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Alkaline thermal-stability SGNH family esterase EstD1 and gene thereof

A thermostable, family-friendly technology, applied in the field of genetic engineering, which can solve the problems of difficult purification, long fermentation period of natural strains, and difficulty in mass production.

Active Publication Date: 2015-09-09
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, natural strains have a long fermentation cycle, low enzyme production, difficult purification, high production costs, and difficulty in mass production, which limits their popularization and application.

Method used

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  • Alkaline thermal-stability SGNH family esterase EstD1 and gene thereof
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  • Alkaline thermal-stability SGNH family esterase EstD1 and gene thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: Cloning of SGNH family esterase gene EstD1

[0046] Extract the genomic DNA of thermophilic Alicyclobacillus D-1: CTAB lysis method, the specific steps are: centrifuge the fresh bacterial liquid cultured in the liquid for 12 hours, and add 800 μL of solution Ⅰ (20mM Tris, pH8.0, 2mM Na 2 -EDTA, final concentration 20mg / mL lysozyme), incubate at 37°C for 30min, add 100μL 10% SDS, mix up and down, add 10μL 10mg / mL proteinase K, keep at 37°C for 30min, add 150μL 5M NaCl and 150μL 10% CTAB solution was mixed upside down, kept at 65°C for 20min, divided into 600μL tubes, then added 600μL of phenol / chloroform / isopropanol (volume ratio 25:24:1) for extraction, and centrifuged at 12000rpm at 4°C for 10min. Take 300 μL of the supernatant and extract it again in 300 μL of chloroform / isopropanol (volume ratio 24:1) to remove impurities, centrifuge at 12000 rpm for 10 min at 4°C, then take the supernatant and add 2 times the volume of absolute ethanol and 1 / 10 volume ...

Embodiment 2

[0053] Embodiment 2: Preparation of recombinant SGNH family esterase EstD1

[0054] Take the recombinant plasmid pEASY TM - BL21(DE3) strain of E2-EstD1 and pEASY only TM -The empty plasmid BL21(DE3) strain of E2 was inoculated in LB (containing 100 μg / mL Amp r ) culture medium, shake rapidly at 37°C for 16h. Then this activated bacterial solution was inoculated into fresh LB (containing 100 μg / mL Amp r ) medium, cultured with rapid shaking for about 2–3 hours (OD 600 After reaching 0.4-0.6), add IPTG with a final concentration of 0.7mM for induction, and continue shaking culture at 20°C for about 20h. Centrifuge at 12000rpm for 10min to collect the bacteria. With an appropriate amount of pH7.0 citric acid-Na 2 HPO 4 After suspending the bacteria in the buffer solution, the bacteria were disrupted by ultrasonic in a low-temperature water bath. The crude enzyme solution concentrated in the above cells was centrifuged at 12,000rpm for 10min, the supernatant was aspirated...

Embodiment 3

[0055] Example 3: Characterization of the purified recombinant SGNH family esterase EstD1

[0056] (1) Activity analysis of recombinant SGNH family esterase EstD1

[0057] The activity of the purified recombinant SGNH family esterase EstD1 was determined by the p-nitrophenol method: take four 1.5mL centrifuge tubes, numbered 1, 2, 3, and 4 respectively, and the test tubes 1, 2, and 3 are Experimental group (3 repetitions), No. 4 is the control group. Add 420 μL of 50 mM Tris-HCL buffer at pH 8.0 and 30 μL of 10 mM substrate pNPC to four centrifuge tubes respectively 2 , after preheating at 37°C for 5 minutes, add 50 μL enzyme solution of appropriate dilution to tubes 1, 2, and 3 every 10 seconds, add the same amount of water to control tube 4, react at 37°C for 5 minutes, and add to tubes 1 and 3 every 10 seconds. 2. Add 50μL 0.1M Na to the experimental tube 2 CO 3 To terminate the reaction, add an equal volume of stop solution to the No. 4 control tube and immediately ins...

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Abstract

The invention discloses alkaline thermal-stability SGNH family esterase EstD1. The amino acid sequence of the alkaline thermal-stability SGNH family esterase EstD1 is one of the amino acid sequence (1) shown in SEQ ID NO.1 and the amino acid sequence (2), wherein one or more pieces of amino acid are added or replaced or lacked or inserted, shown in SEQ ID NO.1. The invention further discloses a gene EstD1 of the alkaline thermal-stability SGNH family esterase EstD1, the nucleotide sequence of the gene EstD1 is one of the nucleotide sequence (1) shown in SEQ ID NO.2 and the nucleotide sequence (2), wherein one or more pieces of nucleotide are added, replaced, lacked or inserted, shown in SEQ ID NO.2, the nucleotide sequence (3) formed through hybridization with the nucleotide sequence (1) or nucleotide sequence (2) under strict conditions and the nucleotide sequence (4) which is different from the nucleotide sequence (1), the nucleotide sequence (2) and the nucleotide sequence (3) due to the degeneracy of a genetic codon. The alkaline thermal-stability SGNH family esterase EstD1 can be used for deacetylation of 7-ACA.

Description

technical field [0001] The invention relates to an alkaline thermostable SGNH family esterase EstD1, its coding gene, and its application in 7-ACA deacetylation, belonging to the technical field of genetic engineering. Background technique [0002] SGNH family esterase (esterase, EC 3.1.1.1) is a class of hydrolytic enzymes with broad substrate specificity, including lipase, protease, thioesterase, aromatic esterase, lysophospholipase, carbohydrate esterase, acyl transfer enzyme activity. This family was identified around 1995, but its members are still rarely characterized. They differ from most of the α / β hydrolase superfamily esterases that contain the typical conserved sequence of GxSxG, but contain the unique conserved sequence of GDSL , is a new family of esterases. The esterases of the GDSL family contain 5 conservative sequence blocks (five consensus sequence blocks I-V), and 4 relatively conservative catalytic sites Ser, Gly, Asn and His in the 5 conservative sequ...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N15/75C12N15/74C12N15/81C12N1/21C12N1/19C12P35/06C12R1/19
Inventor 丁俊美于婷婷黄遵锡李俊俊慕跃林杨云娟
Owner YUNNAN NORMAL UNIV
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