Indole alkaloids production by Catharanthus roseus protoplasts with artificial cell walls containing of guluronic acid rich alginate gel

When low molecular weight alginate (guluronic acid (G)-oligomers, M.W. less than or equal to 2,000) was added to Catharanthus roseus cell culture, intracellularly stored ajmalicine was released into the broth. Addition of agarose and high molecular weight alginate did not promote the release of ajmalicine. C. roseus protoplasts were immobilized in guluronic acid rich (G-rich) alginate gel beads (artificial cell wall having a kind of elicitor function) and used for ajmalicine production. Immobilized protoplasts could be cultivated in a shake flask at low osmotic pressure, without disruption. The extracellular ajmalicine production by the protoplasts immobilized in alginate was much higher than that by the cells immobilized in alginate and protoplasts immobilized in agarose. On the 7 d of cultivation, cell wall regeneration in the immobilized protoplasts was detected under a fluorescence microscope. This implies that prevention of cell wall regeneration is a prerequisite for long term process with protoplasts. When 30 mM CaCl2 was added to the broth, active protoplasts were maintained for 15 d with neither cell wall regeneration nor inhibition of indole alkaloid production. The specific productivities of various indole alkaloids (ajmalicine, catharanthine and tryptamine) by the immobilized C. roseus protoplasts in the presence of CaCl2 in the broth were much higher than those of the immobilized cells.