Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant r194a

A hydroxysteroid and dehydrogenase technology, applied in bacteria, genetic engineering, oxidoreductase, etc., can solve the problems of expensive auxiliary reagents, difficult to recover, and low stereoselectivity.

Active Publication Date: 2019-10-08
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the current chemical methods have achieved certain results, there are often disadvantages in chemical methods such as limited types and numbers of catalysts, low stereoselectivity, expensive auxiliary reagents, and difficult recovery.

Method used

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  • Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant r194a
  • Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant r194a
  • Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant r194a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Preparation of Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant

[0043] 1. Mutant Gene Synthesis

[0044] Original sequence: codon-optimized wild-type CA 7α-HSDH gene sequence (see patent publication CN102827848A), the nucleotide sequence of which is shown in SEQ ID NO:4.

[0045] By comparing the similarities and differences between wild-type CA 7α-HSDH and homologous enzyme proteins from the primary structure to the high-order structure, the site that affects the enzymatic properties is the 194th amino acid of wild-type CA 7α-HSDH , the amino acid is arginine, and the corresponding nucleotide sequence is codons 580-582.

[0046] Changed the codons 580-582 of the wild-type CA 7α-HSDH gene sequence from CGC to GCC, and replaced the original arginine with alanine to obtain a CA 7α-HSDH mutant named CA 7α-HSDH R194A The nucleotide sequence of the mutant is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:2.

[0047] 2. Constr...

Embodiment 2

[0115] Example 2. Determination of R194A Mutant Enzyme Kinetic Parameters

[0116] 1. Preparation of NADPH standard curve

[0117] 0.005, 0.01, 0.02, 0.03, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4 and 0.5 mM NADPH solutions were respectively prepared using reaction buffer (50 mM Tris-HCl, 200 mM NaCl, pH 8.5). After zeroing with the above blank solvent, add NADPH solutions of various concentrations into 2mL cuvettes respectively, and measure the light absorption value OD at 340nm 340 . With the concentration of NADPH solution as the abscissa and the corresponding light absorption value as the ordinate, a standard curve was drawn.

[0118] The result is as Figure 5As shown, the obtained standard curve equation is y=2.5771x+0.0045, R 2 =0.9995. 2. Determination of enzyme activity

[0119] Add 958μL reaction buffer (50mM Tris-HCl, 200mM NaCl, pH8.5), 20μL 50mM NADP to a 2mL cuvette + solution, 2 μL of the CA 7α-HSDH R194A mutant solution (1.77 mg / mL) prepared in Example 1, aft...

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Abstract

The invention relates to hydroxy steroid dehydrogenase, in particular to a clostridium sardiniense 7alpha-hydroxy steroid dehydrogenase mutant R194A. The amino acid sequence of the mutant is shown in SEQ ID NO:2, and the mutant is prepared by changing the 194 amino acid of 7alpha-hydroxy steroid dehydrogenase with the amino acid sequence of SEQ ID NO:1 into Ala from Arg. The catalytic efficiency of the mutant for substrates NADP+ and TCDCA is 3.34 times that of a wild type. The mutant R194A has great application potential in the industrial production process of biologically converting CDCA / TCDCA to obtain UDCA / TUDCA.

Description

technical field [0001] The invention relates to hydroxysteroid dehydrogenase, in particular to a mutant R194A of Clostridium sardinia 7α-hydroxysteroid dehydrogenase. Background technique [0002] The asymmetric reduction of carbonyl groups has always been one of the hotspots in chemical reaction research. Although the current chemical methods have achieved certain results, they often have disadvantages such as limited types and numbers of catalysts, low stereoselectivity, expensive auxiliary reagents, and difficult recovery. The enzymatic reaction not only has high efficiency, chemoselectivity, regioselectivity but also high stereoselectivity. The enzymatic reaction mediated by hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) has relatively strict stereoselectivity and "relaxed" substrate specificity. For example, as early as the early 1980s, scientists have begun to use 7α-, 7β-HSDH produced by microorganisms to combine epimerization with chenodeoxycholi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21
Inventor 王伯初娄德帅祝连彩谭君王玥
Owner CHONGQING UNIV
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