Chi-Chung Peng

Size and stability of reconstituted sesame oil bodies.

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil‐body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil‐body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross‐linked by glutaraldehyde or genipin.

Gene Family of Oleosin Isoforms and Their Structural Stabilization in Sesame Seed Oil Bodies

Oleosins are structural proteins sheltering the oil bodies of plant seeds. Two isoform classes termed H- and L-oleosin are present in diverse angiosperms. Two H-oleosins and one L-oleosin were identified in sesame oil bodies from the protein sequences deduced from their corresponding cDNA clones. Sequence analysis showed that the main difference between the H- and L-isoforms is an insertion of 18 residues in the C-terminal domain of H-oleosins. H-oleosin, presumably derived from L-oleosin, was duplicated independently in several species. All known oleosins can be classified as one of these two isoforms. Single copy or a low copy number was detected by Southern hybridization for each of the three oleosin genes in the sesame genome. Northern hybridization showed that the three oleosin genes were transcribed in maturing seeds where oil bodies are being assembled. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and sesame oleosin isoforms. The results indicated that reconstituted oil bodies could be stabilized by both isoforms, but L-oleosin gave slightly more structural stability than H-oleosin.

Immobilization of Clostridium cellulolyticum d-Psicose 3-Epimerase on Artificial Oil Bodies

The rare sugar D-psicose possesses several fundamental biological functions. D-Psicose 3-epimerase from Clostridium cellulolyticum (CC-DPEase) has considerable potential for use in D-psicose production. In this study, CC-DPEase was fused to the N terminus of oleosin, a unique structural protein of seed oil bodies and was overexpressed in Escherichia coli as a CC-DPEase-oleosin fusion protein. After reconstitution into artificial oil bodies (AOBs), refolding, purification, and immobilization of the active CC-DPEase were simultaneously accomplished. Immobilization of CC-DPEase on AOB increased the optimal temperature but decreased the optimal pH of the enzyme activity. Furthermore, the AOB-immobilized CC-DPEase had a thermal stability and a bioconversion rate similar to those of the free-form enzyme and retained >50% of its initial activity after five cycles of enzyme use. Thus, AOB-immobilized CC-DPEase has potential application in the production of d-psicose at a lower cost than the free-form enzyme.