| Summary: | 博士 === 國立臺灣海洋大學 === 生物科技研究所 === 96 === A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments, respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His-Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy of fluorescence and circular dichroism, chitin binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU-(GlcNAc)2 or 4MU-(GlcNAc)3 soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589 toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids.
The functional and structural significance of the C-terminal region of Bacillus licheniformis chitinase was explored using C-terminal truncation mutagenesis. Comparative studies between full-length and truncated mutant molecules included initial rate kinetics, fluorescence and CD spectrometric properties, substrate binding and hydrolysis abilities, thermostability, and thermodenaturation kinetics. Kinetic analyses revealed that the overall catalytic efficiency, kcat/Km, was slightly increased for the truncated enzymes toward the soluble 4-methylumbelliferyl-N-N’-diacetyl chitobiose or 4-methylumbelliferyl-N-N’-N’’-triacetyl chitotriose or insoluble α-chitin substrate. By contrast, changes to substrate affinity, Km, and turnover rate, kcat, varied considerably for both types of chitin substrates between the full-length and truncated enzymes. Both truncated enzymes exhibited significantly higher thermostabilities than the full-length enzyme. The truncated mutants retained similar substrate-binding specificities and abilities against the insoluble substrate but only had approximately 75% of the hydrolyzing efficiency of the full-length chitinase molecule. Fluorescence spectroscopy indicated that both C-terminal deletion mutants retained an active folding conformation similar to the full-length enzyme. However, a CD melting unfolding study was able to distinguish between the full-length and truncated mutant molecules by the two phases of apparent transition temperatures in the mutants. These results indicate that up to 145 amino acid residues, including the putative C-terminal chitin-binding region and the fibronectin (III) motif of B. licheniformis chitinase, could be removed without causing a seriously aberrant change in structure and a dramatic decrease in insoluble chitin hydrolysis. The results of the present study provide evidence demonstrating that the binding and hydrolyzing of insoluble chitin substrate for B. licheniformis chitinase was not dependent solely on the putative C-terminal chitin-binding region and the fibronectin (III) motif.
In summary, roles of C-terminal region from V. parahaemolyticus and B. licheniformis chitinases played in the chitin hydrolysis were demonstrated in different versions as those previously reported in many other chitinases even they are belonged in the same chitinase family.
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