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.
Image
Examples
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.
PUM
| Property | Measurement | Unit |
|---|---|---|
| absorbance | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- IPC
- C12N9/18; C12N15/55; C12N15/70; C12P7/62
- CPC
- C12N9/18; C12P7/62; C12Y301/01
- Inventors
- 叶茂; 邓毛程