Characterization of an Antifungal and Cryoprotective Class I Chitinase from Table Grape Berries (Vitis vinifera Cv. Cardinal)

• Published in: Journal of agricultural and food chemistry 2009-10, Vol.57 (19), p.8893-8900
• Main Authors: Fernandez-Caballero, Carlos, Romero, Irene, Goñi, Oscar, Escribano, M. Isabel, Merodio, Carmen, Sanchez-Ballesta, M. Teresa
• Format: Article
• Language: English
• Subjects: air temperature; Amino Acid Sequence; antifungal properties; Biological and medical sciences; biomarkers; Botrytis cinerea; carbon dioxide; Chemical Aspects of Biotechnology/Molecular Biology; chitinase; Chitinases - genetics; Chitinases - metabolism; Chitinases - pharmacology; cold; cryoprotectants; Cryoprotective Agents; enzyme inactivation; Escherichia coli; Escherichia coli - genetics; Food industries; Freezing; Fruit - enzymology; Fruit and vegetable industries; fruit crops; Fundamental and applied biological sciences. Psychology; Fungicides, Industrial - pharmacology; Gene Expression; gene expression regulation; L-Lactate Dehydrogenase - metabolism; lactate dehydrogenase; messenger RNA; Molecular Sequence Data; peels; plant pathogenic fungi; Plant Proteins; protein structure; protein synthesis; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Sequence Alignment; solubility; table grapes; Vitis - enzymology; Vitis vinifera
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf9016543

Gene expression of a class I chitinase (Vcchit1b) in the skin of table grapes was analyzed as a molecular marker for changes induced at low temperature and also to study the effect of high CO2 levels modulating transcript levels at 0 °C. An active recombinant VcCHIT1b was overexpressed in Escherichia coli, and as the protein was produced as insoluble inclusion bodies, it was solubilized and refolded. The purified recombinant chitinase showed an optimum pH of 6.0 and a temperature of 50 °C, retaining activity at 0 and −10 °C. Purified chitinase exerted in vitro antifungal activity against Botrytis cinerea. Furthermore, recombinant chitinase was able to cryoprotect lactate dehydrogenase against freeze/thaw inactivation. However, the recombinant VcCHIT1b did not show any antifreeze activity when the thermal hysteresis activity was measured using differential scanning calorimetry.