A Study On Expression Of Lipase In Pichia Pastoris And Its Application In Conversion Of Waste Cooking Oil

Using lipase to change waste cooking oil into biodiesel is effective for the green and resource utilization of food waste. In the current work, a recombinant Pichia pastoris which can express lipase was cosntructed. Using recombinant lipase instead of traditional chemicals as catalyst, and green nontoxic ethanol instead of methanol as acyl receptor, the waste cooking oil was changed into biodiesel through the transesterification reaction.proROL from Rhizopus oryzae CICC3005 was cloned into the vectors pGAPZαA and subsequently, electrotransformed into Pichia pastoris X-33. A recombinant strain with the highest enzyme activities was isolated. The characteristics of the recombinant proROL was carried out.The results showed that the highest lipase activity was 426.6 U/mL when using olive oil as substrate. The optimum pH for the recombinant proROL was pH 8.0 and the lipase was stable from pH 6.0 to 8.0. The optimum temperature for the recombinant proROL was 30℃, and the lipase was stable from 30 to 35 ℃. The enzyme activity was enhanced by some metal ions,such as Mn²⁺, Zn²⁺, Na+ and K+, and some surfactants, such as 0.001% Triton X-100 and 0.01% SDS. The recombinant proROL was stable in methanol and methylbenzene and n-hexane. The enzyme activity was relatively higher using pnp-caprylate（8C） and pnp-myristate（14C） as substrate.Using waste cooking oil as raw material, absolute ethyl alcohol as acyl receptor, and recombinant lipase as catalyst, the waste cooking oil was changed into biodiesel through the transesterification reaction. To find out the optimal reaction condition, we compared the productivity of biodiesel among different water content, alcohol oil molar ratio and amount of recombinant lipase. The results showed that the highest biodiesel yield was reached 49%, when water content was 5%, alcohol oil molar ratio was 4:1, and amount of recombinant lipase was 10%.In this work, we constructed a recombinant Pichia Pastoris which can secrete lipase and evaluated characteristics of the enzyme. We also used waste cooking oil as raw material, recombinant lipase instead of traditional chemicals as catalyst, and green nontoxic ethanol instead of methanol as acyl receptor to produce biodiesel. It would reduce the usage of chemicals, save cost, and reduce pollution. Our study might shed light on the foundation for future research about industrial biodiesel production.