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P450BM3 mutant, and application of P450BM3 mutant in hydroquinone synthesis using benzene or phenol as substrate

A P450BM3, hydroquinone technology, applied in the application, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of difficult hydroquinone yield, low hydroquinone yield, poor operation safety, etc. The effect of strong industrial application potential, short reaction time and good regioselectivity

Active Publication Date: 2019-01-04
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, the yields of the two kinds of bacteria catalyzing the synthesis of hydroquinone are very low (1g / L), and the key enzymes that play a catalytic role in the bacteria have not been reported, leading to their modification to increase the yield of hydroquinone. It is very difficult to produce
[0006] To sum up, the existing chemical method and "chemical-biological" method have problems such as cumbersome steps, poor operation safety, many by-products, and low product yield. There are technical bottlenecks

Method used

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  • P450BM3 mutant, and application of P450BM3 mutant in hydroquinone synthesis using benzene or phenol as substrate
  • P450BM3 mutant, and application of P450BM3 mutant in hydroquinone synthesis using benzene or phenol as substrate
  • P450BM3 mutant, and application of P450BM3 mutant in hydroquinone synthesis using benzene or phenol as substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Site-directed mutagenesis of cytochrome P450-BM3 monooxygenase

[0043] The gene of wild-type cytochrome P450-BM3 monooxygenase (gene sequence shown in SEQ ID NO.2) was subjected to site-directed mutagenesis by using the large primer PCR mutagenesis technique.

[0044] Taking the 328th amino acid mutation as an example, first design a pair of primers for the mutation site.

[0045] The primers used were:

[0046] Upstream primer: 5'-CTTAATTGCGGGACACGAAACAACAAGTGGTC-3',

[0047] Downstream primer: 5'-CGCAGG aaa AGTTGGCCATAAGCGCAGC-3', where the underlined sequence is the mutation site.

[0048] The PCR system (20 μL) is: 0.5-20 ng template, 1 μL (10 μM) of each pair of mutant primers, 10 μL Prime STARMax DNA polymerase, and sterilized distilled water to make up to 20 μL.

[0049] The PCR reaction program is: (1) denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 sec, (3) annealing at 55°C for 15 sec, (4) extension at 72°C for 50 sec, and a to...

Embodiment 2

[0058] Example 2: Expression, purification and enzyme activity determination of cytochrome P450-BM3 monooxygenase mutants

[0059] The expression strain of the cytochrome P450-BM3 monooxygenase mutant obtained in Example 1 was inoculated in LB medium containing kanamycin (50 μg / mL) (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), shake culture overnight at 37°C, take 500 μL of the bacterial solution and insert it into 200 mL of TB medium (peptone 12 g / L, yeast powder 24 g / L, glycerol 0.4%, KH 2 PO 4 2.31g / L, K 2 HPO 4 12.54g / L), placed in a 500mL Erlenmeyer flask at 37°C and 220rpm for shaking culture, when the absorbance of the culture solution OD 600 When it reaches 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.2mM for induction, and the induction temperature is 25°C. After induction for 14 to 20 hours, the culture medium is centrifuged , the cells were collected and washed twice with 0.1 M potassium phosphate buffer (pH 8.0) to o...

Embodiment 3

[0060] Example 3: Combinatorial Mutations of Cytochrome P450-BM3 Monooxygenases

[0061] On the basis of the mutation points V78F, A82F and A328F obtained by single-point saturation mutation, the three mutation sites of V78F, A82F and A328F were respectively combined to further obtain the combined mutants P450V78F / A82F, P450V78F / A328F, P450A82F / A328F and P450V78F / A82F / A328F.

[0062] Inoculate the recombinant Escherichia coli containing the combined mutation recombinant expression plasmid into LB medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L) containing kanamycin (50μg / mL), and culture with shaking at 37°C Overnight, take 500 μL of bacterial solution and insert it into 200mL TB medium (peptone 12g / L, yeast powder 24g / L, glycerol 0.4%, KH 2 PO 4 2.31g / L, K 2 HPO4 12.54g / L), placed in a 500mL Erlenmeyer flask at 37°C and 220rpm for shaking culture, when the absorbance of the culture solution OD 600 When it reaches 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) wi...

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Abstract

The invention provides a mutation site remarkably associated with the specific enzyme activity of a cytochrome P450BM3 monooxygenase, a cytochrome P450BM3 monooxygenase mutant, a coding gene of the mutant, a recombinant plasmid containing the cytochrome P450BM3 monooxygenase mutant gene, a recombinant transformant containing the cytochrome P450BM3 monooxygenase mutant gene, a preparation method ofthe mutant, and application of the mutant in hydroquinone synthesis using benzene or phenol as a substrate. Compared with a wild cytochrome P450BM3 monooxygenase, the P450BM3 mutant provided by the invention shows high activity and extremely high regioselectivity; and through the mutant, pure hydroquinone can be more effectively prepared, and the production cost can be greatly reduced, so that higher applicability for industrial hydroquinone production is achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a mutation site significantly associated with the specific enzyme activity of cytochrome P450BM3 monooxygenase, a mutant of cytochrome P450BM3 monooxygenase and its coding gene, containing P450BM3 The recombinant expression carrier and the recombinant expression transformant of the monooxygenase mutant gene, the preparation method of the mutant, and the application of the mutant in the synthesis of hydroquinone with benzene or phenol as the substrate. Background technique [0002] Hydroquinone (HQ) is an important intermediate in the chemical and pharmaceutical fields. It can be used as a photographic developer, an antioxidant in rubber and gasoline, a raw material for the manufacture of dyes and pharmaceutical compounds, and a basic raw material for the preparation of biologically active natural products and functional materials such as polymer liquid crystals ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12P7/22
CPCC12N9/0081C12P7/22C12Y114/15006
Inventor 李爱涛周航宇余小娟王斐
Owner HUBEI UNIV
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