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

A technology of protease and high temperature resistance, applied in the field of fermentation engineering, can solve the problems of unstable enzyme production conditions, difficult control of fermentation conditions, poor high temperature resistance, etc. short effect

Inactive Publication Date: 2019-12-20
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing screening strains of Pichia pastoris are high-yielding strains obtained through multi-generational mutagenesis, which have problems such as unstable enzyme production conditions, easy back mutation, difficult control of fermentation conditions, and long fermentation period. The high temperature resistance of enzyme production is poor

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The breeding method of embodiment 1 Pichia pastoris.

[0036] (1) Insert Pichia pastoris into the seed shake flask medium, and the medium components are 2% soluble starch, 0.16% yeast powder, 0.08% peptone, 0.2% sodium chloride, 0.2% magnesium sulfate, dihydrogen phosphate Potassium 0.04%, agar powder 1.4%, methanol 1.5%, the balance is water, cultivate to logarithmic phase, centrifuge and wash with HM hypertonic solution, prepare bacterial suspension with HM hypertonic solution, add lysozyme, centrifuge, wash and centrifuge solution, prepared into protoplasts;

[0037] (2) Add protoplasts to the hypertonic solution to make a bacterial suspension, dilute it, spread it on the regeneration plate, and then add a layer of culture medium prepared from the hypertonic solution on the upper layer, and the consumption of the culture medium prepared from the hypertonic solution 20% of the lower layer, cultivated in an incubator until colonies grow to obtain a protoplast suspensi...

Embodiment 2

[0041] Example 2 Induced expression and detection of Pichia pastoris AN-PK-9K-GS115

[0042] (1) Transfer the obtained Pichia pastoris AN-PK-9K-GS115 to the YPD plate, then select a single colony and transfer it to the fermentation medium, and culture it for 20-24 hours at 30°C and 180rpm as a seed solution.

[0043] Fermentation medium: 85% phosphoric acid 26.7mL / L, CaSO 4 0.93g / L,K 2 SO 4 18.2g / L, MgSO 4 ·7H 2 O14.9g / L, KOH 4.13g / L, glycerol 40g / L, methanol 15g / L.

[0044] (2) the seed liquid is inserted in the 50L fermentor that contains 35L BSM inorganic salt medium with the inoculum size of 10%, adjusts pH value at 6, and temperature is 30 ℃, and regulating stirring speed is 600rpm, and dissolved oxygen amount DO is controlled at 10%, along with the carrying out of fermentation, the dissolved oxygen amount rises to 30% and continues for 30-60 minutes, adjust the stirring speed to be 800rpm, and supplement methanol once (the replenishment amount is 1.5% of the tota...

Embodiment 3

[0047] Example 3 Enzymatic hydrolysis of soybean meal with high temperature resistant protease

[0048] First, mix the enzymolysis base material soybean meal with water, the amount of water added is by weight, the ratio of dry enzymolysis base material to water is 1:1, adjust the pH to 6, and then add high-temperature-resistant protease crude oil at a temperature of 55°C. Enzyme, added in an amount of 300U / g dry enzymolysis base material; re-fermentation and enzymolysis, the time is 9h, and finally liquid enzymolysis protein is obtained, or dry enzymolysis protein is obtained after drying. In this example, the degradation rate of protein in the soybean meal reaches 95%, and the protein small peptide content is 65%, accounting for 70% of the total protein of the dry enzymatic hydrolyzed protein.

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Abstract

The invention discloses high-temperature-resistant proteases, a bacterial strain breeding method thereof and an application of the high-temperature-resistant proteases to enzymolysis, and belongs to the technical field of fermentation engineering. Pichia pastoris is classified and named as pichia pastoris AN-PK-9K-GS115, the preservation No. is CCTCC NO: M 2019593, the preservation date is 30 July, 2019, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuchang Luojia Mountain, Wuhan. According to a breeding method, chemical mutagenesis substances are added, the process is controllable, the high-temperature-resistant proteases can enable the degradation rate of vegetable protein and protein in yeast culture to achieve 80% or above, and the content of products namely small peptide achieves 40-70%. The enzyme is used for treating plant protein and the yeast culture, so that the protein quality can be improved, the nutrient value can be increased, the function properties of solubility, foamability and the like can be improved, and the protein is easy to digest and absorb by animal bodies.

Description

technical field [0001] The invention relates to the technical field of fermentation engineering, in particular to a high-temperature-resistant protease and its strain selection method and application in enzymolysis. Background technique [0002] 75% of industrial enzyme preparations are proteolytic enzymes, and protease is the enzyme with 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 soybean meal protease strains include physical and chemical factors single or combined mutagenesis, protoplast technology, genetic engineering technology and so on. [0003] Existing screening strains of Pichia pastoris are high-yielding strains obtained through multi-generational mutagenesis, which have problems such as unstable enzyme production conditions, easy back mutation, difficult control of ferment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N15/01C12N9/60A23K10/14A23K10/37C12R1/84
CPCA23K10/14A23K10/37C12N1/16C12N9/60C12N15/01C12N1/165C12R2001/84Y02P60/87
Inventor 彭楠常章兵田建平梁运祥
Owner HUAZHONG AGRI UNIV
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