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Preparation method and application of novel halophilic archaea extracellular protease

A technology of extracellular protease and halophilic archaea, applied in the field of genetic engineering, can solve the problem that protease cannot adapt to high salinity industrial processing environment, etc., and achieve the effect of excellent enzymatic characteristics, good stability and high stability

Active Publication Date: 2020-09-25
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, many proteases derived from bacteria cannot adapt to the high-saline industrial processing environment. Although the proteases derived from halophilic archaea can tolerate saline-alkali environment, most of them require a high concentration of 2-5M NaCl to maintain the stability of the enzyme

Method used

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  • Preparation method and application of novel halophilic archaea extracellular protease
  • Preparation method and application of novel halophilic archaea extracellular protease
  • Preparation method and application of novel halophilic archaea extracellular protease

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Experimental program
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Effect test

Embodiment 1

[0056] Acquisition of hly encoding gene hly for novel halophilic archaeal extracellular protease;

[0057] Based on a halophilic archaea Haladaptatus sp.DYF 46 (separated by the inventor from Dongying Salt Field, Shandong, China, preserved in China General Microorganism Culture Collection and Management Center, patent strain preservation number: CGMCC 19759, preservation date: 2020.4.29) The whole genome, open reading frame prediction and gene annotation results were used to screen suspected extracellular protease genes. The consistency between the sequence and the known hydrolase gene sequence in the database was compared by Blastp (http: / / blast.ncbi.nlm.nih.gov / ). The hly gene obtained through database comparison analysis has a size of 1191bp and a base composition of 238A (19.98%), 176T (14.78%), 395C (33.17%) and 383G (32.16%), and its nucleotide sequence is as shown in SEQ ID No. :1 shown. The size of the encoded protein is 396 amino acid residues, and its amino acid se...

Embodiment 2

[0061] Construction and induced expression of recombinant expression plasmid of hly;

[0062] The gene hly obtained in the present invention is cloned into an expression vector, and a recombinant expression strain is constructed for inducible expression. Based on the ORF analysis of NCBI ORF Finder, the gene open reading frame sequence was obtained, and the upstream primer hly-NcoI-F (5'-ATAccatggCAAGGAAAGCCAATGGCG-3', NcoI) and the downstream primer hly-XhoI-R (5'- ATActcgagGTTGTCGCTGGAGTCGAG-3', XhoI), PCR amplification to obtain the target fragment (1206bp). The expression plasmid was constructed by restriction endonuclease cloning, that is, the PCR product was double-digested with restriction enzymes NcoI and XhoI, and the purified fragment was ligated with the plasmid pET28a that had been double-digested with NcoI and XhoI to obtain the recombinant plasmid pET28a-hly. The 3' end of the insert (NcoI and XhoI sites) contains 6×His tag. CaCl 2 The transformation method wa...

Embodiment 3

[0065] Purification and refolding of recombinant protein Hly;

[0066] Resuspend the bacteria collected by low temperature centrifugation in 30mL cell lysate (8M urea, 10mM CaCl 2 , 50 mM Tris-HCl, pH 8.0) and sonicated on ice. The supernatant was collected by low temperature centrifugation. The mature enzyme was obtained from the supernatant by the on-column renaturation method (that is, the protein was purified by nickel affinity chromatography and the protease was renatured in vitro in a high-salt environment). The specific operation is as follows:

[0067] 1) Add β-mercaptoethanol with a final concentration of 2mM to the cell lysate of 10 times the column bed volume (CV), and equilibrate the nickel column.

[0068] 2) Add 2mM final concentration of β-mercaptoethanol to the supernatant and load the sample.

[0069] 3) Add 5CV buffer I (2mM β-mercaptoethanol, 8M urea, 40mM imidazole, 10mM CaCl 2 , 50mM Tris-HCl, pH 8.0) to elute foreign proteins.

[0070] 4) Add 5CV re...

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Abstract

The invention belongs to the field of genetic engineering, and relates to a preparation method and application of novel halophilic archaea extracellular protease. The preparation method comprises thefollowing steps: firstly, obtaining a coding gene hly based on a genome of halophilic archaea DYF 46; connecting the hly to a vector by a molecular cloning technology to construct a recombinant plasmid; transforming the recombinant plasmid into a prokaryotic host, culturing the transformed recombinant host cells in an LB liquid culture medium containing kanamycin, centrifugally collecting thalli at low temperature after culture and inducible expression, resuspending in a cell lysis solution, ultrasonically crushing, centrifugally collecting supernatant for recombinant protein purification; andpurifying protein by nickel affinity chromatography, obtaining mature enzyme by a column renaturation method, and purifying by gel filtration chromatography to obtain high-purity Hly. The Hly obtained by the preparation method has excellent enzymatic characteristics, and can be applied to production and processing processes of complex and salt-containing degraded proteins such as detergents, wastewater treatment, pharmacy and environmental remediation.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a preparation method and application of a novel halophilic archaea extracellular protease. Background technique [0002] The discovered serine protease precursors from halophilic archaea all have signal peptide, propeptide, catalytic domain and C-terminal extension (CTE). The precursor molecule is secreted out of the cell through the TAT pathway, and then the signal peptide is cleaved, and the N-terminal propeptide helps the enzyme to form a correct conformation before being cleaved, and finally forms a mature extracellular protease. Mature extracellular proteases consist of a catalytic domain and a CTE, and the CTE may be related to maintaining the stability of the enzyme in a high-salt environment or binding to a macromolecular substrate. [0003] At present, many bacterial-derived proteases cannot adapt to high-saline industrial processing environments. Although...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/70
CPCC12N9/50C12N15/70
Inventor 崔恒林赵阳洁侯靖
Owner JIANGSU UNIV
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