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Linoleate isomerase mutant and application thereof

A technology of linoleic acid isomerase and mutants, applied in the direction of isomerase, cis-trans isomerase, application, etc., can solve the problems of unsatisfactory enzyme activity and industrial application, and achieve good Industrial application prospects, the effect of improving enzyme activity

Active Publication Date: 2017-06-13
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some studies have used genetic engineering technology to construct engineering strains with PAI-producing activity, but the enzyme activity is not ideal enough to reach the level of industrial application

Method used

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  • Linoleate isomerase mutant and application thereof
  • Linoleate isomerase mutant and application thereof
  • Linoleate isomerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Wild-type linoleic acid isomerase (PAI; Gene Bank No. AX062088), but the enzyme activity of the wild-type linoleic acid isomerase is low, which cannot well meet the requirements of industrial applications.

[0032] The wild-type linoleic acid isomerase amino acid sequence is shown in SEQ ID NO: 1, and the PET28a recombinant plasmid containing the wild-type linoleic acid isomerase gene (nucleotide sequence shown in SEQ ID NO: 2) ( With a histidine tag) as a template, saturation mutation was carried out at the M62 and S295 sites. Therefore, use oligo7 software to design blunt-end primers, and then perform PCR site-directed mutagenesis. The primers are shown in Table 1, wherein M62-R and S295-R are reverse primers, and the rest are forward primers. The primers are the same, and the first three bases in the forward primer are mutation sites. In this PCR mutation, KOD-Plus-Neo DNA polymerase kit (purchased from Toyobo Technology Co., Ltd., Shanghai) was used.

[0033] Tabl...

Embodiment 2

[0047] 1. Expression of linoleic acid isomerase and its mutants

[0048] (1) Transform the plasmid containing the wild-type linoleic acid isomerase gene and the mutant plasmid obtained in Example 1 into Escherichia coli JM109 (DE3) competent cells, and then spread it on an LB solid plate and incubate at 37°C for 10 h Afterwards, the growth status of the bacteria was observed, and the recombinant transformants were picked and inoculated in LB medium, and cultured at 37° C. for 18 hours to obtain recombinant transformants.

[0049] (2) Inoculate the recombinant transformant in LB liquid medium containing 5ml of kanamycin (50μg / ml), place it in a shaker at 37°C and 200rpm, and culture it until the OD600 reaches about 0.4-0.5, and obtain seed liquid.

[0050] (3) The seed liquid was inoculated in the self-inducing medium with an inoculation amount of 1% by volume, and cultured at 25° C. for 48 hours. The bacterial solution was broken up by ultrasonic waves until no cells were de...

Embodiment 3

[0057] Example 3 Detection of Enzyme Activity of Linoleic Acid Isomerase and Mutants thereof

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Abstract

The invention discloses a linoleate isomerase mutant and application thereof, and the linoleate isomerase mutant is obtained by mutation of 62th-site methionine of linoleate isomerase with an amino acid sequence as shown in SEQIDNO:1 into alanine or glycine, and mutation of 295th-site serine into alanine, leucine or valine. The enzyme activity for catalysis of linoleic acid for preparation of trans-10,cis-12 conjugated linoleic acid of the linoleate isomerase mutant can be greatly improved compared with that of a wild-type enzyme, especially during double mutation such as M62A / S295L, M62A / S295A, M62A / S295V, M62G / S295L, M62G / S295A and M62G / S295V, the enzyme activity can be increased by about 10 times, and the linoleate isomerase mutant has good prospects for industrial application.

Description

[0001] (1) Technical field [0002] The invention relates to a mutant of linoleic acid isomerase and its application. [0003] (2) Background technology [0004] Conjugated linoleic acid (CLA) is a general term for octadecadienoic acid with conjugated double bonds, which has anti-cancer, anti-oxidation, anti-cardiovascular disease and weight loss effects. The US FDA approved CLA as a food additive in 2008, European countries in 2012, and my country approved CLA as a new resource food in 2009. The market demand is huge. [0005] Ruminant meat, milk and their products are natural food sources of CLA, but the content is low. CLA can also be chemically produced by base-catalyzed isomerization reaction using linoleic acid (LA) as a substrate, but the products are two isomers of c-9,t-11CLA and t-10,c-12CLA It is difficult to separate and purify, and the product is mixed with fatty acid cyclization by-products that are a safety hazard for consumption. Some microorganisms in nature...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12P7/64
CPCC12N9/90C12P7/6427C12Y502/01005
Inventor 陈雪君崔茂林陈振明周硕赖敦岳
Owner HANGZHOU NORMAL UNIVERSITY
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