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Novel esterase and its application

A new type, esterase technology, applied in the field of genetic engineering

Inactive Publication Date: 2012-10-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been no research reports on the discovery of new heat-resistant esterases from Turpan soil samples using metagenomic technology

Method used

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  • Novel esterase and its application
  • Novel esterase and its application
  • Novel esterase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Establishment of metagenomic library and acquisition of positive clones, gene cloning and expression

[0049] 1. Extraction of total DNA:

[0050] Weigh 10g of Turpan soil sample, add 13.5mL DNA extraction buffer (0.1M Tris, 0.1M EDTA-Na, 0.1M Na 3 PO 4 , 1.5M NaCl, 1%CTAB, pH 8.0), shake vigorously for 5min, add 1.5ml 20%SDS, bathe in 65℃ water for 2h, centrifuge at 5000rpm for 5min, take the supernatant, extract twice with equal volume of chloroform, and centrifuge at 16000rpm 10min, take the supernatant, add 0.6 times the volume of isopropanol, place at room temperature for 2h, centrifuge at 16000rpm for 20min, discard the supernatant, add 10mL of pre-cooled 70% ethanol to the precipitate, centrifuge at 16000rpm for 10min, collect the DNA precipitate, air-dry, and use an appropriate amount of supernatant Dissolved in pure water.

[0051] 2. DNA purification by kit method: follow the instructions of the gel recovery kit.

[0052] 3. Metagenomic electroph...

Embodiment 2

[0072] The enzymatic property research of embodiment 2 recombinant esterase Est112

[0073] 1. Optimum reaction temperature and thermal stability of recombinant esterase

[0074] The crude enzyme solution of recombinant esterase is subjected to enzymatic reaction at 30-75°C, the reaction system is 410 μL, and its composition is 400 μL potassium phosphate solution containing 40 μM p-nitrophenyl acetate (pH7.5) and 10 μL enzyme solution. The reaction system was reacted at 60° C. for 4 minutes, and then the absorbance of p-nitrophenol released during the process was measured at a wavelength of 405 nm, and a blank control without adding enzyme solution was made at the same time. The enzyme activity was measured to obtain the optimum reaction temperature (the highest enzyme activity was recorded as 100%). In 50mM potassium phosphate buffer (pH 7.5), the recombinant esterase enzyme solution was placed at different 40°C, 45°С, 50°C, 55°С, 60°С and 65°C for 40 hours, and the residua...

Embodiment 3

[0077] Example 3 Degradability determination of recombinant esterase Est112 to AHLs

[0078] 1. Reaction system

[0079] Add 1 μmol of C4 -HSL,C 6 -HSL,C 8 -HSL,C 10 -HSL,C 12 -HSL and 3OC 8 - Dissolve HSL in 50 μL of methanol, and add 950 μL of 50 mM potassium phosphate buffer to prepare 1 mM AHLs substrate solution, mix 31 μg of recombinant enzyme powder with 1 ml of 1 mM substrate, react at 30°С for 30 minutes, and perform HPLC Detection, while fire extinguishing enzyme powder as a blank control.

[0080] 2. HPLC analysis

[0081] The chromatographic conditions are as follows:

[0082]

[0083] Through HPLC detection analysis, recombinant esterase to C 4 -HSL,C 6 -HSL,C 8 -HSL,C 10 -HSL,C 12 -HSL and 3OC 8 The degradation rates of -HSL reached 64.7%, 96.2%, 100%, 100%, 100%, and 94.3%, respectively, indicating that it has broad substrate specificity and high ability to degrade AHLs (the degradation profile is shown in 4).

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Abstract

The invention discloses DNA of a novel esterase. The novel esterase DNA has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.3. The invention also discloses an expression vector containing the novel esterase gene, a recombinant esterase and application of the recombinant esterase in degradation of homoserinelactone signal molecules. The novel esterasehas efficient soluble expression in an Escherichia coli expression system and good thermal stability, and has a strong degradation effect on homoserinelactone signal molecules.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a novel esterase and its application. Background technique [0002] Esterase (EC3.1.1.1) refers to a class of important biological enzymes that can catalyze the hydrolysis of ester bonds. It is widely used in the fields of food industry, pharmaceutical industry, daily chemical industry, and biological protection. It has broad applications in quorum sensing quenching. application prospects. [0003] In recent years, studies have found that the pathogenic process of many human, animal and plant pathogenic bacteria is closely related to quorum sensing. The expression of pathogenic bacteria virulence genes, in addition, pathogenic bacteria have the ability to resist the host's immune defense system and drug action through the quorum sensing system. Therefore, destroying the quorum sensing system of pathogenic bacteria is a new anti-infection strategy to achieve the pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/10C12N15/70
Inventor 刘玉焕范新炯
Owner SUN YAT SEN UNIV
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