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A novel deep-sea thermostable alkaline esterase and its application

An esterase, alkaline technology, applied in the field of genetic engineering

Active Publication Date: 2022-07-15
SECOND INST OF OCEANOGRAPHY MNR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still few patents on alkaline bacteria esterase authorized in China

Method used

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  • A novel deep-sea thermostable alkaline esterase and its application
  • A novel deep-sea thermostable alkaline esterase and its application
  • A novel deep-sea thermostable alkaline esterase and its application

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Embodiment 1

[0024] The acquisition of embodiment 1 esterase gene e6

[0025] Based on deep-sea sediment-derived bacteria Croceicoccus marinus E4A9 TWhole genome, open reading frame prediction and gene annotation results to screen lipid hydrolase-related genes. Sequences were aligned by Blastx (http: / / blast.ncbi.nlm.nih.gov / ) for homology to known esterase gene sequences in databases. The e6 gene was obtained by database alignment analysis, with a size of 1338 bp and a base composition of 204A (15.25%), 206T (15.40%), 464C (34.68%) and 464G (34.68%). Its nucleotide sequence is as shown in SEQ ID No. :1 shown. The size of the encoded protein is 445 amino acid residues, and its amino acid sequence is shown in SEQ ID No.2. The gene sequence was searched for homology in GenBank, and the highest similarity was the esterase in the same family (Erythrobacteraceae) bacteria Erythrobacter sp.QSSC1-22B, the similarity was 63%, which was in the GenBank database The registration number is OBX19645...

Embodiment 2

[0028] Example 2 Construction of recombinant expression plasmid and recombinant strain of esterase gene e6

[0029] The esterase gene e6 obtained by the present invention is cloned into an expression vector to construct a recombinant expression strain. Based on the open reading frame sequence of the esterase gene obtained by the ORF analysis of NCBIORF Finder, the upstream primer E6F (5'-TCGC) to amplify the whole esterase gene was designed. GGATCC ATGCCGCCTTCGCCCAC-3', BamHI) and downstream primer E6R (5'-TCCG CTCGAG CTAGTTGCTGGCGGTTTCCTC-3', XhoI), PCR amplification confirmed the full-length sequence of the gene. The expression plasmid was constructed by the method of enzyme digestion and cloning, that is, the PCR product was double-digested with BamHI and XhoI, and the purified fragment was ligated with the plasmid pSMT3 double-digested by BamHI and XhoI, using CaCl 2 Transform into E.coli DH5α, and select positive clones for kanamycin resistance. Plasmids of positive ...

Embodiment 3

[0030] Embodiment 3 utilizes recombinant expression strain to express recombinant esterase gene e6

[0031] Transfer 3 ml of the constructed recombinant expression strain to 100 ml of LB liquid medium containing 20 μg / ml kanamycin and 34 μg / ml chloramphenicol, and culture with shaking at 37°C to OD 600 When it reached 0.6, IPTG was added with a final concentration of 0.5 mM to induce expression, and then it was transferred to 25°C for 8 hours with shaking at 150 r / min. The 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 sonicated on ice. The supernatant was collected by cryogenic centrifugation, using NTA-Ni 2+ The expressed protein was purified by affinity column chromatography. The expressed recombinant protein contains 6×His tag at the N-terminus, which can be affinity adsorbed to the layer suction column, and the eluate is collected by gradient elut...

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Abstract

The invention discloses a novel thermostable alkaline esterase E6 derived from deep-sea bacteria and its encoding gene and application. The present invention relates to esterase gene e6 from deep-sea bacteria Croceicoccus marinus E4A9 T , after the heterologous expression of the esterase gene, when the substrate is p-nitrophenol butyrate (C4), the catalytic activity is the highest, and the enzymatic activity is 75.2 U / mg. The optimum pH for esterase E6 esterase-catalyzed hydrolysis was 11.0; after incubation at 40, 50 and 60 ℃ for 60 min, it still maintained more than 50% activity; in the presence of organic solvents and metal ions, the enzymatic activity was higher. The esterase has the characteristics of thermal stability and alkalinity, and can be used in industrial production under high temperature and high alkali conditions in industrial fields such as fine chemicals, pharmaceuticals, washing and wastewater treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a novel thermostable alkaline esterase derived from deep-sea bacteria, its encoding gene and its application. Background technique [0002] The ocean is a huge treasure house of microbial resources, containing a large number of novel genes with diverse types and unknown functions. In recent years, in order to meet people's growing production and living needs, some applied enzymes that can adapt to extreme reaction conditions have played an important role in industrial production. , salt tolerance, heavy metal tolerance and other new functional genes have also attracted more and more attention. Lipid hydrolase is a class of enzymes that can catalyze the hydrolysis and synthesis of ester compounds. It has wide application potential in the fields of chiral drug synthesis, food processing and food flavor improvement, oil hydrolysis, degreasing of leather spinning raw m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/18C12N15/55C12P7/52C12P7/54C12P7/40C12P7/6418C12R1/01
CPCC12N9/18C12P7/40C12P7/52C12P7/54C12P7/6418C12Y301/01001C12R2001/01C12N1/205
Inventor 霍颖异许学伟戎振王春生
Owner SECOND INST OF OCEANOGRAPHY MNR
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