A fungal nitrilase mutant with improved catalytic activity and thermal stability and its construction method
A nitrilase and thermal stability technology, applied in the field of genetic engineering, can solve the problems of insufficient catalytic efficiency, poor thermal stability, etc., to improve catalytic activity and thermal stability, improve enzyme activity and thermal stability, and improve production. The effect of efficiency
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Embodiment 1
[0022] The present invention uses known information to conduct bioinformatics analysis on the current fungal nitrilase gene, and establishes the important functions of the 128th isoleucine Ile and the 161st asparagine Asn near the active center for catalytic characteristics. Using the recombinant plasmid carrying the fungal nitrilase gene as a template, the template comes from the Escherichia coli host E. coliDH5α capable of methylation, and using the oligonucleotide sequence with the mutation site as a primer to carry out saturation mutation, using Reverse PCR technology, amplifying the 128-position and 161-position mutant plasmids.
[0023] The sequences of the saturation mutation primers are as follows:
[0024]
[0025] NNN represents the mutation site, and the site was mutated into other 19 amino acids to construct a saturated mutation library.
[0026] The reverse PCR reaction system is:
[0027]
[0028] The PCR program conditions were set as follows: pre-denatu...
Embodiment 2
[0030] The linear mutant plasmid recovered from rubber tapping was digested with Dpn I restriction endonuclease, and the enzyme digestion conditions were warmed at 37°C for 0.5 h. The enzyme digestion reaction system is as follows:
[0031]
[0032] The digested products were detected by agarose gel electrophoresis, and the remaining products were used in subsequent experiments.
Embodiment 3
[0034]The digested linear mutant plasmid was directly transformed into Escherichia coli E. coli DH5α host cells by heat shock at 42°C, spread on LB plates containing kanamycin resistance, and cultured at 37°C for 10-12 h. Pick a single colony, insert it into LB liquid medium for culture, extract the plasmid, carry out enzyme digestion and PCR verification. The plasmids of positive clones were selected and sent to Shanghai Sangon for sequencing. Those with correct sequencing results were transformed into Escherichia coli E. coli Rosetta-gami (DE3) expression hosts, cultured on LB plates containing kanamycin and chloramphenicol resistance overnight at 37°C, and positive transformants were screened, that is, fungal nitrile hydrolysis Enzyme mutants. 0.5 mM IPTG was used to induce enzyme production, and the cells were collected by centrifugation to make a bacterial suspension, and the enzyme activity of the mutant was detected, such as figure 1 shown. The mutant free cells were...
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