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Background  Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using  Rhizopus oryzae  lipase as catalyst. However, the high cost of  R. oryzae  lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from  R. oryzae  reached 1334 U/mL in  Pichia pastoris , which is still not optimistic for industry applications.    Results  The prosequence of lipases from  Rhizopus  sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from  R. oryzae  was constructed by replacing the prosequence with that from the  R. chinensis  lipase and expressed in  P. pastoris . The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C.    Conclusions  This is the first report on improving the expression level of the lipase from  R. oryzae  by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase from  R. oryzae  as an economic catalyst make this study a promising one for biodiesel applications.

High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production