A cryoprotective and cold-adapted 1,3- beta-endoglucanase from cherimoya (Annona cherimola) fruit.

摘要

A 1,3- beta-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO₂-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3- beta-glucanase with a M_r of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 degrees C and it retained 40% maximum activity at 5 degrees C. The purified 1,3- beta-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 degrees C. Kinetic studies revealed low k_cat (3.10 +or- 0.04 s--1) and K_m (0.32 +or- 0.03 mg ml--1) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k_cat at both 37 and 5 degrees C, and a low E_a (6.99 kJ mol--1) within this range of temperatures. In vitro functional studies indicated that the purified 1,3- beta-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD₅₀ was 8.7 mug ml--1 (394nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO₂-treated cherimoya tissues, these results suggest that 1,3- beta-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO₂.