Method for catalytically synthesizing scopolamine and recombinant bacterial strain

A technology of recombinant strain and scopolamine, which is applied in the field of bioengineering, can solve the problems of complex process flow, environmental pollution, long cycle and the like, and achieves the effect of wide application prospect.

Inactive Publication Date: 2013-03-13
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long cycle required by the traditional breeding method; the method of chemical synthesis has many problems such as complex process, high cost and environmental pollution; the method of plant cell suspension culture has problems such as low content of tropane alkaloids and poor stability.

Method used

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  • Method for catalytically synthesizing scopolamine and recombinant bacterial strain
  • Method for catalytically synthesizing scopolamine and recombinant bacterial strain
  • Method for catalytically synthesizing scopolamine and recombinant bacterial strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 The method for preparing the full-length cDNA of Anisodus acutangulus H6H cDNA containing three parts of scopolamine 6-beta-hydroxylase in Escherichia coli

[0031] Ligate the amplified product obtained after PCR amplification of the plasmid template and primers to the pMD18-T vector, transform Escherichia coli DH5α to obtain E. coli clones, and pick single-clonal colonies to screen positive clones by PCR; extract the plasmid for further enzyme digestion verify. The results showed that the gene of three-point hyoscyamine 6-β-hydroxylase (hereinafter referred to as AaH6H) had been cloned into pMD18T.

[0032] The Escherichia coli DH5α was purchased from Shanghai Yingjun Biotechnology Co., Ltd.;

[0033] The pMD18-T vector was purchased from Bao Biological Engineering (Dalian) Company;

[0034] The described carrying out PCR amplification of the plasmid template and the primer set refers to: carrying out PCR amplification with the plasmid containing the full l...

Embodiment 2

[0037]Example 2 Construction of prokaryotic expression vector pGEX4T-1-AaH6H, pET30a-AaH6H

[0038] Based on the above pMD18T, the AaH6H gene in pMD18T- was constructed on pGEX4T-1 and pET30a vectors. Specifically, BamH I / Sal I double digestion of pMD18T-AaH6H, pGEX4T-1 and pET30a; recovery of genes and large vector fragments; ligation transformation, picking of single clone colony PCR screening of positive clones; extraction of plasmids for further digestion verification. The results showed that the gene had been successfully constructed into the prokaryotic expression vectors pGEX4T-1 and pET30a, thereby obtaining the prokaryotic expression vectors pGEX4T-1-AaH6H and pET30a-AaH6H containing AaH6H.

[0039] The pGEX4T-1 and pET30a plasmids were purchased from Shanghai Meiji Biotechnology Co., Ltd.

Embodiment 3

[0040] Embodiment 3 obtains recombinant bacterial strain bacterium colony

[0041] The obtained expression plasmid vector was transformed into Escherichia coli BL by heat shock method 21 (DE 3 ) competent cells to obtain recombinant Escherichia coli colonies.

[0042] The Escherichia coli BL 21 (DE 3 ) was purchased from Shanghai Yingjun Biotechnology Co., Ltd.;

[0043] The Escherichia coli BL 21 (DE 3 ) Competent cells were prepared in the following way: pick BL on the reactivated LB solid medium plate 21 (DE 3 ) single colony in 50ml LB liquid medium, cultured with vigorous shaking at 37°C until OD600=0.3, transferred the bacteria to a sterile, ice-precooled 80mL centrifuge tube, placed on ice for 10min, and made the culture Cool to 0 °C. 5,000rpm, 4°C, centrifuge for 5min, pour out the culture medium as much as possible; add 30ml pre-cooled 0.1M CaCl 2 Suspend the cells in the solution; place on ice for 20 minutes; centrifuge at 5,000 rpm, 4°C for 5 minutes. Car...

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Abstract

The invention belongs to the technical field of biological engineering and discloses a recombinant bacterial strain and a method for catalytically synthesizing scopolamine by using the bacterial strain. The recombinant bacterial strain comprises exogenous acutangular anisodus root hyoscyamine 6 beta-hydroxylase genes and can convert a hydroxylase substrate into the scopolamine, wherein the conversion efficiency is 80 percent. The invention provides a method for producing the scopolamine with high medical value. The scopolamine can meet the important clinical requirement and is a novel high-quality medicinal resource.

Description

technical field [0001] The invention relates to the field of bioengineering, specifically bioengineering synthesis of alkaloids with high medicinal value, which is a kind of alkaloids with high medicinal value obtained by transforming and transforming E. The Scopolamine Method. Background technique [0002] Alkaloids (Alkaloids) are one of the important active ingredients in Chinese herbal medicine. They are a type of nitrogen-containing basic organic compounds that exist in living organisms (mainly plants). Natural alkaloids have strong physiological activities, including hyoscyamine and scopolamine. , Anisodamine and Anisodamine. These alkaloids have strong anticholinergic physiological activity and mainly act on the parasympathetic nervous system. Among them, hyoscyamine and scopolamine have been widely used for a long time as the most common anticholinergic drugs and antispasmodic analgesics. However, the resource plants rich in scopolamine in nature are less than sco...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/19
Inventor 开国银刘源云倪晓宁郭莺莺
Owner SHANGHAI NORMAL UNIVERSITY
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