High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme.
Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae. For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219. We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysis 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 two 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 spectra and determination of disulfide bonding. This suggests that protein folding of the recombinant lipase occurred correctly in yeast.
Kohno
M
M
Tsukuba R & D Center, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara-Mura, Ibaraki, Tsukuba-gun, 300-2497, Japan. kouno@ji.tkc.fujioil.co.jp
Enatsu
M
M
Yoshiizumi
M
M
Kugimiya
W
W
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D005800:Genes, Fungal; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D008049:Lipase; D014357:Trypsin