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Alkaline protease mutant PROK-M with increased specific activity and thermal stability, and coding gene and applications thereof

A technology of thermostability and protease, which is applied in the field of agricultural biology, can solve the problems of enzyme activity and thermostability.

Active Publication Date: 2019-05-07
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

[0003] In industrial applications, not only alkaline protease is required to be fast, efficient and low cost, but also alkaline protease is often required to be able to adapt to higher temperatures, but the enzyme activity and thermal stability of existing alkaline proteases often fail to meet the above requirements

Method used

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  • Alkaline protease mutant PROK-M with increased specific activity and thermal stability, and coding gene and applications thereof
  • Alkaline protease mutant PROK-M with increased specific activity and thermal stability, and coding gene and applications thereof
  • Alkaline protease mutant PROK-M with increased specific activity and thermal stability, and coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Preparation of alkaline protease mutant recombinant vector pPIC9r-PROK-M

[0051] The biological source of the wild-type alkaline protease of the present invention is a fungus derived from Rosa phyllosa (Parengyodontium album).

[0052] The sequence fragment of the alkaline protease wild type without the signal peptide was cloned into the expression vector pPIC-9r, and the recombinant vector was named pPIC9r-PROK; using the recombinant vector pPIC9r-PROK as a template, it was amplified by primers carrying mutation sites, A recombinant vector carrying the mutant sequence was obtained and named pPIC9r-PROK-M.

[0053] The same method was used to construct the recombinant expression vector of the gene encoding the signal peptide sequence.

[0054] Table 1 alkaline protease mutant PROK-M specific primers

[0055]

Embodiment 2

[0056] Embodiment 2 prepares alkaline protease mutant

[0057] The obtained recombinant plasmid pPIC9r-PROK-M was transformed into Pichia pastoris GS115 to obtain recombinant yeast strain GS115 / PROK-M. Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL of 0.5% methanol for precipitation. The BMMY medium was resuspended, and placed again at 30°C, 220rpm to induce culture. Add 0.5 mL of methanol every 12 hours to keep the concentration of methanol in the bacterial solution at 0.5%, and take the supernatant for enzyme activity detection.

[0058] The supernatant of the recombinant protease expressed in the shake flask was collected, concentrated through a 5kDa membrane bag, and at the same time the medium was replaced with a low-salt buffer, and then f...

Embodiment 3

[0060] The activity analysis of embodiment 3 alkaline protease mutant and wild type

[0061] 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: 1 mL of reaction system includes 500 μL of appropriately diluted enzyme solution, 500 μL of substrate, reacted for 20 min under optimal conditions, 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, and aspirated Add 2.5 mL of sodium carbonate (0.4 mol / L) to 500 μL of the supernatant, 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).

[0062] 1. Comparison of opti...

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Abstract

The invention belongs to the technical field of agricultural biology, and specifically relates to an alkaline protease mutant with increased specific activity and thermal stability, and a coding geneand applications thereof. The amino acid sequences of the alkaline protease mutant are shown as SEQ ID No.3. Compared with the specific activity of a wild type, the specific activity of the mutant canbe enhanced 15%; the optimum temperature of enzymatic reaction can be maintained unchanged; the optimum pH value can be reduced 0.5; and compared with the wild type, the thermostability of the mutantcan be enhanced under conditions of 55 DEG C and 60 DEG C. Thus, compared with the activity of the wild type, the alkaline protease mutant PROK-M can be higher in activity and more high temperature resistant.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to an alkaline protease mutant with improved specific activity and thermal stability, its coding gene and application. Background technique [0002] Protease refers to a class of enzyme molecules that degrade proteins and polypeptides by cleaving peptide bonds. It is widely distributed in microorganisms, animals and plants. Among them, microorganisms are the main source of proteases for industrial use. According to the optimal pH of protease, it can be divided into three categories: acid protease, alkaline protease and neutral protease. Among them, the molecular weight of alkaline protease is small, between 26-34kDa, its optimum pH range is 9.5-10.5, and it is relatively stable under the condition of pH5.0-10.0. The active center of alkaline protease is generally serine, which is easily inhibited by diisopropylfluorophosphate and phenylmethylsulfonate chloride, ...

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 涂涛罗会颖任雅馨黄火清姚斌苏小运王亚茹王苑柏映国孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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