Beta-glucosidase is among the suite of enzymes produced by white rot fungi (WRF) to biodegrade plant biomass. This study investigated the enzymatic activities and kinetic properties of β-glucosidase from seventeen WRF comprised of the following species from various geographical locations: Pleurotus ostreatus, Auricularia auricular, Polyporus squamosus, Trametes versicolor, Lentinula edodes, and Grifola frondosa. All the WRF studied showed β-glucosidase activities. Significant variations in protein and carbohydrate contents were also recorded. Beta-glucosidase activities after 30 min of incubation ranged from 6.4 μg (T. versicolor) to 225 μg (G. frondosa). The calculated kinetic constant (Km) ranged from 0.47 μM (A. auricular-1120) to 719 μM (L. edodes-7). The Vmax depending on the kinetic transformation model ranged from 0.21 μg·min-1 (T. versicolor) to 9.70 μg·min-1 (G. frondosa-28). Beta-glucosidase activities also exhibited pH optima between 3.5 and 5.0 while temperature optima were between 60°C and 70°C with some media exhibiting a secondary temperature peak at 90°C attributable to the presence of thermostable isoenzyme. WRF if appropriately screened and purified can be harnessed to potentially improve the bio-conversion of cellulose to glucose and also facilitate efficient plant biomass biodegradation and production of useful plant bio-products.
White rot fungi (WRF) are microorganisms of great interest that secrete complex suites of nonspecific extracellular ligninolytic enzymes, i.e., lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13), and laccase (EC 1.10.3.2) to biodegrade lignin [
The β-glucosidase family (EC 3.2.1.21) is a widespread group of enzymes that catalyze the hydrolysis of a broad variety of glycosides [
This study focused on investigating β-glucosidase activities and kinetic properties of selected WRF in submerged fermentation and the effects of different genera and geographical origins on the activities. The aim was also to enhance our understanding of the variabilities in biochemical characteristics and catalytic properties of β-glucosidase among selected fungal genera. White rot fungi showing best activities can be used individually or in combination with other organisms for plant biomass conversion to fermentable sugars and other bioproducts.
1) Coomassie Plus—The Better BradfordTM Assay Kit (Pierce Rockford, IL); 2) concentrated sulfuric acid (H2SO4); 3) phenol (5%) prepared by diluting 55.6 mL 90% w/w phenol in about 800 mL distilled deionized H2O, then adjusted to a final volume of 1 L with distilled deionized H2O and stored at 4˚C; 4) acetate buffer (50 mM, pH 5.0) prepared by dissolving 6.8 g sodium acetate trihydrate crystals in about 700 mL distilled deionized H2O then titrated to pH 5.0 with 99% glacial acetic acid and adjusted to a final volume (1 L) with distilled deionized H2O; 5) sodium carbonate (0.2 M) prepared by dissolving 21.2 g in distilled deionized H2O. Glucose standard solution prepared by dissolving 1.0 g anhydrous glucose (Sigma Co.) in distilled deionized water; 6) p-Nitrophenyl β-D-glucopyranoside (β-PNPGLU) (5 mM) prepared by dissolving 1.51 g (β-PNPGLU) (Sigma Chemical Co., St. Louis, MO) in about 800 mL sodium acetate buffer (50 mM, pH 5.0) and adjusted to the final volume (1 L) with the same buffer and stored at 4˚C; and 7) p-Nitrophenol standard solution prepared by dissolving 1.0 g p-nitrophenol (PNP) in distilled deionized H2O.
White rot fungi isolates from the order Agaricales, Auriculariales and Aphylophorales (
Actively growing mycelia of each isolate were carefully scraped off the surface of potato dextrose agar plates and used to inoculate 100 mL sterile liquid cultivation medium in 250 mL Erlenmeyer flask under sterile conditions. The medium was composed of 5 g soluble starch, 0.1 g yeast extract, and 10 mL sawdust extract and made-up to 1 L with distilled deionized water (ddH2O). Inoculated flasks were incubated at 23˚C ± 2˚C for 14 days after which the mycelia mat was carefully removed and the remaining liquid from each of the triplicates per strain was pooled, filtered through a 0.22 µm acetate filter, then transferred into sterile bottles and kept at 4˚C until used.
Carbohydrate content of the filtrate was measured using the phenol-sulfuric acid method for total carbohydrate determination [
Protein concentration was determined as described by Bradford [
The β-glucosidase activity in extracts was assayed using p-Nitrophenyl-β-D-glucopyranoside (β-PNPGLU) as substrate. Two mL of 5 mM β-PNPGLU (prepared in 50 mM
#MBFBL, Mushroom biology and fungal biotechnology laboratory at North Carolina A & T State University, Greensboro, NC.
sodium acetate buffer, pH 5.0) was placed in a 15-mL centrifuge triplicate tubes to which was added 0.5 mL WRF extract and incubated at 37˚C for 30 min. The reaction was stopped using 1.5 mL 0.5 M Na2CO3 and the absorbance measured against a blank (sodium acetate) at 400 nm after 30 min. Each experiment had a control consisting of substrate without enzyme that was also subjected to the same experimental conditions. A PNP standard was prepared containing 0, 20, 40, 60, 80 and 100 µg of PNP.
The kinetic parameters were assessed by estimating β- glucosidase activities at various β-PNPGLU concentrations (0.02, 0.05, 0.1, 0.5, 1.0, 1.5 mM). The MichaelisMenten constant (Km) and maximum reaction rate (Vmax) were calculated using both linear and non-linear transformations of the Michaelis-Menten equation. The measurements to determine the temperature effects and temperature coefficients (Q10) for β-glucosidase activities were at 10˚C intervals (between 10˚C and 100˚C) while the activation energy (Ea), was determined using the Arrhenius equation. Two milliliters of 5 mM β-PNPGLU (prepared in 50 mM sodium acetate buffers at pH ranging from 3 to 7 in 0.5 increments) was used to assess the effects of pH on the β-glucosidase activities.
Growth media after the static cultivation of 17 WRF isolates spanning six fungal genera [Pleurotus, Auricularia, Polyporus, Trametes, Lentinula, and Grifola] (
†Standard deviation (n = 3). §ANOVA (Means with the same letters within the same columns are not significantly different).
Protein content in WRF enzyme production media ranging between 2.8 and 24.0 µg·mL−1 have been reported [
Beta-glucosidase activities, estimated as PNP released after 30 min of incubation with β-PNPGLU at 37˚C indicated that G. frondosa-28 (commercial isolate) exhibited the highest activities. Trametes versicolor-122 (wild isolate from NC) showed the least total β-glucosidase activities; however, there were wide ranges in β-glucosidase activities within each genus, except in the Pleurotus strains. The range of β-glucosidase activities measured within genera after 30 min of incubation were; Pleurotus (10.3 - 12.5 µg), Grifola (39.1 - 225 µg), Lentinula (7.2 - 22.6 µg), Auricularia (16.1 - 24 ug), Polyporus (15 - 21 µg), and Trametes (6.4 - 41.6 µg). There were also differences in β-glucosidase activities between genera ranging from 6.4 (T. versicolor-122) to 225 µg (G. frondosa-28). Morais et al. [
Beta-glucosidase activities were not correlated with the protein contents possibly because protein in the crude extracts could include other proteins not associated with β-glucosidase activities. The variations in crude secretions could be explained by the fact that the protein contents of the extracts included other enzymes and amino acids not related to the enzyme activities under investigation. It has been reported that when enzymes are assayed from crude extracts, the results may not suggest a complete degradation or hydrolysis of the substrate [
All the fungal β-glucosidase activities assayed fitted the Michaelis-Menten kinetic model (plots not shown) which has been reported to account for the kinetic properties of several enzymes. The Michaelis-Menten constant Km (indicative of enzyme-substrate affinity) and Vmax (maximum velocity at enzyme saturation) for β-glucosidase activities in the media were calculated using both the linear and non-linear regression fit of the Michaelis-Menten equation. The average Km value using the three linear transformations ranged from 0.47 (A. auricula-1120) to 719 µM (L. edodes-7), while Vmax ranged from 0.21 µg·min−1 (T. versicolor-122) to 9.63 µg·min−1 (G. frondosa-28). The lower Km values usually indicate higher enzyme-substrate affinity [
Vmax (µg·min−1); Km (µM). Numbers next to each species are assigned identification by Mushroom Biology and Fungal Biotechnology Laboratory (MBFBL).
The pH profile of each fungal cultivation media for β-glucosidase activities showed that the extracts exhibited pH optima between 3.5 and 5.0 (
In this study, it was noted that the increase in product formed was relatively gradual with increasing temperature until the optimum temperature was reached, after which the enzyme denatured rapidly and consistently with the temperature profiles of other studies [
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Jabbar et al. [
Significant quantitative and qualitative differences exist among the β-glucosidase activities of WRF. The enzymes also exhibited different response patterns to pH and incubation temperature conditions. Thus screening the WRF responsible for the biodegradation of most plant biomass for their β-glucosidase biocatalytic potentials offers an attractive tool for plant biomass transformation. If appropriately screened and purified, they can be harnessed to potentially improve the bioconversion of cellulose to glucose and also facilitate efficient plant biomass biodegradation to useful bio-products.
This work is a contribution of the Winfred Thomas Agricultural Research Station, Alabama A&M University, Normal, AL. Trade or manufacturers’ names mentioned are for information only and do not constitute endorsement, recommendation, or exclusion by Alabama A&M University and collaborating universities. This research was supported in part by USDA, Evans-Allen Grant # ALAX 011.