Improved Enzyme from Transgenic Pichia pastoris Applicable for Industrial-Scale Biodiesel Production

Ying Li, from China Agricultural University, and her team have previously cloned a lipase gene from Rhizomucor miehei and expressed it in Pichia pastoris strain GS115. It produced the enzyme RML, which catalyzed methanolysis of soybean oil. However, the enzyme activity and amount of enzyme produced were not enough for industrial application. Li and her team now aim to improve the properties of RML to apply it to biofuel production.

Pichia pastoris strain X-33 was improved by adding propeptide to the target gene, optimizing its signal peptide and varying the number of target gene copies inserted. The highest enzyme activity were observed in the strain with two copies of the target gene. The enzyme it produced was termed Lipase GH2. This enzyme had improved thermostability as well as methanol and ethanol tolerance. The conversion rate of microalgae oil to biofuel was above 90% after 24 hours of reaction at 30°C.

Results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased enzyme production and enhanced the enzyme's properties. The modified enzyme is potentially applicable for industrial-scale biodiesel production.