Carbonyl reductase gene, and preparation method and application of immobilized carbonyl reductase
A carbonyl reductase and reductase technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of unstable application of free enzymes and easy environmental interference.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0037] Example 1 Gets dehydrogenase rabbit liver alcohol related embodiment
[0038] Save our laboratory rabbits liver crude enzyme catalyzes the synthesis of chiral tolvaptan. The crude enzyme solution after ammonium sulfate fractionation, DEAE anion exchange method, Hi Trap Sephadex 6B size exclusion chromatography, ultrafiltration separation four purification methods. Purified enzyme solution obtained is then separated by polyacrylamide gel electrophoresis. Cut stained bands do proteomic detection.
[0039] Results: The purified enzyme solution before and after the protein by SDS-PAGE electrophoresis as figure 1 (a) and figure 1 (b). Can be observed from the drawing to the molecular weight of the purified protein is mainly in the front range 14.4-97.4kDa, after purification, in addition to a majority of the molecular weight of larger proteins, there remains a molecular weight of active enzyme mainly in the range of 23-53kDa. Greatly reduces the scope of screening, provides a gr...
Embodiment 2
[0043] Example 2 Construction and Screening of high expression recombinant strain of carbonyl reductase
[0044] The CM10 report may be annotated sequence carbonyl reductase, PrimerPremier5 using primer design software, designed as follows:
[0045] Table 2 lists the primers
[0046]
[0047] Dissecting a rabbit, the rabbit liver in RNAwait immediately remove liquid efficiently protected against degradation RNAase RNA. Remove 1cm 3 Volume of liver tissue in a mortar (DEPC water treated) while liquid nitrogen was added crushed, then clean station using RNA extracted liver tissues Trizol method. Using a kit for reverse transcription of the genome of rabbit liver acquired. Rabbit liver genomic reverse transcription as a template, with primers carbonyl reductase gene has been cloned design. The object is then ligated into the pET28a vector and the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells. After transformation, take appropriate bacteria plated on LB ...
Embodiment 3
[0061] Example 3 Inducing expression and purification of carbonyl reductase RLSR5
[0062] The monoforma containing recombinant expression plasmids falls overnight in LB containing 100 μg / ml ampicillin, inoculated in 2L induced medium, 37 ° C, 150 rpm / min culture to OD 600 The value was 0.6, which was added to the end concentration of 0.5 mmol / L, and induced 12h at 28 ° C. 6000 rpm / min, 15min centrifugal collected bacteria, resuspended with appropriate volume of phosphate buffer, ultrasonic crush (working 2S, stop 5 s, 40 min, 10 ° C, 30% power). The supernatant was taking the supernatant (12000 rpm / min, 30nin), and the supernatant was precipitated to the protein sample through SDS-PAGE polyacrylamide gel electrophoretic detection, recording and analyzed the results.
[0063] Use histrap TM HP (GE Healthcare, Piscataway, NJ, USA) and protein purification system ( Purification of recombinant protein RLSR5 purified by PURE; GE Healthcare. The concentration of recombinant ...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Particle size | aaaaa | aaaaa |
| Molecular weight | aaaaa | aaaaa |
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More