Natural substrate assay for chitinases using high-performance liquid chromatography: a comparison with existing assays.

The determination of kinetic parameters of chitinases using natural substrates is difficult due to low Km values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)4, we have developed an assay for the determination of kcat and Kmvalues of chitinases. Product concentrations as low as 0.5μM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20×25cm) using spectrophotometric detection at 210nm. By means of this assay, kcat and Kmvalues for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33±1s−1 and 9±1μM and 28±2s−1 and 4±2μM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)2 yielded a kcat value of 18±2s-1 and a Km value of 30±6μM. For two ChiB mutants containing a Trp → Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)2 assays yielded rather similar Km values (5-fold difference at most) but showed dramatic differences in kcat values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.