BIOCHEMICAL STUDIES ON SOME LIGNOCELLULOLYTIC ENZYMES INVOLVED IN SACCHARIFICATION OF CASSAVA (Manihot esculenta Crantz) PEELS FOR OPTIMIZED FERMENTABLE SUGARS PRODUCTION

Abstract

The utilization of lignocellulosic biomass for biofuel production requires the activity of some hydrolytic enzymes for the saccharification of carbohydrate polymers. Being the limiting stage of the bioconversion process, it must be optimized with enzymes of desirable qualities. In this research, optimally produced saccharifying enzymes were purified and studied for the degradation of cassava peels, an agro-waste biomass into fermentable sugars. The carbohydrate profile of the peels was analyzed using HPLC fitted with UV-detector. Microorganisms isolated from the composting peel were identified by morphology and molecular gene sequence analysis before being screened for lignocellulolytic activity. The submerged fermentation conditions for the production of key lignocellulolytic enzymes (endo-1,4-glucanase, exo-1,4-glucanase, β-D-glucosidase and endo-1,4-xylanase) by viable microorganisms were optimized including biomass pretreatment. Further, optimally produced enzymes were subjected to ammonium sulphate precipitation and purified to apparent homogeneity in a two-step process comprising anion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-100 prior to biochemical studies. After initial screening, production of endo-1,4-glucanase and exo-1,4-glucanase by Fusarium oxysporum CPOA-2 both reached maximum on the 4th day of incubation at pH 5.0 and temperature 35 °C and 37 °C, respectively. Optimal production of β-D-glucosidase by F. oxysporum CPOA-2 was at pH 5.5 and 37 °C on the 6th day. Penicillium citrinum CPJO-6 optimally produced endo-1,4-xylanase on day 4 at 42 °C and pH 5.0. The combination of yeast extract, ammonium sulphate and tryptone as nitrogen source favoured the production of the lignocellulolytic enzymes. Cellobiose, sucrose and xylose were good inducers for the cellulases while endoxylanase production was improved by xylan. As determined by SDS-PAGE, the subunit molecular mass of the homogenously purified β-D-glucosidase, endo-1,4-glucanase, exo-1,4- glucanase and endo-1,4-xylanase were 86.5 kDa, 65 kDa, 46 kDa and 25.4 kDa, respectively. The interaction studies reveal a concerted hydrolytic action among the cellulolytic enzymes with varying degree of synergy (1.02 - 1.44), remarkably increasing to 1.58 when endo-1,4-xylanase was added to the enzyme cocktail. Further studies on the rate limiting enzyme, β-D-glucosidase, showed that the F. oxysporum CPOA-2 β-D-glucosidase (foxBGL) has a native molecular weight of 136.2 kDa (determined by gel filtration on a calibrated Sephadex G-100 column) and exhibited maximum activity at pH 5.5 and 50 °C. The purified foxBGL was acidic-thermostable, retaining over 50% of initial activity after 120 min of incubation at 70 °C. The enzyme activity was stimulated by Na+, K+, Cu2+, Mn2+, Mg2+ and Co2+ but inhibited by Pb2+, Al3+, EDTA and SDS. This foxBGL was very active on pNPG, cellobiose and lactose; moderately active on salicin and maltose but showed very little activity on methylcellulose, α-cellulose, carboxymethyl cellulose, Avicel® and trehalose. The Km and Vmax were 0.72 mM and 2.17 mM/min, respectively for pNPG and 0.85 mM and 1.25 mM/min for cellobiose. Glucose competitively inhibited foxBGL with Ki value of 7.4 mM. An optimized enzyme cocktail comprising cellulolytic and xylanolytic enzymes was developed for efficient saccharification of cassava peel as a low-cost feedstock for the production of fermentable sugars. The purified enzymes possess remarkable properties for suitable application in biotechnological industries.