Characterization of Chitinase from White-Rot Fungus <i>Irpex lacteus </i>NK-1

Chitin is a cell wall structural component of many fungi and is important for mycelium growth. Therefore, enzymes like chitinase which break down chitin, are likely important in fungi during morphological changes. <i>Irpex lacteus</i>, a white-rot fungus isolated from wood-rotting fungi, produces several chitinases. Although it produces a range of chitinases, there are currently no characterization reports exploring them, despite the interest and body of published works evaluating carbohydrate degrading enzymes. In this study, <i>Il</i>Chi18C was cloned and recombinantly produced using <i>Pichia pastoris</i> as a host. Properties of purified <i>Il</i>Chi18C were determined, revealing an optimal pH of 5.0 and temperature of 50 °C when using <i>p</i>NP-<i>N,N</i>'-diacetyl-β-D-chitobioside (<i>p</i>NP-(GlcNAc)₂) as a substrate. It is activated in the presence of metal ions such as Mg²⁺, Ca²⁺, and Mn²⁺, but inhibited by DMSO, EtOH, and SDS. The Km and Vmax of <i>Il</i>Chi18C for this substrate are 3.48 mM and 5.46 µM min^-1, respectively. Using <i>p</i>NP-(GlcNAc)₂ and chitin powder as substrates, <i>Il</i>Chi18C predominantly exhibited exo-type chitinase activity, releasing chitobiose from the non-reducing ends of chitin chains. It was also observed that this enzyme acts on the fruiting body of <i>Flammulina velutipes</i>, releasing chitobiose as the main product.