Cyanohydrins lyase and preparing method and application thereof

A lyase and cyanohydrin technology, applied in the field of cyanohydrin lyase, can solve the problems of insufficient enzyme activity, difficult to reach, low soluble protein content, etc.

Active Publication Date: 2016-10-19
弈柯莱生物科技(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Effenberger et al. reported the recombinant expression of MeHNL in E. coli in E. coli in 1996, but most of the proteins were inclusion bodies and the soluble protein content was low (Angew 1996,35,437)
Chen Shuhua and others cloned the MeHNL gene into the plasmid pPic9K in 2001, and realized the expression in yeast (Acta Bioengineering, 2001, 17(1), 78), but the enzyme activity is still not high enough to meet the requirements of practical application

Method used

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  • Cyanohydrins lyase and preparing method and application thereof
  • Cyanohydrins lyase and preparing method and application thereof
  • Cyanohydrins lyase and preparing method and application thereof

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preparation example Construction

[0102] The preparation method of S-cyanohydrin

[0103] The present invention also provides a kind of preparation method of S-cyanohydrin, described method comprises steps:

[0104] (1) contacting the mutant cyanohydrin lyase of the present invention with a reaction substrate to carry out a catalytic reaction, thereby generating the S-cyanohydrin;

[0105] (2) Separating and purifying the S-cyanohydrin product.

[0106] In a preferred embodiment of the present invention, in the step (1), the reaction substrate is m-phenoxybenzaldehyde and acetone cyanohydrin (or, hydrogen cyanide).

[0107] In a preferred embodiment of the present invention, in the step (1), the temperature of the catalytic reaction is 0-20°C.

[0108] The main advantages of the present invention are:

[0109] (1) The mutated cyanohydrin lyase of the present invention has catalytic activity much higher than that of the wild type cyanohydrin lyase, which is about twice that of the wild type;

[0110] (2) Th...

Embodiment 1

[0113] Construction of embodiment 1 mutant

[0114] According to the protein sequence 1 (SEQ ID NO.: 1) of MeHNL reported by Wajant, according to the codon preference of Escherichia coli, MeHNL DNA sequence 2 (SEQ ID NO.: 2) was synthesized and cloned into plasmid pET28 (purchased from Invitrogene company) NdeI-HindIII site. Using the plasmid as a template, primers were designed, see sequence 3 and sequence 4. Using a random mutagenesis kit, by changing the MnSO 4 concentration to carry out multiple reactions, thereby introducing 1 to 3 point mutations into the MeHNL gene, so that 1-3 amino acids in the amino acid sequence of the MeHNL enzyme are replaced. To amplify the enzyme encoding MeHNL, primers SEQ ID No.3 and SEQ ID No.4 were used as forward and reverse primers, respectively. Both primers contained sites compatible with the PCR-amplified MeHNL gene fragment obtained by site-directed recombination cloning using Gateway Technology. The gel electrophoresis profile of ...

Embodiment 2

[0123] Example 2 strain construction and high-density fermentation

[0124] Synthesize a tryptophan promoter containing sequence 6 (SEQ ID NO.: 6), and connect it to the NcoI and NdeI sites of pET28a, and then connect the coding polynucleotides of sequence 1 and 5 in Example 1 to the aforementioned The NdeI-XhoI site of the Escherichia coli plasmid pTrp2-MeHNL1 and pTrp2-MeHNL5 containing the tandem tryptophan promoter (plasmid map as shown in Figure 4 shown). The plasmid was transformed into Escherichia coli JM109 (purchased from Invitrogene), and the corresponding strains were obtained on Kana resistance plates, inoculated into LB medium, cultured overnight at 37°C, and preserved with 20% glycerol.

[0125] The strains were inoculated in a 1L shake flask filled with 200mL LB medium, and cultured at 37°C, 180-220rpm for 10-16h. The above-mentioned cultivated seeds were inoculated in a 3L upper tank fermentation medium (M9) in a ratio of 10% (v / v) (glucose 4g / L, disodium hy...

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Abstract

The invention provides a cyanohydrins lyase and a preparing method and application thereof. Specifically, a mutational cyanohydrins lyase is obtained. Experiment results prove that the catalysis activity of the mutational cyanohydrins lyase is 200% or more that of wild mutational cyanohydrins lyase.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a cyanohydrin lyase and its application. Background technique [0002] Cyanohydrin lyase is a very useful industrial enzyme in chemical production. Its natural activity is to catalyze the cleavage of cyanohydrin and release hydrocyanic acid. Cyanohydrin lyase can catalyze the reverse reaction, that is, the addition of HCN to aldehydes and ketones to obtain optically active α-cyanohydrin products. [0003] Natural S-cyanohydrin lyase exists in a few plant tissues such as rubber, cassava and sorghum, and its abundance is low, making it difficult to purify. In 1995, Wajant isolated cassava cyanohydrin lyase MeHNL from cassava using a five-step purification method (Plant Sci., 1995,108,1); White et al. used a three-step method to extract MeHNL from cassava leaves, using salt The way of analysis and dialysis has obtained enzyme liquid, but the stereoselectivity th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00
CPCC12N9/88C12P13/004C12Y401/02037
Inventor 田振华罗煜丁时诚瞿旭东
Owner 弈柯莱生物科技(集团)股份有限公司
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