Potent cryoprotective activity of cold and CO2-regulated cherimoya (Annona cherimola) endochitinase

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO2-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a Mr of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 °C, and it retained about 30% of its maximum activity at 5 °C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 °C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high kcat (6.93 s−1) value for the fluorogenic substrate 4-MU-(GlcNAc)3, reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high kcat at 37 and 5 °C, and a low Ea (11.32 kJ mol−1). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze–thaw inactivation of lactate dehydrogenase. The PD50 was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO2-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO2 concentrations.