7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 triangle C6 and application thereof

A hydroxysteroid and dehydrogenase technology, applied in application, enzyme, genetic engineering and other directions, can solve the problems of product purification and separation difficulties, affecting product purity, etc.

Active Publication Date: 2021-02-05
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The content of TCDCA in chicken gall powder is 42.58%, but the content of TCA accounts for 4.743%. TUCA appears in the product after biocatalytic synthesis, which affects the purity of the product and increases the difficulty of purification and separation of the later product.

Method used

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  • 7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 triangle C6 and application thereof
  • 7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 triangle C6 and application thereof
  • 7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 triangle C6 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of 7α-hydroxysteroid dehydrogenase (J-1-1) mutant

[0036] 1. Design and synthesis of mutant genes

[0037] The amino acid sequence of wild-type 7α-hydroxysteroid dehydrogenase J-1-1 (7α-HSDH J-1-1) is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:3. The gene of 7α-HSDH J-1-1 was isolated from feces samples of healthy black bears in Sichuan Black Bear Protection and Breeding Base in our laboratory. The full length of its open reading frame is 786bp, encoding 261 amino acids. The isolation and cloning method of the gene is described in the patent application text of the application number 2017113648196 submitted on December 18, 2017, and the title of the invention is "7α-hydroxysteroid dehydrogenase and its coding gene and application". The entire content of the patent application is incorporated herein.

[0038] The amino acid sequence (261aa) of wild-type 7α-hydroxysteroid dehydrogenase J-1-1 is as follows:

[0039] M...

Embodiment 2

[0110] Example 2. Determination of Enzyme Activity of 7α-Hydroxysteroid Dehydrogenase Mutant J-1-1 ΔC6

[0111] 1. Preparation of NADPH standard curve

[0112] 0 mM, 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM NADPH (Sigma-Aldrich, Cat. No.: 10621692001) solutions were respectively prepared using reaction buffer (50 mM Tris-HCl, pH 8.0). After zeroing with the above reaction buffer (50mM Tris-HCl, pH 8.0), add NADPH solutions of various concentrations into 2mL cuvettes respectively, and measure the light absorption value OD at 340nm at room temperature 340 . Take the concentration of NADPH solution as the abscissa, and the corresponding 340nm light absorption value as the ordinate, and draw a standard curve. The result is as Figure 4 As shown, the obtained standard curve equation is y=2.79559x-0.0003, R 2 = 0.9999.

[0113] 2. Enzyme activity assay

[0114] (1) with ddH 2 O respectively prepare 50mM NADP + Coenzyme (Sigma-Aldrich, Cat. No.: N5755), 50mM CDCA (Sigma-Aldrich, Ca...

Embodiment 3

[0121] Example 3. Application of 7α-hydroxysteroid dehydrogenase mutant J-1-1 △C6 to catalyze conversion of chicken bile powder

[0122] 1. HPLC-ELSD method to detect product conversion

[0123] ① Detection method

[0124] Column temperature 40°C; flow rate 0.8mL / min; mobile phase A: 50mM ammonium acetate formic acid solution (pH4.5); mobile phase B: methanol; detector: ELSD (evaporative light scattering detector, Agilent, 1260Infinity ELSD, GB14460009 ), impactor: off; atomizer temperature 80°C; N 2 The flow rate is 1.6L / min. According to the following table linear gradient elution.

[0125] 50mM ammonium acetate formic acid solution (pH4.5): Accurately weigh 3.854g of ammonium acetate and dissolve in 1L of ultrapure water, and adjust the pH to 4.5 with formic acid.

[0126] Gradient Elution of Bile Acid Detection by HPLC-ELSD

[0127]

[0128] ②Standard curve drawing

[0129] Accurately weigh TCDCA and TUDCA standard substances, use a volumetric flask to prepare its...

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Abstract

The invention relates to hydroxysteroid dehydrogenase, in particular to a 7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 triangle C6 and application thereof. The amino acid sequence of the 7 alphahydroxysteroid dehydrogenase mutant is shown as SEQ ID NO: 2, and the 7 alpha-hydroxysteroid dehydrogenase mutant is obtained by truncating 6 amino acids at the C end of 7 alpha-hydroxysteroid dehydrogenase with the amino acid sequence shown as SEQ ID NO: 1. The mutant has substrate selectivity, can specifically catalyze CDCA and taurine or glycine conjugates thereof, has no catalytic activity onCA and taurine or glycine conjugates thereof, can be used for efficiently synthesizing TUDCA from complex substrate chicken gall powder without generating toxic substances, namely by-products TUCA. The invention has huge application potential in the process of obtaining TUDCA through specific biotransformation of TCDCA.

Description

technical field [0001] The present invention relates to hydroxysteroid dehydrogenase, in particular to the mutant J-1-1 △ C6 of 7α-hydroxysteroid dehydrogenase (J-1-1) and its catalytic preparation of TUDCA with complex substrate chicken bile powder as raw material in the application. 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, there are often disadvantages in chemical methods 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 a high degree of stereoselectivity. The enzymatic reaction mediated by hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) has relatively strict stereoselectivity and "not" strict substrate specificity. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/02C12R1/19
CPCC12N9/0006C12N15/70C12P33/02C12Y101/01159
Inventor 潘银平王伯初祝连彩唐士金
Owner CHONGQING UNIV
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