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Clostridium sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S

A technology of hydroxysteroids and dehydrogenases, applied in bacteria, genetic engineering, oxidoreductases, etc., can solve problems such as low stereoselectivity, difficult recovery, limited types and numbers of catalysts, etc.

Active Publication Date: 2017-01-04
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 sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S
  • Clostridium sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S
  • Clostridium sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S

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Experimental program
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Effect test

Embodiment 1

[0036] Example 1. Preparation of Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant

[0037] 1. Mutant Gene Synthesis

[0038] 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.

[0039] 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 145th amino acid of wild-type CA 7α-HSDH , the amino acid is threonine, and the corresponding nucleotide sequence is codons 433-435.

[0040]The codons 433-435 of the wild-type CA 7α-HSDH gene sequence were changed from ACT to AGT, and the original threonine was replaced with serine to obtain a CA 7α-HSDH mutant, named CA 7α-HSDH T145S mutant , its nucleotide sequence is shown in SEQ ID NO: 3, and its amino acid sequence is shown in SEQ ID NO: 2.

[0041] ...

Embodiment 2

[0062] Example 2. Determination of T145S Mutant Enzyme Kinetic Parameters

[0063] 1. Preparation of NADPH standard curve

[0064] Using reaction buffer (50 mM Tris-HCl, 200 mM NaCl, pH 8.0), 0.1, 0.2, 0.3 and 0.4 mM NADPH solutions were prepared respectively. 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.

[0065] The results are shown in Figure 3, the obtained standard curve equation is y=2.7868x-0.0002, R 2 = 0.9999.

[0066] 2. Enzyme activity assay

[0067] Add 958μL of reaction buffer (50mM Tris-HCl, 200mM NaCl, pH 8.0), 20μL of 50mM NADP to a 2mL cuvette + . 2 μL of the CA 7α-HSDH T145S mutant (2.46 mg / mL) prepared in Example 1, after zero adjustment, 20 μL of 50 mM substrate TCDCA was adde...

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Abstract

The invention relates to hydroxysteroid dehydrogenase and particularly relates to a clostridium sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S. The amino acid sequence of the clostridium sardiniense 7alpha-hydroxysteroid dehydrogenase mutant T145S is shown by SEQ ID No:2 and obtained by changing the 145th-site amino acid of the 7alpha-hydroxysteroid dehydrogenase with an amino acid sequence of SEQ ID No:1 from Thr into Ser. The mutant is 4.85 times of a wild type in the catalysis efficiency against the substrates TCDCA and NADP<+> and has a huge application potential in a biotransformation process of CDCA / TCDCA for obtaining UDCA / TUDCA.

Description

technical field [0001] The invention relates to hydroxysteroid dehydrogenase, in particular to a mutant T145S 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 "not" strict 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 chenodeoxych...

Claims

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Application Information

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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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