title: A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit.
abstract: A 1,3-β-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-β-glucanase with a Mr of 22.07kDa 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°C and it retained 40% maximum activity at 5°C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50°C. Kinetic studies revealed low k(cat) (3.10±0.04 s(-1)) and Km (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°C, and a low Ea (6.99 kJ mol(-1)) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-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 μg 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 CO2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO2.
authors.affiliation: Grupo Biotecnología y Calidad Posrecolección, Departamento de Caracterización, Calidad y Seguridad, Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN-IF (CSIC), José Antonio Novais 10, Ciudad Universitaria, E-28040 Madrid, Spain.
authors.forename: Oscar
authors.initials: O
authors.lastname: Goñi
authors.forename: María T
authors.initials: MT
authors.lastname: Sanchez-Ballesta
authors.forename: Carmen
authors.initials: C
authors.lastname: Merodio
authors.forename: María I
authors.initials: MI
authors.lastname: Escribano
mesh_terms: D031041:Annona / Q000201:enzymology*; D002245:Carbon Dioxide / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry*; D003080:Cold Temperature; D003451:Cryoprotective Agents / Q000737:chemistry; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D002245:Carbon Dioxide; D007770:L-Lactate Dehydrogenase; D002480:Cellulase