A laccase from Bacillus pumilus cota constructed by genetic engineering
A technology of Bacillus pumilus and laccase, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of low enzyme catalytic activity, poor substrate specificity, and low natural expression of CotA laccase
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Embodiment 1
[0014] Example 1 Expression and purification of wild Bacillus pumilus CotA laccase.
[0015] The recombinant expression strain CotA / pColdII / BL21 (DE3) constructed from glycerol tube inoculation earlier stage was cultivated overnight in LB liquid medium (containing 100mg / L ampicillin), and the seeds were inserted into LB liquid fermentation medium (containing 100mg / L ampicillin) by 2% inoculum. 100mg / L ampicillin). After culturing Escherichia coli at 37°C for 2 hours, add 0.3mM IPTG at a final concentration for induction, and continue fermentation and culture in a shaker at 15°C for 24 hours, then centrifuge the fermentation broth at 4°C and 8000rpm for 10 minutes to remove the supernatant and collect the bacteria. The collected cells were resuspended in phosphate buffer, and after resuspension, the cells were crushed with an ultrasonic cell disruptor to release intracellular proteins. After the crushing was completed, the crushed liquid was centrifuged at 4°C and 8000 rpm for ...
Embodiment 2
[0017] Example 2 CotA laccase mutant construction and preparation
[0018] (1) Site-directed mutation
[0019] Using the previously constructed recombinant plasmid pColdII-CotA containing the B. pumilus CotA laccase gene as a template, the 386th position of leucine (Leu) in the laccase was mutated into tryptophan (Trp), which was named L386W.
[0020] The site-directed mutagenesis primers for introducing the L386W mutation are:
[0021] Forward primer 5'-TATGGGCGCCCTATTT GG TTACTAGATAAC-3' (the underline is the mutation site)
[0022] reverse primer 5'- CC AAATAGGGCGCCCATATTTATCTTGTGT-3' (underline is the mutation site)
[0023] Using the above primers, the recombinant plasmid pColdII-CotA was used as a template to carry out PCR reaction. All reactions were carried out in a 50 μL system, and the reaction conditions were: 95°C pre-denaturation for 3 minutes, followed by 25 cycles (95°C for 20 s, 57°C for 20 s, 72°C for 6 min), after the cycle, 72°C extension for 7 minute...
Embodiment 3
[0026] Example 3 Enzyme activity analysis of CotA laccase mutants.
[0027] (1) Definition of enzyme activity unit
[0028] When using the ABTS method to measure the enzyme activity of laccase, the amount of enzyme required to convert 1 μmol of substrate per minute is defined as an activity unit.
[0029] (2) Enzyme Activity Determination Steps
[0030] Preheating: Take 2.4mL pH4.0 citrate buffer solution in a test tube, add 0.5mL ABTS solution (the final concentration of ABTS is 0.5mM) into the test tube and place it in a 37°C water bath to preheat for 2min.
[0031] Reaction: Add 0.1mL sample enzyme solution and shake evenly.
[0032] Measurement: Use a spectrophotometer to measure the kinetics of the uniformly oscillating sample, measure the change in OD value per second within 30s at a wavelength of 420nm (the reaction rate is a uniform reaction) and calculate the enzyme activity.
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