Effects of C-Terminal Domain Truncation on Enzyme Properties of Serratia marcescens Chitinase C

Abstract

A chitinase gene (SmChiC) and its two C-terminal truncated mutants, SmChiCG426 and SmChiCG330 of Serratia marcescens, were constructed and cloned by employing specific polymerase chain reaction (PCR) techniques. SmChiCG426 is derived from SmChiC molecule without its C-terminal chitin-binding domain (ChBD) while SmChiCG330 is truncated from SmChiC by its C-terminal deletion of both ChBD and fibronectin type III domain (FnIII). To study the role of the C-terminal domains of SmChiC on the enzyme properties, SmChiC, SmChiCG426, and SmChiCG330 were expressed in Escherichia coli by using the pET-20b(+) expression system. The His-tag affinity-purified SmChiC, SmChiCG426, and SmChiCG330 enzymes had a calculated molecular mass of 51, 46, and 36 kDa, respectively. Certain biochemical characterizations indicated that the enzymes had similar physicochemical properties, such as the optimum pH (5), temperature (37 °C), thermostability (50 °C), and identical hydrolyzing product (chitobiose) from both the soluble and insoluble chitin substrates. The overall catalytic efficiency k cat /K M was higher for both truncated enzymes toward the insoluble α-chitin, whereas the binding abilities toward the insoluble α-chitin substrate were reduced moderately. The fluorescence and circular dichroism (CD) spectroscopy data suggested that both mutants retained a similar folding conformation as that of the full-length SmChiC enzyme. However, a CD-monitored melting study showed that the SmChiCG330 had no obvious transition temperature, unlike the SmChiC and SmChiCG426.