Biodiesel, a nontoxic,cleaning, renewable and biodegradable fuel, is expected as a substitute for conventional fossil diesel. There are three main approaches to produce biodiesel, alkali-catalysis processing, enzymatic-catalysis processing and supercritical processing. With the unique property of energy-saving and environment-friendly, enzymatic-catalysis appears a great potential for industrial application. The main bottleneck of this technology is high cost and low stability of the lipase, as well as the inactivation of lipase by methanol and so on. To settle the problem, several methods have been used including the fixed-bed bioreactor, enzyme immobilized processing, whole-cell biocatalyst, changing addition method of methanol, developing of novel acyl acceptor, enhancing methanol resistance of lipase. The main problems and the relative strategy research of the enzymatic-catalysis technology were sum up.

Lipase gene from Penicillium expansum(lip07) was cloned and over-expressed in Pichia pastoris.a random mutant named ep8,which contained a single amino acid substitution,was obtained by using the lip07 as an error-prone PCR template in previous study.ep8 shows higher thermostability than that of lip07,To further improve the thermostability of the lipase,the Lys of wild-type(lip07) and mutant(ep8) in 202 were substituted by Ala using the Overlap extension PCR technique respectively.The mutant genes(lip07-K202A and ep8-K202A) were subcloned into pAO815,and then transformed into the Pichia pastoris GS115 for extracelluar expression,respectively.15% SDS-PAGE analysis indicated that the molecular mass of PEL-ep8-K202A and PEL-lip07-K202A are both about 28kDa,which is same with the wild-type lipase.The Tm of PEL-ep8-K202A is 41.66℃,2.63℃ higher than that of the wild-type(39.03℃) and 1.21℃ higher than the random mutant(PEL-ep8:40.45℃);the Tm of single mutant(PEL-lip07-K202A) is 37.08℃,2℃ lower than that of the wild-type lipase.