title: 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.
authors.affiliation: Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan, fpl5505@mail.ntou.edu.tw.
authors.forename: Fu-Pang
authors.initials: FP
authors.lastname: Lin
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Wu
authors.forename: Hung-Nien
authors.initials: HN
authors.lastname: Chen
authors.forename: Hui-Ju
authors.initials: HJ
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002686:Chitin / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010940:Plant Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012706:Serratia marcescens / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D010940:Plant Proteins; D002686:Chitin; D002688:Chitinases; C119825:chitinase C