Extracellular production of active Rhizopus oryzae lipase by Saccharomyces cerevisiae

The gene encoding Rhizopus oryzae lipase (ROL) was successfully expressed, and extracellularly active ROL was produced by Saccharomyces cerevisiae. The genes encoding the mature region (mROL) and the mROL having the pro sequence (ProROL) were fused to the gene encoding the pre- or prepro-alpha-factor leader region, and expressed under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), which is strongly functional in S. cerevisiae. Efficient lipase activity was observed in the culture medium when the gene encoding ProROL was expressed. The recombinant lipase (rROL) produced by S. cerevisiae was purified by ethanol precipitation, butyl-Toyopearl 650M chromatography, and Sephacryl S-100 HR gel filtration to a single band by native-PAGE. However, the single band was found to consist of two proteins with different molecular masses (35 kDa and 46 kDa) on SDS-PAGE. The N-terminal analysis showed that 35 kDa protein was processed in the prosequence, and attached 28 amino acids of the C-terminal part of the prosequence, named rPro28ROL, while the 46 kDa protein had the entire pro-region, named rProROL, The preference of rROL for a series of triglycerides was determined using the titrimetric assay. rROL showed higher activity on saturated substrates having C-8-, C-10-, C-12-, and C-14- fatty acid chains in triglycerides. Extracellular lipase activity reached 2880 U/l at 120 h of cultivation in YPD medium.