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Yeast engineering bacteria for producing trypsin in high-yield manner and construction method of yeast engineering bacteria

A technology of trypsin and yeast engineering, applied in the field of genetic engineering, can solve the problems of low protein expression and low activity of trypsin, and achieve the effects of optimized process, low production cost and increased yield

Inactive Publication Date: 2015-02-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The outstanding problems of heterologous expression of trypsin are low protein expression and low trypsin activity

Method used

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  • Yeast engineering bacteria for producing trypsin in high-yield manner and construction method of yeast engineering bacteria
  • Yeast engineering bacteria for producing trypsin in high-yield manner and construction method of yeast engineering bacteria
  • Yeast engineering bacteria for producing trypsin in high-yield manner and construction method of yeast engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of recombinant plasmid pPIC9K-TLExmtI containing mature trypsin gene (TLExmtI)

[0028] Using the nucleic acid sequence shown in sequence 1 in the patent CN 103173367 A as a template, F primer (sequence shown in SEQ ID NO.6), R primer (sequence shown in SEQ ID NO.7) as primers, obtained by PCR , named R123I mutant, that is, the nucleotide sequence shown in SEQ ID NO.3 was obtained. according to figure 1 The construction method shown, with the gene sequence of the trypsin gene ExmtI shown in SEQ ID NO.3, the thioredoxin TrxA shown in SEQ ID NO.4 as a template, design fusion primers (containing enterokinase digestion on the primers) Amino acid site DDDDK base sequence of these five amino acids), using the fusion PCR method, the TrxA gene is fused to the 5' end of the mature trypsin gene (ExmtI), and four asparagus are inserted before the TrxA gene and the protease gene (ExmtI) amino acid and lysine (D 4 K), promptly obtain the nucleotide fragment...

Embodiment 2

[0030] Example 2 Construction of High Yield Mature Trypsin Yeast Engineering Bacteria

[0031] The Pichia pastoris expression plasmid pPIC9K-α-factor-TLExmtI was linearized with SalI, and the pichia pastoris GS115 competent cells were transformed by electric shock. The specific method is as follows:

[0032] 1) Inoculate pichia pastoris GS115 activated on YPD plate in 25mL / 250mL Erlenmeyer flask, and culture overnight at 30°C; inoculate 1% of the above culture solution into 50mL / 500mL Erlenmeyer flask, and the culture cell concentration OD600 is 1.3-1.5;

[0033] 2) Centrifuge at 5000r / min at 4°C for 10min to collect the cells, suspend the cells in 50mL and 25mL of sterile water respectively; 3) resuspend the above cells in 5mL of 1M sorbitol, centrifuge at 5000r / min at 4°C for 10min to collect the cells;

[0034] 4) Resuspend the above cells in 500 μL of 1M sorbitol, aliquot into 80 μL / 1.5mL EP tubes for electrotransformation of competent cells;

[0035] 5) Mix 20 μL of linear...

Embodiment 3

[0039] Example 3 Recombinant Pichia pastoris 3L tank fermentation

[0040] The engineering bacterium constructed in Example 2 is used as a production strain (the recombinant bacterium constructed in the patent CN 103173367 A is used as a control strain), and after the YPD plate is activated, 50mL / 250mL seed medium is inoculated, and cultivated at 30°C and 220r / min for 24h as Fermentation culture seed liquid. 10% inoculated with 800mL / 3L fermentation medium, pH 5.5, cultured in stages at 30°C: 0-19h, 500rmp / min culture, dissolved oxygen dropped from 100% to about 8%, and then increased to about 60%; 19-34h , the rotation speed gradually increased to 1000rmp / min, and 50% glycerol was fed exponentially, DO began to drop to about 7%, and then rose to 79.1%; 34-144h, 1.8% (V / V) methanol was fed to induce trypsin production.

[0041] Seed medium (g / L): 20 peptone, 10 yeast extract, 20 glucose.

[0042] Fermentation medium (g / L): glycerol 40; K 2 SO 4 18; KOH 4.13; MgSO4 7H 2 O...

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Abstract

The invention discloses a yeast engineering bacteria for producing trypsin in a high-yield manner and a construction method of the yeast engineering bacteria, belonging to the genetic engineering field. According to the constructed yeast engineering bacteria, trypsin of thioredoxin is blended at the front expression end, and four aspartic acids and lysine (D4K) are inserted between the thioredoxin and the trypsin; the trypsin amidase activity (BAPNA (nitroaniline)) is 47.4U / mL and the esterase activity (BAEE (N-benzoyl-L-arginine-ethylester)) is 2667U / mL, the problems of trypsinogen activation and low yield are solved, and the trypsin production method by the yeast engineering bacteria has the advantages of high yield, simplified process and industrial application convenience.

Description

technical field [0001] The invention relates to a yeast engineering bacterium with high trypsin production and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] Trypsin, as an important type of serine protease that was discovered earlier, was first found in the intestinal digestive juice of mammals. In the field of food, it can improve the drying rate of protein in baked food, operability, and shorten the mixing time of dough; improve the quality of dried eggs and egg products; tenderize, hydrolyze blood protein, and restore bone protein in meat processing; In fish meat processing, fish meat is hydrolyzed, viscosity is reduced, fish skin is removed, and fish roe is processed. [0003] In the field of medicine, its main functions are: it can decompose and thin pus, sputum, blood clots, etc., facilitate drainage and drainage, accelerate wound purification, and promote the regeneration of granulation tissue. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/76C12N1/19C12N15/81C12R1/84
CPCC12N9/6427C12Y304/21004
Inventor 康振陈坚堵国成张云丰令桢民
Owner JIANGNAN UNIV
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