Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Cloning and application of a new hydroxylase (cytochrome P450) gene of amycolatopsis sp.CGMCC1149

A technology of hydroxylase and amino acid, which is applied in application, genetic engineering, oxidoreductase, etc., can solve the problems of bacteria poisoning, low expression level, limiting the production of statins in Wuxi, etc., and achieve the effect of overcoming the toxic effect

Inactive Publication Date: 2016-04-20
JIANGNAN UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the expression level of cytochrome P450 in Amycolatopsissp. is relatively low, and lovastatin has a certain toxic effect on the bacteria, these all limit the increase of Wuxi statin production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Cloning and application of a new hydroxylase (cytochrome P450) gene of amycolatopsis sp.CGMCC1149
  • Cloning and application of a new hydroxylase (cytochrome P450) gene of amycolatopsis sp.CGMCC1149

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: Construction of expression plasmid

[0015] Design a pair of primers according to the nucleotide sequence of SEQIDNO.1

[0016] P-F: 5′-GAC GAATTC ATGACCCAGACGGTTCC-3';

[0017] P-R: 5′-TAG AAGCTT TTACCAGGTGACCGGCAG-3';

[0018] In order to facilitate the connection of the PCR fragment to the plasmid pEtac, EcoRI and HindIII restriction sites were introduced at the 5' ends of the two primers, respectively, and the underline "_" indicated the introduced restriction sites.

[0019] The PCR program was: 94°C pre-denaturation for 5 min; 94°C denaturation for 1 min, 65°C annealing for 1 min, 72°C extension for 2 min, 30 cycles; 72°C extension for 10 min. After the product was recovered, it was connected to the T vector, transformed into E.coliJM109, and positive transformants were screened using LB resistance plates (with a final concentration of 50ug / mL kanamycin), the plasmid was extracted, and the correctness of the plasmid was verified. The plasmid an...

Embodiment 2

[0020] Example 2: Induced expression of cytochrome P450

[0021] Insert the E.coliDH5α(pEtac-p450) transformant into LB (containing a final concentration of 50ug / mL kanamycin) medium, and culture at 37°C until OD 600 About 0.4-0.6, add IPTG to a final concentration of 0.1 mM, and continue to induce culture at 25°C for 8 hours. Collect the cells by centrifugation, wash the cells twice with 1×TE (pH8.0) buffer, wash the cells with an appropriate amount of wall-breaking buffer (50mM Tris-HCl buffer (pH7.4), containing 20% ​​glycerol, 2mMDTT, 0.1mM EDTA) Suspend the bacteria and perform ultrasonic crushing. The crushing conditions are: power 300W, working for 1s, interval of 3s, and total time of 10min. Centrifuge at 10,000 r / min at 4°C for 15 minutes, collect the supernatant as crude cytochrome P450 enzyme solution, and obtain pure cytochrome P450 after purification.

Embodiment 3

[0022] Example 3: Establishment of lovastatin in vitro hydroxylation system

[0023] In order to overcome the unfavorable factors such as the relatively low expression level of CYP in Amycolatopsissp. and the toxic effect of lovastatin on the bacteria and to efficiently apply CYP, an in vitro hydroxylation system of lovastatin was established for the in vitro hydroxylation product I of lovastatin synthesis. The reaction system is: a total volume of 200 μL, pure CYP, 0.26 mM NADH, ferredoxin reductase (0.04 U), 320 μg ferredoxin, 0.23 mM lovastatin, 100 mMPBS buffer (pH 7.4) to make up 200 μL. React in a water bath at 30°C and 150r / min for 6h, terminate the reaction at 105°C for 5min, and detect the hydroxylated product I of lovastatin.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to Amycolatopsis sp. CGMCC1149 novel hydroxylase (cytochrome P450) gene cloning, and an application of the above gene, and belongs to the fields of the microbiology and the molecular biology. The invention also relates to a novel cytochrome P450 (CYP) gene sequence. The full length of the gene comprises 1212bps, and the gene codes 403 amino acids. A lovastatin hydroxylation system is established and a new method for synthesizing wuxistatin through the biotransformation of the above enzyme is disclosed in order to effectively apply the CYP and realize the high expression of the CYP.

Description

technical field [0001] The invention relates to the cloning of the hydroxylase cytochrome P450 gene in the process of synthesizing Wuxi statin by Amycolatopsissp. Background technique [0002] Elevated cholesterol content is the main cause of hyperlipidemia. In the process of cholesterol synthesis, hydroxymethylglutaryl coenzyme A (HMG-COA) reductase is the key rate-limiting enzyme. HMG-COA reductase inhibitor can inhibit its enzyme activity, make it unable to play a normal role, reduce the generation of cholesterol, and achieve the purpose of lowering blood lipids. [0003] Statins are competitive inhibitors of HMG-COA reductase and are the first choice among lipid-lowering drugs. This experiment discloses a method for producing Wuxi statin in the patent No.: 200410044893.6, patent name: Amycolatopsis and its biotransformation technology of lovastatin into Wuxi statin. The inhibitory effect of Wuxistatin on HMG-COA reductase is 4 times that of lovastatin, and the semi-let...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12P17/06C12R1/01
Inventor 诸葛斌方慧英霍孝雨诸葛健宗红
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com