Low-temperature organic solvent tolerant lipase derived from bacillus mojavensis
A lipase and amino acid technology, applied in the field of low-temperature organic solvent-resistant lipase, can solve the problem of no bacillus lipase, etc., and achieve the effect of broad organic solvent tolerance, good tolerance, and good heat resistance
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Embodiment 1
[0055] Example 1: Cloning of lipase gene
[0056] 1. Acquisition of conserved sequences
[0057] According to the conserved Oxyanion hole of the lipase gene and the conserved sequences of the Activity center P(V / I)V(M / L)(V / L)HG and AHS(M / Q)GG, the designed degenerate primers are shown in Table 1:
[0058] Table 1 Primer Information
[0059]
[0060]
[0061] The genome of Bacillus mojave was extracted by SDS method (Fang Weiguo, Chinese Journal of Applied and Environmental Biology, 2002, 8(3): 305-307). Upstream primers and downstream primers were randomly combined, and there were 12 primer pairs in total. Using the genome as a template, the following degenerate PCR reactions were performed using the above primer pairs. PCR system: rTaq 0.5uL, 10× PCR buffer 5uL, dNTP mix 8uL, primer 11uL, primer 21uL, genome 0.3uL, water 34uL. Reaction conditions: 94℃ for 1s, 94℃ for 1min, 37℃ for 1s, 37℃ for 30s, 72℃ for 2min20s, 72℃ for 1min, 35 cycles (95℃ for 1min, 52℃ for 1min, ...
Embodiment 2
[0076] Example 2: Construction of lip2-2 recombinant Escherichia coli
[0077] 1. Cloning of lip2-2 Expressed Gene
[0078] Using the Bacillus mojave genome as a template, PCR was performed using the cloning primers lip-U / lip-D in the table. The PCR reaction system was shown in Example 1. 30s, 1min at 72°C), 10min at 72°C, and hold at 4°C.
[0079] 2. Enzymatic cleavage of genes and vectors and refinement of enzymatic cleavage products
[0080] The above PCR product was recovered by gel, and the PCR product and plasmid pET24a were double digested with EcoRI and Hind III. The digestion system: 20.0uL gel recovery product (or vector), 21.0uL ddH 2 O, 5.0uL 10×M Buffer, 2.0uL EcoRI and 2.0uL HindIII, digested overnight at 37°C. (2 tubes of 50uL restriction enzyme digestion system and 2 tubes of vector were used for the recovery product of gel injection), and the restriction enzyme digestion product was purified with Omega PCR product purification kit.
[0081] 3. ligation ...
Embodiment 3
[0087] Example 3: Induction and expression of recombinant Escherichia coli and isolation and purification of target protein
[0088] 1. Inducible expression of recombinant bacteria
[0089] After inoculating a loop of BL21 (pET24a-lip2-2) into LB liquid medium containing kanamycin for about 8 hours, inoculate BL21 (pET24a-lip) in 50mL LB liquid medium ( containing 50 μg / mL kanamycin), 37 ° C, 200 rpm shaking culture, while the empty host BL21 (DE3) as a control. Waiting for OD 600 Increase to about 0.4-0.6, add IPTG to a final concentration of 1.0 mM, and culture at 37° C. and 200 rpm for 5 h to induce the expression of exogenous proteins.
[0090] 2. Lipase activity determination method
[0091] Low temperature lipase activity was determined by colorimetric method. Taking p-nitrophenyl palmitate (p-NPP) as the substrate, the enzyme activity was calculated by the amount of p-nitrophenol (p-NP) produced by the enzymatic hydrolysis of unit volume of enzyme solution in unit...
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