Biochemical Properties of a Novel Cold-Adapted GH19 Chitinase with Three Chitin-Binding Domains from Chitinilyticum aquatile CSC-1 and Its Potential in Biocontrol of Plant Pathogenic Fungi

GH19 (glycoside hydrolase 19) chitinases play crucial roles in the enzymatic conversion of chitin and biocontrol of phytopathogenic fungi. Herein, a novel multifunctional chitinase of GH19 (CaChi19A), which contains three chitin-binding domains (ChBDs), was successfully cloned from Chitinilyticum aquatile CSC-1 and heterologously expressed in Escherichia coli. We also generated truncated mutants of CaChi19A_Delta I, CaChi19A_Delta I Delta II, and CaChi19A_CatD consisting of two ChBDs and a catalytic domain, one ChBD and a catalytic domain, and only a catalytic domain, respectively. CaChi19A, CaChi19A_Delta I, CaChi19A_Delta I Delta II, and CaChi19A_CatD exhibited cold adaptation, as their relative enzyme activities at 5 degrees C were 40.7, 51.6, 66.2, and 82.6%, respectively. Compared with CaChi19A and other variants, CaChi19A_Delta I Delta II demonstrated a higher level of stability below 50 degrees C and retained relatively high activity over a wide pH range of 5-12. Analysis of the hydrolysis products revealed that CaChi19A and CaChi19A_Delta I Delta II exhibit exoacting, endoacting, and N-acetyl-beta-d-glucosaminidase activities toward colloidal chitin. Furthermore, CaChi19A and CaChi19A_Delta I Delta II exhibited inhibitory effects on the hyphal growth of Fusarium oxysporum, Fusarium redolens, Fusarium fujikuroi, Fusarium solani, and Coniothyrium diplodiella, thereby illustrating effective biocontrol activity. These results indicated that CaChi19A and CaChi19A_Delta I Delta II show advantages in some applications where low temperatures were demanded in industries as well as the biocontrol of fungal diseases in agriculture.