BjCHI1 from Brassica juncea displays both chitinase and agglutination activity

Abstract

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.

利用畢赤酵母表達系統表達芥菜幾丁質酶基因BjCHI1及其兩個衍生基因BjCHI2和BjCHI3,獲得相應的蛋白質。經FPLC純化后 ,測定了 3種蛋白質的幾丁質酶活性 ,發現它們均能降解CM chitin RBV和膠狀幾丁質。以CM chitin RBV為底物時的Km值分別為 0 .799mg mL、0 .5 44mg mL和 0 .793mg mL ,差別甚微。而以膠狀幾丁質為底物時的Km值分別為 0 .2 81mg mL、0 .388mg mL和 1.6 43mg mL ,表現一定的差別 ,說明幾丁質結合域影響了酶對不溶性底物的親和力。 3種蛋白中 ,只有BjCHI1在 33μg mL以上濃度具有凝集素活性 ,而BjCHI2和BjCHI3的濃度即使高達 80 0 μg mL也無凝集素活性 ,表明 2個幾丁質結合域是BjCHI1具有凝集素活性的必需條件 ,這是植物中發現的第一個兼有幾丁質酶和凝集素活性的蛋白質