acid protease Bs2688 and gene and application thereof

An acid protease and gene technology, applied in the field of agricultural biology, can solve the problems of low catalytic efficiency and low activity of acid protease

Pending Publication Date: 2018-11-27
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 a fungal-derived acid protease Bs2688, which has the characteristics of good pH stability, high temperature resistance, and easy production and fermentation

Method used

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  • acid protease Bs2688 and gene and application thereof
  • acid protease Bs2688 and gene and application thereof
  • acid protease Bs2688 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1 prepares protease Bs2688 mutant

[0050] 1. Cloning of protease coding gene Bs2688

[0051]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 Transfer to a new 50mL centrifuge tube containing 15ml CTAB lysate, gently invert up and down to mix well, place in a 65°C water bath for 3 hours, and invert up and down gently once 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 ...

Embodiment 2

[0070] Embodiment 2. Verify the enzymatic performance of recombinant protease

[0071] 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: at pH 3.0 and 55°C, 1 mL of reaction system includes 500 μL of appropriate diluted enzyme solution, 500 μL of substrate, react for 10 min, add 1 mL of trichloroacetic acid (0.4 mol / L) to terminate the reaction; The system was centrifuged at 12000 rpm for 3 minutes, 500 μL of supernatant was absorbed, 2.5 mL of sodium carbonate (0.4 mol / L) was added, and 500 μL of Folin’s 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 generate 1 μmol of tyrosine per minute is 1 activity unit (U).

[0072] 1. Optimal pH and pH stability of...

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Abstract

The invention belongs to the technical field of agro-biology and particularly relates to fungus-originated acid protease Bs2688 and gene and application thereof. An amino acid sequence of the acid protease is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention provides a new protease gene; proteinases with good character are produced by means of genetic engineering; the new protease gene is applicable to the industries, such as feed, food and medicine.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a fungus-sourced acid protease Bs2688 and 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 the optimum pH value varies slightly w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/58C12N15/57C12N15/81C12R1/84
CPCC12N9/58C12N15/815
Inventor 姚斌罗会颖郭玉杰涂涛苏小运王苑黄火清柏映国王亚茹孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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