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Lipase mutant and uses thereof

A mutant and lipase technology, applied in the field of lipase mutants and their applications, can solve the problems of low enantioselectivity and achieve the effect of improving enantioselectivity

Active Publication Date: 2014-06-25
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Utilize biocatalytic method to synthesize ibuprofen compound can be carried out by reaction such as hydrolysis, esterification, oxidation, reduction etc. that various biocatalysts catalyze, wherein by The kinetic resolution reaction catalyzed by lipase to prepare optically pure ibuprofen compounds is one of the feasible routes. reported (Green Chem, 2011, 13, 2607.), but Candida antarctica lipase B has almost no or only very low enantioselectivity for ester derivatives of ibuprofen compounds (Enzyme Microb Tech, 2006, 39, 924; J Org Chem, 1994, 59, 4410; Biotechnol Bioeng, 2001, 75, 559 .), which needs to improve the existing method route to improve the enantioselectivity of lipase to ibuprofen compounds to the ideal level

Method used

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  • Lipase mutant and uses thereof
  • Lipase mutant and uses thereof
  • Lipase mutant and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Extraction of Genomic DNA from Candida Antarctica (cgmcc 2.2366) and Cloning of Wild Type Lipase B Gene

[0050] After culturing Candida antarctica cgmcc 2.2366 with YPD liquid overnight, the fermentation broth was centrifuged in a 1mL centrifuge tube at 3000r / min for 5min, and the supernatant was discarded to collect the cells, which could be repeated several times to obtain a sufficient amount of cells; Resuspend the DNA lysate, add 0.4g acid-washed glass beads, then add 200μL phenol: chloroform: isoamyl alcohol, cover the tube cap tightly to prevent phenol from leaking when vortexing; c) Vortex at high speed for 3min; d ) Centrifuge at 12000 r / min for 5 minutes, transfer the supernatant (about 200 μL) to a new centrifuge tube; e) Add 700 to the supernatant and add pre-cooled absolute ethanol, and place it in anhydrous at -20°C for 1 hour, 12000 r / min Centrifuge for 15 minutes to precipitate DNA, pour off ethanol, use 800 μL ethanol for precipitation, susp...

Embodiment 2

[0051] Example 2 Polymerase Chain Reaction (PCR)

[0052] Using the extracted Candida Antarctica genomic DNA as a template for PCR reaction, the reaction system is as follows:

[0053] TaKaRaTaq (5 U / μL) 0.25 μL 10×PCR Buffer (Mg 2+ Free) 5 μL MgCl 2 (25mM) 3μL dNTP mixture (2.5 mM each) 4μL template DNA genome 1μL Primer 1 / 3 (10μM) 2μL Primer 2 / 4 (10 μM) 2μL Sterile distilled water Make up to 50 μL

[0054] Amplification program: 94°C: 10 Min, (94°C: 30s, 45°C: 30s, 72°C: 30s) 35 cycles, 72°C, 10min.

[0055] Primer 1: CALB- Eco R I-f: 5Cal B- GAATTCC TACCTTCCGGTTCGGACC-3

[0056] Primer 2: CALB- not I-r: 5CalB-Not I-rC GCGGCC GCTCAGGGGGTGACGATGCC-3;

[0057] Restriction endonuclease cut sites are underlined;

[0058] The DNA fragments amplified by PCR were purified using a gel extraction kit. Containing the pPICZα A plasmid E. coli Top 10' was cultured overnight in LB liquid medium at 37°C...

Embodiment 3

[0061] Example 3 E. coli Preparation and transformation of DH5α competent cells

[0062] a) Take 0.4mL from the seed culture medium and inoculate it into 20mL LB liquid medium for 3h; b) 3000r / min, enrich 2mL of bacteria in 1.5mLEP tube twice in 5min, discard the supernatant; c) add 100 Discard the ice-cold TSS solution, re-suspend the bacteria, and ice-bath for 30 minutes; d) Add 20 μL of connection solution (empty plasmid pPICZα A enzyme-digested fragment, target fragment enzyme-digested fragment and connection solution) and gently swirl to mix well, and ice-bath for 30 minutes; e ) heat shock at 42°C for 60s, ice bath for 2min, and add 600 μL LB liquid medium. Cultivate at 37°C and shake at 150r / min for 1h; f) Take 150μL of each and spread on LLB (ZeocinTM) resistance plate.

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Abstract

The invention relates to the technical field of biology, and relates to a lipase mutant and uses thereof. The mutant is obtained from wild type candida antarctica lipase B (CALB) through mutation. The invention particularly relates to the lipase mutant, a preparing method thereof, and a method of preparing S-configuration ibuprofen type compounds with optical activity through catalysis of hydrolysis of ester derivatives of the ibuprofen type compounds by utilization of the lipase mutant and through kinetic resolution. The wild type candida antarctica lipase B is subjected to mutation to obtain the modified lipase. The lipase is expressed in pichia pastoris engineering bacteria. The lipase comprises an amino acid sequence having at least 85% of identity with the SEQ ID NO.02. The 189 site residue corresponding to the SEQ ID NO.02 of the lipase is an aromatic amino acid residue and the 190 site residue corresponding to the SEQ ID NO.02 of the lipase is a nonpolar amino acid residue. By subjecting the wild type candida antarctica lipase B to mutation, the enantioselectivity of the lipase to the ibuprofen type compounds is improved.

Description

technical field [0001] The present invention relates to the field of biotechnology, and relates to a lipase mutant and its application. The mutant is obtained through mutation from wild-type Candida antarctica lipase B (CALB), and specifically relates to a lipase mutant And its preparation method and using the lipase mutant to catalyze the hydrolysis of the ester derivatives of ibuprofen compounds, kinetic resolution to obtain the optically active S Method of configuring ibuprofen-like compounds. Background technique [0002] Lipase, namely triacylglycerol acyl hydrolase, is a special ester bond hydrolase, which can catalyze the hydrolysis reaction of oil at the oil-water interface to generate fatty acid and glycerol, monoglyceride or diester. Lipase is usually used to catalyze the hydrolysis reaction of ester compounds in the aqueous phase system, catalyze the esterification and transesterification of alcohol compounds in the non-aqueous phase system, and catalyze the amin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/19C12P7/40C12R1/84
CPCC12N9/20C12P7/40C12Y301/01003
Inventor 游松秦斌张新梁萍穆矛王潇莹马国振金旦妮
Owner SHENYANG PHARMA UNIVERSITY
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