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

Cytochrome P450 BM3 mutant enzyme and application thereof in biocatalytic synthesis of p-hydroxybiphenyl

A P450BM3, cytochrome technology, applied in the field of bioengineering, can solve the problems of cumbersome operation, low yield, unenvironmental protection, etc., and achieve the effect of mild reaction conditions and stable catalytic effect.

Pending Publication Date: 2022-06-28
湖北中农华威生物工程有限公司
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has problems such as low yield, environmental protection, and cumbersome operation.

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
  • Cytochrome P450 BM3 mutant enzyme and application thereof in biocatalytic synthesis of p-hydroxybiphenyl
  • Cytochrome P450 BM3 mutant enzyme and application thereof in biocatalytic synthesis of p-hydroxybiphenyl
  • Cytochrome P450 BM3 mutant enzyme and application thereof in biocatalytic synthesis of p-hydroxybiphenyl

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Construction of cytochrome P450 BM3 mutant enzyme expression vector and engineering bacteria

[0034] 1. Acquisition of P450 BM3 gene

[0035] The Bacillus megaterium purchased by the laboratory from the strain collection center in the early stage was subcultured, and the genome was extracted with a genome extraction kit. The extraction steps were carried out according to the product instructions.

[0036] According to the P450 BM3 gene sequence published on the NCBI website (as shown in SEQ ID NO: 1), Snapgen software was used to assist the design of PCR amplification primers. The synthesis of primers was completed by Shanghai Shenggong, as shown in Table 1.

[0037] Table 1 - P450 BM3 PCR primers

[0038]

[0039]

[0040] Using the extracted genome as a template, the target gene was amplified with the 2×Taq Plus PCRMasterMix kit produced by Tiangen Biochemical Technology Co., Ltd. The PCR reaction conditions are as follows: to a PCR reaction system...

Embodiment 2

[0065] Example 2 Protein expression and purification

[0066] The embodiment of the present invention provides that the mutants obtained in Example 1 (cytochrome P450 BM3 mutant F87G and cytochrome P450 BM3 mutant E267V) are expressed and purified in Escherichia coli to obtain cytochrome P450 BM3 mutant enzymes (mutant F87G and Mutant E267V), specifically comprises the following steps:

[0067] 1. Media and buffers used in protein expression and purification

[0068] LB medium: 10g / L tryptone, 10g / L sodium chloride, 5g / L yeast extract;

[0069] Wash buffer: 10 mM imidazole, 50 mM K 2 HPO 4 -KH 2 PO 4 , 300mM NaCl, 10% glycerol, pH 8.0;

[0070] Elution buffer: 200 mM imidazole, 50 mM K 2 HPO 4 -KH 2 PO 4 , 300mM NaCl, 10% glycerol, pH 8.0;

[0071] Storage buffer: 50mM K 2 HPO 4 -KH 2 PO 4 , 300 mM NaCl, 10% glycerol, pH 8.0.

[0072] 2. Protein expression

[0073] The glycerol bacteria containing the recombinant plasmid were inoculated into 6 mL of LB medium ...

Embodiment 3

[0087] Embodiment 3, adopt biocatalytic method to catalyze biphenyl to synthesize p-hydroxy biphenyl

[0088] In this example, biphenyl is used as the substrate to synthesize p-hydroxybiphenyl through enzymatic catalysis. The total volume of the reaction system in this example is 1 ml, and the reaction system of other specifications can be adjusted accordingly with reference to this reaction system.

[0089] 1. Substrate preparation

[0090] Dissolve the substrate biphenyl with ethanol to obtain a stock solution with a final concentration of 40 mM;

[0091] 2. Preparation of reaction buffer

[0092] The reaction buffer was 50 mM K 2 HPO 4 -KH 2 PO 4 buffer, pH 8.0.

[0093]3. Catalytic reaction

[0094] In the buffer system, add the enzyme (mutant F87G or mutant E267V) at a final concentration of 0.3 mg / mL, then add 10 μL of the substrate biphenyl (dissolved in ethanol) to a final concentration of 400 μM, and add NADPH at a final concentration of 250 μM , the total vol...

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 provides a cytochrome P450BM3 mutant enzyme and application of the cytochrome P450BM3 mutant enzyme in biocatalytic synthesis of p-hydroxybiphenyl, the cytochrome P450BM3 mutant enzyme is obtained by carrying out amino acid mutation on the 87th site and the 267th site of an amino acid sequence of wild type cytochrome P450BM3 monooxygenase, and the method specifically comprises the following steps: A, the cytochrome P450BM3 mutant enzyme is point mutation of a single site, the point mutation comprises: E267V or F87G; the cytochrome P450BM3 mutant enzyme is E267V and F87G, or the cytochrome P450BM3 mutant enzyme is mutated at two sites, namely E267V and F87G. The hydroxylation activity of the cytochrome P450 monooxygenase mutant P450BM3 provided by the invention on biphenyl is improved by nearly 1.5 times compared with the activity of wild type CYP, and the cytochrome P450 monooxygenase mutant P450BM3 has a relatively stable catalytic effect. And the reaction conditions are mild, and no high-temperature and high-pressure environment exists.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a cytochrome P450 BM3 mutant enzyme and its application in biocatalytic synthesis of p-hydroxybiphenyl. Background technique [0002] p-Hydroxybiphenyl is a white needle-like or flake-like solid with a molecular formula of C 12 H 10 O, molecular weight is 170, CAS number is 92-69-3, melting point is 164.5°C, boiling point is 306.5°C, and it is almost insoluble in water. It can be used as dyestuff; intermediate of resin and rubber. The red light sensitized and green light sensitized dyes synthesized with it are one of the main raw materials for color films. It can also be used as an analytical reagent, such as the colorimetric determination of acetaldehyde and lactic acid, and the quantitative determination of muramic acid. Deoxyribonuclease I inhibitor. Intermediates for dyes, resins and rubbers, fungicides, solubilizers for water-soluble paints. Its synthesis is mai...

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
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N15/70C12P7/22
Inventor 魏有福
Owner 湖北中农华威生物工程有限公司
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