Characterisation of a chitinase from Pseudoalteromonas sp. DL-6, a marine psychrophilic bacterium

In this study, we isolated a new psychrophilic bacterium, Pseudoalteromonas sp. DL-6 from marine sediments, which grew well on chitin-containing plates at 4°C. One endo-type chitinase gene, chiA, was cloned from the genomic DNA of this bacterium and heterologously expressed in Escherichia coli BL21 (DE3). ChiA showed very high catalytic activity, even at 4°C, and exhibited maximal activity on a chitinous substrate at pH 8.0 and 20°C. Kinetic studies indicated that ChiA has a greater catalytic efficiency on 4-methylumbelliferyl-β-D-N,N',N″-triacetylchitotriose[4-MU(GlcNAc)3] than on 4-methylumbelliferyl-β-D-N,N'-diacetylchitobioside[4-MU(GlcNAc)2]. Electrospray ionisation mass spectrometry (ESI-MS) analysis showed that the hydrolysis products of powdered chitin after ChiA digestion consisted of a series of chitin oligomers with different degrees of polymerisation. The ChiA mode of action was also examined using (GlcNAc)2-6 as a substrate, and the results suggested that ChiA is a non-processive endo-type chitinase.