Biochemical characteristics of C‐terminal region of recombinant chitinase from Bacillus licheniformis– implication of necessity for enzyme properties

Abstract

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, k(cat)/K(m), 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 alpha-chitin substrate. By contrast, changes to substrate affinity, K(m), and turnover rate, k(cat), 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.