Lipase mutant deriving from talaromyces thermophilus, coding gene and application thereof
A technology for encoding genes and mutants, applied in the application field of 2-carboxyethyl-3-cyano-5-methylhexanoic acid, can solve the problem of restricting large-scale industrial applications, harsh production conditions, and the amount of organic solvents used Large and other problems, to achieve the effect of good application prospects
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Embodiment 1
[0030] Example 1 Construction of Lipase Mutation Library
[0031] The pET-28b-TTL plasmid, which was cloned from the lipase gene of Talaromyces thermophilus (GenBank accession No.JF414585.1) and linked to pET-28b(+) as a template (nucleotide sequence SEQ ID NO.2 ), using T7promoter and T7terminator as primers (Table 1) for PCR amplification, and randomly introducing mutations. PCR reaction system (50μL): template 0.5~20ng, 1×Taq Buffer (without Mg 2+ ), 0.2mM dGTP, 0.2mM dATP, 0.1mM dCTP, 0.1mM dTTP, 0.2mMMnCl 2 , Primers T7promoter and T7terminator each 0.2μM, Taq DNA polymerase 5U. PCR conditions (1) 95°C pre-denaturation for 3 minutes; (2) 95°C denaturation for 30s; (3) 55°C annealing for 30s; (4) 72°C extension for 1 minute, steps (2) to (4) total 30 cycles; (5) ) Finally, extend at 72°C for 10 minutes and store at 4°C. After the PCR product was analyzed by agarose gel electrophoresis, the gel was cut and recovered, and it was digested with NcoI and Xhol, ligated with the s...
Embodiment 2
[0034] Example 2 High-throughput screening of mutants
[0035] Pick the single colony in Example 1 and culture it in a 96 deep-well plate, add 200μL LB medium (containing 50μg / mL kanamycin) to each well plate, and incubate at 37°C for 2.5h to OD 600 When it is about 0.6, add the final concentration of 0.1mM IPTG to induce, and induce culture at 28℃ for 20h. Centrifuge the bacteria in the 96-deep well plate for 15 min (5000 rpm, 4°C), discard the supernatant, and resuspend the bacteria in Tris-HCl (20 mM, pH 8.0) buffer as the biocatalyst for the reaction. The bacterial suspension was added to each well of a 96 shallow-well plate, 200 μL per well, then the substrate CNDE (final concentration 100 mM) was added, and 0.01% bromothymol blue was used as an indicator, and reacted at 30°C for 1-3 hours.
[0036] According to the speed of the color change, the bacteria with increased vitality were selected (using the wild-type strain as the control), and the catalytic activity of CNDE was v...
Embodiment 3
[0037] Example 3 Site-directed mutation and screening
[0038] In order to further improve the enzyme activity of the mutants, site-directed mutagenesis is used to continue to screen for better complex mutants. Using the expression plasmid pET-28b-L259F as a template, site-directed mutagenesis was performed through whole plasmid amplification. The PCR system (50μL) is: template 0.5-20ng, 5×PrimeSTAR Buffer 10μL, dNTP Mixture 4μL, mutation primer (Table 1) each 1μL, PrimeSTAR DNA Polymerase 0.5μL. PCR conditions (1) 95°C pre-denaturation for 3 minutes; (2) 95°C denaturation for 15 seconds; (3) 55°C annealing for 10 seconds; (4) 72°C extension for 5 minutes, steps (2) to (4) total 30 cycles; (5) ) Finally, extend at 72°C for 10 minutes and store at 4°C. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, and then introduced into E.coli BL21(DE3), and spread on an LB plate containing 50μg / mL kanamycin. The catalytic activity of each mutant on CNDE was...
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