Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca

• A putative chitinase (TfChi18A) from Thermobifida fusca was cloned and overexpressed in E. coli with very good yields.
• TfChi18A has optimum activity at pH 6.0–8.0 and 40–45 °C and the apparent Tm is 57.5 °C.
• TfChi18A degrades both α- and β-chitin but not colloidal chitin.
• TfChi18A has non-processive endo-acting chitinase activity, but may not be a true chitinase.

Thermobifida fusca is a well-known cellulose-degrading actinomycete, which produces various glycoside hydrolases for this purpose. However, despite the presence of putative chitinase genes in its genome, T. fusca has not been reported to grow on chitin as sole carbon source. In this study, a gene encoding a putative membrane-anchored GH18 chitinase (Tfu0868) from T. fusca has been cloned and overexpressed in Escherichia coli. The protein was produced as SUMO fusion protein and, upon removal of the SUMO domain, soluble pure TfChi18A was obtained with yields typically amounting to 150 mg per litre of culture. The enzyme was found to be relatively thermostable (apparent Tm = 57.5 °C) but not particularly thermoactive, the optimum temperature being 40–45 °C. TfChi18A bound to α- and β-chitin and degraded both these substrates. Interestingly, activity towards colloidal chitin was minimal and in this case, substrate inhibition was observed. TfChi18A also cleaved soluble chito-oligosaccharides and showed a clear preference for substrates having five sugars or more. While these results show that TfChi18A is a catalytically competent GH18 chitinase, the observed catalytic rates were low compared to those of well-studied GH18 chitinases. This suggests that TfChi18A is not a true chitinase and not likely to endow T. fusca with the ability to grow on chitin.

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