Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase

An esterase and gene technology, which is applied to salt-tolerant esterase and its encoding gene and application fields, can solve problems such as esterase gene cloning, and achieve the effect of good application value and good application potential.

Active Publication Date: 2016-03-02
GUANGDONG IND TECHN COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no esterase gene has been cloned from the environmental metagenomic library of traditional condiments in my country

Method used

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  • Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase
  • Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase
  • Salt-tolerant esterase, coding gene of salt-tolerant esterase and application of salt-tolerant esterase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Obtaining of esterase gene and verification of the function of synthesizing short-chain aromatic ester compounds

[0039] 1. Acquisition of esterase gene

[0040] From the environmental samples of traditional fermented food (soy sauce, fermented bean curd, soybean paste, etc.) in my country, a fermented food environmental metagenomic library was constructed. A medium containing 100 μg / ml ampicillin, 1% glycerol triphosphate, 1% glucose and 0.5% amino acid complex was used as a selection medium to screen esterase-positive clones from the library. Copy the cells in the library onto a solid selective medium plate with a replica inoculation needle, culture at 37°C for 3 days, and screen for positive clones with hydrolysis circles around the colonies.

[0041] Extract the plasmid of the above-mentioned positive clone and send it to a sequencing company for sequencing to obtain a nucleic acid sequence of esterase, which consists of 948 bases, and its nucleic acid s...

Embodiment 2

[0053] Embodiment 2 Recombinant esterase EST_115 substrate specificity analysis

[0054] 100 mM Tris-HCl buffer (pH 7.0), 1 mM substrate, 10 μl of purified enzyme solution was added, and the absorbance value A405nm was measured at 35°C. Determination of substrates: p-nitrophenol acetate (C2), p-nitrophenol butyrate (C4), p-nitrophenol hexanoate (C6), p-nitrophenol octanoate (C8), p-nitrophenol octanoate (C8), p-nitrophenol Nitrophenol Caprate (C10), p-Nitrophenol Dodecanoate (C12). The result is as figure 1 As shown, the recombinant esterase EST_115 has catalytic activity on p-nitrophenol esters with shorter acyl carbon chains (C2, C4, C6 and C8), and the substrate with the highest catalytic activity is p-nitrophenol butyrate (C4) , followed by p-nitrophenol acetate (C2) and p-nitrophenol hexanoate (C6).

Embodiment 3

[0055] Example 3 Recombinant Esterase EST_115 Salt Tolerance Analysis

[0056] The purified enzyme solution was placed at room temperature under 5%, 10%, 15% and 18% NaCl solutions respectively, and samples were taken at different time points to measure the enzyme activity. The method for measuring enzyme activity is: 100mM Tris-HCl buffer solution (pH7.2), with 1mM p-nitrophenol butyrate as substrate, add 20 μl of sample enzyme solution, and measure the absorbance value A405nm at 35°C. The result is as figure 2 As shown, the recombinant esterase EST_115 can still maintain 40% of the enzyme activity when stored at 18% NaCl concentration for 30 days. The results showed that the recombinant esterase EST_115 had strong salt tolerance.

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Abstract

The invention provides a salt-tolerant esterase, a coding gene of the salt-tolerant esterase and application of the salt-tolerant esterase. An amino acid sequence of a protein molecule of the esterase is shown as SEQ ID NO:2, and a nucleotide sequence of an esterase gene is shown as SEQ ID NO:1. The new esterase gene is obtained by screening from a Chinese traditional food fermentation environment metagenomic library, excellent enzymatic characteristics of a coding protein of the gene are detected, a catalytic hydrolysis / synthesis temperature range is 25-50 DEG C, and a pH value is 5.5-8.0. The esterase has a main advantage that the excellent enzymatic characteristics of the esterase protein can be kept under the condition of 10-18% NaCl. Catalysis of acetic acid, propanoic acid, pentanoic acid, hexanoic acid, ethyl alcohol, propyl alcohol, butyl alcohol, pentyl alcohol and hexyl alcohol to realize synthesis of corresponding short-chain aromatic ester compounds such as ethyl acetate, propyl propionate and butyl pentanoate is realized, and esterification rates can reach 80%-90% mostly. The salt-tolerant esterase can be applied to industrial biosynthesis of short-chain aromatic ester flavorings and has significant economic and social values in foods and daily chemical products.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a salt-tolerant esterase, its coding gene and application. Background technique [0002] Esterase (Esterases, EC3.1.1.X) is a general term for enzymes that catalyze the hydrolysis and synthesis of ester bonds. During hydrolysis, they catalyze ester bonds to produce glycerol and fatty acids; For esters and other fragrance substances. Esterase is a class of widely used hydrolytic enzymes, which are widely used in food processing and food flavor improvement, oil hydrolysis, degreasing of leather and silk spinning raw materials, wastewater treatment, washing industry and pharmaceutical industry. In recent years, new functions and new uses of esterases have been continuously discovered and expanded, which makes esterases have higher research value and broader application prospects. Esterase widely exists in animals, plants and microorganisms. An...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12P7/62
CPCC12N9/18C12P7/62C12Y301/01
Inventor 叶茂邓毛程顾宗珠李平凡张远平
Owner GUANGDONG IND TECHN COLLEGE
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