7[alpha]-hydroxysteroid dehydrogenase (HSDH) mutant St-2-2 [delta]C10 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: 2021-01-05
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 (HSDH) mutant St-2-2 [delta]C10 and application thereof
  • 7[alpha]-hydroxysteroid dehydrogenase (HSDH) mutant St-2-2 [delta]C10 and application thereof
  • 7[alpha]-hydroxysteroid dehydrogenase (HSDH) mutant St-2-2 [delta]C10 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of 7α-hydroxysteroid dehydrogenase (St-2-2) mutant

[0036] 1. Design and synthesis of mutant genes

[0037] The amino acid sequence of wild-type 7α-hydroxysteroid dehydrogenase St-2-2 (7α-HSDH St-2-2) is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:3. The gene of 7α-HSDH St-2-2 was isolated from feces samples of healthy black bears in Sichuan Black Bear Conservation and Incubation Base in our laboratory. The full length of its open reading frame is 789bp, encoding 262 amino acids. The isolation and cloning method of this gene is described in the patent application text of the application number 2019106381160 submitted on July 15, 2019, and the invention title is "7α-hydroxysteroid dehydrogenase and its encoding gene and application", which is referred to as The entire content of the patent application is incorporated herein.

[0038] The amino acid sequence (262aa) of wild-type 7α-hydroxysteroid dehydrogenase St-2-2 i...

Embodiment 2

[0114] Example 2. Determination of Enzyme Activity of 7α-Hydroxysteroid Dehydrogenase Mutant St-2-2 ΔC10

[0115] 1. Preparation of NADPH standard curve

[0116] 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 . With the concentration of NADPH solution as the abscissa and the corresponding 340nm light absorption value as the ordinate, a standard curve is drawn. The result is as Figure 4 As shown, the obtained standard curve equation is y=2.79559x-0.0003, R 2 = 0.9999.

[0117] 2. Enzyme activity assay

[0118] (1) with ddH 2 O respectively prepare 50mM NADP + Coenzyme (Sigma-Aldrich, catalog number: N5755), 50mM CDCA (Sigma-Aldr...

Embodiment 3

[0125] Example 3. Application of 7α-hydroxysteroid dehydrogenase mutant St-2-2△C10 catalytic conversion of chicken gall powder

[0126] 1. HPLC-ELSD method to detect product conversion

[0127] ① Detection method

[0128] Column temperature 40°C; flow rate 0.8mL / min; mobile phase A: 50 mM ammonium acetate formic acid solution (pH4.5); mobile phase B: methanol; detector: ELSD (evaporative light scattering detector, Agilent, 1260 Infinity ELSD, GB14460009), impactor: off; atomizer temperature 80°C; N 2 The flow rate is 1.6L / min.

[0129] 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.

[0130] According to the following table linear gradient elution.

[0131] Gradient Elution of Bile Acid Detection by HPLC-ELSD

[0132]

[0133] ②Standard curve drawing

[0134] Accurately weigh TCDCA and TUDCA standard substances, use a volumetric flask to pr...

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Abstract

The invention relates to hydroxysteroid dehydrogenase (HSDH), in particular to a 7[alpha]-HSDH mutant St-2-2 [delta]C10 and application thereof. The amino acid sequence of the 7[alpha]-HSDH mutant isshown as SEQ ID NO: 2, and the 7[alpha]-HSDH mutant is obtained by truncating 10 amino acids at the C end of 7[alpha]-HSDH with the amino acid sequence shown as SEQ ID NO: 1. The mutant has substrateselectivity, can specifically catalyze CDCA and taurine or glycine conjugates thereof, has no catalytic activity on CA and taurine or glycine conjugates thereof, can be used for efficiently synthesizing TUDCA from complex substrate chicken gall powder, does not generate toxic substances, namely by-product TUCA, and has huge application potential in the process of obtaining the TUDCA through specific biotransformation of the TCDCA.

Description

technical field [0001] The present invention relates to hydroxysteroid dehydrogenase, in particular to the mutant St-2-2 △C10 of 7α-hydroxysteroid dehydrogenase (St-2-2) and its catalyzed 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, 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 a high degree of stereoselectivity. The enzymatic reaction mediated by hydroxysteroid dehydrogenase (Hydroxysteroid dehydrogenase, HSDH) has relatively strict stereoselectivity and "not" strict substrate specificity. It has been pointe...

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 祝连彩周明海唐茁洲敖芳林王伯初潘银平唐士金
Owner CHONGQING UNIV
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