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Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification

A technology of Bacillus megaterium and recombinant plasmid, applied in the field of molecular biology

Active Publication Date: 2012-04-04
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been no research reports on the directional modification of the cytochrome P450 enzyme gene of the Bacillus megaterium ALA2 strain and the high expression of the recombinant enzyme

Method used

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  • Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification
  • Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification
  • Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification

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Experimental program
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Embodiment 1

[0029] Embodiment 1: Cloning of Bacillus megaterium P450 gene and recombinant plasmid pET-20b- cyp and pTrc99A- cyp build

[0030] 1.1 Extraction of Genomic DNA of Bacillus megaterium ALA2 strain (Refined Molecular Biology Experiment Guide (Fourth Edition) Beijing: Science Press, 2005)

[0031] 1.1.1 Cultivation of Bacillus megaterium

[0032] Bacillus megaterium ( Bacillus megaterium ) is available from the United States Department of Agriculture, NRRL No. B-21660.

[0033]Bacillus megaterium grows on solid medium (LLB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, agar 15 g / L), pick single colonies to liquid LLB medium . Incubate in a shaker at 37°C for 8-12 h, and collect the cells. The strains were stored at -80°C in LLB medium containing 10% wt glycerol.

[0034] Extraction of Genomic DNA from Bacillus megaterium

[0035] A. Cell Preparation and Lysis

[0036] (1) Bacillus megaterium ALA2 was cultured on a shaker at 37°C for 8 h, and 5 mL o...

Embodiment 2

[0069] Embodiment 2: Directional transformation of P450 gene and recombinant plasmid pTrc 99A - cypI build

[0070] 2.1 Directed transformation of Bacillus megaterium cytochrome P450 enzyme gene

[0071] The active center site of an enzyme protein has an important influence on the catalytic performance of an enzyme. The active center of the enzyme is a three-dimensional space structure composed of several active points, in which the T269 residue is located at the end of the heme domain I-helix, and the molecular oxygen O 2 The combination and activation play an important role. (Yeom H et al , Biochemistry 34 (1995): 14733)

[0072] With the help of Swiss-pdbViewer4.03 software (Arnold K et al , Bioinformatics, 22,195; Schwede T et al , Nucleic Acids Research 31: 3381), with the nucleotide sequence of the original enzyme cyp As a benchmark, it is hoped that the purpose of improving enzyme activity can be achieved by changing the ability of cytochrome P450 enzyme...

Embodiment 3

[0083] Example 3: Recombinant plasmid pTrc99A- cypI Transformation of Escherichia coli and preparation of recombinant enzyme

[0084] 3.1 Electric shock transformation of Escherichia coli

[0085] Electric shock transformation of Escherichia coli High voltage electric transformation is currently the most efficient method for transforming Escherichia coli with plasmid DNA. Prepare Escherichia coli TOP10 competent cells according to conventional methods, aliquot 50 μL into one tube, and store at -80°C.

[0086] Specific steps of electroconversion:

[0087] (1) Take 1 μL of the recombinant plasmid pTrc99A- cyp I Add to 50 μL of melted E. coli competent cell TOP10, and mix well.

[0088] (2) Adjust the electric shock meter so that the electric pulse is 25 μF, the voltage is 2.5 kV, and the resistance is 200 Ω.

[0089] (3) Transfer the transformation mixture to a pre-cooled electroconversion cell, blot the outer surface of the cell, and then put it into the sample chamb...

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Abstract

The product relates to Bacillus megaterium ALA2 cytochrome P450 enzyme gene and methods of recombinant plasmid construction and enzyme purification and the nucleotide sequence cypI of the gene is mentioned in SEQIDNO.1. Through escherichia coli expression carrier selection and gene directed modification, the expression level of recombinant protein substantially is increased by a selected pTrc99A carrier and is 3.8 times higher than the expression level of recombinant protein with a selected pET20b carrier, and the enzyme activity reaches 700U / mL. The modified coding Bacillus megaterium ALA2 cytochromes P450 enzyme gene cypI is inserted in pYrc99A and transformed into escherichia coli. Then the enzyme activity expressed in escherichia coli raises 32% in comparison with that of original gene cyp and reaches 1020U / mL (figure-2).

Description

[0001] technical field [0002] The invention belongs to the technical field of molecular biology, and relates to a method for constructing a gene encoding Bacillus megaterium cytochrome P450 enzyme and a recombinant plasmid thereof, as well as methods for cloning, site-directed mutation and high-efficiency expression and purification thereof. It specifically relates to the bioinformatics analysis of the cytochrome P450 enzyme gene of the original bacillus megaterium, the directional transformation of the gene, and the preparation of the recombinase. [0003] technical background [0004] Cytochrome P450 (cytochrome P450 or CYP) is a superfamily of heme-thiolate proteins involved in the metabolism of endogenous substances and exogenous substances including drugs and environmental compounds. Cytochrome P450 enzymes are widely distributed and have been found in various animals, plants, fungi and bacteria so far. (Estabrook R W, 1996, FASEB J, 10:202-204) [0005] Genetic di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/63C12N15/66C12N9/02C12N1/21C12R1/19
Inventor 李迅王婷王亮亮
Owner NANJING FORESTRY UNIV
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