High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis

An application method and a crude enzyme technology of protease, applied in the field of feed and health food, can solve the problems of gene mutation, inability to correctly translate exogenous protease gene, etc., and achieve the effects of convenient purification, easy digestion and absorption, and improved protein quality.

Inactive Publication Date: 2014-08-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the prior art are: gene mutations are prone to occur in the process of using genetic engineering technology to construct

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: It is a high temperature resistant protease embodiment of the present invention. The thermostable protease has activity and high thermal stability under the condition of 20-80°C. At 65°C, the activity can remain unchanged for 3 hours. % or more activity; it is active in the range of pH 6-11, and the enzyme activity preservation rate within 24 hours is 100% when the pH is 7-9; the high-temperature-resistant protease producing strain is transformed by Pichia pastoris through genetic engineering . .

Embodiment 2

[0034] Example 2: It is a Pichia pastoris that is used to ferment and produce the high-temperature-resistant protease described in Example 1 of the present invention, and its classification is called Pichia pastoris MMpk-GS115 ( Pichia pastoris MMpk-GS115 ), the preservation number of the preservation center is: CCTCC NO: M 2014171, the preservation date is: April 28, 2014, the preservation unit is: China Center for Type Culture Collection (CCTCC), address: Wuhan University, Luojiashan, Wuchang, Wuhan; Pichia pastoris MMpk-GS115 Morphological and physiological and biochemical characteristics include: it is milky white, and the bacterial body is oval or round in microscopic examination, which is methanol-trophic bacteria.

[0035] The Pichia pastoris used for fermenting and producing high-temperature-resistant protease, after methanol-feeding fermentation, the protease activity of the fermentation broth is greater than or equal to 50000U / mL, less than or equal to 100000U / mL,...

Embodiment 3

[0036] Example 3: It is the method for breeding high temperature resistant protease strains of the present invention, the steps are:

[0037] A. Prepare white lateral tooth mold ( Engyodontium album ), used as a source of thermostable protease gene sequence;

[0038] B. Obtain the target gene sequence by using overlap extension PCR technology Fpk , that is, using primers with complementary ends to make the PCR product form an overlapping chain, so that in the subsequent amplification reaction, the amplified fragments from different sources are overlapped and spliced ​​together through the extension of the overlapping chain;

[0039] C. Insert the target gene into the expression vector: insert into the constructed conventional Pichia expression vector p9kNTS, p9kMFⅡ:

[0040] D. Recombination and linearization of the above expression vector;

[0041] E. Electrotransformation of Pichia pastoris MMpk-GS115: after recombination and linearization, electrotransformation;

[00...

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Abstract

The invention relates to a high-temperature-resistant protease, a strain breeding method thereof and an application method of the high-temperature-resistant protease to enzymolysis. The high-temperature-resistant protease has activity and relatively high thermal stability at the temperature of 20-80 DEG C and is produced through pichia pastoris fermentation. Pichia pastoris which is used for fermenting to produce the high-temperature-resistant protease is classified and named as pichia pastoris MMpk-GS115. The strain breeding method comprises the steps of preparing engyodontium album, constructing a pichia pastoris expression vector, and the like. The application method of the high-temperature-resistant protease to enzymolysis comprises the following steps of firstly, preparing a crude high-temperature-resistant protease from the high-temperature-resistant protease; then, carrying out enzymolysis on an enzymolysis base material by using the crude high-temperature-resistant protease. The high-temperature-resistant protease has the advantages of capability of generating extracellular proteases, high enzymatic activity, short fermentation time and wide specificity of protease cutting sites; in addition, after being subjected to enzymolysis, vegetable proteins such as rice dregs, bean pulp, wine distiller grains, broussonetia papyrifera and the like and yeast cultures contain 10-50% of small peptides, can be improved in quality and increased in nutritional value and are easier to digest and absorb by animals.

Description

technical field [0001] The invention relates to a high-temperature-resistant protease and its strain breeding method and its application method in enzymolysis, in particular to a high-temperature-resistant protease produced by Pichia pastoris and its strain breeding method and its application in plant protein and yeast culture The application method in enzymolysis is especially suitable for feed and health food industries, and belongs to the technical field of fermentation engineering. Background technique [0002] 75% of industrial enzyme preparations are proteolytic enzymes, and proteases account for the largest proportion of industrial enzymes, accounting for about 60% of the world's total annual sales. Proteases are widely used in industries such as feed, detergent, food, medicine and leather manufacturing. The breeding methods of protease strains include physical and chemical factors single or compound mutagenesis, protoplast technology, genetic engineering technology ...

Claims

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Application Information

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IPC IPC(8): C12N9/60C12N1/19C12N15/57C12N15/81C12P21/06C12R1/84
CPCC12N9/60C12P21/06
Inventor 葛向阳彭楠方玉美赵述淼李俊迟晨潘超姜超梁运祥
Owner HUAZHONG AGRI UNIV
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