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Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application

A trypsin and thermal stability technology, applied in the field of genetic engineering, can solve the problems of long fermentation period of Streptomyces, difficult fermentation control, low yield, etc., and achieve the effects of easy industrial application, simple extraction process and high product purity

Inactive Publication Date: 2013-06-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the production of Streptomyces-derived trypsin is restricted by factors such as the long fermentation period of Streptomyces, the low enzyme activity expressed by wild bacteria, and the complex regulation of trypsin gene expression.
[0003] There are many studies on the construction and expression of recombinant genetically engineered strains of trypsin derived from animals, but due to differences in the regulation of transcription and translation in the expression of eukaryotic proteins by prokaryotic organisms, most of the exogenously expressed trypsin is inclusion body protein , the trypsin coding gene of the mouse is fused with the bacterial alkaline phosphatase promoter (phoA) in E.coli, and the exogenous expression of trypsin is realized by adding exogenous kinase to activate trypsinogen, but it needs The addition of exogenous kinase is activated, and the yield is low, while the activity of Streptomyces trypsin does not need to add any exogenous kinases. The research on Streptomyces trypsin started earlier, but it focused on enzymatic properties and physiological and biochemical properties Aspects of research
The research on the exogenous expression of trypsin from Streptomyces mainly focuses on the physiological and biochemical effects of trypsin expressed in different Streptomyces hosts, and the fermentation cycle of Streptomyces is generally long, and fermentation control is relatively difficult, and The proteolysis function of Streptomyces itself will also affect the production of the target protease
However, the action temperature of trypsin in practical applications is usually 37°C. Under this condition, trypsin has a high enzyme catalytic efficiency, but it will be slowly inactivated due to thermal factors, resulting in a decrease in its effectiveness
At present, there has been no report on the thermal stability modification of trypsin, but in recent years, there have been many reports at home and abroad on N-terminal fusion short peptides to improve enzyme thermal stability. Improve the thermal stability of RNaseHI

Method used

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  • Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application
  • Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application
  • Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application

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Effect test

Embodiment 1

[0028] Example 1: Amplification of the trypsin gene with improved thermostability

[0029] The gene of thermostable tyrpsin (thermostable tyrpsin) is derived from the coding sequence of the tryptase gene in Streptomyces griseu (GenBank Accession No. M64471), in which the 639th to 1307th positions are encoded SprT gene. After analyzing the codon bias of the gene, the leading short peptide YVEF was fused at its 5′ end, and the α-factor signal peptide from P. pastoris was fused again on the fusion fragment to obtain the gene Exmt, and in the final BamHI and Not I restriction sites are added to the 5' and 3' ends of the fragment respectively, and the nucleotide sequence of the Exmt gene is shown in SEQ ID NO.1.

Embodiment 2

[0030] Embodiment 2: Construction of recombinant plasmid pPIC9K-Exmt

[0031] The in vitro amplified fragment of the Exmt gene and the expression vector pPIC9K were subjected to double digestion with BamH I and EcoR I, respectively, and ligated with T4 ligase after recovery. The ligation reaction system is (10 μL): target gene fragment 2 μL, carrier DNA 2 μL, 10×T4 ligase Buffer 1 μL, T4 DNA ligase 1 μL, ddH 2 04 μL.

[0032] The ligation product was transformed into competent Escherichia coli JM109 for transformation. The conversion method is as follows:

[0033] (1) Under sterile conditions, take 200 μL of competent cells and place them in a sterile microcentrifuge tube;

[0034] (2) Add 1-2 μL of recombinant plasmid to each tube, rotate gently to mix the contents, and place on ice for 30 minutes;

[0035] (3) Heat shock at 42°C for 90sec (accurate), do not shake the centrifuge tube;

[0036] (4) Quickly transfer the centrifuge tube to an ice bath to cool the cells for ...

Embodiment 3

[0040] Embodiment 3: the construction of the trypsin yeast engineered bacterium that thermostability improves

[0041] The expression vector pPIC9K-Exmt was linearized by cutting with Sal I. Enzyme digestion system (50 μL system): recombinant plasmid 10 μL, Buffer 5 μL, Sal I 3 μL, ddH 2 O32 μL. Water bath at 37°C for 3 hours, purify and recover the linearized product with a PCR product purification kit, and transform Pichia pastoris GS115 by electric shock method, the specific method is as follows:

[0042] (1) Inoculate a loop of activated Pichia pastoris GS115 in 25mL YPD liquid medium, and culture overnight at 28°C to 30°C with shaking;

[0043] (2) Transfer the above culture solution into 100mL YPD (500mL Erlenmeyer flask) liquid culture medium, shake culture at 28℃~30℃, measure every 1h, and cultivate until the cell concentration OD 600 1.3~1.5;

[0044] (3) Cool in ice water for more than 10 minutes;

[0045] (4) Collect the bacteria by centrifugation at 4°C and 80...

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Abstract

The invention discloses an engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application, and belongs to the genetic engineering field. A trypsin gene obtained through in-vitro amplification is fused with a leading short peptide YVEF, and is wholly connected to a Pichia pastoris GS115 chromosome to construct engineered Saccharomyces cerevisiae efficiently secreting and expressing recombinant trypsin, and the recombinant trypsin having an improved stability is obtained after purification, and the heat stabilities of the recombinant trypsin at 40DEG C, 50DEG C and 60DEG C are 1.77, 2.6 and 31 times wild trypsin respectively, so the problem of the low stability of trypsin is solved. The production of trypsin through applying the engineered Saccharomyces cerevisiae has the advantages of high output, simple technology, intelligible heredity and application background of engineered Saccharomyces cerevisiae, and convenient industrial application.

Description

technical field [0001] The invention relates to a yeast engineering bacterium producing thermostable recombinant trypsin and an application thereof, belonging to the field of genetic engineering. Background technique [0002] So far, enzyme preparations have been widely used in tanning, food, fermentation and textile industries. The application of enzymes in the leather industry was initially limited to enzymatic depilation and enzymatic softening. With the continuous development and progress of science and technology, the scope of application of enzyme preparations in the tanning industry has also been continuously expanded and expanded, and they are gradually applied to the tanning preparation section and even the entire tanning process. There are various indications that the research and development of enzyme-based tanning techniques will surely give birth to an ecological concept of tanning technology—biological tanning. Among them, pancreatin is widely used in the tan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N9/76C12R1/84C12R1/545
Inventor 陈坚令桢民马腾博堵国成康振李江华
Owner JIANGNAN UNIV
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