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

Abstract

Cellulase-producing microorganisms produce low titres of β-glucosidases with low tolerance to glucose. This study aimed to improve production, purify, and characterize a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7. β-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 β-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% β-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 β-glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-β-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.

Hyper β-glucosidase producer Beauveria bassiana SAN01—optimization of fermentation conditions and evaluation of saccharification potential

“…4b showed that Bgl production was favored at lower concentrations of both NaCl and KCl, while Bgl production decreased as both the concentrations were steadily increasing corroborating their negative effects on the Bgl production. Interaction between incubation time and all other tested variables indicated that as incubation time increased, Bgl production These results are corroborated by previous studies where enzyme production was also noted to have steeply declined beyond the optimum incubation time [19][20][21]. Interactions between incubation time and NaCl (CE) were more statistically significant amongst all other tested interactions in this study with the observed p value of 0.0003 (<0.05) (Fig.…”

“…However, the conventional optimization approach fails to account for the interplay between the variables utilized and their overall impact on the required output; hence, there is a growing preference for statistical techniques such as response surface methodology (RSM) in the optimization of fermentation processes [20]. This statistical approach decreases the overall number of required experiments and better elucidates the relationships between various elements and the fermentation output [21]. RSM includes Plackett-Burman design (PBD) which efficiently identifies the most influential fermentation components out of many and the central composite design (CCD) which ascertains the ideal concentration of individual components, evaluates their interaction, and also assesses their impact on the response [22].…”

Biochemical and in silico structural properties of a thermo-acid stable β-glucosidase from Beauveria bassiana

“…Concurring with these results, unpurified BGL from a different B. bassiana strain was previously shown to be active under moderately acidic conditions [ 24 ], in addition, most fungal BGLs have been reported to be active at pH 4.0–6.0. Recent examples include the BGL from Aspergillus fumigatus [ 26 ] and Neofusicoccum parvum [ 27 ]. The BGL in this study retained >60 residual activity after 4 h incubation at pH 4.0, 5.0, and 6.0, respectively ( Fig.…”

Optimisation of β-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

“…The optimal conditions provided by the manufacturer for the commercial cellulase enzyme for optimal activity were 5 U/mg enzyme in (pH 5.0) buffer at 50 °C with 2 h of incubation. Therefore, 0.05 g of microcrystalline cellulose (Sigma, St. Louis, MO, USA) and 5 U/mg of enzyme in sodium phosphate buffer (pH 5.0) were incubated at 50 °C for 2 h. To test the effectiveness of β -glucosidase supplementation in the cellulase cocktail, 0.25 U/mg of purified β -glucosidase from a previous study by Singh et al [ 50 ] was supplemented in the commercial cellulase cocktail and incubated at optimal conditions as described above.…”