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A kind of partial glyceride lipase mutant and its application

A partial glyceride and mutant technology, which is applied in the field of preparation of partial glyceride lipase mutants, can solve the problems of complicated side reactions, easy inactivation of enzymes, low product yield and the like, and achieves avoiding side reactions, improving half-life, Reduce the effect of separation and purification steps

Active Publication Date: 2020-06-19
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved in the present invention is to provide a partial glyceride lipase mutant with improved thermal stability and hydrogen peroxide tolerance, to overcome the complicated side reactions, low product yield, and Problems such as easy inactivation of enzymes in high-concentration oxidizing environments

Method used

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  • A kind of partial glyceride lipase mutant and its application
  • A kind of partial glyceride lipase mutant and its application
  • A kind of partial glyceride lipase mutant and its application

Examples

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Embodiment 1

[0032] The construction of embodiment 1 partial glyceride lipase mutant

[0033] Using the partial glyceride lipase plasmid pGAPZαA-PCL derived from Penicillium as a template, the mutant primers were designed, and the plasmid containing the mutant gene was amplified by PCR. After the PCR amplification product was confirmed by 1% agarose gel electrophoresis, an appropriate enzyme digestion system was designed, and DpnI was added to remove the original template with methylation. After digestion for 4 hours, the digested product was purified by PCR, and the purified product was confirmed by nucleic acid electrophoresis. The obtained purified product was mixed with Escherichia coli competent cells DH5α, placed on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and quickly placed on ice for 5 minutes. Add the competent cells obtained in the previous step to 1ml LB medium and activate at 37°C and 180rpm for 50min. After activation, the competent cells were centrifuged at ...

Embodiment 2

[0047] Embodiment 2 Expression and protein purification of partial glyceride lipase mutant

[0048] Expression of the mutant: the plasmid of the partial glyceride lipase mutant was linearized by DNA restriction endonuclease BlnI at 37°C, and the digested product was confirmed by nucleic acid electrophoresis after 5 hours of digestion. After the mutant plasmid was cut completely, the resulting enzyme-digested product was purified by PCR, and finally eluted with sterile water. The purified product was electrotransferred into Pichia pastoris X-33, and the transformation solution was spread on a YPD plate containing 100 μg / mL bleomycin (Zeocin), cultured at 37°C for 48 hours, and the single colony grown was the expression strain. A single colony of the partial glyceride lipase mutant was picked and inoculated in 50ml YPD medium for 24 hours at 37°C and 200rpm, and then further inoculated into 500ml YPD medium for expansion. After 72 hours, the fermented culture solution was centr...

Embodiment 3

[0055] Embodiment 3 Determination of thermal stability of partial glyceride lipase mutant

[0056] The PCL purified in Example 2 and its mutants Y84R, M209A, I260E and I260R were incubated at 45°C at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 , 12h sampling, placed on ice to prevent further loss of enzyme activity, accurate determination of residual enzyme activity. In this experiment, the method of measuring the content of peroxyacid was used to measure the peroxidase activity of partial glyceride lipase PCL and its mutants.

[0057] Definition of enzyme activity: Under certain conditions, the amount of enzyme required to oxidize 1 μmol MCD per minute is an enzyme activity unit, expressed in U. It is known that MCD has a maximum absorbance value at 290nm, and the corresponding enzyme activity can be calculated by comparing the detected absorbance value with the MCD standard curve.

[0058] The reaction system is: 0.1M pentanoic acid pH5.0 buffer (containing 180μM MCD, 9...

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Abstract

Provided is a partial glyceride lipase mutant obtained by means of the site-directed mutagenesis of a partial glyceride lipase derived from penicillium. One or more following mutation modes are adopted for a mutation site of the partial glyceride lipase: (1) tyrosine at the 84-th site of the protein is mutated to arginine; (2) methionine at the 209-th site of the protein is mutated to alanine; and (3) isoleucine at the 260-th site of the protein is mutated to glutamic acid or arginine. By means of the mutant, the thermal stability and hydrogen peroxide tolerance of partial glyceride lipase are greatly improved. Compared with a wild partial glyceride lipase, the mutant has significantly improved capability of catalyzing esters to be subjected to an epoxidation reaction, and the yield of target products is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method and application of a partial glyceride lipase mutant with improved thermal stability and hydrogen peroxide tolerance. Background technique [0002] As we all know, epoxides are an important class of organic synthesis intermediates, which can be introduced into various types of groups by reacting with halogen-containing, nitrogen, sulfur and other nucleophiles to synthesize many high-value compounds. The traditional method of preparing epoxides often uses chemical methods, using some toxic and expensive chemical catalysts, such as silica-supported titanium, methyl rhenium trioxide, etc., adding hydrogen peroxide or oxygen molecules as oxidants to generate the desired epoxy. Therefore, the preparation of epoxides by chemical methods has a certain toxic effect on the environment and limits the actual industrial production. Biocatalytic epoxidation reaction is a type...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12P7/64
CPCC12N9/20C12P7/6445C12Y301/01003
Inventor 王永华蓝东明袁红谭熙钰杨博
Owner SOUTH CHINA UNIV OF TECH
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