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Method for efficiently expressing acid protease and application thereof

A technology of acid protease and fusion protein, which is applied in the field of high-efficiency expression of acid protease, and can solve the problems of low activity of acid protease, limited application of acid protease, reduction of catalytic efficiency, etc.

Active Publication Date: 2021-06-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the enzymatic activity of acid protease is not high, and the difference in pH, temperature, etc. between the optimum action conditions of the enzyme itself and the catalyzed environmental conditions leads to a decrease in the catalytic efficiency of the enzyme, thereby limiting the application of acid protease in industry

Method used

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  • Method for efficiently expressing acid protease and application thereof
  • Method for efficiently expressing acid protease and application thereof
  • Method for efficiently expressing acid protease and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The construction of embodiment 1 recombinant Pichia pastoris

[0035] (1) Construction of recombinant plasmid pPIC9K-en-αSP-acidprotea

[0036] The enhanced α-signal peptide (en-αSP) whose nucleotide sequence is shown in SEQ ID NO.4 is connected to the N-terminus of the acid protease shown in SEQ ID NO.2, and artificially synthesized, Add Bam HI and Not I restriction sites at the beginning and the end respectively, and integrate the sequence connected with the restriction sites into the cloning vector pUC57 to form the plasmid pUC57-enα-SP-acidprotea. The plasmid pUC57-enα-SP-acidprotea was double-digested with Bam HI and Not I to obtain a 1428bp sequence containing enhanced α-signal peptide and acid protease and recovered, and then the vector pPIC9K was obtained by double-digestion with Bam HI and Not I The 8998bp carrier sequence was recovered, and the sequence containing the enhanced α-signal peptide and acid protease was connected with the carrier sequence with DNA...

Embodiment 2

[0042] Embodiment 2 Recombinant Pichia pastoris shakes flask fermentation to produce acid protease

[0043] From the plate containing recombinant Pichia pastoris GS115 / pPIC9K-en-αSP-acidprotea obtained in Example 1, pick 10 single colonies and inoculate them in 25mL liquid medium YPD, and cultivate them at 30°C and 220rpm 24h, room temperature 6000rpm, centrifuge 5min, collect the cells, discard the supernatant, resuspend the cells with 30mL liquid medium YP, induce expression at 28°C, 220rpm, add methanol to the final concentration of 1% every 24h to continue the induction, shake After 72 hours of bottle induction, the flask was shaken to obtain a fermentation broth.

[0044] The recombinant strain P. pastoris GS115 / pPIC9K-acidprotea obtained in Example 1 containing the original α-signal peptide was used as a control strain, and the induction culture was carried out at the same time.

[0045] When the final flask was shaken, the enzyme activity of the control strain using th...

Embodiment 3

[0046] The enzymatic performance of embodiment 3 recombinant acid proteases

[0047] (1) Purification of acid protease

[0048] The fermentation broth obtained in Example 2 was centrifuged at 4°C at 8000r / min for 10min, and the fermentation supernatant was collected to obtain a crude enzyme solution, using 20mmol / L NaH 2 PO 4 -Na 2 HPO 4 (p H 6.5) buffer solution and 10kDa ultrafiltration membrane bag were used to dialyze the crude enzyme solution. The dialyzed crude enzyme solution was collected, centrifuged at 4°C and 12000r / min for 30min, and the supernatant was collected.

[0049] The protein was purified using HitrapTM Q FP (5mL) anion exchange chromatography column. Use the AKTA protein purifier to separate and purify the protein of the crude enzyme solution. The column purification conditions are as follows: use 10 times the column volume of buffer A (20mmol / LNaH2PO4-Na2HPO4 buffer solution, pH 6.5) to equilibrate the anion column, and use 1mL / min Injection at a f...

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Abstract

The invention discloses a method for efficiently expressing acid protease and application thereof, and belongs to the field of gene engineering. The expression enzyme activity of the recombinant pichia pastoris P.pastoris GS115 / pPIC9K-en-alpha SP-acidprotea provided by the invention reaches 5395U / mL and is improved by 109% compared with the enzyme activity of a control strain containing original alpha-signal peptide; the acidic protein disclosed by the invention is in a pH range of 1-5, the enzyme can maintain the enzyme activity of 60% or more, the residual enzyme activity is 80% or more after the enzyme is treated at 20-40 DEG C for 30 minutes, and the acid protease can be suitable for industries such as food, feed or brewing and the like. According to the technical scheme provided by the invention, the acid protease suitable for industrial application can be produced by utilizing a genetic engineering means.

Description

technical field [0001] The invention relates to a method for high-efficiency expression of acid protease and application thereof, belonging to the field of genetic engineering. 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 with diff...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/58C12N9/64C12N15/62C12N15/81C12N1/19C12G1/022C12R1/84C12R1/865
CPCC12N9/58C12N9/64C12N15/815C12G1/0203C07K2319/02Y02P60/87
Inventor 吴丹郑璞陈鹏程魏梦园
Owner JIANGNAN UNIV
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