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Mutation-modified bacillus megaterium ALA2 cytochrome P450 enzyme, and preparation method and application thereof

A technology of Bacillus megaterium and cytochrome, applied in the field of molecular biology, can solve problems such as unfavorable flow to FAD, affect enzyme catalytic efficiency, reduce electron transfer efficiency, etc., and achieve the effect of increasing utilization efficiency

Inactive Publication Date: 2018-12-25
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to Electronics in P450 BM-3 Flow profile analysis in monooxygenase, Trp 1046 The indolyl side chain is relatively large in size, which causes steric hindrance to the activity of the isoalloxazine ring of FAD, which is not conducive to the flow of electrons from NADPH to FAD, reduces the efficiency of electron transfer, and thus affects the catalytic efficiency of the enzyme

Method used

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  • Mutation-modified bacillus megaterium ALA2 cytochrome P450 enzyme, and preparation method and application thereof
  • Mutation-modified bacillus megaterium ALA2 cytochrome P450 enzyme, and preparation method and application thereof
  • Mutation-modified bacillus megaterium ALA2 cytochrome P450 enzyme, and preparation method and application thereof

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Experimental program
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Embodiment 1

[0020] Embodiment 1: Analysis and directional transformation of Bacillus megaterium P450 gene

[0021] 1.1 Determination of mutation sites

[0022] Directed Modification of Cytochrome P450 Enzyme Gene of Bacillus megaterium

[0023] Submit cytochrome P450 via SWISS-MODEL server (http: / / swissmodel.expasy.org / ) BM-3 Amino acid sequence information for the enzyme monooxygenase, resulting in cytochrome P450 BM-3 Among the locally resolved structures of the enzyme monooxygenase, its NADPH-P450 reductase flavin-binding domain and NADPH complex crystals have good resolution ( PDB ID: 4DQL), can be used for the analysis of coenzyme specificity. With the help of Swiss-pdbViewer4.03 software (Arnold K, Bordoli L, Kopp J T. The SWISS-MODEL workspace: a web-basedenvironment for protein structure homology modeling [J]. Bioinformatics, 2006, 22 (2): 195 -201.; Schwede T, Kopp J, Guex N, et al. SWISS-MODEL: An automated proteinhomology-modeling server. [J]. Nucleic Acids Research, 2003,...

Embodiment 2

[0044] Embodiment 2: the preparation of recombinant enzyme and the mensuration of enzymatic activity

[0045] 2.1 Preparation of recombinant enzyme

[0046] Since the recombinant plasmid pTrc99A-cypt contains a His-tag tag, it was purified by His·Bind Purification Kit (Novagen) to obtain a purified recombinant enzyme. Specific operation process:

[0047] A. Processing of samples

[0048] (1) Resuspend the washed bacteria with 8 mL of 1×Binding Buffer, and break the wall by ultrasonic.

[0049] (2) After breaking the wall, centrifuge at 13,000 g for 30 min, and take the supernatant as the sample.

[0050] B. Handling Columns

[0051] (1) Take 1mL packing material and pack it into the column.

[0052] (2) Wash the column with 3 mL of sterile water.

[0053] (3) Wash the column with 5mL of 1×Charge Buffer.

[0054] (4) Wash the column with 3mL of 1×Binding Buffer.

[0055] C. Loading

[0056] (1) Add the sample to the column and control the flow rate to about 6 drops per...

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Abstract

The invention discloses a mutation-modified bacillus megaterium ALA2 cytochrome P450 enzyme, and a preparation method and application thereof. The amino acid sequence is shown as SEQ ID NO. 1. Throughdirectional transformation of P450BM-3 monooxygenase genes, the utilization efficiency of recombinant protein to coenzymes NADH is improved greatly by three selected mutation points R966L / K972L / W1046H, 2.4 times of the utilization efficiency of original enzymes to the coenzymes NADH, and the enzyme activity reaches 11415 U / mg.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a mutation-modified Bacillus megaterium ALA2 cytochrome P450 enzyme and a preparation method and application thereof. technical background [0002] Derived from Bacillus megaterium (Bacillus megaterium) cytochrome P450 BM-3 (also known as CYP102A1) is a soluble monooxygenase, which can catalyze the terminal hydroxylation of long-chain fatty acids and the epoxidation of double bonds of unsaturated fatty acids under physiological conditions. (Munro AW, Girvan HM, McLean KJ: Cytochrome P450--redoxpartner fusion enzymes.Biochim Biophys Acta 2007,1770(3):345-359.) Research found that P450 BM-3 Monooxygenase is mainly composed of two functional domains: N-terminal P450 domain and C-terminal NADPH-P450 reductase domain. (Degtyarenko KN: Structural domains of P450-containing monooxygenasesystems. Protein engineering 1995, 8 (8): 737-747. Guengerich FP, Martin MV, Sohl CD, Cheng...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0042C12N15/70C12Y106/02004
Inventor 李迅王亮亮卢敏王飞
Owner NANJING FORESTRY UNIV
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