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Preparation method of S-cyanohydrin lyase and product thereof

A lyase and cyanohydrin technology, applied in the field of preparation of S-cyanohydrin lyase, can solve problems such as difficulty in meeting practical application requirements, insufficient enzyme activity, and low soluble protein content.

Active Publication Date: 2021-10-22
JIANGXI KEYUAN BIOPHARM
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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 Escherichia coli in 1996, but most of the proteins were inclusion bodies and the soluble protein content was low (Angew 1996, 35, 437)
Cheng 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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  • Preparation method of S-cyanohydrin lyase and product thereof
  • Preparation method of S-cyanohydrin lyase and product thereof

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Experimental program
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Effect test

Embodiment 1

[0028] The specific design principle of the present invention is to obtain various mutant cyanohydrin lyases by constructing random mutation and point saturation mutation libraries, and high-throughput screening.

[0029] The source of the S-cyanohydrin lyase mutant is wild-type cyanohydrin lyase, and its specific synthesis method is: the cyanohydrin lyase wild gene is shown in Seq ID No.1, or the cyanohydrin lyase mutant gene is shown in As shown in Seq ID No.2, restriction sites NdeI and HindIII were added at both ends, and the DNA was digested and purified and inserted into the expression vector pET26b(+) to obtain a recombinant plasmid, which was transformed into E.coliBL21(DE3) for construction into recombinant expression strains. The above recombinant strains were cultured in LB medium to OD 600 After = 1.0, 0.2mM IPTG was induced and cultured at 30°C for 4-5 hours, and the wild-type or mutant daughter cells were collected by centrifugation, and the crude enzyme solutio...

Embodiment 2

[0031] The preparation method of wild-type cyanohydrin lyase mutant is as follows:

[0032] The cyanohydrin lyase wild gene sequence of cassava (Manihot esculenta) is shown as Seq ID No.1, and the point of introduction is introduced by error-prone PCR and / or based on the enzyme protein structure / simulated substrate docking Saturation mutation followed by high-throughput screening to obtain the wild-type mutant of cyanohydrin lyase in our invention, as shown in Seq ID No.2.

[0033] The protein sequence after transcription and translation of the cyanohydrin lyase wild gene of cassava (Manihot esculenta) is shown in Seq ID No.3, and the protein sequence translated by the wild-type cyanohydrin lyase mutant is shown in Seq ID No.4. Compared with the wild type cyanohydrin lyase mutant, glutamic acid at position 50 is mutated to glycine, tryptophan at position 128 is mutated to alanine, and lysine at position 226 is mutated to proline The difference, Seq ID No.4 represents the poss...

Embodiment 3

[0036] The steps for high-throughput screening of cyanohydrinase are as follows: at 25°C, add the following reagents in sequence in a 96-well plate, 130 μL of 100 mM potassium phosphate-citrate buffer (pH 5.0), 20 μL of diluted lyase solution, and finally add For 50 μL of phenytocyanine substrate solution, use a microplate reader to read the change in absorbance at 280 nm for 5 minutes, and the change in absorbance represents the level of enzyme activity.

[0037] Phenylethanol substrate solution: use 100 mM potassium phosphate-citrate buffer (pH 3.5) to prepare a concentration of 8 microliters per milliliter of buffer.

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Abstract

The invention provides a preparation method and application of a novel S-cyanohydrin lyase mutant which is derived from plants. Specifically, the cyanohydrin lyase with various mutations is obtained by constructing a random mutation and point saturation mutation library and high-throughput screening, and the catalytic activity of the cyanohydrin lyase mutants is 200%-500% of that of wild cyanohydrin lyase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method and application of S-cyanohydrin lyase. 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. The enzyme solution was obtained by means of dialysis and dialysis, but the stereoselectivity applied ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70
CPCC12N9/88C12N15/70C12Y401/02047
Inventor 曹金辉宗匡喻海亮曾鹏刘建明陈文欢
Owner JIANGXI KEYUAN BIOPHARM
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