High-level expression of Rhizopus niveus lipase in the yeast Saccharomycescerevisiae and structural properties of the expressed enzyme

Rhizopus niveus lipase (RNL) has a unique structure consisting of two nonco valently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression syst em for the production of recombinant lipase in the yeast Saccharomyces cere visiae. For the present study, we developed a more efficient expression sys tem using the strain ND-12B and the multicopy-type plasmid pJDB219. We puri fied two types of recombinant lipases, each to a single peak by gel-filtrat ion HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysi s of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type). Moreover, there were tw o species within each type of lipase. One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed. Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism s pectra and determination of disulfide bonding. This suggests that protein f olding of the recombinant lipase occurred correctly in yeast. (C) 1999 Acad emic Press.