The recent sequencing of the Thermomyces lanuginosus SSBP genome by our group has revealed four putative family 18 chitinases. In this study, three novel chitinase genes (chit2, chit3 and chit4) and the previously-reported chit1 gene were cloned from T. lanuginosus SSBP. chit1, encoding a 44.1 kDa protein, and chit2, encoding a 36.6 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit1 and Chit2 enzymes exhibited optimum activity at pH 5.0 and pH 4.0, respectively. Chit1 had optimal activity at 50 degrees C and retained 56% of its activity at 60 degrees C after 30 min, while Chit2 was optimally active at 40 degrees C and retained 71% of its activity at 50 degrees C after 60 mm. Both enzymes produced chitobiose as the major product using different substrates. Chit2 displayed antifungal activity against Penicillium verrucosum and Aspergillus niger. These activities could be useful in the environmental degradation of chitinous wastes as well as for biotechnological applications. (C) 2014 Elsevier Ltd. All rights reserved.