A kind of esterase with degrading chiral ester activity and its encoding gene and application
A coding and gene technology, applied to esterase with chiral ester degradation activity and its coding gene and application field, can solve the problems of low efficiency and high cost, and achieve the effect of strong application prospect and high enzyme activity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0063] Example 1: Acquisition and Sequence Analysis of Antarctic Soil-derived Esterase Encoding Gene Sequence
[0064] Source of strain: The gene E19-3664 of the Antarctic microbial esterase of the present invention comes from the soil of site E19 near the Antarctic Great Wall Station.
[0065] Specific steps are as follows:
[0066] 1.1 Construction of subcloning library
[0067] The large fragment plasmid fosmid in E. coli EPI300 clone E19-3664 was extracted with the BAC / PAC DNA extraction kit from OMEGA company according to its instructions. The extracted fosmid was then partially digested with the restriction enzyme Sau3AI (purchased from Fermentas) to obtain a DNA fragment of 2,000-5,000 bp, which was ligated into the BamHI-digested and dephosphorylated pUC19 plasmid (purchased). from NEB Corporation). The ligation reaction solution was electroporated into E.coli Top10 competent cells, coated with LB solid plates containing 100 μg / ml ampicillin and 1% (v / v) tributyrin ...
Embodiment 2
[0072] Example 2: Cloning, heterologous expression and isolation and purification of esterase
[0073] 2.1 Amplification of gene sequences by PCR
[0074] (1) Design two specific primers according to the sequence of gene E19-3664:
[0075] 3664F: AAGAAGGAGA TATACATATG GCGCACACCC CCTGGCCTGCCAG (SEQ ID NO. 3);
[0076] 3664R: TCGAGTGCGG CCGCAAGCTT CGAAGACTTT CCACCTGTGTAGC (SEQ ID NO. 4);
[0077] Primers were synthesized by Jinan Boshang Biotechnology Co., Ltd.
[0078] (2) with 3664F and 3664R as primers, with the fosmid where the gene E19-3664 is located as a template, amplify the target gene fragment with FastPfuDNA polymerase (purchased from Transgen company);
[0079] PCR reaction conditions were: pre-denaturation at 95 °C for 2 min; then denaturation at 95 °C for 20 sec, annealing at 50 °C for 20 sec, extension at 72 °C for 1 min, after 30 cycles; extension at 72 °C for 10 min.
[0080] (3) 1 wt% agarose gel electrophoresis was performed on the PCR amplification produc...
Embodiment 3
[0102] Example 3: Characterization of Antarctic Esterase E19-3664
[0103] 3.1 Substrate specificity analysis
[0104] Different carbon chain length pNP ester substrates C2, C4, C8, C10, C12, C14 (purchased from Sigma) were prepared with isopropanol.
[0105] The standard response is:
[0106] 20μl of 10mM substrate and 960μl of 50mM Tris-HCl (pH 8.0) were preheated at 40°C for 3min, 20μl of enzyme solution was added, and the reaction was carried out at 40°C. After 5min, 100μl of 20wt% SDS (sodium dodecyl sulfate) was added to stop The OD value at 405 nm was measured, and the reaction without the enzyme solution was used as a blank control. Standard curves were drawn with different concentrations of pNP (purchased from Sigma).
[0107] Enzyme activity was defined as the amount of enzyme required to catalyze the hydrolysis of pNP ester substrates to produce 1 μM pNP per minute at a certain temperature as one unit of enzyme activity (U). The results showed that the esterase ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More