Organic solvent-tolerant lipase mutant and application thereof

An organic solvent and lipase technology, applied in the field of genetic engineering and enzyme engineering, can solve problems such as application limitations and inactivation, and achieve the effects of prolonging life, improving production efficiency, and saving production costs

Active Publication Date: 2019-11-19
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The substrates of lipase are often water-insoluble, and have better solubility in organic phases, but in organic solvents, most of the enzyme activities are inhibited or even inactivated, so its application in biocatalysis is restricted. very restrictive

Method used

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  • Organic solvent-tolerant lipase mutant and application thereof
  • Organic solvent-tolerant lipase mutant and application thereof
  • Organic solvent-tolerant lipase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Construction of embodiment 1 mutant expression vector

[0026] According to the QuikChange program of Stratagene Company, mutant R191A, Q201A, V203A, V199F, H139R, V48D and P252D primers were designed on the QuikChange primer design website (http: / / www.genomics.agilent.com / primerDesignProgram.jsp). The lipase MAS1 gene was subjected to site-directed mutation in the adopted site-directed mutation (QuikChange Site-directed Mutagenesis). Specifically as shown in Table 1:

[0027] Table 1 Reaction system for site-directed mutation of lipase gene

[0028]

[0029] The PCR reaction conditions were: pre-denaturation at 98°C for 3 minutes; denaturation at 94°C for 10 seconds, extension at 68°C for 4 minutes, 20 cycles; 72°C for 7 minutes. PCR products were checked by 1% agarose gel electrophoresis. After the PCR product was confirmed by detection, DpnI was added to remove the original template strand with methylation. The enzyme digestion system is as follows: enzyme dig...

Embodiment 2

[0031] Embodiment 2 mutant enzyme protein preparation and purification

[0032] The pET22b expression vector containing MAS1 lipase (the amino acid sequence is SEQ ID NO: 1) and its mutant gene is transformed into BL21 (DE3) strains, and induced expression:

[0033] (1) Inoculate 200 μL of positive cloned glycerol bacteria into 50 mL of low-salt LB (containing 100 μg / mL ampicillin) liquid medium, and culture overnight at 37° C. and 200 rpm for 15 to 18 hours.

[0034] (2) Inoculate the seed liquid into 500mL LB (containing 100μg / mL ampicillin) liquid medium according to the inoculum size of 2%, culture at 37°C and 200rpm to OD600 to 0.6-0.8, and then add 1M IPTG solution until the final concentration of IPTG is 0.4 mM, 15°C, 200rpm induction culture for 18h.

[0035] Purification of recombinant protein after induction:

[0036] (1) Bacteria collection: Pre-cool the bacterial solution on ice for about 15 minutes, then centrifuge at 4°C and 10,000 rpm for 10 minutes, and disca...

Embodiment 3

[0039] Example 3 Screening of tolerance to organic solvent mutants

[0040]The enzyme activity was determined by alkaline titration. The specific method was: add 4mL olive oil polyvinyl alcohol emulsion and 5mL buffer solution to a 100mL Erlenmeyer flask with a stopper, preheat for 5min under a constant temperature water bath shaker, and then add 1mL to the experimental group to dilute appropriately. For the control group, 1 mL of the buffer solution in which the protein was added was added, and after 5 minutes of reaction at 200 rpm, 15 mL of 95% ethanol was added to terminate the reaction. After the reaction, add 2 drops of phenolphthalein solution and titrate with 0.05mol / L NaOH standard solution. Under certain reaction conditions, the amount of enzyme required to catalyze the hydrolysis of the substrate to generate 1 μmol of fatty acid per minute is defined as 1 enzyme activity unit, expressed in U.

[0041] The influence of organic solvents on enzyme stability: Add metha...

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Abstract

The invention discloses an organic solvent-tolerant lipase mutant and an application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. An enzyme mutation library is designed and constructed based on enzyme protein structure analysis, the organic solvent tolerance enhanced lipase mutant is obtained by screening, and a pichia pastoris engineered strain whichcan efficiently express the mutant is constructed and obtained. Compared with the wild type, the organic solvent tolerance enhanced lipase mutant has significantly enhanced tolerance to organic solvents, residual vitality of the mutant is still kept to be 75% after treatment with 50% ethanol for 2 h, and the residual vitality is about 3 times that of the wild type. The lipase mutant can be appliedin preparation and processing of biodiesel and flavor esters, and has good stability, and a service life can be prolonged in the application process, so that the production cost is saved, and production efficiency is improved.

Description

technical field [0001] The invention relates to the fields of genetic engineering and enzyme engineering, in particular to an organic solvent-tolerant lipase mutant and application thereof. Background technique [0002] Lipase, triacylglycerol acyl hydrolase, catalyzes the hydrolysis of natural substrate oils to produce fatty acids, glycerol and mono- or di-glycerides. The basic unit of lipase is only amino acids, usually only one polypeptide chain. Its catalytic activity is solely determined by its protein structure (Schmid et al., 1998). Lipase has a wide range of applications and has become the third largest industrial enzyme on the market. Lipase can catalyze reactions such as lipolysis, transesterification, and ester synthesis, and is widely used in industries such as feed additives, oil processing, food, medicine, and daily chemicals. [0003] Lipase can catalyze the hydrolysis of ester bonds in an aqueous environment, and can catalyze transesterification, esterific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12P7/64C12R1/84
CPCC12N9/20C12N15/815C12P7/649C12Y301/01003Y02E50/10
Inventor 王永华蓝东明赵泽鑫杨博
Owner SOUTH CHINA UNIV OF TECH
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