Nitrilase mutant, gene, carrier, engineering bacteria and application

A technology of nitrilase and mutants, applied in genetic engineering, hydrolase, application, etc.

Active Publication Date: 2019-02-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although some progress has been made in the catalytic production of (R)-mandelic acid using nitrilase, there are still many problems in the industrial production of the nitrilase found, such as high concentration of substrate inhibition, and many reaction parameters require further improvement

Method used

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  • Nitrilase mutant, gene, carrier, engineering bacteria and application
  • Nitrilase mutant, gene, carrier, engineering bacteria and application
  • Nitrilase mutant, gene, carrier, engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Gene synthesis of parent nitrilase

[0027] Parental nitrilase gene (GeneBank: AY487562), in order to express the protein with His-tag after the gene is connected to the vector pET28b, its stop codon was excised, and the codon preference of E.coli was used as a reference pair Its sequence was optimized and NcoI was introduced at the start codon to insert glycine after methionine. The sequence of the newly designed nitrilase gene (tegi) is shown in SEQ ID NO.1 (the amino acid sequence is shown in SEQ ID NO.2). The gene synthesis was entrusted to Shanghai Xuguan Biotechnology Development Co., Ltd. After the gene synthesis Linked into the expression vector pET28b.

Embodiment 2

[0028] Embodiment 2: Construction of mutant library

[0029] Using the parental sequence of the nucleotide sequence shown in SEQ ID NO.1 obtained in Example 1 as a template, the following nucleotide sequences (Table 1) were used as primers to carry out PCR amplification respectively to obtain the nucleotide sequence shown in SEQ ID NO.2 respectively. The 190th serine of the amino acid sequence is mutated to alanine, leucine, valine, glycine, threonine, cysteine, arginine, lysine, histidine, glutamic acid mutant.

[0030] Table 1: Primers

[0031]

[0032]

[0033] The PCR reaction system is:

[0034]

[0035] The PCR program was set as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 7 min, 30 cycles; final extension at 72°C for 10 min, and storage at 4°C.

[0036] After the PCR, 20 μL of the PCR product was taken and detected by 0.9% agarose gel electrophoresis. For the positive PCR prod...

Embodiment 3

[0040] Example 3: Screening of Mutant Library

[0041] (1) Pick the single bacterium colony cultivated in Example 2 into 50mL of LB liquid medium (containing 50ug / mL kanamycin), put it into 37°C, and cultivate it for 10h under the condition that the shaker speed is 150rpm, and then take the seeds The solution was transferred to 100mL LB liquid medium with an inoculum of 2% volume concentration, and cultured at 37°C and the shaker speed was 150rpm. When the concentration of the bacteria was OD 600 When reaching 0.6-0.8, add IPTG with a final concentration of 0.5mM. Then put into 28 DEG C, 150rpm shaker and cultivate for 8 hours, centrifuge to collect the thalline, and then wash the thalline twice with 0.85% physiological saline. Recombinant Escherichia coli bacteria confirmed as positive mutations by sequencing were stored in glycerol tubes (final concentration of glycerol was 15%).

[0042] (2) Determination of enzyme activity and definition of enzyme activity

[0043] Weig...

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PUM

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Abstract

The invention discloses a nitrilase mutant, gene, carrier, engineering bacteria and application in preparation of R-mandelic acid. The nitrilase mutant is obtained by carrying out site-specific mutagenesis on a 190th serine of an amino acid sequence shown in SEQ ID NO.2. The nitrilase mutant (with the amino acid sequence shown in SEQ ID NO.12) disclosed by the invention has the advantages that specific enzyme activity is improved by 3.0 times and enantiomer selectivity is increased to more than 99% from 94% compared with parent nitrilase (with the amino acid sequence shown in SEQ ID NO.2). Results show that the nitrilase mutant (with the amino acid sequence shown in SEQ ID NO.12) can completely hydrolyze 100mM mandelonitrile within 30min with a small amount of wet thalli (10g / L), and enantiomer selectivity is more than 99%, so that the nitrilase mutant disclosed by the invention can be applied to industrialized production of a medical intermediate R-mandelic acid.

Description

(1) Technical field [0001] The invention relates to a nitrilase, in particular to a nitrilase mutant obtained by gene mutation and its application in preparing a pharmaceutical intermediate (R)-mandelic acid. (2) Background technology [0002] Nitrilase (EC 3.5.5.1): An important class of industrial enzymes in the nitrilase superfamily, which contains a specific spatial structure. The protein structural unit is composed of an α-β-β-α sandwich structure. Nitrile hydrolysis This structure of the enzyme has been confirmed by X-ray diffraction technique. According to the crystal structure analysis of worm NiFhit, its active site contains Glu-Lys-Cys 3 residues, Brenner group speculated that the entire nitrilase family may be catalyzed by Glu-Lys-Cys 3 residues. The reaction mechanism of nitrilase is as follows figure 1 As shown, nitrilase first attacks the CN covalent bond to form an intermediate covalently bonded between the enzyme and the substrate, then adds a molecule of w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P7/42C12R1/19
CPCC12N9/78C12P7/42C12Y305/05001
Inventor 薛亚平郑裕国焦标华登恩
Owner ZHEJIANG UNIV OF TECH
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