Optimisation of ß-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis.

Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of ß-glucosidases. In this study, ß-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher ß-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. ß-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for ß-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of ß-glucosidase resulted in a 19.8% increase in glucose yield.

Screening for cellulases and preliminary optimisation of glucose tolerant ß-glucosidase production and characterisation.

The search for a novel microbial producer of cellulases including a glucose tolerant ß-glucosidase is a challenge as most are inhibited by their product glucose. This study aims to screen for cellulolytic fungi using qualitative and quantitative screening methods. Primary screening revealed 34 of 46 fungal isolates with ß-glucosidase activity. Eleven and 13 of these also displayed endoglucanase and exoglucanase activities, respectively. During secondary screening, this number was reduced to 26 ß-glucosidase producers with 13 also having endoglucanase and exoglucanase activities. Isolate C1 displayed enhanced production of ß-glucosidases in the presence of 0.05 M glucose (69% higher activity). Optimisation of growth conditions for ß-glucosidase production by one variable at a time experiments improved production for (isolates) PS1 (64%), MB5 (84%), and C2 (69%). Isolate PS1 identified as Chaetomella sp. BBA70074 displayed the highest tolerance to glucose, retaining 10% of ß-glucosidase activity in the presence of 0.8 M glucose. Tolerance to glucose increased to 14% when produced under optimal conditions. ß-Glucosidase had a molecular weight of 170 kDa with a pH and temperature optima of 6 and 70°C, respectively. Future studies will include optimisation of the production of the glucose tolerant enzyme by Chaetomella sp. BBA70074.

A glucose tolerant ß-glucosidase from a newly isolated Neofusicoccum parvum strain F7: production, purification, and characterization.

Cellulase-producing microorganisms produce low titres of ß-glucosidases with low tolerance to glucose. This study aimed to improve production, purify, and characterize a ß-glucosidase from a newly isolated Neofusicoccum parvum strain F7. ß-Glucosidase production was significantly enhanced by a sequential statistical modelling approach from 1.5-fold in Plackett-Burman design to 2.5 U/ml in the Box-Behnken design compared to the preliminary one variable at a time experiments (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20 °C, 175 rpm, 0.5% glycerol and 1.5% casein in pH 6.0 buffer. Three ß-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular mass of approximately 65 kDa demonstrated the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% ß-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65 °C for one hour which was then reduced to 40% which remained stable for another 90 min. The ß-glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-ß-D-glucopyranoside were 1.18 mM and 28.08 µmol/min, respectively indicating high affinity for the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for this enzyme in industrial application.