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Higher-activity partial glyceride lipase mutant and application thereof

A partial glyceride and mutant technology, which is applied in the field of partial glyceride lipase mutants, can solve problems such as lack, increased cost input, and product price rise, and achieve good activity.

Active Publication Date: 2014-03-12
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can effectively reduce the content of partial glycerides in oils, they need to use two different types of lipase preparations, which increases the cost input in the production process, resulting in an increase in the price of the final product
Using partial glycerol lipase with high activity to hydrolyze monoglycerides and diglycerides to remove partial glycerides is a feasible method, but this type of lipase reagent is still lacking

Method used

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  • Higher-activity partial glyceride lipase mutant and application thereof
  • Higher-activity partial glyceride lipase mutant and application thereof
  • Higher-activity partial glyceride lipase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Construction of lipase SMG1 wild-type Pichia pastoris expression vector

[0036] According to Genbank's spherical Malassezia partial glycerol lipase smg1 gene (accession number: XM_001732152), Shanghai Sangon Bioengineering Co., Ltd. was entrusted to use the artificial synthesis method and optimize the sequence according to the codon preference of Pichia pastoris (sequence SEQ ID NO: 1). After gene synthesis, use primers SMG For: 5'-GGGGTACCAGCAGTATTTACGCCCGTGGCCG-3' and SMG Rev: 5'ACGCGTCGACTTAATGAGCACCAACCTGAGCT-3' to amplify the mature peptide sequence, PCR amplification conditions: 94°C for 5min; 94°C for 20s, 53°C for 30s, 72°C 80s at ℃, 25 cycles; 7min at 72℃. After the PCR amplification product was purified by the DNA purification kit, the purified gene fragment and plasmid pGAPZαA were digested with restriction enzymes Kpn I and Sal I respectively, ligated, and transformed into E. coli DH5α competent cells . Spread on LB (containing 25ug / ml zeocion) plate. T...

Embodiment 2

[0038] Construction of lipase SMG1278 amino acid site mutant

[0039] The mutants SMG1Phe278Gly and SMG1Phe278Ile were constructed by site-directed mutagenesis technology, and the target mutation sites were introduced by the overlap extension PCR method. Using the SMG1 wild-type gene pGAPZαA-SMG1 as a template, the site-directed mutagenesis reaction conditions were as follows:

[0040] Amplify the mutated upstream fragment:

[0041] 10X pfu buffer:

5

d NTP:

2.5

SMG for:

2.5

Phe278Ile rev:

2.5

Pfu DNA polymerase:

1.5

Double distilled water:

40

PGAPZαA-SMG1:

2

[0042] Amplify the mutated downstream fragment:

[0043] 10X pfu buffer

5

[0044] dNTPs:

2.5

Phe278Ile for:

2.5

3' AOX:

2.5

Pfu DNA polymerase:

1.5

Double distilled water:

40

PGAPZαA-SMG1:

2

[0045] Amplify full-length gene fragments with...

Embodiment 3

[0051] Recombinant expression and purification of lipase SMG1 and its mutants

[0052] The mutant expression plasmid was linearized by the restriction endonuclease Bln I, and then electroporated into Pichia pastoris X-33 competent cells. Spread the transformation liquid on a YPD (100ug / ml Zeocin) plate, culture it at 30°C for 3 days, pick a single colony on the plate and ferment it in 100ml YPD medium for 72 hours, centrifuge and concentrate the fermentation liquid for SDS-PAGE detection, and obtain Recombinant strains capable of expressing lipase SMG1 mutant positive.

[0053] Inoculate the wild-type strain and each lipase mutant strain into 100ml of YPD medium, shake culture at 30°C and 200rpm for 48-72 hours, and collect the fermentation supernatant (10,000rpm, centrifuge for 20min, 4°C).

[0054] The fermentation supernatants of the SMG1 wild type and its mutant strains were suction-filtered with a 0.22um filter membrane. Afterwards, 10KD membrane cells (Vivaflow200, Sar...

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Abstract

The invention discloses a higher-activity partial glyceride lipase mutant and application thereof. The mutant is an enzyme mutant prepared by carrying out site-directed mutagenesis on Malassezia globosa partial glyceride lipase, wherein the mutant site is Phe at the 278th site, and the Phe at the 278th site is mutated into Ile or Gly. The obtained mutant Phe278Ile or Phe278Gly has higher partial glyceride hydrolysis activity, and is more suitable for application in biochemical engineering industry. The partial glyceride hydrolysis activities of the modified SMG1Phe278Ile and SMG1Phe278Gly mutants are enhanced to different degrees, which are respectively 1.84 times and 1.68 times of the wild type SMG1 lipase hydrolysis activity; and the modified SMG1Phe278Ile and SMG1Phe278Gly mutants have higher partial glyceride activity, and can be used for removing partial glyceride in grease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a partial glyceride lipase mutant with improved activity and application thereof. Background technique [0002] Lipase (EC3.1.1.3) is triacylglycerol acyl hydrolase, which can catalyze the hydrolysis of natural substrate oils to produce fatty acids, glycerol and mono- or di-glycerides. Partial glyceride lipase is a lipase that only hydrolyzes monoglycerides and diglycerides, but not triglycerides. Lipase participates in a wide range of catalytic reactions, including catalyzing lipolysis, transesterification, ester synthesis and other reactions, and is widely used in feed additives, oil processing, food industry, biomedicine, daily chemical industry and other fields. [0003] The specific substrate selectivity of partial glyceride lipase can be utilized to selectively remove monoglycerides and diglycerides in oils. Patent JP62061590 discloses a method of preparing hard cream with low ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/81
CPCC12N9/20C12Y301/01003
Inventor 王永华蓝东明刘璐王卫飞杨博
Owner SOUTH CHINA UNIV OF TECH
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