Production and Purification of Hydrolytic Enzymes and Enhanced Enzymatic Saccharification of Pine Needles – A Challenging Waste of Temperate Forests

In the present study, pine needles waste was used as the cost- effective carbon source for hydrolytic enzymes production by a potential fungal strain Trichoderma sp. R4 for the further purification process. The culture filtrate was subsequently partially purified by ammonium sulfate precipitation at 40% saturation level of laccase, 40% CMCase, 60% FPase, 50% β-glucosidase and 70% xylanase with purification fold of 3.06 (laccase), 2.20 (cellulase) and 1.59 (xylanase) with 82.94, 62.43 and 63.24 % recovery yield respectively. Gel exclusion chromatography was done for the purification of hydrolytic enzymes with 5.36, 5.51 and 6.33 purification fold and 45.77, 61.33 and 60.23 % recovery yield for laccase, cellulase and xylanase respectively. The molecular mass of purified laccase (40.0 KDa), cellulase – CMCase (45.0 KDa), FPase (31.0 KDa), β-glucosidase (29.0 KDa) and xylanase (65.0 KDa) was obtained by using SDS-PAGE. The maximum enzymatic degradation of pine needles was obtained in purified fractions of enzymes of Trichoderma sp. R4 with a release of 76.75 mg/g reducing sugars.