Cloning, expression, purification and biochemical characterization of the recombinant chitinase enzyme encoded by CTS2 in the budding yeast

Chitin is a polymer of 13-1,4-linked N-acetylglucosamine (GlcNAc) and plays a central role in the assembly of the fungal cell wall. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. Chitinases are classified into three categories, endochitinases, exochitinases and N-acetylglucosaminases, according to the manner in which the enzyme cleaves the chitin polymer. Saccharomyces cerevisiae has two chitinase-encoding genes, CTS1 and CTS2. However, whether Cts2p shows a chitinase activity remains unknown. In this study, we have cloned, expressed and purified the recombinant Cts2p protein from bacterial cells. We have demon-strated that Cts2p has a higher chitobiosidase (exochitinase) activity than endochitinase activity, but no N-acetylglucosaminase activity. The optimal temperature for the chitobiosidase activity of Cts2p is 37 degrees C.