Production of cell wall accumulative enzymes using immobilized protoplasts of Catharanthus roseus in agarose gel

Catharanthus roseus cells and protoplasts were used for production of peroxidase and alpha-galactosidase which are accumulated in the cell wall. Only 4% (0.026 U ml(-1)) of the total peroxidase was secreted into the broth by cultured cells while in cultured protoplasts, 45% (0.12 U ml(-1)) was secreted. Protoplasts were protected against the physical and osmotic stresses by immobilizing them in 3% agarose gel (high mass transfer, non-electric charge, low gelation temperature). In order to increase peroxidase production, the immobilized protoplasts were cultivated in shake cultures at low osmotic pressure (12.3 to 16.4 atm) without disruption. During batch peroxidase production, the total activities obtained with free cells at 4.9 atm, free protoplasts at 19.3 atm, and immobilized protoplasts at 12.3 atm were 0.17, 2.54, and 5.16 U, respectively. When four repeated-batch cultures of the immobilized protoplasts were performed at 16.4 atm, 11.8 U of peroxidase was obtained. This system was also useful for the production of alpha-galactosidase.