Toshimitsu Nakashima

Culture Conditions for Intracellular Lipase Production by Rhizopus chinensis and Its Immobilization within Biomass Support Particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells. It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity. The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells. The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.

Cell aggregation as a trigger for enhancement of intracellular lipase production by a Rhizopus species

Abstract Immobilization of Rhizopus chinensis cells in various Biomass support particles (BSPs) made from polyurethane, nylon, polyester, stainless steel, polyvinyl alcohol, and cellulose enhanced intracellular lipase activity as much as several-fold that of freely suspended cells irrespective of the BSP material. In freely suspended cultivation the overall intracellular lipase activity of free cells, consisting of pellet cells and pulp-like cells, increased with an increase in the ratio of pellet cells to pulp-like cells. It was thus considered that cell aggregation seems to be a principal factor in the enhancement of intracellular lipase formation by Rh. chinensis . Also, other Rhizopus species such as Rh. javanicus, Rh. delemar, Rh. oligosporus, Rh. niveus, Rh. japonicus and Rh. oryzae were immobilized in both polyurethane and cellulose BSPs, In all species used, intracellular lipase production was significantly enhanced compared to results without immobilization. Cell aggregation therefore seems to be a common trigger for the enhancement of intracellular lipase production by Rhizopus species.

Intracellular lipase production by Rhizopus chinensis using biomass support particles in a circulating bed fermentor

Abstract To utilize Rhizopus chinensis cells directly as a catalyst for industrial interesterification of oils and fats, the mass production of acetone-dried cells immobilized in Biomass Support Particles (BSPs) which show high intracellular lipase activity was examined using a circulating bed fermentor (CBF). First, the effects of aeration rate and number of BSPs were investigated in batch culture. The results revealed that the aeration rate had a significant influence on the formation of intracellular lipase. Therefore, a comparatively low aeration rate (4 vvm) capable of attaining good circulation of BSPs in the CBF was adequate for the lipase production. The effects of meat extract (main component of medium) feeding on intracellular lipase production was also examined by constant-feed rate fed-batch cultures. The results indicated that the specific feed rate of meat extract q s controlled in the range of 0.04–0.08 (g-meat extract)(g-cell) −1 (h) −1 might enhance intracellular lipase activity, whereas the cellular growth in BSPs was independent of the meat extract feed rate. Therefore, the fed-batch culture in which the specific feed rate of meat extract q s was controlled at 0.05 by varying the meat extract feed rate stepwise using a microcomputer was carried out, and the intracellular lipase activity of immobilized cells could be enhanced 1.3–1.5 times that obtained in batch cultivation. The specific feed rate of meat extract q s is an effective index for the production of intracellular lipase of Rh. chinensis .

Transesterification of phospholipids by acetone-dried cells of aRhizopus species immobilized on biomass support particles

Acetone-dried cells of aRhizopus species, immobilized on biomass support particles were applied to the transesterification between phosphatidylcholine and heptadecanoic acid. All eight species ofRhizopus tested exhibited good transesterification performance, with the immobilized cells ofR. niveus, R. delemar, andR. javanicus showing particularly high reaction rates which were equal to or exceeded that of a commercially available immobilized lipase preparation, Lipozyme IM60.

Continuous interesterification of oils and fats using dried fungus immobilized in biomass support particles

Abstract The industrial feasibility of an interesterification process using acetone-dried fungus (as a lipase catalyst) immobilized in biomass support particles (BSPs) was examined by continuous interesterification between olive oil and methyl stearate, where the water content of the reaction mixture (Cw) was controlled at a given value. The Cw affected not only the inactivation rate of lipase in the cells but also the production rate of the by-product (diglycerides). The optimal Cw was determined as about 100 ppm. The half-life of lipase in the cells was about 1200 h at the optimal Cw, suggesting that the interesterification process using the immobilized fungus is industrially feasible.

Purification and Characterization of Lipase from Rhizopus chinensis Cells

Lipase from Rhizopus chinensis cells was purified and characterized. The molecular mass of purified lipase was 28.4 kDa and the optimal temperature and pH for its activity were 37 degrees C and 5.5, respectively. Purified lipase exhibited high hydrolytic activity against fatty acid methyl esters such as methyl caprylate, methyl laurate, and methyl palmitate. Freeze-dried lipase catalyzed the transesterification between olive oil and methyl laurate in n-hexane.

Effects of aeration rate on intracellular lipase production by Rhizopus chinensis immobilized within biomass support particles in a circulating bed fermentor

Abstract The effects of the aeration rate on intracellular lipase production by Rhizopus chinensis immobilized in biomass support particles (BSPs) was examined using circulating bed fermentors (CBFs) of three sizes. The minimum aeration rate that achieved good circulation of BSPs in CBF was appropriate for BSPs-CBF intracellular lipase production.

A Novel Interesterification Process for Fats and Oils Using Acetone-Dried Fungus Immobilized in Biomass Support Particles

Lipase not only hydrolyzes triglycerides but also catalyzes transesterification under micro-aqueous condition. Among lipase reactions the production of cocoa butter substitute by transesterification has recently drawn industrial attention. Since lipases are expensive as compared with amylases and proteases, however, the development of a more economical transesterification process is desirable.