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CYP119-T213G enzyme and purification method thereof

A CYP119-T213G, 7.CYP119-T213G technology, applied in the field of CYP119-T213G enzymes and their purification, can solve the problems of reduced catalytic efficiency and achieve the effects of improving epoxidation reactions, high corresponding selectivity, and wide application value

Active Publication Date: 2014-09-24
LUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Several amino acid mutations of A, V, S, F, and W have been carried out at position 213. Only the catalytic efficiency of T213A is close to that of the wild-type enzyme, and the catalytic efficiency of other mutants is significantly reduced.

Method used

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  • CYP119-T213G enzyme and purification method thereof
  • CYP119-T213G enzyme and purification method thereof
  • CYP119-T213G enzyme and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 CYP119-T213G enzyme expression vector construction

[0018] Mutation of CYP119 enzyme: The primer sequences of the mutation site designed according to the pET30a-CYP119-T213G vector are: SEQ ID No.3 and SEQ ID No.4. Site-directed mutagenesis was performed using the QuickChange Lighting Site-directed Mutagenesis Kit, and E. coli DH5α was transformed. Select positive clones for sequencing to confirm whether the mutation is completed and the accuracy of the mutated site.

[0019] SEQ ID No.3: Upstream primer for amplifying CYP119-T213G enzyme

[0020] 5'-TTCTCATAGCGGGTAATGAG ACAACTAACTTAATATCAAA-3', the bold underlined site is the mutation site.

[0021] SEQ ID No.4: Downstream primer for amplifying CYP119-T213G enzyme

[0022] 5'-TTTGATATTAAGTTAGTTGT CTCATTACCCGCTATGAGAA-3', the bold underlined site is the mutation site.

Embodiment 2

[0023] A large amount of expression of embodiment 2 CYP119-T213G enzyme

[0024] The pET30a-CYP119-T213G plasmid was transformed into BL21(DE3)plysS Escherichia coli competent cells. Select positive single colonies, inoculate them in 5 mL double-antibody LB liquid medium, and culture overnight at 37°C with shaking. Take 2 mL of the overnight cultured bacterial liquid and put it in 1 L of double-antibody TB culture medium. Add 250 μl / L Trace Element, culture at 37°C with shaking until OD0.6. Add 0.4mM IPTG to a final concentration of 0.4mM, induce at 32°C for 45h.

Embodiment 3

[0025] The purification of embodiment 3CYP119-T213G enzyme

[0026] Take the bacteria liquid fermented in Example 2, centrifuge at 12000 rpm, 4°C for 10 min, discard the supernatant, add 20 mL pH7.4, 50 mMPBS solution to fully suspend the bacteria. Place the sample on ice water, and use an ultrasonic cell breaker with 50% power, 3s-3s, 20 minutes to break it twice. After cell destruction, heat in a water bath at 55°C for 15 minutes, centrifuge at 12,000 rpm at 4°C for 40 minutes, and take the supernatant as the crude enzyme solution.

[0027] Then the crude enzyme solution is purified, and the equilibrium solution I is eluted for 5 column volumes; the crude enzyme solution filtered by a 0.22 μm filter membrane is put on the column; the equilibrium solution I is washed for 5 column volumes; the equilibrium solution II is washed for 5 column volumes; Wash 6 column volumes with eluent I; wash 5 column volumes with eluent II, and collect the eluate.

[0028] The collected eluate...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a CYP119-T213G enzyme and a purification method thereof. The invention aims to provide a CYP119 enzyme with higher catalytic efficiency. According to the technical scheme, the amino acid sequence of the CYP119-T213G enzyme is disclosed as SEQ ID NO.2. The invention also relates to a vector for expressing the CYP119-T213G enzyme. The invention also relates to a purification method of the CYP119-T213G enzyme. The invention provides a CYP119 enzyme with higher catalytic efficiency, which has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to CYP119-T213G enzyme and a purification method thereof. Background technique [0002] The CYP119 enzyme (cytochrome P450 119) comes from the acidophilic and thermophilic Sulfolobus solfataricus isolated from the crater of Yellowstone Park, so the enzyme has the functions of acid resistance and high temperature resistance. CYP119 enzyme can catalyze the epoxidation reaction of substrates such as lauric acid and styrene, which is green and has a high application prospect. It has been reported in the literature that CYP119 can catalyze the hydroxylation of lauric acid and the epoxidation of styrene under the conditions of hydrogen peroxide or pseudomonas redoxin-reductase (Pd / PdR) and coenzyme NADPH. In the catalase pathway of styrene epoxidation reaction catalyzed by CYP119 enzyme at normal temperature, the catalytic constant is Kcat=0.6min -1 (Laura S. Koo et al...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
CPCC12N9/0081C12Y114/15006
Inventor 张春王钦李静郭建敏杜曦何芳
Owner LUZHOU MEDICAL COLLEGE
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