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Modified 7beta-hydroxysteroid dehydrogenase and application thereof

A hydroxysteroid and dehydrogenase technology, applied in the field of enzyme engineering, can solve the problems of unsafe control, low selectivity, and difficulty in industrial scale-up production.

Pending Publication Date: 2020-11-17
SUZHOU LEAD BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Among them, the reduction of 7-keto-lithocholic acid (7-keto-LCA) needs to use metal sodium or Pd / C catalytic hydrogenation, the selectivity is low, and the industrial scale-up production is not easy to control and unsafe

Method used

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  • Modified 7beta-hydroxysteroid dehydrogenase and application thereof
  • Modified 7beta-hydroxysteroid dehydrogenase and application thereof
  • Modified 7beta-hydroxysteroid dehydrogenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Materials and methods

[0113] Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and details of gene cloning operations can be found in the aforementioned Sambrook et al., 1989.

[0114] i) Reagents and instruments:

[0115] DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa Company;

[0116] The plasmid extraction kit was purchased from Axygen;

[0117] 7-Ketolithocholic acid was purchased from McLean, product number A832268, with a purity of 98%;

[0118] Oxidized nicotinamide adenine dinucleotide phosphate (NADP) was purchased from Aladdin, product number N113163, with a purity of 97%.

[0119] ii) Vector and strain: the expression vector used was pET-30a(+), the plasmid was purchased from Novagen, and the host cell used was Escherichia coli BL21(DE3), which was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0120] iii) Sequencing...

Embodiment 2

[0132] Example 2, Preparation and detection of mutants of 7β-HSDH of Clostridium sp.Marseille

[0133] Using the nucleic acid encoding 7β-HSDH (SEQ ID NO: 1) of Clostridium sp. Marseille as a template, the mutant was prepared according to the method of Example 1. The obtained mutants are shown in Table 1. The activity of SEQ ID NO:1 and its mutants was determined according to the method in the vi) of Example 1, and the results are shown in Table 1, wherein relative activity refers to the activity of mutants / SEQ ID NO:1.

[0134] Table 1

[0135]

[0136]

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PUM

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Abstract

The invention relates to a modified 7beta-hydroxysteroid dehydrogenase (7beta-HSDH). Specifically, compared with initial 7beta-HSDH, the modified 7beta-HSDH has an improved activity of catalyzing reduction of 7-keto-lithocholic acid or a derivative thereof to produce ursodeoxycholic acid or a derivative thereof. The invention also relates to a polynucleotide encoding the modified 7beta-HSDH, a vector and host cells expressing the modified 7beta-HSDH, and a method for producing the ursodeoxycholic acid and the derivative thereof by using the modified 7beta-HSDH and the host cells.

Description

technical field [0001] The present invention relates to the field of enzyme engineering. In particular, the present invention relates to a modified 7β-hydroxysteroid dehydrogenase (7β-HSDH) and its use in the production of ursodeoxycholic acid or its derivatives. Background technique [0002] Ursodeoxycholic acid (UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile. Analogues, choleretic effect. It is clinically used to treat gallstones, cholestatic liver disease, fatty liver, various types of hepatitis, toxic liver disorders, cholecystitis, cholangitis and biliary dyspepsia, bile reflux gastritis, eye diseases, etc. [0003] UDCA can be obtained from the gall of bears in captivity, but the yield is low, the source is limited, and there are animal protection problems. Therefore, the artificial synthesis of UDCA is of great significance. The synthesis methods of UDCA mainly include total chemical synthesis and chemical-enzy...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P33/06C12P33/00C12R1/19
CPCC12N9/0006C12Y101/01201C12P33/06C12P33/00C12N15/70
Inventor 谢新开杜好勉
Owner SUZHOU LEAD BIOTECH CO LTD