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Marine bacterium derived thermal-stable and salt-tolerant SGNH family hydrolytic enzyme and application

A hydrolytic enzyme, consistent technology, applied in the field of genetic engineering, can solve problems such as unclear function

Active Publication Date: 2018-04-10
SECOND INST OF OCEANOGRAPHY MNR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the reported SGNH family hydrolases are derived from eukaryotes, and the family of hydrolases from bacteria is less, and their functions are not clear

Method used

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  • Marine bacterium derived thermal-stable and salt-tolerant SGNH family hydrolytic enzyme and application
  • Marine bacterium derived thermal-stable and salt-tolerant SGNH family hydrolytic enzyme and application
  • Marine bacterium derived thermal-stable and salt-tolerant SGNH family hydrolytic enzyme and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Acquisition of hydrolase gene aline4

[0042] Based on the complete genome, open reading frame prediction and gene annotation results of the bacteria Altererythrobacter indicus DSM18604 isolated from the mangrove wild rice nodule soil, the lipohydrolase-related genes were screened. The homology between the sequences and the known hydrolase gene sequences in the database was compared by Blastp (http: / / blast.ncbi.nlm.nih.gov / ). The aline4 gene was obtained through database comparison analysis, with a size of 573bp and a base composition of 139A (24.26%), 115T (20.07%), 149C (26.00%) and 170G (29.67%), and its nucleotide sequence is as shown in SEQ ID No :1 shown. The size of the encoded protein is 190 amino acid residues, and its amino acid sequence is shown in SEQ ID No.2. Homology search was performed on the AlinE4 protein sequence in GenBank, and the highest amino acid sequence identity with it was non-cultured source esterase, the identity was 71%, and its...

Embodiment 2

[0045] Construction of the recombinant expression plasmid and recombinant strain of embodiment 2 gene aline4

[0046] The gene aline4 obtained in the present invention is cloned into an expression vector to construct a recombinant expression strain. Based on the gene open reading frame sequence obtained by the ORF analysis of NCBIORF Finder, the upstream primer aline4F (5'-TCGC GGATCC ATGGGCGAATCGCGC-3', BamHI) and downstream primer aline4R (5'-TCCG CTCGAG TCACTTCTTCGCAGGCAGCGCC-3', XhoI), PCR amplification confirms the full-length sequence of the gene. The expression plasmid was constructed by enzyme digestion cloning, that is, the PCR product was double-digested with BamHI and XhoI, and the purified fragment was ligated with the plasmid pSMT3 that had been double-digested with BamHI and XhoI. 2 The transformation method was transformed into E.coli DH5α, and positive clones were screened for kanamycin resistance. A plasmid extraction kit (Omega, USA) was used to extract ...

Embodiment 3

[0047] Example 3 Utilize the recombinant expression strain to express the recombinant gene aline4

[0048] Transfer 3ml of the constructed recombinant expression strain to 100ml of LB liquid medium containing 20μg / ml kanamycin and 34μg / ml chloramphenicol, and shake at 37°C until OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.5mM to induce expression, transfer to 20°C and shake at 150r / min for 16h. The bacterial cells were collected by low-temperature centrifugation, resuspended in NTA-10 solution (500 mM sodium chloride, 10 mM imidazole, 20 mM Tris hydrochloric acid, pH 8.0), and subjected to sonication on ice. Centrifuge at low temperature to collect supernatant, use NTA-Ni 2+The expressed protein was purified by affinity column chromatography. The expressed recombinant protein contains a 6×His tag at the N-terminal, which can be affinity-adsorbed to the layer suction column, and is eluted through gradient elution with different concentrations of imida...

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Abstract

The invention discloses novel marine bacterium derived thermal-stable and salt-tolerant organic solvent SGNH family hydrolytic enzyme AlinE4 as well as a coding gene and an application of the hydrolytic enzyme. The hydrolytic enzyme aline4 provided by the invention comes from Altererythrobacter indicus DSM18604, and a nucleotide sequence of the hydrolytic enzyme aline4 is shown as SEQ ID NO.1. Thehydrolytic enzyme gene provided by the invention, after undergoing heterogeneous expression, has esterase activity, and the maximum catalytic activity, namely 25.8U / mg, is achieved when p-nitrophenolbutyrate (C4) is taken as a substrate. The optimum catalytic hydrolysis pH and temperature of the AlinE4 are 7.5 and 40 DEG C; the AlinE4, when bred at 40 DEG C, 50 DEG C and 60 DEG C for 60min, canstill keep the activity above 50%; and with the presence of an organic solvent and metal ions, relatively high enzymatic activity is kept. The hydrolytic enzyme has the characteristics of being thermal-stable and alkaline; and the hydrolytic enzyme is applicable industrial production in such fields as detergents, wastewater treatment, fine chemical engineering, pharmacy, environmental remediationand the like under the conditions of high temperature and containing salt and organic solvents.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a novel thermostable and salt-tolerant SGNH family hydrolase derived from marine bacteria, its coding gene and its application. Background technique [0002] SGNH family hydrolases are a class of hydrolases containing four strictly conserved catalytic residues serine, glycine, asparagine and histidine in four conserved sequence regions. SGNH family hydrolases are widely found in eukaryotes and prokaryotes, and have various activities such as lipase, protease, thioesterase, aromatic esterase, lysophospholipase, carbohydrate esterase, acyltransferase, etc., and have a wide range of substrates It is a kind of hydrolase, which can participate in various physiological functions such as bacterial virulence and plant growth and development, morphogenesis and defense. The broad substrate spectrum and functional diversity make this family of hydrolases have a wide range of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55
CPCC12N9/16
Inventor 许学伟霍颖异洪利国吴月红崔恒林王春生
Owner SECOND INST OF OCEANOGRAPHY MNR
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