Lipase gene COLIP and lipase encoded by same

A lipase gene and coding technology, applied in the field of genetically engineered bacteria, can solve problems such as premature termination of expression, low protein expression, and impact on gene expression, and achieve the effect of reducing production costs

Inactive Publication Date: 2014-11-19
WUHAN POLYTECHNIC UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the different preferences of codon usage in different hosts, codons that can be efficiently transcribed in a host are very likely to become low-frequency codons in Pichia pastoris, which is one of the main limiting factors for gene expression; 2) The secondary structure of mRNA, the more complex the secondary structure, the lower the protein expression; 3) GC content and distribution, usually the higher the GC content, the more affected the gene expression, and the low GC content is easy to cause expression 4) The site vulnerable to protease attack or protease action site in the enzyme molecule affects the stability of the gene expression product
Due to the influence of the above factors and the combined effect of many other factors, when the heterologous lipase gene is expressed in Pichia pastoris, the expression amount and the activity of the expression product are not high, and it is difficult to meet the needs of industrial production.

Method used

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  • Lipase gene COLIP and lipase encoded by same
  • Lipase gene COLIP and lipase encoded by same
  • Lipase gene COLIP and lipase encoded by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Design and synthesis of embodiment 1 lipase gene COLIP sequence

[0029] With the aid of BioEdit software, the amino acid sequence of lipase was deduced to ensure that the active center and specific spatial structure characteristics of lipase remained unchanged, and the protease action site in lipase was eliminated to prevent its degradation. With the aid of DNA2.0 software (https: / / www.dna20.com / ), the amino acid sequence was converted into a nucleotide sequence, and a lipase gene COLIP was designed. During the design process, the following influencing factors were mainly improved: for synonymous codons of the same amino acid, the high-frequency codons of Pichia pastoris were used to replace the original low-frequency codons (among them, in the amino acid sequence of lipase, there are 21 amino acids There are synonymous codons, and the usage frequency of the synonymous codons in Pichia pastoris is shown in Table 1); the complex mRNA secondary structure is eliminated, a...

Embodiment 2

[0034] The construction of embodiment 2 Pichia pastoris recombinant genetically engineered bacteria GS115 (COLIP)

[0035] Firstly, the synthetic lipase gene COLIP was connected to pMD18-T simple vector (from Bao Biological Engineering Co., Ltd., product number: 3271). Each 10 μL ligation system contained 1 μL pMD18-T simple vector, 5 μL buffer, 2 μL lipase gene COLIP, 2 μL water, and ligated overnight at 4°C. The ligation mixture was transformed into Escherichia coli (from the China Center for Type Culture Collection, the collection number is CCTCCAB93006), and the recombinant plasmid pMD-COLIP containing the lipase gene COLIP was obtained after detection by PCR. The transformation system of Escherichia coli is: 10 μL of ligation system and 100 μL of Escherichia coli competent cells. The transformation process was: put the ligation mixture on ice for 15 min, then place the ligation mixture at 42°C for 80 sec; then place it on ice for 2 min; add 0.8 mL of LB liquid medium to ...

Embodiment 3

[0039] Fermentation of embodiment 3 Pichia pastoris recombinant genetically engineered bacteria GS115 (COLIP)

[0040] Seed culture: inoculate the activated recombinant Pichia pastoris genetically engineered strain GS115 (COLIP) into 50 mL (250 mL Erlenmeyer flask) seed medium YPD, and cultivate at 28° C. and 250 r / min for 20 hours. The formula of the seed medium YPD is: formula: 1% yeast extract powder, 2% peptone, 2% glucose.

[0041] Fermenter medium: glucose 40g / L, KH 2 PO 4 20g / L, CaSO 4 1g / L, (NH4) 2 SO 4 5g / L, MgSO 4 .7H 2 O10g / L, K 2 SO 4 14g / L, CuSO 4 0.002g / L, MnSO 4 .H 2 O0.003g / L, ZnCl 2 0.007g / L, FeSO 4 .7H 2 O0.007g / L, biotin 0.004g / L.

[0042] Fermentation tank fermentation: under the conditions of the fermentation tank, the above-mentioned seed liquid was inserted into a 10L fermentation tank (the volume of the fermentation medium was 8L) according to the inoculation amount of 5% (V / V), and after culturing at 28°C for 24 hours, 400mL of 20wt was ...

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Abstract

The invention belongs to the field of artificial synthetic gene, and particularly relates to a lipase gene COLIP, a lipase encoded by the same and gene engineering bacteria for expressing the lipase gene COLIP. The invention discloses the lipase gene COLIP having the nucleotide sequence shown in SEQIDNO.1, and the lipase encoded by the lipase gene has the amino acid sequence shown in SEQIDNO.2. Compared with the prior art, the obtained lipase gene COLIP can be ultra-efficiently expressed in pichia pastoris, and the constructed pichia pastoris gene engineering bacteria GS115 (COLIP) can be used for mass production of the lipase. Fermentation results show that under a condition of a 10 L fermentation tank, fermentation is carried out for 120 hours, the enzyme activity of the lipase in a fermentation broth can be stabilized at 45000 U/mL or more, and the protein content in the fermentation broth is stabilized at 3.2 g/L or more.

Description

technical field [0001] The invention belongs to the field of artificially synthesized genes, and in particular relates to a lipase gene COLIP, a lipase coded therefor, and a genetically engineered bacterium expressing the lipase gene COLIP. Background technique [0002] Lipase (EC3.1.1.3, also known as triacylglycerol acyl hydrolase) is an important industrial enzyme, widely used in feed, food, leather, washing, oil chemical industry, synthesis of prodrugs, chiral drugs The split and industrial fields such as bioenergy. Lipase plays an important role in lipid metabolism and is the most basic enzyme for fat digestion and utilization. It can hydrolyze fat into free fatty acids, glycerol and monoglycerides for animal absorption and utilization. The digestive function of young animals with a single stomach is not fully developed, the secretion of endogenous digestive enzymes is insufficient, and the rate of fat digestion and absorption is low. Adding lipase in the diet can tar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/20C12N15/81C12N1/19C12N15/66C12R1/84
Inventor 杨江科詹志春周文静毛玲缪礼鸿
Owner WUHAN POLYTECHNIC UNIVERSITY
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