| Although the catalytic properties of natural Penicillium expansum lipase(PEL) had its own characterics, it could not commendably satisfy its indrustrial application because of the shortcomings of thermal stability, organic solvent tolerance, and so on. In this study, the specific sites of PEL were mutated by site saturation mutagenesis and then the PEL mutants were screened successfully by tributyrin emulsion plate.. The mutants were expressed in procaryotic expression system(E. coli BL21) Then, the PEL mutants were expressed in Eukaryotic expression system(Pichia pastoris GS115) to analyze their enzymatic properties, aiming to improve their thermal stability and organic solvent tolerance, which were used as a better enzyme source for their further industrial application. The main research results were as follows:1. Construction of the mutant libraries and screening of the high-activity lipases.The 92 site of PEL was mutated by designed degenerate primers to construct one mutant library which was expressed in prokaryotic expression system and screened by tributyrin emulsion plate. As a result, a PEL mutant (D92E) was obtained. The experimental results showed that its thermal stability was higher than that of the wild-type PEL. On the basis of the foregoing screening results, another three sites of PEL was mutated using semi-rational design method. As a result, three corresponding mutants T66E, N73Q and D151E were obtained.2. Expression of the mutants and analysis of their enzymatic propertiesThe screened PEL mutants was cloned into expression plasmid pPIC3.5k to perform the construction of recombinant plasmids pPIC3.5k-PEL-D92E, pPIC3.5k-PEL-T66E, pPIC3.5k-PEL-N73Q and pPIC3.5k-PEL-D151E, which were linearized by 5al I and transformed into Pichia pastoris GS115 to be expressed by methanol induction. Further more, the enzymatic properties of the PEL mutants was identified. The main results were as follows:(1) The thermal stability of the PEL mutant D92E was relatively higher than that of the wild-type PEL.The residual enzyme activity of the mutant D92E was 70.28% while that of the wild-type PEL was only 31.72%after treatment at 50℃ for 10 min.The thermal. stability of themutant D151E was increased slightly compared with the wild-type PEL and that of the mutants T66E and N73Q was decreased significantly. (2) The organic solvent tolerance of the mutants D92E and D151E were relatively higher than that of the wild-type PEL. After ethanol treatment at 37℃ for 10 min, the residual enzyme activity of the mutants D92E and D151E was 88.32% and 95.47% respectivity while that of the wild-type PEL was 62.06%. The organic solvent tolerance of the mutants T66E and N73Q was decreased significantly compared with that of the wild-type PEL. |
