A Ca2⁺-responsive GH37 neutral trehalase from Lentinula edodes: Cloning, heterologous expression and biochemical characterization.

Trehalases (EC 3.2.1.28) hydrolyze trehalose into two glucose molecules and play important roles in fungal trehalose metabolism. Although GH37 neutral trehalases have been characterized in yeasts and filamentous fungi, their biochemical properties in basidiomycetes remain poorly understood. In this study, we cloned LeNTH1, a putative GH37 trehalase gene from Lentinula edodes, expressed it in Pichia pastoris, and characterized the purified recombinant enzyme. The deduced protein comprised 745 amino acid residues and contained a predicted N-terminal signal peptide. The purified recombinant form preferentially hydrolyzed trehalose, showed maximal activity at pH 7.0 and 30 °C, and was stable over a broad pH range. Pre-incubation with CaCl₂ enhanced rLeNTH1 activity in a concentration-dependent manner, reaching approximately 3.3-fold the control activity after treatment with 10 mM CaCl₂. CaCl₂ also increased thermal stability: after incubation at 50 °C for 120 min, approximately 45% of the initial activity remained in the presence of 5 or 10 mM CaCl₂, whereas no more than 13% remained at 0-2.5 mM CaCl₂. These results indicate that added Ca2⁺ enhances both the activity and thermal stability of rLeNTH1, although direct Ca2⁺ binding was not demonstrated. In fruiting-body crude extracts, neutral trehalase activity increased during maturation, whereas LeNTH1 transcript levels remained nearly constant. Because the activity assay was not specific to LeNTH1, its contribution to this increase remains unresolved. These findings provide evidence for biochemical diversity among basidiomycete GH37 trehalases.