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

A 1,3-beta-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO(2)-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 +/- 0.04 s(-1)) and K(m) (0.32 +/- 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 PD(50) was 8.7 mu g ml(-1) (394 nM), 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 CO(2)-treated cherimoya tissues, these results suggest that 1,3-beta-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO(2). (C) 2011 Elsevier Ltd. All rights reserved.