How Can RuBisCO Be Released from the Mesophyll Cells of Green Tea Residue?

Although ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been obtained from green tea residue mesophyll cells (TRMCs), its intact release has not yet been achieved. To release RuBisCO, this study employed a combination or sequential treatments using urea, β-mercaptoethanol, sodium dodecyl sulfate (SDS), and enzymes. Factors that hindered RuBisCO release from TRMCs were investigated through SDS-PAGE analysis, protein release quantification, and electron microscopy techniques. Alkali treatment of TRMCs at 95 °C facilitated protein release, while also causing protein modification or degradation. Conversely, the combined treatment of β-mercaptoethanol with urea and/or SDS could effectively disrupt the disulfide bonds, hydrogen bonds, and/or hydrophobic interactions within the cells, leading to the release of 40% or more of the proteins. This treatment showed strong electrophoretic bands at 55 and 15 kDa, indicating that RuBisCO was completely released. No protein was released during the treatment with SDS and pepsin/papain/alkaline protease, indicating that RuBisCO was hindered by the presence of cellulose and hemicellulose. Sequential treatment with SDS and Viscozyme L dissolved TRMC lignocellulose without releasing RuBisCO, suggesting the low solubility of RuBisCO. Overall, the presence of lignocellulose in the cell wall and the low solubility of RuBisCO were identified as key factors hindering its release from the TRMCs.