Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases.
Results: Gene P1724 was found in a chitin-enriched microbial community from a wetland on the Qinghai-Tibetan plateau using the metagenomic approach. The translated protein sequence of P1724 was predicted to have two GH18 catalytic domains and showed very low similarities with any known and hypothetical chitinases. The gene sequence of P1724 , its N-terminal GH18 domain ( P1724nGH18 ), or C-terminal GH18 domain ( P1724cGH18 ) was cloned and expressed in Escherichia coli BL21 (DE3). Using colloid chitin as substrate, these purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C; the activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ ; all three chitinases showed both chitotiosidase and endochitinase activities, and could hydrolyze chitosan as well. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity 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. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18, which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin.
Conclusion: Compared to most microbial chitinases that contain only one catalytic domain, P1724 contains two and was firstly discovered by the metagenomic approach. P1724, its N-terminal and C-terminal catalytic domains were heterologously expressed and characterized. These three recombinant chitinases are phylogenetically distant to any chitinases studied so far, have unique hydrolytic mode, high catalytic efficiency and so new promising candidates for chitinases in applications. In addition, this study broadens the knowledge of unknown chitinases in nature and shows a natural strategy for enzyme engineering by adding other catalytic domain(s).