Mutant lipase with improved thermal stability and preparation method and application thereof
A technology of thermal stability and lipase, applied in the field of enzyme engineering, can solve problems such as decreased expression level, and achieve the effects of improving expression level, strong thermal stability, and increasing pH reaction range
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Embodiment 1
[0058] Embodiment 1: Construction of mutant lipase expression plasmid
[0059] Using pPICZαA-Lip2 (refer to Chinese patent application 201610279266.3 "A Thermostable Lipase and Its Preparation Method and Application" for the construction method) was used as a template to perform reverse PCR, and two reverse PCRs were required for each pair of disulfide bonds introduced. Using 122C-F and 122-R in Table 1 as primers, the primers for the first amplification of lipase 4sEx were obtained.
[0060] Table 1 Summary of mutation primers
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[0063] Note: The bold slash is the mutation site
[0064] The PCR amplification conditions are: 94°C for 2min; 94°C for 10s, 66°C for 30s, 68°C for 5min, 10 cycles. The reaction system is shown in Table 2 below.
[0065] Table 2 PCR reaction system
[0066] Upstream primer (10μM) 1.5μL Downstream primer (10μM) 1.5μL KOD-Plus High Fidelity Enzyme 1μL Template (50ng / μL) 1μL double distill...
Embodiment 2
[0073] Example 2: Electrotransformation of Pichia pastoris with linearized plasmid, screening of transformants and screening of enzyme production
[0074] After the positive transformants with correct sequencing were amplified and cultured overnight in LLB+Zeocin (Zeocin concentration: 25 μg / ml) liquid medium, the plasmids were extracted, linearized with PmeⅠ, purified and recovered, and the linearized products were linearized with a total of 5 μg of plasmids Electroporation transformation with X33 Pichia Competent Mix. Competent preparation of Pichia pastoris refers to the operation manual of Invitrogen Company. The electroporation program was set according to the parameters recommended by Bio-Rad.
[0075] Immediately after electroporation, add 1mL 1mol / L sorbitol solution, incubate and recover the bacterial solution at 30°C for 1 hour, spread evenly on YPDS+Zeocin (Zeocin concentration is 100μg / ml) resistance plate for screening, and select a single colony to obtain mutati...
Embodiment 3
[0076] Embodiment 3: the fermentation of recombinant engineering bacterial strain
[0077] Refer to Invitrogen’s Pichia Expression Kit Operation Manual and slightly modify it. The modified content is as follows: inoculate a single colony of the mutant engineered strain into 2 mL of YPDS-Zeocin (Zeocin concentration is 100 μg / mL) liquid medium for purification and culture overnight, centrifuge The cells were resuspended in BMGY liquid medium and cultured overnight, then inoculated into 50 mL of BMMY liquid medium, cultured at 25°C and 280 r / min for 96 hours, and supplemented with methanol every day to a final concentration of 1% by volume.
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