Carboxylesterase D-1CarE5 from thermophilic Alicyclobacillus tengchongensis strain, and gene thereof

A carboxylesterase and gene technology, applied in the field of genetic engineering, can solve the problems of limited popularization and application, difficulty in mass production, low enzyme production by natural strains, etc.

Inactive Publication Date: 2012-11-21
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Carboxylesterase is an important pesticide-degrading enzyme. However, natural strains have low enzyme production, are difficult to produce in large quantities, and have high production costs, which limit their popularization and application.

Method used

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  • Carboxylesterase D-1CarE5 from thermophilic Alicyclobacillus tengchongensis strain, and gene thereof
  • Carboxylesterase D-1CarE5 from thermophilic Alicyclobacillus tengchongensis strain, and gene thereof
  • Carboxylesterase D-1CarE5 from thermophilic Alicyclobacillus tengchongensis strain, and gene thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Carboxylesterase Gene D-1CarE5 clone

[0036] Alicyclobacillus thermophilic D-1 genomic DNA: CTAB lysis method, the specific steps are: centrifuge the fresh bacterial liquid cultured in liquid for 12 hours, and add 800 μL solution Ⅰ (20 mM Tris, pH8.0, 2 mM Na 2-EDTA, final concentration 20 mg / mL lysozyme), incubated at 37°C for 30 min, added 100 μL 10% SDS and mixed up and down, then added 10 μL 10 mg / mL proteinase K, incubated at 37°C for 30 min, added 150 μL μL of 5 M NaCl and 150 μL of 10% CTAB solution were mixed upside down, incubated at 65°C for 20 min, added an equal volume of phenol / chloroform / isopropanol (volume ratio 25:24:1) for extraction, at 4°C Centrifuge at 12000 rpm for 10 min, take the supernatant and extract it again in chloroform / isopropanol (volume ratio 24:1) to remove impurities, centrifuge at 12000 rpm for 10 min at 4 °C, take the supernatant and add 2 times the volume of anhydrous Ethanol and 1 / 10 volume of pH5.2 3M NaAc, precipit...

Embodiment 2

[0043] Example 2: Preparation of carboxylesterase D-1CarE5 [a3]

[0044] pET -D-1CarE5 of Escherichia coli BL21(DE3) strain and pET-only - Empty plasmid for 28a(+) Escherichia coli BL21(DE3) strain was inoculated in LB (50 μg / mL Kan) medium with 0.1% inoculum, and shaken rapidly at 37 °C for 16 h. Then inoculate the activated bacterial solution into fresh LB (50 μg / mL Kan) culture solution with 1% inoculum, and culture with rapid shaking for about 2–3 h (OD 600 After reaching 0.6–1.0), add IPTG at a final concentration of 0.7 mM for induction, and continue shaking culture at 20 °C for about 20 h or at 26 °C for about 8 h. Centrifuge at 12000 rpm for 5 min to collect the cells. After suspending the cells with an appropriate amount of pH7.0 Tris-HCl buffer solution, the cells were disrupted by ultrasonic waves in a low-temperature water bath. The above intracellular concentrated crude enzyme solution was centrifuged at 13,000 rpm for 10 min, the supernatant was aspira...

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Abstract

The invention relates to a carboxylesterase D-1CarE5 having an amino acid sequence represented by SEQ ID NO.1 and a gene thereof, and provides a coding gene D-1CarE5 of the carboxylesterase, a recombinant vector of the carboxylesterase gene D-1CarE5, and a recombinant strain of the carboxylesterase gene D-1CarE5. The carboxylesterase has the following properties: the carboxylesterase has an optimum temperature of 40DEG C and an optimum pH value of 7.38 when alpha-naphthyl acetate is used as a substrate; the carboxylesterase can be activated by metal ions comprising Mn<2+> and Zn<2+> when the final concentrations of the metal ions are 1mM, and can be strongly inhibited by Cu<2+>, Ag<+>, Hg<2+>, Pb<+> and the like; the carboxylesterase keeps basically stable at 35DEG C for 50min; the activity of the carboxylesterase after processed with buffer solutions having a concentration of 1/15mol/L and pH values of 5.91, 7.38 and 8.04 for 80min is above 40%, 90% and 45% respectively; and simultaneously the carboxylesterase can hydrolyze beta-naphthyl acetate. The carboxylesterase can be applied to pesticide residual or pesticide pollution treatment.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a carboxylesterase D-1CarE5 derived from Alicyclobacillus thermophiles and its gene. Background technique [0002] Carboxylesterases (Carboxylesterases, E C3.1.1.1) are a class of non-specific esterases that can catalyze the hydrolysis of carboxylic acid esters to generate carboxylic acids and alcohols. They belong to the α / β hydrolase family of lipases. Their amino acid sequence Contains Ser-Asp-His triplet structure, Ser, Asp, His constitute the catalytic active center of carboxylate lipase (Nardini, M, et al., Current Opinion in Structural Biology . 1999, 9(6):732-737.). [0003] Carboxylate lipase widely exists in animals, plants and microorganisms (Melloney J., et al., J of Molecular Catalysis B : Enzymatic . 2005, 32:261-270.), wherein microbial-derived carboxylesterase is an important industrial enzyme, which has important application value in catalyzi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12N1/19C12R1/01
Inventor 黄遵锡谢振荣周峻沛李俊俊唐湘华杨云娟许波
Owner YUNNAN NORMAL UNIV
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