An extracellular acidic and thermostable chitinase (HgChi) from thermophilic Humicola grisea was purified and characterized. Enhancement in chitinase production (Qp = 2.9662 Ul-1 h-1) was achieved through derivation of optimum fermentation conditions via central composite design. H. grisea observed to produce various isoforms of chitinase, among which the major expressed form has molecular mass of about 50 kDa. Purified chitinases exhibited optimal activity at pH 3.0 and 70 °C. Chitinase showed notable stability at increasing temperatures. Half-life of chitinase is 169.06 min at optimum temperature. Chitinase has effectively catalyzed N-acetyl chitobiose (GlcNAc)2, and N-acetyl chitotriose (GlcNAc)3 and colloidal chitin. Purified chitinase from H. grisea showed high affinity towards colloidal chitin as evident by its comparatively lower Km value. Presence of metal ions viz. Mn2+, Co2+, NH4+ and Mg2+ significantly increased the chitinase activity. Thin layer chromatography (TLC) analysis revealed the significant hydrolyzing competence of HgChi for colloidal chitin, (GlcNAc)3 and (GlcNAc)2 into oligomers and N-acetyl-d-glucosamine (GlcNAc). Thermostable chitinase appeared as potential candidate for efficient conversion of chitin to bioactive oligosaccharides at industrial scale.
Keywords: Chitinase; H. grisea; Kinetics; Optimization; Purification; Thermodynamics.
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