This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7±0.7 × 106 cells g-1 fresh wt for G. dura and 1.2±0.78×106 cells g-1 fresh wt for G. verrucosa when incubated at 25°C for 4-6 h duration. The young growing tips maximally released the protoplasts having a size of 7-15 μm in G. dura and 15-25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25-35 μm, presumably from cortical region, were also observed in G. verrucosa.
- Commercial enzymes
- Protoplast production