Cholesterol oxidase gene, engineering bacterium and application thereof

A technology of cholesterol oxidase and genetic engineering strains, applied in the direction of genetic engineering, oxidoreductase, application, etc., can solve the problems of low utilization rate of degraded sterols, low yield, low substrate conversion rate, etc., to improve production efficiency and product quality Quality, improved utilization, environmental friendliness

Active Publication Date: 2011-08-31
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Most mycobacterium strains and Rhodococcus degrade sterols often have a low substrate conversion rate, resulting in low yields of AD or ADD products, which have become the biggest bottleneck in industrial applications.
[0008] Therefore, it is necessary to study this to overcome the problem of low utilization rate of mycobacteria and rhodococcus to degrade sterols, and to achieve a large amount of important intermediates AD or ADD

Method used

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  • Cholesterol oxidase gene, engineering bacterium and application thereof
  • Cholesterol oxidase gene, engineering bacterium and application thereof
  • Cholesterol oxidase gene, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 , Mycobacterium NwIB-00 cholesterol oxidase gene acquisition, molecular identification and construction of Escherichia coli for heterologous expression

[0037] This example only takes the PCR method as an example. By analyzing the whole genome information of mycobacterium NwIB-01, the complete cholesterol oxidase gene is obtained, and PCR amplification primers are designed to clone cholesterol oxidase from mycobacterium NwIB-00. The DNA sequence of cho-1 (as shown in SEQ ID NO: 1, the corresponding amino acid sequence is shown in SEQ ID NO: 2) and the DNA sequence of cho-2 (as shown in SEQ ID NO: 3, the corresponding amino acid sequence As shown in SEQ ID NO: 4), the specific process is as follows:

[0038] 1.1. Acquisition of full-length cholesterol oxidase gene

[0039] It is currently known that the enzymes involved in cholesterol metabolism in NwIB-00 are: 3-sterone-Δ1-dehydrogenase gene (KSDD) and 3-sterone-9α-hydroxylase gene (KSH), through the softw...

Embodiment 2

[0057] Example 2 , Construction of cho-2 and cho-1 deletion genetically engineered strains in mycobacterium NwIB-00

[0058] This example takes the knockout of cho-2 as an example to illustrate the main technical means and methods of homologous recombination double crossover knockout commonly used in mycobacteria, and the knockout of cho-1 is completed by this method.

[0059] The cho-2 knockout plasmid in mycobacterium NwIB-00 was constructed, and the plasmid was electrotransformed into mycobacteria competent cells. Kanapenicillin and hygromycin B were used for preliminary screening of recombinants, and then rescreening was carried out on sucrose plates to obtain gene knockout recombinant NwIB-00.CHO2-405, and the above recombinants were verified by PCR method. The gene deletion double exchange operation can refer to the prior art (gene replacement using preserved DNA Bhavna G. Gordhan and Tanya Parish).

[0060] The specific steps for implementation are as follows:

[00...

Embodiment 3

[0072] Example 3 , Mycobacterium NwIB-00 and NwIB-01 transformation of steroidal compounds and result analysis method

[0073] Use 1%-10% surfactant, polymer or organic solvent (such as Tween80, ethanol, silicone oil, soybean oil, etc.) to aid the dissolution of the steroidal compound. Two-stage or three-stage cultivation is adopted, and the final transformation medium is inoculated with 1% to 20% of seeds, and the steroid compound can be added at any time. The conditions for steroid conversion are: culture temperature 25-37°C, high dissolved oxygen value, pH controllable between 5.0-8.0, and the end time of the conversion reaction can be determined by thin-layer chromatography (TLC) or gas chromatography (GC) analysis and conversion results. After the reaction, the steroidal conversion product can be extracted three times with the same volume of organic solvents such as ethyl acetate and chloroform, and the reaction liquids are combined and dried in vacuum for further anal...

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Abstract

The invention provides a cholesterol oxidase gene, cholesterol oxidase coded by the cholesterol oxidase gene, an expression vector containing a gene sequence of the cholesterol oxidase gene, a gene engineering recombinant strain containing the expression vector and a method for preparing the cholesterol oxidase. The cholesterol oxidase recombinant strain provided by the invention can be used for performing 3-site hydroxyl dehydrogenation or sterol metabolism on 3-hydroxyl steroids and can also be used for degrading sterols, so that the problem of low utilization ratio of mycobacteria and rhodococcus for degrading sterols in the sterol medicine industry is solved.

Description

technical field [0001] The present invention relates to the field of genetic engineering, more specifically to the technical field of cholesterol oxidase, in particular to two kinds of mycobacterium cholesterol oxidase genes and related engineering bacteria and their application. Background technique [0002] Steroids, also known as steroids, are tightly bound by 17 carbon atoms to form a structure with cyclopentane polyhydrophenanthrene as the parent nucleus. Its structural general formula is as shown in formula I, [0003] [0004] It consists of three six-membered rings and one five-membered ring, which are called A, B, C and D rings respectively, and those with * are chiral carbon atoms. Generally, there are methyl groups on the C10 and C13 positions of the mother nucleus, and there may be hydroxyl (-OH), keto (-C=O) or alkyl groups on the C3, C11, and C17 positions, and some positions of the A ring or B ring contain double Key, there are often side chains of differ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/63C12N1/21C12P33/02C12R1/32
Inventor 魏东芝王风清姚抗陶欣艺
Owner EAST CHINA UNIV OF SCI & TECH
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