A kind of leucine dehydrogenase mutant and its preparation method and application
A technology for leucine dehydrogenase and mutants, which is applied in the field of leucine dehydrogenase mutants and their preparation, and can solve problems such as difficulty in catalyzing aliphatic ketones
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] Example 1: Bacteria screening on a 96-well plate
[0050] According to the conservation of the sites near the pocket, degenerate codon primers containing other possible amino acids were designed, and the transformants on the plate were selected and cultured in a 96-deep well plate, each well containing 200 μL LB medium (kan concentration of 50 μg / mL), and then placed on a constant temperature shaker at 37°C and 250 rpm for shaking culture overnight. Afterwards, 50 μL of seed solution was drawn from each well and transferred to another 96-deep well plate corresponding to the position, and each well contained 400 μL of LB medium (kan concentration was 50 μg / mL). Incubate for 3-4 hours at 37°C with shaking on a constant temperature shaker at 250 rpm. Next, 50 μL of 2 mM IPTG was added to induce the expression of LeuDH, and cultured at 25° C. on a constant temperature shaker at 250 rpm for 18 hours. Pipette 100 μL of the induced bacterial solution to measure OD. Afterwar...
Embodiment 2
[0051] Example 2: Expression and purification of mutant enzymes K77S / N270L / L52S and K77S / N270L / T143C
[0052] The recombinant Escherichia coli was overexpressed in a shake flask, and when the OD reached 0.6, IPTG with a final concentration of 0.3mM was added to induce expression, and then cultured in a constant temperature shaker for 16h (25°C, 180rpm). Crush using a sonicator on ice. Afterwards, centrifuge at 12000rpm for 20min at 4°C, and the supernatant is used for protein purification or extraction of crude enzyme solution. For protein purification, the supernatant after ultrafiltration through a 0.22 μm filter membrane was loaded on a Ni-NTA gel column, and was eluted in Tris- HCl (500 mM) buffer, most of the protein was collected in the eluate. The eluate was analyzed by SDS gel electrophoresis. Use a MWCO 30000da filter column to remove imidazole by ultrafiltration. Purified enzymes were collected in phosphate buffered saline. The protein concentration of the purif...
Embodiment 3
[0053] Example 3: Catalytic activity of mutant enzymes K77S / N270L / L52S and K77S / N270L / T143C
[0054] At a temperature of 25° C., a 200 μL reaction system contains: a certain amount of pure enzyme, 20 mM substrate, 0.2 mM NADH, 2M ammonium chloride ammonia water buffer solution (pH 9.5). After incubation for 2 minutes, add appropriate enzyme solution to start the reaction for 1 minute and measure the change of absorbance at 340nm. Enzyme activity was calculated based on the reduction of NADH.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More