Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922
financial support in form of project fellowship provided by
Keywords: β-glucosidase, Melanocarpus sp., purification, substrate specificity, transglycosylation.
This study reports the purification
and characterization of β-glucosidase from a newly isolated thermophilic
fungus, Melanocarpus sp. Microbial Type Culture Collection
(MTCC) 3922. The molecular weight of β-glucosidase was determined
to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively,
and pI of ~ 4.1. It was optimally active at
Cellulose, which constitutes the highest proportion of municipal and plant wastes, represents a major source of renewable energy and raw materials. Therefore, the utilization of cellulosic wastes to produce energy is potentially of great importance. Cellulases bring about the hydrolysis of cellulose, a homo-polymer of β-1,4 linked glucose units that comprises of amorphous and crystalline regions, by synergistic action of its constituent enzymes. These enzymes include; a) β-1,4-endoglucanase (1,4-β-D-glucan 4-glucanohydrolase; EC 18.104.22.168, cellulase), which cleaves internal β-1,4-glycosidic bonds, b) cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase; EC 22.214.171.124, cellulase 1,4-β-cellobiosidase), an exo-acting enzyme which releases cellobiose from reducing and non reducing ends of cellulose and c) β-glucosidase (β-D-glucoside glucohydrolase; EC 126.96.36.199, cellulase 1,4-β-glucosidase) that hydrolyzes cellobiose to glucose (Bhat and Bhat, 1997). β-glucosidase is generally responsible for the regulation of the whole cellulolytic process and is a rate-limiting factor during enzymatic hydrolysis of cellulose, as both endoglucanase and exoglucanase activities are often inhibited by cellobiose (Harhangi et al. 2002). Thus, β-glucosidase not only produces glucose from cellobiose, but also reduces cellobiose inhibition, allowing endoglucanase and exoglucanase enzymes to function more efficiently.
β-Glucosidase from Aspergillus supplemented to Trichoderma reesei cellulase preparations, plays an important role for cellulose saccharification on an industrial scale (Reczey et al. 1998). In recent years, interest in β-glucosidase has gained momentum owing to their ability to catalyze transglycosylation reactions. These types of reactions have great importance in wine industry because of its ability to improve the aroma of wines. The glycosylated precursor such as terpenes (Caldini et al. 1994) is found in mango, passion fruits and grapes (Sarry and Gunata, 2004) and β-glucosidases are more effective and specific than acid hydrolysis process for liberating terpenol (i.e., volatile alcohols) from terpenylglucoside. These glycosidically bound volatiles also have interest in the food, cosmetic and tobacco industries (Jerković and Mastelić, 2004). Saccharomyces cerevisiae cannot utilize cellulosic materials; therefore for the direct conversion of cellulose to ethanol various cellulase and β-glucosidase genes have been expressed in S. cerevisiae (Van Rensburg et al. 1998). A recent U.S. patent 5,454,389 reports that a crude cellulase having high ratio of β-glucosidase activity to filter paper units provide improved efficiency of deinking (Yang et al. 1999).
Although, there are number of reports on the production of β-glucosidase from yeast (S. cerevisiae, Pichia etchellsii) and mesophilic fungi (Trichoderma harzianum and Aspergillus sp.). Recent reports suggest that thermophilic fungi (Thermoascus aurantiacus, Chaetomium thermophile, Humicola insolens, Sporotrichum thermophile) and hydrocarbon utilizing novel fungus Cladosporium resinae are also good sources of β-glucosidase (Pandey and Mishra, 1997; Iwashita et al. 1998; Van Rensburg et al. 1998; Oh et al. 1999; Maheshwari et al. 2000; Parry et al. 2001; Yun et al. 2001). Recently, we have reported endoglucanase and β-glucosidase production from a rare thermophilic fungus Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922 (Kaur et al. 2006), a fungus initially reported to be devoid of cellulases (Maheshwari and Kamalam, 1985). This study for the first time reports the purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922.
thermophilic fungus isolated from composting soil and identified as
Melanocarpus sp. MTCC 3922 was employed in this study. The
fungus was grown and maintained on yeast potato soluble starch agar
(YpSS) of following composition (% w/v): starch, 1.5; yeast extract,
0.4; KH2PO4, 0.2; K2HPO4,
0.23; MgSO4.7H2O, 0.05; citric acid, 0.057 and
agar, 2.0. The pH of medium was adjusted to 7.0. The fungus was cultured
the preparation of inoculum, the culture was grown in 500 ml Erlenmeyer
flask containing 100 ml glucose-urea medium of the following composition
(% w/v); glucose, 1.0; yeast extract, 1.0; KH2PO4,0.6; K2HPO4, 0.04; MgSO4.7H2O,
0.05; urea, 0.05. During shake flask culturing few glass beads (
fermentation (SSF) was carried out in 500 ml Erlenmeyer flasks that
chromatography (DEAE Sepharose). The concentrated sample was centrifuged
(10,000 x g for 20 min) and loaded on DEAE-Sepharose (FF) column (24
exchanger (PBE 94). β-Glucosidase was further purified using
column (10 x
was assayed using p-nitrophenyl- β-D-glucopyranoside (pNPG) by
micro titer plate method as described (Parry et al.
2001). A reaction mixture (100 μl) containing 25 μl
of enzyme, 25 μl of pNPG (
Filtration, SDS-PAGE and IEF. The homogeneity and molecular mass
of β-glucosidase was determined by gel filtration and SDS-PAGE.
The gel filtration of purified β-glucosidase (10 μg) and
standard protein markers (Bangalore GENEI,
focusing (IEF) was performed according to the instructions provided
by Novex, using 5% acrylamide gel containing 2.4% broad pH range (3.5-10.0)
ampholine carrier ampholyte. The cathode buffer contained lysine 2.9%
(w/v) and arginine 3.5% (w/v), whereas, phosphoric acid (
and pH optima and stability. The temperature profile of purified
β-glucosidase was obtained by determining the activity on pNPG
between 30 and
of metal ions and other reagents. β-Glucosidase was incubated
specificity. Substrate specificity of β-glucosidase was determined
by using pNP- β-glucopyranoside, pNP-α-D-glucopyranoside,
pNP-β-galactopyranoside, oNP-β-D-galactopyranoside, oNP-β-D-xylopyranoside,
pNP- β-D-xylopyranoside and pNP-cellobioside, (
Effect of mono/disaccharides on β-glucosidase. The effect of mono/disaccharides (1 mg/ml) on β-glucosidase activity was studied using pNPG as a substrate.
Effect of alcohols. The effect of methanol, ethanol and propan-2-ol (0-100% v/v) on the hydrolysis of pNPG were studied using the pNPG assay.
A reaction mixture (150 μl) comprising of purified β-glucosidase
enzyme (1 μg), different concentrations (20-100% v/v) of methanol
(50 μl) and acetate buffer (
All experiments were performed in triplicate and results are given as mean value. The standard error ranged between 1-5%.
new strain of Melanocarpus sp. MTCC 3922, isolated from
the composting soil, grew profusely as white, cottony hyphal mass
extracellular β-glucosidase from Melanocarpus sp. was purified
by ion exchange chromatography using DEAE-Sepharose (weak anion exchanger)
and PBE 94 (Strong ion exchanger) columns. During DEAE-Sepharose column
chromatography, the major β-glucosidase peak was eluted with
Purified β-glucosidase from Melanocarpus sp. was homogeneous as judged by SDS-PAGE, gel filtration, and IEF-PAGE. The molecular weight of β-glucosidase, estimated by gel filtration and SDS-PAGE, was 102 and 92 kDa, respectively. Further homogeneity was confirmed by IEF that showed single band of β-glucosidase with pI value of ~ 4.1 (Figure 1a and Figure 1b, respectively).
was optimally active at
β-Glucosidase showed enhanced activity in presence of reducing agents, DTT and mercaptoethanol (Figure 4). The presence of monovalent and divalent metal ionsNa+, K+, Ca2+, Mg2+ and Zn2+ also positively influenced the activity of β-glucosidase. The presence of EDTA and SDS did not inhibit the enzyme activity, whereas, CuSO4 inhibited the enzyme activity up to 38.0%.
The action of purified β-glucosidase was tested over different substrates with α and β configurations. The results summarized in Table 2 show that β-glucosidase was maximally active against pNPG. β-Glucosidase activity on pNP- β-D-cellobioside was only 4.69% of that on pNPG. No activity was observed in the presence of the nitro group at the ortho position in oNP-β-D-galactopyranoside, oNP-β-D-xylopyranoside, as well as pNP-β-D-galactopyranoside and pNP-xylopyranoside. β-Glucosidase recognized cellobiose and salicin as substrate, however, enzyme was inactive against CMC (low and high viscosity), Avicel, Solka floc, laminarin and birchwood xylan.
The effect of different mono and disaccharides were studied in presence of pNPG. The results in Table 3 revealed that in the presence of glucose, ~ 47.0% decrease in hydrolysis of pNPG was observed. However, low level of inhibition was observed in presence of xylose, galactose and sucrose.
and Vmax for the hydrolysis of pNPG by β-glucosidase
was determined using 0-
The results in Figure 6 showed that short chain length alcohols, methanol and ethanol at a final concentration of 70% (v/v) increased the activity of β-glucosidase by 1.5 folds, while 70% (v/v) propan-2-ol has no effect on the enzyme activity but propan-2-ol between 80 and 100% (v/v) resulted in the inhibition of the enzyme activity.
The results in Figure 7 showed that in the presence of 20% (v/v) methanol, low transglycosylation activity was observed but as the methanol level was increased to 60% (v/v) there was a steady increase in transglycosylation activity. Further increase in methanol concentration resulted in decrease in activity.
thermophilic fungus, Melanocarpus sp. MTCC 3922, isolated
from composting soil and used in the present study, is a rare fungus
which produced very high amount of β-glucosidase (132.4 U/g of
substrate) when rice straw was used as carbon source under solid-state
fermentation. This is first report on the purification and characterization
of β-glucosidase from Melanocarpus sp. MTCC 3922. Previously,
we have shown that Melanocarpus sp. expressed only one isoform
of β-glucosidase with acidic pI in the presence of rice
straw, when crude filtrate was resolved on IEF gels (Kaur
et al. 2006). The enzyme was purified to homogeneity with specific
activity of 10.04 μmol min-1mg protein-1 and
yield (%) 15.89. The molecular weight of the native β-glucosidase
estimated by gel filtration was 102 kDa, and by SDS-PAGE analysis
was about 92 kDa suggesting that the enzyme is a monomer. This property
is shared with Trichoderma harzianum and Acremonium persicinum
having single protein of molecular weight of 75 kDa and 128 kDa, respectively
(Pitson et al. 1997; Yun et al. 2001).
But it was different from β-glucosidase of Pichia etchellsii
and Thermoascus aurantiacus, which had high molecular weights
and oligomeric nature (Pandey and Mishra, 1997;
Parry et al. 2001). Furthermore, the purity of enzyme
was confirmed by isoelectric focusing which showed a single protein
band. The purified enzyme has acidic pI, which has also been
observed in A.
maximum activity for the enzyme was observed at pH 6.0 and
β-Glucosidase from Melanocarpus sp. was preferentially active against pNP-β-glucopyranoside when compared to cellobiose. Enari and Niku-Paavola (1987) classified β-glucosidases into three major groups according to their substrate specificity; (1) aryl β-glucosidases with a strong affinity for aryl β-glucosides; (2) cellobiases, which only hydrolyze oligosaccharides (including cellobiose); and (3) β-glucosidases that are active with both type of substrates. Our results showed that β-glucosidase purified from Melanocarpus sp. MTCC 3922 was active against both aryl β-glucosides and cellobiose therefore it can be concluded that β-glucosidase from Melanocarpus sp. belong to group 3.
et al. (1988) suggested that the preference of β-glucosidases
for aryl glycosides is due to the high electrophilicity of the aglycone
moiety, which enhances the stability of the ortho or para
nitrophenoxide anion generated during the first step of catalysis.
β-Glucosidase from Melanocarpus sp. showed broad specificity
towards diglycosides. β-Glucosidase from Melanocarpus sp.
was found to hydrolyze cellobiose to a greater and salicin to lesser
extent indicating no steric hindrance with this compound as also observed
in Humicola grisea (Takashima et al.1996)
and Candida peltata (Saha and Bothast, 1996).
Kinetic study revealed that β-glucosidase from Melanocarpus
sp. has lower value of Km (
Presence of methanol and ethanol had a positive influence on the hydrolytic activity of β-glucosidase. In the presence of methanol and ethanol, an increase in enzyme activity was observed, though, the activity decreased with the longer alcohol chains. Activation by alcohol has been earlier observed for β-glucosidase from Thermoascus aurantiacus (Parry et al. 2001), Aspergillus oryzea (Riou et al. 1998), Fusarium oxysporum (Christakopoulos et al. 1994). β-Glucosidase from Melanocarpus sp. showed high transglycosylation activity in the presence of methanol. Transglycosylation was determined following the concept of using secondary reactions of a primary reaction product to induce a recordable signal (Mayer et al. 2001). The pNPG was used as donor and methanol as an acceptor. The primary reaction was catalyzed using purified β-glucosidase enzyme. To monitor the transglycosidase activity, purified endoglucanase (molecular weight ~ 40kDa) was used as a revealing enzyme. The observations indicated the transglycosylation in the presence of methanol by β-glucosidase. Although purified endoglucanase did not cleave pNPG, but enzyme hydrolyzed the polysaccharide formed by transglycosylation and released yellow nitro-phenol as signal indicating putative transglycosylation activity (Mayer et al. 2001). Previously, we have shown purified endoglucanase from Melanocarpus sp. exhibiting processive activity against crystalline cellulose and filter paper (Kaur et al. 2007). Presence of methanol resulted in higher levels of transglycosylation. Parry et al. (2001) have previously shown that methanol enhances the glycosyl-transferase activity of β-glucosidase in thermophilic ascomycete Thermoascus aurantiacus. Similarly Matsumura et al. (1999) also reported direct transglycosylation of xylan and octanol to octyl β-D-xylobioside by purified xylanase of Aureobasidium pullulans.
Expectedly, β-glucosidase from Melanocarpus sp. exhibited synergistic interaction with endoglucanase to increase the efficiency of glucose production from cellulose by converting rice straw to glucose (data not shown) indicating utility of β-glucosidase in enzymatic hydrolysis of cellulosics for subsequent production of ethanol fuel. The observed high activity of β-glucosidase from Melanocarpus sp. on rice straw also makes it a promising candidate for application in bioconversions as well as catalysis of novel compounds through transglycosylation reactions.
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