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Nitrilase mutant and application thereof

A technology of nitrilase and mutants, applied in the direction of hydrolytic enzymes, enzymes, and the introduction of foreign genetic material using vectors, etc.

Active Publication Date: 2015-07-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in the catalytic production of R-o-chloromandelic acid by using nitrilase, in order to achieve the requirements of industrial application, we should further improve the catalytic activity and enantioselectivity of the enzyme.

Method used

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  • Nitrilase mutant and application thereof
  • Nitrilase mutant and application thereof
  • Nitrilase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Gene synthesis of parent nitrilase

[0033] In order to express the protein with the His-tag after the gene of the parental nitrilase (GeneBank: AY487562) was connected to the vector pET-28b, its stop codon was removed, and the codon preference of E. The sequence was sequence 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 to the expression vector pET-28b.

Embodiment 2

[0034] Construction of embodiment 2 mutant library

[0035] Using the parental sequence of the nucleotide sequence shown in SEQ ID NO.1 obtained in Example 1 as a template, the following two pairs of nucleotide sequences were used as primers to perform PCR amplification respectively, and the 132-threonine was mutated into the essence respectively. Amino acid single mutant Thr132-Arg (the nucleotide sequence is shown in SEQ ID NO.3, the amino acid sequence is shown in SEQ ID NO.4), the 189-position phenylalanine is mutated into the single mutant Phe189 of serine -Ser (the nucleotide sequence is shown in SEQ ID NO.5, the amino acid sequence is shown in SEQ ID NO.6) and the single mutant Phe189-Thr (nucleoside The acid sequence is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8).

[0036] Two pairs of oligonucleotide primers are as follows (5'-3'):

[0037] Forward primer (132Thr): CTGAAACCGACCNNNGTGGAGCGTACCCTG

[0038] Reverse primer (132Thr): CAGGG...

Embodiment 3

[0048] Example 3: Screening of Mutant Library

[0049] (1) Primary screening of mutant library

[0050] Pick a single colony cultured in Example 2 and place it in a 96-well plate containing 1 mL of LB culture solution, add Kan (final concentration 50 μg / mL), and culture at 37°C until OD 600 When it reaches 0.6-0.8, add IPTG (final concentration 0.1mM) and induce at 28°C for 12h. Centrifuge at 9000rpm for 10min, remove the supernatant, and obtain bacterial cells. Pick wet bacteria and resuspend with 1mL (pH 7.5, 0.1M) phosphate buffer to prepare bacteria suspension. The reaction was carried out in an Ep tube, in a 1mL system: 900μl (pH 7.5, 0.1M) phosphate buffer (containing 5% methanol at the final volume concentration), 100μL of the above bacterial suspension, with a final concentration of 20mM o-chloromandelonitrile, at a temperature of 40°C , rotate at 1000 rpm, react for 5 min, and stop the reaction with 10 μl of 6M HCl. Enzyme activity in resting cells was measured us...

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Abstract

The invention discloses a nitrilase mutant and its application in R-o-chloromandelic acid synthesis. The mutant is obtained by single mutation or double mutation of the 132nd threonine or 189th phenylalanine on the amino acid sequence shown as SEQ ID NO.2. Compared with non-mutant nitrilase, the enzyme activity is increased by 3.70 times and reaches 1.08U / mg, and the enantioselectivity is up to 99%. Also, the result shows that in a two-phase system (with ratio of toluene to water being 2:8) the nitrilase mutant can catalyze 300mM o-chloromandelonitrile, and the yield is 90.8%. Through fed batch of the substrate, a maximum of 500mM o-chloromandelonitrile can be catalyzed, the yield is 90%, and the enantioselectivity is greater than 99%.

Description

(1) Technical field [0001] The invention relates to a nitrilase, in particular to a mutant nitrilase and its application in the synthesis of R-o-chloromandelic acid. (2) Background technology [0002] Nitrilase (EC 3.5.5.1): a class of important industrial enzymes in the nitrilase superfamily, with αββα folding structure and catalytic triplet Glu-Lys-Cys, generally exists in the form of homologous multimer, and sub- The number of bases is generally an even number, and the molecular weight of its single subunit is mostly between 30-45kDa. Its main function is to hydrolyze nitrile compounds to generate corresponding carboxylic acids. According to the different catalytic substrates, nitrilases can be divided into three categories. , namely fatty nitrilase (aliphatic nitrilase), aromatic nitrilase (aromatic nitrilase, mainly hydrolyze aromatic nitrile, heterocyclic nitrile) and aryl fatty nitrilase (arylaliphatic nitrilase). Utilizing the catalytic activity of nitrilase is an e...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/70C12N1/21C12P41/00C12P7/42
CPCC12N9/78C12P7/42C12Y305/05001
Inventor 薛亚平郑裕国施成赐徐喆柳志强
Owner ZHEJIANG UNIV OF TECH
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