A kind of engineering bacteria for producing recombinant aspartic protease and its application

An amino acid, silkworm chrysalis protein technology, applied in the application, genetic engineering, recombinant DNA technology and other directions, can solve the problems of low expression, low efficiency, limited production of aspartic protease, etc., and achieves improved hydrolysis degree, improved solubility, excellent The effect of hydrolysis potential

Active Publication Date: 2022-07-22
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In summary, most of the aspartic protease-producing strains are obtained through natural mutagenesis combined with large-scale screening. Great difficulties in culture, which limit the production of aspartic proteases and thus require new expression systems

Method used

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  • A kind of engineering bacteria for producing recombinant aspartic protease and its application
  • A kind of engineering bacteria for producing recombinant aspartic protease and its application
  • A kind of engineering bacteria for producing recombinant aspartic protease and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of aspartic protease encoding gene

[0031] Using Af293 derived from Aspergillus fumigatus Af293 as the parent, PCR was used to amplify the aspartic protease encoding gene SEQ ID NO.

Embodiment 2

[0032] Example 2 Preparation of aspartic protease

[0033] The expression vector pPIC9γ was double digested (EcoR I+Not I), and the gene encoding aspartic protease was double digested (EcoR I+Not I), and then the mature high specific activity xylan was digested The gene fragment of the enzyme mutant was connected with the expression vector pPIC9γ to obtain a recombinant plasmid containing the high specific activity xylanase mutant gene and transformed into Pichia pastoris GS115 to obtain a recombinant yeast strain.

[0034] Take the GS115 strain containing the recombinant plasmid, inoculate it into a 1L Erlenmeyer flask of 300mL BMGY medium, and place it at 30°C, 220rpm shaker for 48h; then centrifuge the culture solution at 3000×g for 5min, discard the supernatant, and use 100mL of 0.5 Resuspend in BMMY medium with % methanol, and place it again at 30°C, under the condition of 220rpm to induce culture. 0.5 mL of methanol was added every 12 h to keep the methanol concentratio...

Embodiment 3

[0035] Example 3 Activity analysis of recombinant aspartic protease

[0036] Casein method: The specific method is as follows: The protease activity of AF293 was determined using casein as a substrate. Under the given pH and temperature conditions, 1 mL of reaction system consists of 500 μL of 1% casein and 500 μL of enzyme solution, and incubated for 10 minutes. The reaction was stopped by adding 1 mL of 10% trichloroacetic acid (TCA), centrifuged at 12,000 x g for 5 min, and then the supernatant (500 μL) was added to 2.5 mL of 0.4 M Na 2 CO 3 To the composed test tube, Folin and Ciocalteu reagent (500 μL) were added last. The mixture was further incubated at 40°C for 20 minutes and the absorbance was measured at 680nm.

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Abstract

An engineering bacterium for producing recombinant aspartic protease and its application, obtained from fungi by PCR Aspergillus fumigatusAf293 aspartic protease gene Af293 , and will Af293 Constructed on pPIC9γ plasmid to obtain recombinant plasmid Pic9γ‑Af293 , and transformed into Pichia pastoris GS115 to obtain recombinant genetically engineered bacteria. The recombinant aspartic protease expressed by the genetically engineered bacteria is stable at pH 4.0-5.0 and temperature 45-55 DEG C, and the specific activity is 8408.9±305.6 U / mg. In the application of hydrolyzed silkworm pupa protein, the recombinant aspartic protease showed excellent hydrolysis potential. After 6 hours of hydrolysis, the degree of hydrolysis was increased by 50±6.1%, and the solubility was increased by 80%. The stability characteristics of the recombinant aspartic protease and The hydrolysis properties enhance its potential for industrial applications, especially in the food and feed industries.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, in particular to an engineering bacterium for producing recombinant aspartic protease and its application. Background technique [0002] Aspartic proteases (EC 3.4.23) are an important class of proteolytic enzymes characterized in that their active center consists of two catalytic aspartic acid residues (Coates L et al. 2001. Biochemistry, 40 ( 44):13149-13157.). Most aspartic proteins are active at acidic pH and are also known as acid proteases. Based on their special catalytic properties, they have important applications in the pharmaceutical, food and beverage and feed industries. [0003] The main source of aspartic protease is microorganisms. The reported strains of aspartic protease mainly include Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillium, Rhizopus, etc. and their variants and mutants. Wait. The commercial acid protease producing bacte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/57C12N9/62C12P21/06C12R1/84
CPCC12N9/62C12N15/815C12P21/06C12Y304/23
Inventor 王俊里查德·安萨·赫尔曼游帅白致远
Owner JIANGSU UNIV OF SCI & TECH
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