Ouyang Shi-Wen, Zhao Kai-Jun, Feng Lan-Xiang, Chye Mee-Len, Ram Sathishkvmar
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Sheng Wu Gong Cheng Xue Bao. 2002 Sep;18(5):572-7.
The proteins encoded by the Brassica juncea chitinase gene BjCHI1 and its derived genes BjCHI2 and BjCHI3 were expressed by Multi-copy Pichia expression system. The chitinase activity of FPLC purified BjCHI1, BjCHI2 and BjCHI3 were tested and the results showed that all the three proteins degraded both CM-chitin-RBV and colloidal chitin. The Km values of BjCHI1, BjCHI2 and BjCHI3 for CM-chitin-RBV were estimated as 0.799 mg/mL, 0.544 mg/mL and 0.793 mg/mL, respectively. When the colloidal chitin was used as substrate, the Km values were 0.281 mg/mL, 0.388 mg/mL and 1.643 mg/mL, respectively, indicating chitin-binding domain can increase affinity of chitinase to insoluble substrate. In the agglutination activity assay, only BjCHI1 shows activity when the protein concentration was more than 33 micrograms/mL, while BjCHI2 and BjCHI3 without agglutination activity even when the concentration was increased as high as 800 micrograms/mL. This means that the two chitin-binding domains in BjCHI1 are essential for agglutination and BjCHI1 is the first protein which shows both chitinase and agglutination activity identified so far in plants.
通过多拷贝毕赤酵母表达系统表达了芥菜几丁质酶基因BjCHI1及其衍生基因BjCHI2和BjCHI3编码的蛋白质。对经快速蛋白质液相色谱（FPLC）纯化的BjCHI1、BjCHI2和BjCHI3的几丁质酶活性进行了检测，结果表明这三种蛋白质均能降解羧甲基几丁质-红甜菜病毒（CM-chitin-RBV）和胶体几丁质。BjCHI1、BjCHI2和BjCHI3对CM-chitin-RBV的米氏常数（Km值）分别估计为0.799 mg/mL、0.544 mg/mL和0.793 mg/mL。当以胶体几丁质为底物时，Km值分别为0.281 mg/mL、0.388 mg/mL和1.643 mg/mL，表明几丁质结合结构域可增加几丁质酶对不溶性底物的亲和力。在凝集活性测定中，只有当蛋白质浓度超过33微克/毫升时BjCHI1才表现出活性，而BjCHI2和BjCHI3即使浓度增加到800微克/毫升也没有凝集活性。这意味着BjCHI1中的两个几丁质结合结构域对凝集至关重要，并且BjCHI1是迄今为止在植物中鉴定出的首个同时具有几丁质酶和凝集活性的蛋白质。
