Departamento de Ciencia y Tecnología de Productos Vegetales, Instituto del Frio, IF-CSIC, Ciudad Universitaria, Madrid, Spain.
J Agric Food Chem. 2009 Oct 14;57(19):8893-900. 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 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.
葡萄果皮中 I 类几丁质酶（Vcchit1b）的基因表达作为低温诱导变化的分子标记，并研究高 CO2 水平对 0°C 下转录水平的调节作用。在大肠杆菌中过表达活性重组 VcCHIT1b，由于该蛋白作为不溶性包涵体产生，因此进行溶解和重折叠。纯化的重组几丁质酶在 pH6.0 时表现出最佳活性，在 50°C 时具有最高活性，在 0°C 和-10°C 时仍保持活性。纯化的几丁质酶对灰葡萄孢具有体外抗真菌活性。此外，重组几丁质酶能够在体外保护乳酸脱氢酶免受冷冻/解冻失活。然而，当使用差示扫描量热法测量热滞活性时，重组 VcCHIT1b 并没有表现出任何抗冻活性。
