Functionality of a class I beta-1,3-glucanase from skin of table grapes berries

We have analyzed gene expression of a class I β-1,3-glucanase (Vcgns1), in the skin of red table grapes (Vitis vinifera cv. Cardinal) as a marker for changes in response to low temperature, and also to assess how high CO2 levels (20% CO2 plus 20% O2) modulated its transcript accumulation at 0 °C. To elucidate the possible physiological role of class I β-1,3-glucanase in table grapes as a cryoprotectant, and/or an antifreeze, we also report on recombinant expression of Vcgns1. The results indicate that storage at 0 °C for 3 days increased Vcgns1 mRNA levels and β-1,3-glucanase activity in the skin of non-treated grapes. However, the accumulation of the transcripts, and the level of glucanase activity, were lower in the skin of grapes after 3 days of CO2 treatment, as well as when treated fruit were transferred to air. By using heterologous expression of the Vcgns1 cDNA in Escherichia coli, we showed that it encoded a protein with glucanase activity with an optimum pH and temperature of 6 and 45 °C, respectively, and a high stability at 0 °C. Furthermore, the purified VcGNS1 exhibited in vitro cryoprotective activity for the freeze labile l-lactate dehydrogenase (LDH) enzyme. In contrast, when the thermal hysteresis activity (THA) of the recombinant VcGNS1 was measured, using differential scanning calorimetry (DSC), the results indicated that it did not show antifreeze activity. The high stability of the recombinant protein at 0 °C, and its cryoprotective activity shown in this work suggest that VcGNS1 may participate in the response of table grape to combat low temperature conditions.