| Penicillium expansion PF898 is a mutant strain obtained by many generation mutagenesis, can produce an alkaline lipase at a high level. Both for broadening the industrial applicability and for furthering understanding of the relationship between structure and function, we have applied protein engineering to improve the characterise of the Penicillum expansum lipase (PEL).Mutants of D92P, G98A, N148T, E113V, P163G, P163L, P163H, P163W, P163F, P163R of PEL gene were obtained by in vitro site-directed mutagenesis using overlap extension PCR. The recombinant plasmid PEL-pPIC3.5K containing mutant genes were cloned into Escherichia coli TG1 strain and expressed in Pichia pastoris GSl 15 strain. The comparison results of the muatants with wild-type PEL showed that:(1) The optimum temperature for PEL-D92P-GS was higher by 5℃ than that of the wild type PEL-GS, while the thermostability of the mutant was similar to the wild type, the substitution of Pro for Asp at position 92 might introduce a pyrrolidine ring at a loop, and an increase of the frequency of proline occurrence at loop could make the protein structure more rigid, thus enhanced the optimum temperature of PEL.(2) Tribulyrin was hydrolysed by PEL-G98A-GS, but nearly no activity was detected towards olive oil. It showed that the 98th Gly was essential for the lipase activity towards olive oil.(3) The catalytic activity of PEL-N148T-GS was 109.03 U mg-1 at 40℃, which was 115% that of the wild type at the same conditions. The optimum temperature of mutant was the same to the wild type while it is sensitive to temperature.(4) The optimum temperature of the mutant PEL-E113V-GS (45℃) was higher by 5℃ than that of the wild type PEL-GS, while the thermostability of the mutant was some decreased to the wild type.(5)SDS-PAGE detection showed that the expression product PEL-P163G-GS, PEL-P163L-GS, PEL-P163H-GS, PEL-P163W-GS, PEL-P163F-GS, PEL-P163R-GS were different from PEL-GS, and their yield decreased dramatically.
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