A kind of decarboxylase and its application

A decarboxylase and carboxyl technology, applied in the field of decarboxylase, can solve problems such as inactivity, and achieve the effect of important industrial application value

Active Publication Date: 2020-06-09
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3,4-dihydroxyphenylpyruvate has two more hydroxyl groups than phenylpyruvate, which has a greater steric hindrance effect, and the known phenylpyruvate decarboxylase has no activity on it

Method used

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  • A kind of decarboxylase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] This example is the cloning of the decarboxylase gene described in the present invention and the construction of Escherichia coli engineering bacteria.

[0020] 1. Extraction of Proteus mirabilis ATCC 25933 DNA

[0021] The Proteus mirabilis ATCC 25933 strain was cultured in LB medium for 12 hours, centrifuged at 12,000rpm for 10 minutes to obtain the bacterial cells, and the bacterial genome DNA extraction kit (TaKaRa Company) was used to extract the total genomic DNA of the bacterial cells according to the operation, and stored in the refrigerator for later use.

[0022] 2. Competent preparation of Escherichia coli

[0023] (1) Inoculate E. coli DH5α and BL21(DE3) into 250 mL shake flasks containing 20 mL LB medium respectively, and culture overnight at 37° C. and 200 rpm.

[0024] (2) Inoculate in 50mL LB medium according to 1% inoculum amount, and cultivate to OD at 37°C 600 About 0.6 (about 2 ~ 3h).

[0025] (3) Transfer the bacterial solution to a 50 mL pre-coo...

Embodiment 2

[0053] This example is the induced expression and separation and purification of the decarboxylase described in the present invention.

[0054] 1. Add 500 μL of recombinant bacteria solution to 50 mL of LB culture solution. Cultivate at 37°C for 2.5h, and stand at 15°C for 0.5h. Then add 40 μL of 0.5M IPTG, and culture for 24 hours under cold induction at 15°C. Centrifuge the fermentation broth (8000rpm, 10min) to obtain the bacterial cells, redissolve the bacterial cells with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (20mmol / L, pH 7.4), crush them with an ultrasonic breaker, and collect them by centrifugation (8000rpm, 10min) The supernatant was used to obtain a crude enzyme solution.

[0055]2. Use the AKTA avant 150 protein purification system to purify the crude enzyme liquid obtained in step 1 with a nickel column. The elution method is: put the four pipelines A1, A2, B1, and B2 into water, and set the system flow to 20mL / min flow rate, e...

Embodiment 3

[0057] This example is the optimum temperature of the decarboxylase of the present invention. With 3,4-dihydroxyphenylpyruvate (10mM) as the substrate, the substrate and the phosphate buffer solution with pH 7.4 were bathed in water for 15min under different temperature conditions of 20-70°C to measure the activity of decarboxylase and determine the enzyme The optimum reaction temperature is 40°C.

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Abstract

The invention relates to acquisition and cloning and expression of a decarboxylase gene from proteus mirabilis (Proteus mirabilis) and belongs to the field of bioengineering. The decarboxylase is capable of removing carboxyl of 3,4-dihydroxyphenylpyruvic acid to generate 3,4-dihydroxybenzaldehyde. The decarboxylase has important industrial application value.

Description

technical field [0001] The invention clones and expresses a decarboxylase, discloses its nucleotide sequence, amino acid sequence, enzymatic properties and application, and belongs to the field of industrial microorganisms. Background technique [0002] Decarboxylase is usually a thiamine diphosphate-dependent non-oxidative enzyme, which is composed of coenzyme ThDP, Mg 2+ A holoenzyme composed of proteins. [0003] 3,4-Dicarboxyphenylacetaldehyde is an important chemical intermediate, which we found can be obtained by decarboxylation of 3,4-dihydroxyphenylpyruvate. So far, some decarboxylases (Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a keyenzyme of the indole acetic acid pathway.European Journal of Biochemistry, 2003, 270(10), 2322-2331), these decarboxylases can remove the carboxyl group of phenylpyruvate or indolepyruvate to generate the corresponding aldehyde. 3,4-dihydroxyphenylpyruvate has two more hydrox...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/70
CPCC12N9/88C12N15/70C12N2800/101C12Y401/01043
Inventor 蔡宇杰望必莹冯佳婷白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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