Characterization of an antifungal and cryoprotective class I chitinase from table grape berries (Vitis vinifera cv. Cardinal).
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 CO(2) levels modulating transcript levels at 0 degrees 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 degrees C, retaining activity at 0 and -10 degrees 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.
Fernandez-Caballero
Carlos
C
Departamento de Ciencia y Tecnología de Productos Vegetales, Instituto del Frio, IF-CSIC, Ciudad Universitaria, Madrid, Spain.
Romero
Irene
I
Goñi
Oscar
O
Escribano
M Isabel
MI
Merodio
Carmen
C
Sanchez-Ballesta
M Teresa
MT
D000595:Amino Acid Sequence; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D003451:Cryoprotective Agents; D004926:Escherichia coli / Q000235:genetics; D005615:Freezing; D005638:Fruit / Q000201:enzymology*; D005659:Fungicides, Industrial / Q000494:pharmacology; D015870:Gene Expression; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D027843:Vitis / Q000201:enzymology*
D003451:Cryoprotective Agents; D005659:Fungicides, Industrial; D010940:Plant Proteins; D011994:Recombinant Proteins; D007770:L-Lactate Dehydrogenase; D002688:Chitinases; C119825:chitinase C