Nitrilase mutant and application thereof in preparation of benzoylformic acid compounds

A kind of nitrilase, mutant technology, applied in biological field

Pending Publication Date: 2021-12-07
上海晖胧生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nitrilases have been widely used, but nitrilases that can catalyze benzoylnitr

Method used

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  • Nitrilase mutant and application thereof in preparation of benzoylformic acid compounds
  • Nitrilase mutant and application thereof in preparation of benzoylformic acid compounds
  • Nitrilase mutant and application thereof in preparation of benzoylformic acid compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Construction of wild-type nitrilase recombinant expression strain

[0021] Entrusted Beijing Qingke Xinye Biotechnology Co., Ltd. to provide codon optimization and gene synthesis services, derived from the wild-type nitrilase (AfNLase) gene of Alcaligenes faecalis (Alcaligenes faecalis), the gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in Shown in SEQ ID NO.2. This gene was cloned between the restriction sites EcoRI and HindIII on the pET-28a(+) plasmid to obtain the pET-28a(+)-AfNLase recombinant plasmid. The recombinant plasmid pET-28a(+)-AfNLase was transfected into the host Escherichia coli E.coli BL21(DE3) to obtain the recombinant genetic engineering bacteria E.coli BL21(DE3)-pET-28a(+)-AfNLase.

[0022] LB medium was used to activate and cultivate the genetically engineered bacteria E.coli BL21(DE3)-pET-28a(+)-AfNLase. The specific formula of LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized wat...

Embodiment 2

[0044] 1. The cultivation of engineered bacteria

[0045] Inoculate the engineered bacteria containing related genes into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and culture with shaking at 37°C for 12 hours. Transfer to 500 mL of fresh LB liquid medium also containing 50 μg / mL Kan, shake culture at 37 °C until OD600 reaches about 0.8, add IPTG to its concentration of 0.5 mM, and induce culture at 28 °C for 16-18 h. After the cultivation, the culture solution was centrifuged at 4000rpm for 10min, the supernatant was discarded, the bacterial cells were collected, and stored in a -70°C ultra-low temperature refrigerator until use.

[0046] 2. Determination of Enzyme Activity

[0047] The thalli cells collected after the culture were finished were washed twice with 0.25M PB buffer (pH 7.5). Afterwards, the bacteria were resuspended in 0.25M PB buffer (pH 7.5) twice the volume of the fermentation broth, and the cells were disrupted by ultrasonic to obtain a crude en...

Embodiment 3

[0051] Example 3 Genetically engineered bacteria catalyze benzoyl nitrile to generate acetophenone

[0052] Take the fermentation broth of the genetically engineered bacteria E.coli BL21(DE3)-pET-28a(+)-AfNLase-W144A constructed in Example 1, centrifuge at 4000rpm for 10min to collect the thalline, weigh 0.5g of the wet thalline and wash it with 200mL 0.25M Resuspend broken cells in phosphate buffer (pH 7.5) to obtain crude enzyme solution and add it to a 250mL round bottom flask; weigh 2.0g of benzoyl nitrile into the flask, turn on magnetic stirring, and control the reaction temperature in a water bath to 40°C for hydrolysis For the reaction, 4M NaOH was used to control the reaction pH=7.5. After reacting for 8 hours, the contents of benzoylnitrile and acetophenone in the reaction system were detected by liquid chromatography. The substrate conversion rate was 93%, and the concentration of acetophenone acid was 10.6 g / L.

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Abstract

The invention discloses a nitrilase mutant and application thereof in synthesis of benzoylformic acid compounds. The mutant is obtained by performing single mutation or double mutation on tryptophan at the 144th site or tyrosine at the 191st site of an amino acid sequence as shown in SEQ ID NO.2. The invention further discloses a preparation method of the nitrilase mutant. Compared with non-mutated wild nitrilase, the nitrilase mutant disclosed by the invention has the advantages that the enzyme activity (1.2 U/L) is obviously improved and reaches 162.5 U/L; and when benzoyl nitrile is catalyzed to be hydrolyzed to produce benzoylformic acid, the highest raw material conversion rate can reach 96%, and the method has a very great industrial application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a nitrilase, in particular to the application of the nitrilase and its mutants in synthesizing acetophenone acid compounds. Background technique [0002] α-ketoacids are important intermediates in chemical synthesis. A series of unnatural α-amino acids can be prepared through oxidation and ammoniation, and then used to synthesize a variety of important pharmaceutical intermediates. Phenylglyoxylic acid (PGA), also known as benzoylformic acid, belongs to α-keto acid compounds and is an important synthetic building block. More than 50 kinds of chemical raw materials can be directly synthesized from acetophenone . For example, Glycopyrronium Bromide is used to treat stomach acid and gastric ulcer, Cyclandelate is used to treat cerebrovascular disease, an antispasmodic drug Oxybutynin is used to treat polyuria and frequent urination, anticholinergic drug Atropine, etc.; these The drug is ...

Claims

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Application Information

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IPC IPC(8): C12N9/78C12N15/70C12N1/21C12P7/42C12P7/40C12R1/19
CPCC12N9/78C12Y305/05001C12N15/70C12P7/42C12P7/40
Inventor 马飞鸿杜项龙马士忠
Owner 上海晖胧生物医药有限公司
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