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Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof

A technology of acid protease and heat stability, applied in the field of agricultural biology, can solve the problems of low catalytic efficiency and low activity of acid protease

Active Publication Date: 2019-02-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of low enzymatic activity and low catalytic efficiency of existing acid proteases, the present invention provides an acid protease Bs2688 mutant derived from fungi with improved thermostability, which has the characteristics of acid resistance, high temperature resistance, and easy production and fermentation

Method used

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  • Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof
  • Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof
  • Acid protease Bs2688 mutant K203E with improved thermal stability and gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 prepares protease Bs2688 mutant

[0045] 1. Cloning of protease coding gene Bs2688

[0046] The fungus Bispora sp.MEY-1 cultured in liquid for 3 days was centrifuged at 12,000rpm for 10min, the collected mycelium was added to a high-temperature sterilized mortar, quickly ground to powder with liquid nitrogen, and then the ground fungus was transferred Put it into a new 50mL centrifuge tube containing 15ml CTAB lysate, mix it up and down gently, place it in a 65°C water bath for 3 hours, and mix it upside down gently every 20min to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the supernatant and add an equal volume of phenol / chloroform for extraction, and place it at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 min at 4°C. ...

Embodiment 2

[0066] Embodiment 2. Verify the enzymatic performance of recombinant protease

[0067] The activity analysis of the protease of the present invention is carried out by using Folin's phenol reagent chromogenic method. The specific method is as follows:

[0068] Under the conditions of pH 3.0 and 55°C, 1 mL of reaction system includes 500 μL of appropriate diluted enzyme solution, 500 μL of substrate, reacted for 10 min, and added 1 mL of trichloroacetic acid (0.4 mol / L) to terminate the reaction; centrifuged the reaction system at 12000 rpm for 3 min, Aspirate 500 μL of the supernatant, add 2.5 mL of sodium carbonate (0.4 mol / L), and then add 500 μL of Folin’s phenol reagent, develop color at 40°C for 20 minutes and then measure the OD value at 680 nm after cooling. Definition of protease activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate casein to generate 1 μmol of tyrosine per minute is 1 activity unit (U).

[0069] 1. Optimal...

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Abstract

The invention belongs to the technical field of agricultural living things, and particularly relates to a fungal acid protease Bs2688 mutant and a gene and application thereof. An amino acid sequenceof the protease is as shown in SEQ ID No.3. The invention provides the novel protease gene, and the protease good in performance is produced by means of gene engineering, and can be applied to the industries of feed, food, medicines and the like.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to an acid protease Bs2688 mutant derived from fungi with improved thermostability, its gene and application. Background technique [0002] Protease is a class of enzymes that catalyze the hydrolysis of proteins, which are widely found in plants, animals and microorganisms. Compared with animal and plant-derived proteases, microbial-derived proteases have the characteristics of convenient cultivation, simple operation, and high enzyme production, which are convenient for industrialized batch production and large-scale production and application. Therefore, microbial-derived proteases have become an important source of current proteases. [0003] There are many ways to classify protease. According to the pH of protease action, it can be divided into acid protease, alkaline protease and neutral protease. Acid protease is usually stable between pH 2.0 and 6.0, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81C12N1/19C12P21/06C12R1/84C12R1/865C12R1/78
CPCC12N9/58C12N15/81C12N15/815C12P21/06
Inventor 姚斌涂涛郭玉杰罗会颖黄火清王亚茹柏映国王苑苏小运孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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