Cloning, Purification, and Characterization of a Novel Metagenome-Sourced Microbial Chitinase with Dual Catalytic Domains
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: A metagenomic approach was used to discover the chitinases from a wetland on the Qinghai-Tibetan plateau. Gene P1724 was found and predicted to have two GH18 catalytic domains. Gene sequence
... containing P1724 , only its N-terminal GH18 domain ( P1724nGH18 ) or only C-terminal GH18 domain ( P1724cGH18 ) were cloned and expressed in Escherichia coli BL21 (DE3). 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 produced predominantly N, N΄-Diacetylchitobiose from colloid chitin. 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's, which indicated that two GH18 domains of P1724 works synergistically in degrading chitin. Conclusion: Compared to the most of microbial chitinases containing only one catalytic domain, chitinases P1724 with two GH18 catalytic domains was discovered firstly by the metagenomic approach. P1724 is a novel chitinase with unique amino acid sequences and hydrolytic mode, and could be used in cold environments or industries.