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Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant k179m

A hydroxysteroid, K179M technology, applied in the directions of enzymes, genetic engineering, oxidoreductases, etc., can solve the problems of difficult recovery, expensive auxiliary reagents, low stereoselectivity, etc., and achieve the effect of huge application potential.

Active Publication Date: 2020-02-18
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 k179m
  • Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant k179m
  • Clostridium sardinia 7α-hydroxysteroid dehydrogenase mutant k179m

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Preparation of CA 7α-HSDH mutants

[0056] 1. Mutant Gene Synthesis

[0057] 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.

[0058] 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 179th amino acid of wild-type CA 7α-HSDH , the amino acid is lysine, and the corresponding nucleotide sequence is the 535th-537th codon.

[0059] The 535th-537th codon of the wild-type CA 7α-HSDH gene sequence was changed from AAA to ATG, and the original lysine was replaced with methionine to obtain a CA 7α-HSDH mutant, named CA 7α-HSDH The nucleotide sequence of the K179M mutant is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 2.

[0060] 2. Construction of vectors ...

Embodiment 2K179

[0121] Example 2. K179M mutant enzyme activity detection

[0122] Enzyme activity assay:

[0123] At room temperature, add 1958uL 50mM Tris-HCl (PH=8.0) solution, 20uL 50mM NADP to the cuvette + solution, and then add 2uL of the K179M enzyme protein solution obtained in Example 1, mix well and adjust to zero, and finally add 20uL of 50mM TCDCA solution, mix well, and start timing. The change in absorbance at 340 nm is read every minute.

[0124] At the same time, the enzymatic activity of CA7α-HSDH was measured in the same manner as above.

[0125] The results show that the mutant K179M of the present invention catalyzes the epimerization of the 7-hydroxyl group of TCDCA to generate TUDCA intermediate taurine 7-ketolithocholic acid (T7K-LCA). The specific activities of K179M enzyme protein of the present invention and Clostridium sardinia 7α-HSDH enzyme protein to TCDCA are shown in Table 1.

[0126] Specific activity (U / mg) Definition: Under the above reaction conditions,...

Embodiment 5K179

[0130] Embodiment 5. K179M mutant thermostability research

[0131] The enzyme sample (without glycerol) was placed in a metal bath at 50° C. for 20 minutes, and the residual enzyme activity was detected according to the method for measuring enzyme activity in Example 2. Each sample was repeated no less than 3 times.

[0132] The results are shown in Table 2. After heat treatment for 20 minutes, the enzyme activity of wild-type CA 7α-HSDH decreased rapidly, and only 32.33% of the enzyme activity remained, while K179M still retained 45.09% of the enzyme activity. It shows that compared with the wild-type CA 7α-HSDHK179M, it has better thermal stability and is more suitable for industrial applications.

[0133] Table 2. Comparison of thermal stability between wild-type CA 7α-HSDH and 7α-HSDHJ-1-1

[0134]

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Abstract

The invention belongs to the technical field of biologics and in particular relates to a Sardinia clostridium 7alpha-hydroxy steroid dehydrogenase mutant K179M of which the amino acid sequence is as shown in SEQ ID NO:2, and is generated by converting a 179-site amino acid of 7alpha-hydroxy steroid dehydrogenase of which the nucleotide sequence is as shown in SEQ ID NO:1 into methionine. The catalysis ratio activity of the mutant to TCDCA (Taurochenodeoxycholic Acid) is 2.9 times of that of a wild type. The K179M mutant provided by the invention has great application potential in the industrial production process in biological conversion of TCDCA into TUDCA (Tauro Ursodesoxy Cholic Acid).

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a mutant K179M 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 cheno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12P33/02
CPCC12N9/0006C12P33/02C12Y101/01159
Inventor 祝连彩撖菁萱姚恺怡唐士金王伯初季顺林娄德帅谭君
Owner CHONGQING UNIV
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