The discovery and characterization of a novel chitinase with dual catalytic domains from a Qinghai-Tibetan Plateau wetland soil metagenome.

A novel chitinase (P1724) was discovered from a Qinghai-Tibetan plateau microbial metagenome. P1724 contains two GH18 family catalytic domains and is phylogenetically distant from any of the chitinases studied. P1724 and its truncated versions, P1724(∆cGH18) and P1724(∆nGH18), were produced in Escherichia coli and characterized. Using colloidal chitin as substrate, the three recombinant proteins showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive, as they remained active at 4 °C; their chitinase activities were decreased with the presence of Cu2+ and EDTA, but increased with Ba2+ and Ca2+; they all showed both chitobiosidase and endochitinase activities. Compared to P1724(∆nGH18), P1724 and P1724(∆cGH18) shared more similarities in temperature and pH stabilities, NaCl tolerance, and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. kcat/Km value of P1724 was significantly higher than the sum values of P1724(∆cGH18) and P1724(∆nGH18), which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin. This study has not only broadened the understanding of unknown chitinases in nature but also discussed the strategy of adding additional catalytic domains in enzyme engineering.