Maximum Β-glucosidase Activity Research Articles

English

β-glucosidases are found in all domains of life and has the ability to hydrolyse the glycosidic bond between carbohydrates and non-carbohydrates residues making it a potent enzyme from an industrial and agricultural perspective. The aim of the current study was isolation, screening, and identification of bacterial strains having maximum β-glucosidase activity from soil sample of Jacobabad, Pakistan. The isolates were screened primarily, and efficient isolates were confirmed for β-glucosidase production through secondary screening. Among the different isolated strains, 1j2 was selected as the best β-glucosidase producer. The isolate showed 99% similarity with Bacillus stercoris after 16S rRNA sequencing. Different culture conditions were optimized for effective β-glucosidase production. Enhanced level of β-glucosidase was achieved at 40°C, pH 8 at 150 rpm in a β-glucosidase production medium supplemented with a combination of agricultural residues of wheat bran 0.25% (w/v) and orange peel 0.25% (w/v), using 5% inoculum. Bacillus stercoris produced a higher yield (2.89U/mg) of β-glucosidase under optimized conditions. Partial purification of β-glucosidase was achieved after 80% ammonium sulfate precipitation. The enzyme was purified and had shown maximum specific activity of 53.2 U/mg with molecular weight of approximately 36 kDa. In all the tested metals Cu2+ was observed to have inhibitory effect while Ca2+ had not affected the enzyme activity significantly. While organic solvents and surfactants had significantly reduced the β-glucosidase activity. Based on the afore mentioned abilities of purified β-glucosidase from Bacillus stercoris strain it could be a promising player for the industrial applications of lignocellulose hydrolysis found in agricultural residues for the cheap production of biofuels.

On-site enriched production of cellulase enzyme using rice straw waste and its hydrolytic performance evaluation through systematic dynamic modeling.

The application of on-site produced cellulolytic enzymes in place of commercial enzymes towards hydrolytic preparations of reducing sugars using inexpensive lignocellulosic wastes is considered the most efficient strategy to accomplish a cost-effective biofuel production process. Along with improved production, intrinsic and systematic performance evaluation of the produced enzyme during the hydrolysis process through kinetic intervention remains a crucial requirement for achieving the improved performance of the process. With this motivation, the present study primarily deals with the nutritionally optimized production strategy of cellulases from rice straw (RS) waste using Trichoderma reesei (MTCC 164). The highest cellulase production was obtained 8.09 ± 0.32g/l in batch mode at optimized combinations of 3.5% (w/v) RS inducer, 3.0% (w/v) lactose, and 1.5% (w/v) peptone. Production was further improved through pH-regulated (pH 5.5 to 6.5) fed-batch fermentations. The enzyme produced at pH 6 was considered for hydrolysis studies at 4 to 10% (w/w) solid loading due to reasonable exoglucanase, endoglucanase, and maximum β-glucosidase activity levels of 9.3 U/ml, 3.87 U/ml, and 2.65 U/ml respectively. Multi-reaction systematic kinetic modeling was implemented to evaluate enzyme performance during hydrolysis, and the values of inhibitory kinetic parameters (K2r = 7.1 < K1r = 18.5 < K3r = 276.6) suggested that sequential conversion of cellulose to glucose by existing enzyme components was more dominant over direct conversion.

β-Glucosidase from almonds and yoghurt cultures in the biotransformation of isoflavones in soy milk

β-Glucosidase is an enzyme that hydrolyzes the isoflavone bonds and develops the active form of aglycones providing various health benefits. The potential of β-glucosidase from two sources: almonds and soy yoghurt cultures (lactic cultures) to react with soy in the biotransformation of soy isoflavones was studied. This study investigated 1) the optimum amount of culture (1, 2, 3, 4%) and incubation temperature (30, 35, 42 °C) for fermentation of soymilk (SM); 2) the production of β-glucosidase and its ability to react with isoflavones with and without the addition of skim milk powder (SMP). The ability of β-glucosidase to convert isoflavones into aglycones was measured using spectrophotometry and reverse-phase high-pressure liquid chromatography. The results showed that a 1% lactic acid culture at 30 °C produced the highest viable count of lactic acid bacteria (~108 CFU/ml) in fermented SM. Fermentation at 30 °C and 1% inoculation showed the maximum β-glucosidase activity (4.28 U/ml) while 8 U/ml of β-glucosidase from almonds had the highest activity (daidzein: 1.48 g/l and genistein: 0.78 g/l). Among the yoghurt cultures, 2% inoculation in soymilk led to the highest aglycones (daidzein: 1.14 g/l and genistein: 0.50 g/l). Supplementing soymilk with skim milk powder led to aglycone levels similar to β-glucosidase from almond.

Enhancement and bioavailability of phenolic content in Kappaphycus alvarezii through solid substrate fermentation

Known as carrageenan, Kappaphycus alvarezii is a popular species of red edible seaweed which is mainly cultivated for its hydrocolloids. K. alvarezii is a good source of phenolics which are considered to be natural antioxidants that promote myriads of health benefits. Extraction of phenolic compounds using conventional solvent extraction may result in hazards and toxicological effect and low yield of bound phenolics. Therefore, this study was conducted to increase the release of bound phenolics through solid substrate fermentation. In the present study, three different varieties of K. alvarezii were fermented with Aspergillus oryzae through solid substrate fermentation (SSF) at 30 ℃ for 2 to 6 days. The changes of total phenolic content and antioxidant activity due to the effects of SSF were investigated. Results obtained revealed that the highest total phenolic content for green (10.022 mg GAE/g) and purple (4.037 mg GAE/g) varieties of K.alvarezii were on the fourth day of fermentation while for yellow (4.479 mg GAE/g) variety was on the sixth day of fermentation. A remarkable enhancement of antioxidant activity was discovered through DPPH radical scavenging activity on the fourth day of fermentation for all varieties of K. alvarezii tested. Total phenolic content also showed a significant correlation with antioxidant activity of fermented seaweed. Cellulase, xylanase, and β-glucosidase enzymes produced by A.oryzae during SSF were also investigated to see the relationship with the release of phenolic compounds after fermentation. The highest cellulase activity was observed on the fourth day of fermentation. The similar observation was also found for xylanase enzyme in yellow and purple varieties of K. alvarezii , except for green variety where the highest xylanase activity was on the sixth day of fermentation. The maximum β-glucosidase activity in yellow and green varieties was observed on the fourth day of fermentation while purple variety exhibited the maximum β-glucosidase activity on the sixth day. There are positive correlation between the enzymes studied and the polyphenols content in all varieties of K. alvarezii . This study demonstrated that fermented K. alvarezii contained more phenolic content compared to non-fermented seaweed.

Ethanol production by Aspergillus niger US4MTCC9931 and Saccharomyces cerevisiae MTCC174 using different lignocellulosic biomass feed stocks

To investigate the production of cellulases and hemicellulases from Aspergillus niger MTCC9931, solid state fermentation (SSF) was performed using 10 different lignocellulosic materials derived from agrowastes, i.e., rice straw, rice husk, wheat straw, corn cob, sugar cane bagasse, saw dust, banana stalk, Eichhornia, Parthenium stalk, and residual fruit pulp. Among these agrowastes, the maximum yield of reducing sugars (116.46 ± 2.56 g/mL) was observed with residual fruit pulp. Further, the same substrate showed the highest endoglucanase (389.1 ± 0.44 IU/g), MCC-adsorbable endoglucanase (3.4 ± 0.14 IU/g), cellulase (12.0 ± 0.13 IU/g), and FPase (4.9 ± 0.64 IU/g) activities. Sawdust showed the maximum xylanase activity (7478.0 ± 6.51 IU/g), and corncob showed maximum β-glucosidase activity (79.87 ± 1.15 IU/g). The maximum activities of all enzymes were observed at 72 h of SSF under shaking conditions. A. niger MTCC9931 can be concluded to be an important strain for potential applications in saccharification. The enzymatic hydrolysates of the agrowastes were used as substrates for ethanol production by Saccharomyces cerevisiae MTCC174. The maximum yield (35.34 g/L) of ethanol was obtained when residual fruit pulp was used as a substrate. This study has further demonstrated the feasible biotechnological conversion of agrowastes to produce ethanol using both A. niger and S. cerevisiae.

Functional properties of fermented soymilk by Lactobacillus fermentum BM-325

Soy products have attracted much attention recently as carriers for probiotics. Fermentation of soymilk with lactic acid bacteria (LAB) was found to improve the nutritional and bioactivity values of consumed foods. The aim of this study was to evaluate functional properties of fermented soymilk with probiotic LAB strain Lactobacillus fermentum BM-325, based on their growth behavior, enzymes and antioxidant activities. Growth behavior such as viable cell number, pH, total acidity and enzyme activities were determined during fermentation period. Maximum number of viable cell was Log10 CFU·ml-1 12.6±0.9, maximum β-glucosidase activity was 78.9±3.9 U·mg-1 and maximum α-galactosidase activity was 97.7±4.8 U·mg-1, as well as antioxidant activity was IC50 0.04±0.002 mg·mL-1. Angiotensin converting enzyme inhibitory (ACEI) activity was determined in fermented soymilk and in partially purified peptide fraction. The ACEI activity was reached 60% after 20 h fermentation in soymilk. Our results suggested that fermentation of Lactobacillus fermentum BM-325 in soymilk for 4 h could be used to increase bioactivity of soymilk and suitable for the development of functional food.

A Novel Triculture System (CC3) for Simultaneous Enzyme Production and Hydrolysis of Common Grasses through Submerged Fermentation.

The perennial grasses are considered as a rich source of lignocellulosic biomass, making it a second generation alternative energy source and can diminish the use of fossil fuels. In this work, four perennial grasses Saccharum arundinaceum, Panicum antidotale, Thysanolaena latifolia, and Neyraudia reynaudiana were selected to verify their potential as a substrate to produce hydrolytic enzymes and to evaluate them as second generation energy biomass. Here, cellulase and hemi-cellulase producing three endophytic bacteria (Burkholderia cepacia BPS-GB3, Alcaligenes faecalis BPS-GB5 and Enterobacter hormaechei BPS-GB8) recovered from N. reynaudiana and S. arundinaceum were selected to develop a triculture (CC3) consortium. During 12 days of submerged cultivation, a 55–70% loss in dry weight was observed and the maximum activity of β-glucosidase (5.36–12.34 IU) and Xylanase (4.33 to 10.91 IU) were observed on 2nd and 6th day respectively, whereas FPase (0.26 to 0.53 IU) and CMCase (2.31 to 4.65 IU) showed maximum activity on 4th day. Around 15–30% more enzyme activity was produced in CC3 as compared to monoculture (CC1) and coculture (CC2) treatments, suggested synergetic interaction among the selected three bacterial strains. Further, the biomass was assessed using Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). The FTIR analysis provides important insights into the reduction of cellulose and hemicellulose moieties in CC3 treated biomass and SEM studies shed light into the disruption of surface structure leading to access of cellulose or hemicelluloses microtubules. The hydrolytic potential of the CC3 system was further enhanced due to reduction in lignin as evidenced by 1–4% lignin reduction in biomass compositional analysis. Additionally, laccase gene was detected from A. faecalis and E. hormaechei which further shows the laccase production potential of the isolates. To our knowledge, first time we develop an effective endophytic endogenous bacterial triculture system having potential for the production of extracellular enzymes utilizing S. arundinaceum and N. reynaudiana as lignocellulosic feedstock.

Potential use of soybean hulls and waste paper as supports in SSF for cellulase production by Aspergillus niger

Abstract Cellulase has by vast applications in the biofuel, pulp and paper, detergent and textile industries. The three components of the enzyme complex (endoglucanase, exoglucanase and β-glucosidase) can effectively depolymerize the cellulose chains in lignocellulosic substrate. Solid-state fermentation (SSF) by fungi is a preferable production route for cellulase because of its low cost, among other advantages. This work describes the cellulase production by Aspergillus niger NRRL3 grown on SSF. SSF was carried out on soybean hulls and waste paper as supports. The effect of the support on cellulase production was assessed under a completely randomized factorial design. The support-time interaction was significant for all the variables studied. Both materials were characterized in terms of water absorption index and critical humidity point. Samples of culture were analyzed with scanning electron microscopy (SEM) to study spores and fungal growth. Maximum endoglucanase activity was found at 96 h using soybean hulls as support (5914.29 U L−1), being four times higher than that obtained using waste paper at the same fermentation time. The exoglucanase activity in soybean hulls was maximal at 96 h (4551.19 U L−1), being 9.6 times higher than that obtained in waste paper at the same time. The maximum β-glucosidase activity in soybean hulls (984.01 U L−1) was reached at 96 h, being 1.7 times greater than that obtained in waste paper. Besides, the use of soybean hulls provided high volumetric productivities at shorter times, which may decrease production costs considering a scaled process.

Enhancement of β-Glucosidase Activity from a Brown Rot Fungus Fomitopsis pinicola KCTC 6208 by Medium Optimization

β-Glucosidase, which hydrolyzes cellobiose into two glucoses, plays an important role in the process of saccharification of the lignocellulosic biomass. In this study, we optimized the activity of β-glucosidase of brown-rot fungus Fomitopsis pinicola KCTC 6208 using the response surface methodology (RSM) with various concentrations of glucose, yeast extract and ascorbic acid, which are the most significant nutrients for activity of β-glucosidase. The highest activity of β-glucosidase was achieved 3.02% of glucose, 4.35% of yeast extract, and 7.41% ascorbic acid where ascorbic acid was most effective. The maximum activity of β-glucosidase predicted by the RSM was 15.34 U/mg, which was similar to the experimental value 14.90 U/mg at the 16th day of incubation. This optimized activity of β-glucosidase was 23.6 times higher than the preliminary activity value, 0.63 U/mg, and was also much higher than previous values reported in other fungi strains. Therefore, a simplified medium supplemented with a cheap vitamin source, such as ascorbic acid, could be a cost effective mean of increasing β-glucosidase activity.

Investigation of the Germination of Barley and Wheat Grains with a Design of Experiments for the Production of Hydrolases.

The production of hydrolases from cereals has been examined in order to investigate food-derived enzymes as an alternative source to microbial enzymes for the use in food processes. For that, the influence of temperature on the pretreatment, imbibition and germination of barley and wheat grains was determined by measuring the β-glucosidase, β-galactosidase and lipase activities using a design of experiments. The evaluation of the statistical model showed an increase of the β-glucosidase activity with low imbibition and low germination temperature for barley grains and low imbibition and high germination temperature for wheat grains. The maximum β-glucosidase activity in wheat extracts was (585±151) nkat per g of dry mass (dm), while in barley extracts it was (109±15) nkat per g of dm. The maximum β-galactosidase activities in barley and wheat extracts were (34±12) and (63±23) nkat per g of dm, respectively. The maximum lipase activities of (6.7±0.1) and (4.6±4.4) nkat per g of dm in barley and wheat extracts, respectively, were rather low compared to the glycosidase activities. The extracts were also tested for other hydrolase activities (e.g. peptidase and α-amylase activities). The insights obtained enable the basis for the potential use of cereal hydrolases in food processing, which might be attractive to consumers.

Biochemical characterisation of α-glucosidase and β-glucosidase in the alimentary canal of larval Leptinotarsa decemlineata SAY, 1824 (Coleoptera: Chrysomelidae)

ABSTRACT Host plant resistance is an environmentally safe method used for reducing a pest population. Basically, when developing resistant cultivars one needs to study the biochemical characteristics of the digestive enzymes in the insect’s midgut. In this study, the activities of α- and β-glucosidase were determined from Leptinotarsa decemlineata midgut using p-nitrophenyl-α-Dglucopyranoside and p-nitrophenyl-β-D-glucopyranoside as substrates respectively. The results showed that the specific activity of α- and β-glucosidase from 4th instar larvae midguts of L. decemlineata were 5.14 and 5.48 Umg-1 protein respectively. The activity of α-glucosidase was optimal at pH 4, whereas the maximum activity of β-glucosidase in the midgut of L. decemlineata occurred at pH 4-5.5. Both enzymes were stable at pH 3-8 over an incubation time of 8 hours. The respective activities of α- and β-glucosidase were at their highest at 45 °C and 50 °C, but they were not stable at 50 °C during an incubation time of 8 days. Furthermore, our data showed that MgCl2, Tris and urea have a moderate but SDS a severe inhibitory effect on enzyme activity. Biochemical characterisation revealed one and three bands of α- and β-glucosidase activities in the midgut of L. decemlineata respectively.

Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovoranscellulose binding domain and its application in hydrolysis of microcrystalline cellulose

BackgroundThe complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation.ResultsThe glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188.ConclusionsThe hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.

A Statistical Approach for Optimization of Simultaneous Production of β-Glucosidase and Endoglucanase by Rhizopus oryzae from Solid-State Fermentation of Water Hyacinth Using Central Composite Design.

The production cost of β-glucosidase and endoglucanase could be reduced by using water hyacinth, an aquatic weed, as the sole carbon source and using cost-efficient fermentation strategies like solid-state fermentation (SSF). In the present study, the effect of different production conditions on the yield of β-glucosidase and endoglucanase by Rhizopus oryzae MTCC 9642 from water hyacinth was investigated systematically using response surface methodology. A Central composite experimental design was applied to optimize the impact of three variables, namely, substrate concentration, pH, and temperature, on enzyme production. The optimal level of each parameter for maximum enzyme production by the fungus was determined. Highest activity of endoglucanase of 495 U/mL was achieved at a substrate concentration of 1.23%, pH 7.29, and temperature 29.93°C whereas maximum β-glucosidase activity of 137.32 U/ml was achieved at a substrate concentration of 1.25%, pH 6.66, and temperature 32.09°C. There was a direct correlation between the levels of enzymatic activities and the substrate concentration of water hyacinth as carbon source.

Optimisation of β-glucosidase entrapment in alginate and polyacrylamide gels

Immobilisation of β-glucosidase, isolated from Aspergillus niger, by entrapment in both calcium alginate and polyacrylamide gels was studied. A retention of 66% of initial activity was observed in the alginate beads prepared with 3% (w/v) alginate, 0.2 m CaCl 2 and 1 h of treatment. The maximum β-glucosidase activity in polyacrylamide gels (∼55%) was achieved in gels prepared with 20% acrylamide and 1.2% of crosslinking agent (bisacrylamide). β-Glucosidase immobilised in alginate gel did not follow pure Michaelis kinetics, exhibiting substrate inhibition. The K m of this enzyme was larger than that of the free β-glucosidase, suggesting that the alginate network limited the permeation rate of substrate and product. However, β-glucosidase entrapped in polyacrylamide gel showed a similar K m value to that of native enzyme. The pH value for maximum activity of free and immobilised enzymes was 4.0. The pH-activity curves were coincident, except at very low pH values where the enzyme trapped in alginate was more stable.

Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments

β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β-d-glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity (Vmax) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity(Km)was higher in the suspended sediments.VmaxandKmboth increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. Vmax was uninfluenced by whether ground or stream water was perfused through the sediments, but Km was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.

Use of chemostat for enhanced production of β-glucosidase by newly isolated anaerobic cellulolyticClostridium strain RT9

A new anaerobic, mesophilic, spore-forming, cellulolyticClostridium strain, RT9, was isolated from bovine rumen fluid. This strain has the ability to hydrolyze cellulose (Sigma cell-100), carboxymethyl-cellulose (CMC), and cellobiose into ethanol, acetate, butyrate, lactate, acetone, H2, and Co2 as the major fermentation products. The bacterium also exhibited endoglucanase, exoglucanase, and β-glucosidase activities in batch culture during growth on chemically defined medium with cellulose as the sole carbon source. Chemostat experiments were conducted under pH, dilution rate, and carbohydrate limitations to study the effect of various parameters on enzymes and product formation. In chemostat with 0.5% cellobiose as the limiting nutrient, maximum β-glucosidase activity and ethanol production occurred at pH values of 5.5 and 6.5, respectively. At low pH, acid accumulation was higher. β-glucosidase production increased up to 17-fold at a low dilution rate, and ethanol raised twofold. Relative activity of β-glucosidase on a percent basis was maximum at pH 6.5 and 45 °C. In a carbon-limited, continuous-culture experiment, when CMC was used as an inducer in cellobiose-growing culture, β-glucosidase activity was higher than the cellobiose-limited chemostat. On the other hand, enzyme declined twofold, and ethanol production increased in glucose limitation.

The production of β-glucosidase in shake-flasks by Aspergillus wentii

Studies in shake-flasks showed that Aspergillus wentii produces the maximum activity of β-glucosidase among the cultures tested. The activity against cellobiose was about 2–3 fold that against 4NPG. Aspergillus wentii produced a maximum activity of 16.5 U/ml in 14 days on malt extract. It also produced a comparable amount on other simple soluble sugars, which indicates that it is constitutive and does not require an inducer. Peptone was found to the best nitrogen source for β-glucosidase production. Optimum C/N ratio was found to be 7.3. Phosphate, magnesium and trace metals did not play significant roles in the production of β-glucosidase when they were used with malt extract as a carbon source. An inoculum of 6% (v/v) of 20-h-old culture grown on malt extract produced the maxium β-glucosidase activity.

Cell-associated and extracellular cellulolytic enzyme activity in the marine fungus Dendryphiella arenaria

Cellulolytic activity in Dendryphiella arenaria was demonstrated by its ability to release dye from dyed crystalline cellulose and to produce cell-associated and extracellular enzymes when grown on a similar substrate. Exoglucanase was found only in an insoluble form, associated with the hyphae and cellulose particles following sonic disruption of the cells. Endoglucanase was predominantly cell free but with a small amount associated with the cell fractions. High β-glucosidase activity was seen in the filtrate and cell fractions, although in the soluble intracellular fraction its activity disappeared with time. Optimal pH activities for exoglucanase and endoglucanase were demonstrated to be 7.0 and 5.8, respectively. Temperature optima of the enzyme components also differed; maximum exoglucanase activity in the cell-associated fractions and endoglucanase activity in the cell-associated and filtrate fractions occurred at 65 and 60 °C, respectively. Maximum β-glucosidase activity in the filtrate and soluble intracellular fractions occurred at 55 °C and that in the particulate fraction occurred at 60 °C.