydroxylase from this source is a reversible oxidoreductase that catalyzes the reaction between the C17-keto group and the C17-alcohol group on the steroid nucleus. The reversible reduction / oxidation reaction is not suitable for the high-efficiency production of testosterone; ② During the process of catalyzing the conversion of 4-AD into testosterone, it will be accompanied by some by-products of C19 steroids, such as ADD and 1(2)-dehydrotestosterone ( 1(2)-DT), resulting in extremely low accumulation of testosterone, unable to meet the requirements of industrial production

[0007] Some people choose human 17β-hydroxysteroid hydroxylase 3 (17β-HSD3) to catalyze the synthesis of testosterone from 4-AD, such as figure 1 However, the enzyme activity is low, the amount of expression is small, and the reaction requires a large amount of enzyme, resulting in high cost of testosterone enzymatic catalysis, which is not conducive to large-scale preparation

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