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Styrene cyclooxygenase gene and application thereof

A cyclooxygenase and gene technology, applied in the fields of oxidoreductase, genetic engineering, plant gene improvement, etc., can solve problems such as harsh reaction conditions, low selectivity, and difficult separation and purification

Inactive Publication Date: 2014-09-17
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually, there are many shortcomings in the chemical catalytic method, the reaction conditions are harsh, the selectivity is low, the by-products are many, the separation and purification are difficult, and it will also cause environmental pollution.

Method used

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  • Styrene cyclooxygenase gene and application thereof
  • Styrene cyclooxygenase gene and application thereof
  • Styrene cyclooxygenase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1 screening styrene cyclooxygenase gene

[0087] Environmental samples were enriched in culture medium containing M12 inorganic salt (Hartmans et al. (1989) APPl.Environ.Microbiol., 55(11), 2850-2855) and styrene 2mM for 8 days, spread after dilution Cultured on a screening plate (M12+styrene 2mM+4-chloroindole 0.1mM+0.01% yeast powder+1.5% agar powder) at 30°C for 2 days, blue colonies appeared, picked out, and further purified to obtain a single colony.

[0088] The screened strain was cultured with styrene (10mM) as the only carbon source, and the growth state was good.

[0089] The screened strains were grown for 8-10 hours in a medium containing M12, 3 mM styrene, 0.05% yeast powder, 0.05% tryptone, and 0.1% glucose, centrifuged, and the cells were harvested. The wet bacteria were resuspended in pH 7.0 phosphate buffer, added with 0.5mM 4-chloroindole, transformed at 37°C, 240rpm for 20min, and a dark blue substance was generated.

[0090] Therefore, t...

Embodiment 2

[0124] The optimization of embodiment 2 styrene cyclooxygenase expression conditions

[0125] For the construction and expression method of the recombinant E.coli (pETAB), please refer to the instructions. In order to express the recombinant protein with high activity, a single factor preliminary optimization was carried out on the expression temperature and the concentration of the inducer.

[0126] Expression temperature optimization: When the inducer IPTG is 1mM, induce the expression of styAB gene at 37°C, 30°C, 25°C, 20°C, 15°C, and 10°C respectively, take samples at different induction times, refrigerate and centrifuge, harvest the cells, and use 4- Chlorindole was used as the model substrate to detect the activity of StyAB. The result was that the total enzyme activity was the highest at 20°C and 15°C for 18-21 hours of induction, and there was no significant difference in the total activity between the two, so 20°C was chosen as the expression temperature.

[0127] In...

Embodiment 3

[0129] Embodiment 3 Styrene cyclooxygenase whole cell biotransformation condition optimization

[0130] For protein expression, Example 2 was used to optimize the conditions, and the collected wet bacteria were used for whole-cell biotransformation. Preliminary single-factor optimization was carried out on the conversion reaction temperature and pH value.

[0131] Optimum reaction temperature analysis: take an equal amount of freshly cultured wet bacteria, resuspend in phosphate buffer (0.1M, pH6.5), add 10% BEHP and an equal amount of 4-Bromostyrene. Respectively at 25°C, 28°C, 30°C, 32°C, 35°C, 40°C, 45°C, 240rpm shaking reaction for 24h, take the organic phase to detect the epoxy yield by HPLC, and the epoxy yield is the highest at 30°C.

[0132] Optimum reaction pH analysis: Take an equal amount of freshly cultured wet bacteria, resuspend them in 0.1M phosphate buffer with pHs of 6.0, 6.5, 7.0, 7.5, and 8.0, add 10% BEHP and an equal amount of 4-Bromostyrene, The reactio...

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Abstract

The invention belongs to the technical field of biotechnology, and relates to genetic engineering, biological conversion, and biocatalysis technologies. The invention discloses a styrene cyclooxygenase gene of a pseudomonas strain, and an application thereof in catalyzing an asymmetric epoxidation reaction. The styA of the styrene cyclooxygenase gene is 1248bp, and the styB of the tyrene cyclooxygenase gene is 513bp. The styA and the styB respectively code styrene monooxygenase SMO and reductase. The length of basic group connecting the styA and the styB is 53bp. When a recombinant strain established by using the styrene cyclooxygenase gene provided by the invention is subjected to cyclooxygenase protein expression, whole cells or enzymes are used for converting styrene and analogues thereof. The gene provided by the invention is advantage in high conversion rate, mild conditions, suitability for industrialization, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to genetic engineering, biotransformation and biocatalysis technologies, and discloses a styrene cyclooxygenase gene of bacteria belonging to the genus Pseudomonas and its application in catalyzing asymmetric epoxidation reactions. Background technique [0002] Chiral epoxy compounds are important intermediates in organic synthesis, pharmaceutical industry, and perfume industry, and have a wide range of applications. Conventional epoxy compounds are produced using chemical catalysis. Generally, chemical catalytic methods have many disadvantages, such as harsh reaction conditions, low selectivity, many by-products, difficult separation and purification, and environmental pollution. [0003] With the development of biocatalysis technology, the biocatalysis of cyclooxygenase to obtain epoxy compounds has become a focus of attention. In the past two decades, there have been many literature repo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N15/81C12P17/02
Inventor 吴中柳林晖刘艳乔婧
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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