A low-temperature alkaline esterase estsl3 resistant to salt and organic solvents and its gene and application

A technology resistant to organic solvents and alkalinity, applied in the field of genetic engineering, can solve problems such as inability to apply, and achieve the effects of increasing the yield of the target product, reducing energy consumption, and improving flavor

Active Publication Date: 2018-09-18
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Most of the esterases currently obtained come from conventional environments, the optimum temperature is around 40°C, and the optimum pH is neutral to alkaline, which cannot be applied in many industries

Method used

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  • A low-temperature alkaline esterase estsl3 resistant to salt and organic solvents and its gene and application
  • A low-temperature alkaline esterase estsl3 resistant to salt and organic solvents and its gene and application
  • A low-temperature alkaline esterase estsl3 resistant to salt and organic solvents and its gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Esterase gene EstSL3 clone

[0050] Extraction of genomic DNA of halophilic and alkalophilic bacteria: the bacterial genome extraction kit (DP302) of Tiangen Company was used to extract genomic DNA, and the specific operation was carried out in accordance with the instructions of the kit.

[0051] When we cloned the xylanase gene from this bacterium, we obtained the 3' end of the esterase gene. Sequence comparison analysis showed that the similarity of the gene was relatively low, and none of them had been characterized. Therefore, according to the sequence The nucleotide sequence of the 3' end of the esterase gene was designed, and three upstream TAIL-PCR specific primers were designed: the design direction was the direction of the unknown region to be amplified, the position of sp2 was designed to be inside sp1, and sp3 was located inside sp2. The distance between each two primers is not strictly regulated (for the convenience of electrophoresis identi...

Embodiment 2

[0055] Example 2 Preparation of recombinant esterase.

[0056] Introduced at the 5' and 3' ends of the gene, respectively NCOI and Hind III Restriction sites estSL3-m- F and estSL3-m- R is a pair of primers (see Table 1), and the genomic DNA of halophilic and alkalophilic bacteria is used as a template for PCR amplification. The PCR reaction parameters were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 51°C for 30 sec, extension at 72°C for 1 min, 30 cycles, and incubation at 72°C for 5 min. The expression vector pET28a(+) was double digested ( NCOI + Hind III ), and the gene encoding esterase estSL3 double digestion ( NCOI + Hind III ), the cut gene fragment encoding mature esterase was connected to the expression vector pET28a(+) to obtain the esterase gene containing estSL3 The recombinant plasmid pET28a- estSL3 And transform Escherichia coli BL21 (DE3), obtain recombinant Escherichia coli strain BL21 / estSL3 .

...

Embodiment 3

[0058] Example 3 Determination of the properties of recombinant esterase EstSL3

[0059] 1. Activity analysis of recombinant esterase

[0060] The activity of recombinant esterase was determined by colorimetric method to determine the activity of esterase: the specific method is as follows: at pH 9.0, 30°C, 1 mL of reaction system included 100 μL of appropriate diluted enzyme solution, 20 μL of substrate (10 mM), add 880 μL 50mmol / L pH 9.0 phosphate buffer, react for 10 min and immediately set the OD 404 measure its absorbance. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol p-nitrophenol per minute under given conditions.

[0061] 2. Determination of optimum pH and pH stability of recombinant esterase EstSL3

[0062] Optimum pH determination: The purified recombinant esterase EstSL3 was subjected to an enzymatic reaction at 37°C in a 0.1M pH5.0–9.0 buffer. Determination of the pH stability of the enzyme: the enzyme solution was p...

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Abstract

The invention relates to the field of genetic engineering, and provides a salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3, a genome coding gene estSL3 and a recombinant vector and a recombination strain comprising the gene, the esterase EstSL3 comes from halophilic and basophilic bacteria, the amino acid sequence of the esterase EstSL3 is shown as SEQ ID NO.1, and the nucleotide sequence of the genome coding gene estSL3 of the esterase is shown as SEQ ID NO.2. The esterase EstSL3 is adapted to pH9.0 and the reaction temperature of 30DEG C best, and the esterase EstSL3 still has about 70 percent of enzyme activity under 0DEG C, which indicates that the enzyme is a low-temperature esterase. Under the existence of 0.5 to 4.0M NaCl, more than 98 percent of enzyme activity remains, and moreover, under the concentration of 4M and 5M NaCl, the esterase EstSL3 is highly stable. The esterase disclosed by the invention has the characteristics of low temperature, alkalinity, salt tolerance, detergent tolerance and organic solvent tolerance, and can be applied in industrial fields such as fine chemical, pharmacy, detergents and food.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a low-temperature alkaline esterase EstSL3 resistant to salt and organic solvents and its gene and application. Background technique [0002] Esterase (Esterase E.C.3.1.1.1) is a general term for enzymes that catalyze the hydrolysis and synthesis of ester bonds. During hydrolysis, it catalyzes ester bonds to produce alcohols and carboxylic acids, and during synthesis, it dehydrates and condenses carboxyl groups of acids and hydroxyl groups of alcohols to form esters and other fragrance products. During the catalytic reaction process of esterase, it has high catalytic specificity for substrate, its reaction conditions are mild, its by-products are few, and it does not require coenzyme, so it has high application value in industrial production and environmental treatment. [0003] Esterase-producing organisms are widely distributed in nature, ranging from ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12N1/19C12N1/15
CPCC12N9/18C12Y301/01001
Inventor 叶秀云林娟王国增
Owner FUZHOU UNIV
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