The chitinolytic enzyme system of the compost-dwelling thermophilic fungus Thermomyces lanuginosus

Abstract

Chitin, a highly insoluble 1,4- -linked polymer of N-acetyl- -D-glucosamine, is the second-most abundant bio-polysaccharide in nature after cellulose. Most chitinolytic fungi are known to produce more than one kind of chitinase. 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 (chitl, chit2 and chit3) and the previously reported chit4 gene were cloned from Thermomyces lanuginosus SSBP and their gene structures were analysed. chit3, encoding a 36.6 kDa protein, and chit4, encoding a 44.1 kDa protein, were successfully expressed in Pichia pastoris. The recombinant Chit3 and Chit4 enzymes exhibited optimum activity at pH 4.0 and 5.0 and at 40oC and 50°C, respectively. Chit3 was stable at 40oC and retained 71% of its activity at 50°C after 60 min, while Chit4 was stable at 50°C and retained 56% of its activity at 60°C after 30 min. Both enzymes produced chitobiose as the major product using colloidal chitin, chitooligosaccharides and shrimp shell powder as substrates. Of the fungal strains tested, Chit3 displayed antifungal activity against Penicillium sp. and Aspergillus sp. This is the first report on the multi-chitinolytic system of T. lanuginosus and enzyme characterization has shown the potential of the enzymes to be used in degradation of the under-utilized bio-resource chitin.