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Engineering bacterium for producing recombinant aspartic protease and application of engineering bacterium

A technology of aspartic acid and protease, which is applied in application, genetic engineering, recombinant DNA technology, etc., can solve the problems of limited production of aspartic acid protease, low expression, low efficiency, etc., and achieve excellent hydrolysis potential and improved solubility , the effect of increasing the degree of hydrolysis

Active Publication Date: 2021-08-31
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Engineering bacterium for producing recombinant aspartic protease and application of engineering bacterium
  • Engineering bacterium for producing recombinant aspartic protease and application of engineering bacterium
  • Engineering bacterium for producing recombinant aspartic protease and application of engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of Example 1 Aspartic Protease Encoding Gene

[0031] Using Af293 derived from Aspergillus fumigatus Af293 as the female parent, the PCR method was used to amplify the aspartic protease coding gene SEQ ID NO.1, and the cloning method reference (You, et al., 2018).

Embodiment 2

[0032] The preparation of embodiment 2 aspartic acid proteases

[0033] The expression vector pPIC9γ was double-enzymatically digested (EcoR I+Not I), and the gene encoding aspartic acid protease was double-enzymatically digested (EcoR I+Not I), and then the enzyme-digested mature high specific activity xylan The gene fragment of the enzyme mutant is connected with the expression vector pPIC9γ to obtain a recombinant plasmid containing the gene of the high specific activity xylanase mutant and transform it 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 with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000×g for 5min, discard the supernatant, and use 100mL of sediment containing 0.5 The BMMY medium with % methanol was resuspended, and placed again at 30° C., 220 rpm to induc...

Embodiment 3

[0035] The activity analysis of embodiment 3 recombinant aspartic acid proteases

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

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Abstract

The invention relates to an engineering bacterium for producing recombinant aspartic protease and application of the engineering bacterium. The engineering bacterium is characterized in that an aspartic protease gene Af293 from fungus Aspergillus fumigatusAf293 is obtained through PCR, the Af293 is constructed on a pPIC9 gamma plasmid to obtain a recombinant plasmid Pic9 gamma-Af293, and the recombinant plasmid Pic9 gamma-Af293 is transformed into pichia pastoris GS115 to obtain the recombinant gene engineering bacterium. The recombinant aspartic protease expressed by the genetically engineered bacterium is stable when the pH value is 4.0-5.0 and the temperature is 45-55 DEG C, and the specific activity is 8408.9 + / -305.6 U / mg. In the application of hydrolyzing silkworm chrysalis protein, the recombinant aspartic protease shows excellent hydrolysis potential, the hydrolysis degree is improved by 50 + / -6.1% and the solubility is improved by 80% after hydrolysis is performed for 6 hours, and the potential capability of the recombinant aspartic protease in industrial application, especially in food and feed industries, is enhanced by the stability characteristic and hydrolysis performance of the recombinant aspartic protease.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to an engineering bacterium for producing recombinant aspartic acid 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 conditions, also known as acid proteases. Based on their special catalytic properties, they have important application value in the pharmaceutical, food and beverage and feed industries. [0003] The main source of aspartic protease is microorganisms. The reported strains producing aspartic protease mainly include Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Penicillium, Rhizopus, etc. and their variants and mutants. Wait. Commercial ac...

Claims

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Application Information

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Patent Type & Authority Applications(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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