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Acid protease Bs2688 mutant Y282L with improved thermostability and gene and application thereof

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

Active Publication Date: 2019-05-28
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 Y282L with improved thermostability and gene and application thereof
  • Acid protease Bs2688 mutant Y282L with improved thermostability and gene and application thereof
  • Acid protease Bs2688 mutant Y282L with improved thermostability and gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Example 1 Preparation of protease Bs2688 mutant

[0045] 1. Clone the protease encoding gene Bs2688

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

Embodiment 2

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

[0067] The activity analysis of the protease of the present invention is carried out by using the Folin phenol reagent color method. The specific method is as follows: Under the condition of pH3.0 and 55℃, a 1mL reaction system includes 500μL of appropriate diluted enzyme solution, 500μL of substrate, and react for 10min. Add 1mL of trichloroacetic acid (0.4mol / L) to terminate the reaction; The system was centrifuged at 12000 rpm for 3 minutes, 500 μL of supernatant was sucked into 2.5 mL of sodium carbonate (0.4 mol / L), and 500 μL of Phenol reagent was added. The color was developed at 40°C for 20 minutes and the OD value was measured at 680 nm after cooling. Definition of protease activity unit: Under certain conditions, the amount of enzyme required to decompose the substrate casein to produce 1 μmol tyrosine per minute is 1 activity unit (U).

[0068] 1. Optimal pH and pH stability of protease Bs2...

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Abstract

The invention belongs to the technical field of agrobiology, and particularly relates to a fungus-derived acid protease Bs2688 mutant and a gene and application thereof. The amino acid sequence of theprotease provided by the invention is shown in SEQ ID NO. 3. The invention provides a novel protease gene, and the protease with excellent properties is produced by using a genetic engineering meansand is applied to a feed, food, medicine and other industries.

Description

Technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a fungal-derived acid protease Bs2688 mutant with improved thermal stability and its gene and application. Background technique [0002] Proteases are a class of enzymes that catalyze the hydrolysis of proteins, which are widely present in plants, animals and microorganisms. Compared with the protease derived from animals and plants, the protease derived from microorganism has the characteristics of convenient cultivation, simple operation and high enzyme production, which is convenient for industrialized mass production and large-scale production application. Therefore, microbial proteases have become an important source of current proteases. [0003] There are many ways to classify proteases. According to the pH at which the proteases act, they are divided into acid proteases, alkaline proteases and neutral proteases. Acid protease is usually stable between pH 2....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81C12N1/19C12R1/84
Inventor 罗会颖姚斌郭玉杰涂涛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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