| Penicillium expansum PF898 is a mutant strain obtained by many generation mutagenesis, can produce an alkaline lipase at a high level. To research on the gene and protein structure of the lipase from Penicillium expansum PF898(designated PEL), improve its property of cold-activity, we have done the research as follows-.1 .Overexpression of PEL gene in Pichia pastorisThe alkaline lipase gene of Penicillium expansum (PEL) was coloned into the yeast integrative plasmid pPIC3.5K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression products of PEL gene was analysis by SDS-PAGE and olive oil plate, and the result indicated that PEL gene was functionally overexpressed in Pichia pastoris and up to 95% of the secreted protein. Recombinant lipase had a molecular mass of 28kD, showing a range similar to that of PEL, could hydrolyze olive oil and formed clear halos in the olive oil plates. Four different strategies (different media, pH, glycerol and methanol concentration) were applied to optimize the cultivation conditions. The activity of lipase was up to 260u/ml after 72h under the optimal cultivation conditions. It is pointed out that the absence of the expensive biotin and yeast nitrogen base in the medium increased the lipase production. The possible reason of this result is absence of yeast nitrogen base increased the medium pH during cultivation, and PEL shows a higher stability at this condition. The lipase activity of the supernatant from the culture grown at pH 7.0 was higher than the one from the culture in the same medium at pH6.45 can also due to the pH stability of PEL. The results also showed that the methanol and glycerol concentration had a marked effect on the production of lipase.2. Site-directed Mutagenesis of PELAttractive applications of cold-active enzymes in biotechnology would include food processing, additives in detergents (cold washing), biosynthetic processes with volatile intermediates, or environmental bioremediation, we have been attempting to create cold-adapted forms from mesophilic enzyme PEL by Site-directed Mutagenesis usingoverlap extension PCR. According to three-dimensional structure modeling, we select two mutants: P163A and D92L. The mutant recombinant plasmid pPlC3.5K-PEL-P163A was expressed in Pichia pastoris GS115 and SMD1168. Comparative experiments of PEL-P163A-GS with PEL and PEL-GS showed that: I . The optimum temperature of PEL-P163A-GS was 15 C lower than that of the wild type; II. The mutant is sensitive to the temperature, the residual activity is 62% incubated under 35 C for 30min, while the activity of PEL-GS is nearly unchanged in the same condition. The culture temperature influences the yield of PEL-GS and PEL-P163A-GS, it strongly affects the yield of PEL-P163A-GS, Lowering the temperature from 28 to 25 C during the methanol induction phase results in a twofold to threefold increase in yield, the activity reached 270u/ml after 72h at 25 C, pH7.35, and the activity of PEL-GS is 380u/ml after 96h at the same condition. The yield and activity of PEL-P163A are almost the same whether expressed in GS115 or SMD1168, suggested PEL-P163A should be unsensitive to the protease. The mutant PEL-D92L was expressed in Pichia pastoris GS115, SDS-PAGE detection showed that the expression product PEL-D92L-GS is different from PEL-GS, and its' yield decreased dramatically, the themostability of PEL-D92L-GS is also different from the PEL-GS, but their optimum temperatures are same.3. Directed evolution of PEL through random mutagenesisMutagenesis PCR carried out in error-prone conditions was used on the vector pSK-PEL, using the oligos "beginning" and "end", homologous to the 5'and to the 3' ends of the gene of PEL respectively. The conditions for the error-prone PCR are adjusted to 0.2 mutations every 100 amplified bases. The amplified DNA fragment was gel-purified and cloned into vector pPIC3.5K using the SnaB I and EcoR I restric...
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