PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF CELLULASE FROM TRICHODERMA VIRIDE

Abstract

Cellulases from filamentous fungi are important biocatalysts for the enzymatic conversion of cellulose into fermentable sugars and have considerable potential in biomass valorization. The present study was undertaken to purify and biochemically characterize an extracellular cellulase from Trichoderma viride. The crude enzyme preparation was subjected to sequential ammonium sulfate fractionation, dialysis, and DEAE–Sepharose ion exchange chromatography. The crude preparation exhibited a total cellulase activity of 88.99 U and a specific activity of 5.18 U mg⁻¹ proteins. Following purification, fraction F5 showed the highest specific activity of 17.27 U mg⁻¹, corresponding to a 3.33-fold purification, although with a recovery of 1.75%. SDS–PAGE analysis revealed progressive enrichment of the cellulase-containing protein fraction and a prominent band with an apparent molecular mass of approximately 52 kDa. The purified enzyme exhibited maximum activity at pH 5.0 and 50°C, with appreciable activity over broader pH and temperature ranges. KM and Vmax values of purified cellulase from Trichoderma viride were 68.97 µM and 142.86 U/mL respectively. The enzyme showed predominant activity toward cellulose, with a hydrolysis-product profile suggestive of exo glucanase/cellobiohydrolase-like activity. MALDI-TOF peptide mass fingerprinting generated a database match to bacterial ribosomal protein uL2 and molecular mass considered a definitive identification. Overall, the purified T. viride cellulase demonstrated useful acidic pH and moderate-temperature activity and represents a promising enzyme preparation for cellulose hydrolysis and potential lignocellulosic biomass-conversion applications. Further LC–MS/MS-based peptide sequencing and Trichoderma-specific database analysis are required for definitive molecular identification.