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Production of extracellular β-glucosidase by Monascus purpureus on different growth substrates
Process Biochemistry 42 (2007) 904–908 www.elsevier.com/locate/procbio
Short communication
Production of extracellular b-glucosidase by Monascus purpureus on different growth substrates Daniel J. Daroit, Silvana T. Silveira, Plinho F. Hertz, Adriano Brandelli * Laborato´rio de Bioquı´mica e Microbiologia Aplicada, Departamento de Cieˆncia de Alimentos (ICTA), Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, 91501-970 Porto Alegre, RS, Brazil Received 8 November 2006; received in revised form 23 January 2007; accepted 24 January 2007
Abstract Various agro-industrial residues in combination with peptone, NH4Cl and/or soy bran were screened as substrates for extracellular bglucosidase (BGL) production by Monascus purpureus NRRL1992 on submerged fermentations (SmF). Higher BGL production was achieved when the agro-industrial residues were combined with peptone, and the utilization of NH4Cl (inorganic nitrogen source) had not supported high enzyme production. The combination between grape waste and peptone was the best for enzyme production, and was selected as the growth substrate for further investigations. The evaluation of the effects of the medium components on enzyme production showed that the influence of peptone was more important than grape waste. The production of extracellular BGL by M. purpureus was inducible and controlled by carbon (glucose) catabolite repression. # 2007 Elsevier Ltd. All rights reserved. Keywords: Agro-industrial wastes; Cellulase; Enzyme; b-Glucosidase; Monascus purpureus; Submerged fermentation
1. Introduction b-D-Glucosidases (E.C. 3.2.1.21) constitute a heterogeneous group of enzymes that occurs in several organisms, performing a variety of functions. The principal reaction catalyzed by this class of enzymes is the hydrolytic cleavage of b-glycosidic linkages in low-molecular-mass glycosides, and the affinity of BGL for a particular substrate is dependent upon the nature of the enzyme source, physiological function and the location of the enzyme [1,2]. BGLs have been exploited in a variety of biotechnological applications. In the enzymatic saccharification of cellulose, BGL produces glucose by cleaving cellobiose. Since cellobiose inhibits the action of endo- and exoglucanases, BGL also contributes to the efficiency of this process [3]. BGL have been also studied due to its potential to release flavour compounds such as terpenes from odorless non-volatile glycosidic precursors in fruit juices and wines [4], release phenolic compounds with antioxidant, nutraceutical and flavourant properties from their glycosilated forms in fruit and vegetable
* Corresponding author. Tel.: +55 51 3308 6249; fax: +55 51 3308 7048. E-mail address: [email protected] (A. Brandelli). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.012
residues [5], cassava detoxification [6], among other applications [1,2]. The filamentous fungi Monascus are used in Asia for centuries as a source of pigments for the coloring of traditional foods. This genus is subject of constant studies, mainly due to the growing interest for natural pigments to be used in the food industry. Although other metabolic products from Monascus species, like alcohols, organic acids, antimicrobial agents and substances with therapeutic activity have been described [7–9], little information about enzymes from Monascus is available [9]. Large amounts of agro-industrial residues are generated every year from diverse economic activities, increasing the biotechnological interest on the utilization of these residues as substrates (or raw-materials) in biotechnological processes [5,10–12]. The utilization of agro-industrial waste as growth substrates may represent an added value to the industry and meets the increasing consciousness for energy conservation. Submerged fermentations (SmF) are normally used in the fermentation industry because they are easier to handle and control in the fermentation process [13]. In this paper, several agro-industrial byproducts were tested as substrates for BGL production by M. purpureus in submerged fermentations (SmF). Then, the effects of selected substrates were evaluated to establish the best conditions for
D.J. Daroit et al. / Process Biochemistry 42 (2007) 904–908
BGL production. The repression of enzyme production was also investigated. 2. Materials and methods 2.1. Microorganism M. purpureus NRRL1992 was maintained on Sabouraud dextrose agar plates at 4 8C and subcultured periodically. Cultures reactivated by transferring onto fresh Sabouraud agar plates and cultured at 30 8C for 12–14 days were used for inoculum preparation.
905
Table 2 BGL activities on the different treatments for a 22 factorial experiment Run
Grape waste
Peptone
Enzyme activity (U/mL)
1 2 3 4 5 6 7
( 1) (10 g/L) ( 1) (10 g/L) (+1) (50 g/L) (+1) (50 g/L) (0) (30 g/L) (0) (30 g/L) (0) (30 g/L)
( 1) (1 g/L) (+1) (20 g/L) ( 1) (1 g/L) (+1) (20 g/L) (0) (10.5 g/L) (0) (10.5 g/L) (0) (10.5 g/L)
0.542 1.953 0.097 2.780 2.294 2.385 2.352
carried out adding the chosen substrates to the mineral medium, as indicated in Table 2.
2.2. Substrate selection Carboxymethylcellulose (CMC) and agro-industrial residues, including cheese whey powder, pinha˜o (seeds of Araucaria angustifolia) rind, grape waste of wine industry, and soy bran were tested as substrates (20 g/L) for BGL production by M. purpureus in combination with peptone (5 g/L), NH4Cl (2.5 g/ L) and/or soy bran (5 g/L), in mineral medium. The mineral medium contained: K2HPO4 (5.0 g/L), KH2PO4 (5.0 g/L), MgSO47H2O (0.1 g/L), CaCl2 (0.025 g/ L), ZnSO47H2O (0.01 g/L) and MnSO4 (0.01 g/L). The substrate combinations are specified in Table 1. The initial pH of the medium was adjusted to 6.0. Erlenmeyer flasks (125 mL) containing 25 mL of medium were inoculated with 250 mL (1%, v/v) of a conidial and mycelial suspension with OD620 of 0.4. For the preparation of this suspension, 12–14-day-old cultures were scraped from the surface of Sabouraud agar, added to a 0.85% NaCl saline sterile solution, and mixed until a homogeneous solution was obtained. The inoculated flasks were incubated at 27 8C on a rotatory shaker at 100 rpm for 9 days, and the substrate combination which gave the higher BGL activity in the screening tests was further employed in a first-order factorial design.
2.3. First-order factorial design The influence of the concentrations of the selected substrates was evaluated in a first-order factorial design (22) with three replicates in the central points, which means a total of five different treatment combinations [14]. Three levels of each independent variable were chosen, with the upper and lower limits of this set to be in the range described in the literature, and BGL activity was taken as the dependent variable. Table 2 shows the independent variables and their levels, as well as the response evaluated. All data were analyzed with the software Statistica 5.0 (Statsoft, Tulsa, OK, USA). The experiments were Table 1 BGL activity in Monascus purpureus culture supernatant after 9 days of submerged cultivation on different substrates Substrate (%, w/v)
Enzyme activity (U/mL)a
CMC (2.0%) + NH4Cl (0.25%) CMC (2.0%) + peptone (0.5%) Cheese whey powder (2.0%) + NH4Cl (0.25%) Cheese whey powder (2.0%) + peptone (0.5%) Cheese whey powder (2.0%) + soy bran (0.5%) Pinha˜o rind (2.0%) + NH4Cl (0.25%) Pinha˜o rind (2.0%) + peptone (0.5%) Soy bran (2.0%) Soy bran (2.0%) + NH4Cl (0.25%) Soy bran (2.0%) + peptone (0.5%) Grape waste (2.0%) + NH4Cl (0.25%) Grape waste (2.0%) + peptone (0.5%) Grape waste (2.0%) + soy bran (0.5%) Grape waste (2.0%) + NH4Cl (0.25%) + soy bran (0.5%)
0.085 0.014 0.363 0.027 0.202 0.018 1.043 0.029 0.398 0.024 0.066 0.020 0.638 0.044 0.827 0.034 0.779 0.018 1.096 0.039 0.085 0.009 1.682 0.038 0.664 0.024 0.223 0.016
a b ac d b a e f f d a g ef c
Different letters represents statistically different means at p < 0.05. a Means of duplicate cultivations and two enzymatic assays for each cultivation.
2.4. Enzymatic activity At the end of cultivation, or at defined intervals, samples of 500 mL were taken, centrifuged (12,000 g for 5 min), and the supernatant was used as enzyme source. BGL activity was assayed by a modified procedure, based on the method of Hang and Woodams [10]. The reaction mixture (200 mL) contained 90 mL of citrate buffer (250 mM, pH 4.5), 10 mL of p-nitrophenyl-b-D-glucopyranoside (pNPG; 4 mg/mL), and 100 mL of the culture supernatant. After incubation at 37 8C for 30 min, the reaction was stopped by adding 1 mL of cold sodium carbonate buffer (500 mM, pH 10). The activity of bglucosidase was estimated spectrophotometrically by reading the absorbance of the liberated p-nitrophenol at 405 nm (e = 18,700). One unit (U) of b-glucosidase activity was defined as the amount of enzyme required for the hydrolysis of 1 mmol of substrate (pNPG)/min, under the assay conditions.
2.5. Repression studies To evaluate the possible repression of enzyme production, the fungus was grown on mineral medium containing glucose (20 g/L) and peptone (5 g/L). The enzymatic activity was estimated as described above. The concentration of reducing sugars was determined by the 3,5-dinitrosalicylic acid (DNS) method, where 1 mL of DNS solution was added to 100 mL of culture supernatant and incubated at 100 8C for 10 min. After cooling, the absorbance was measured at 570 nm, using glucose as standard.
3. Results and discussion 3.1. Substrate screening for BGL production The results of BGL production by M. purpureus growing on different substrates are shown in Table 1. The combination between agro-industrial residues and peptone or soy bran resulted in higher BGL production when compared with the combination between CMC and peptone. This result is interesting due to the fact that CMC is a highly purified synthetic substrate, becoming expensive for large-scale enzyme production. The utilization of agro-industrial residues as potential substrates for the production of BGL and other cellulase, hemicellulase and pectinase components has attracted much attention [10,11,15,16], since it can contribute to lower the costs of enzyme production and also to reduce the environmental pollution caused by the accumulation of lignocellulosic wastes. It was demonstrated that the best substrate combination for BGL production was the grape waste supplemented with peptone (1.682 U/mL). Fig. 1 shows the pattern of BGL production within 14 days of cultivation and the productivity (U/mL day). Although the extracellular BGL activity continues to grow until
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D.J. Daroit et al. / Process Biochemistry 42 (2007) 904–908
Fig. 1. BGL production (~) by Monascus purpureus on mineral medium containing 20 g/L grape waste and 5 g/L peptone; and productivity (&). Each point is the mean S.E.M. of duplicate cultivations and two assays for each cultivation.
the 14th day of cultivation, a considerable fall in the productivity is observed at this point. In this sense, the cultivation period of 9 days was chosen for extracellular BGL production by M. purpureus. Grapes are the second world’s largest fruit crop, with more than 60 million tonnes produced annually. Grape pomace represents approximately 20% of the weight of processed grapes, which amounts to more than 9 million tonnes per year [17]. Thus, grape residues are abundant and can be employed in composting processes [18], as sources of antioxidant compounds [19], as substrates for enzyme [20], ethanol [21] and citric acid production [22], among other applications. Particularly, grape pomace represents a potentially valuable source of phenolic antioxidants [19], and the combination of fungal growth and BGL production could be explored to liberate free phenols from this substrate, as in the case of cranberry pomace [5]. In this work, inorganic nitrogen sources (NH4Cl) had not supported the production of high levels of BGL (Table 1), a fact also reported for other fungi [23]. On the other hand, some studies have found that inorganic nitrogen sources resulted in equal or higher BGL production than organic ones [16]. The results obtained indicate that the presence of peptone in the culture media played a critical role in the production of high levels of BGL by M. purpureus (Table 1). Organic nitrogen sources are usually used for pigment [8] and enzyme [7,9] production by Monascus species. Particularly, Liu et al. [7] observed that inorganic nitrogen sources resulted in much lower serine carboxypeptidase production by M. purpureus. Among the residues screened as substrates in combination with NH4Cl, soy bran showed higher results for BGL production (Table 1). Also, due to the high enzyme activity achieved when soy bran was utilized as the single substrate, this industrial byproduct was tested as an alternative to the utilization of peptone. Corn steep liquor [15] and soy meal [24] are examples of cheap substrates successfully employed as complex nitrogen sources in BGL production, contributing to lower the costs of enzyme production. However, BGL production on grape waste supplemented with soy bran was only 39% of that obtained on grape waste supplemented with peptone. Nevertheless, the use of soy bran in combination with grape waste showed similar results
to that obtained with peptone supplemented-pinha˜o rind. The lower recalcitrance, the presence of vitamins and minerals, and the essential amino acid content of soy bran, and also the higher lignin content of pinha˜o rind could be responsible for this result. Lignin, a highly insoluble complex branched polymer that surrounds and strengthens the cellulose microfibrils, is highly resistant to biodegradation and protects cellulose and hemicellulose against hydrolysis [3]. Cheese whey powder, in association with NH4Cl or soy bran, was not a good substrate for BGL production. However, the utilization of cheese whey powder and peptone has increased the enzyme production, being the second most effective substrate combination for this purpose (Table 1). Cheese whey is a lactoserich residue of the dairy industry, and lactose has been demonstrated to induce high production of enzymes of the cellulase complex, including BGL, in some fungi [25] and bacteria [26]. Cellulase genes are normally expressed at a low basal level, and the regulation mechanism of enzyme transcription known as carbon catabolite repression is common among fungi grown on easily attainable carbon sources [27]. The growth of fungal species on mineral medium containing glucose generally represses enzyme production, and only after glucose depletion the fungi begins the production of BGL [28]. In agreement with these results, M. purpureus produced a constitutive basal level of BGL in the presence of glucose, and the higher enzyme production started only after glucose depletion (Fig. 2). Similar results were found when M. purpureus was grown on mineral medium containing peptone as the sole carbon and nitrogen source (not shown). Thus, the production of extracellular BGL by M. purpureus seems to be inducible and controlled by carbon (glucose) catabolite repression. 3.2. Effects of medium components on BGL production The results obtained so far point to a positive interaction among the agro-industrial residues tested and peptone, producing higher BGL activity values. The extent of the contribution and the effects of grape waste and peptone on BGL production by M. purpureus in submerged cultures were studied in a 22 factorial
Fig. 2. Repression of BGL production by M. purpureus when grown on mineral medium containing 20 g/L glucose and 5 g/L peptone. (&) Reducing sugar (glucose) concentration, (~) enzyme activity. Each point is the mean S.E.M. of duplicate cultivations and two assays for each cultivation.
D.J. Daroit et al. / Process Biochemistry 42 (2007) 904–908 Table 3 Main effects and interactions analysis for enzyme activity in submerged culture by M. purpureus Factor
Effect (U/mL)
S.E.
t-Value
p-Value
Mean Grape waste (1) Peptone (2) 12
1.77 0.19 2.04 0.63
0.01 0.04 0.04 0.04
101.75 4.15 44.43 13.80
<0.000* 0.053** <0.000* 0.005*
* **
Significant factors ( p < 0.05). Significant factors ( p < 0.06).
design. Table 2 shows the independent variables (grape waste and peptone) and their levels, as well as the enzymatic activity (dependent variable) for each treatment combination. An estimation of main effects of variables on the response is obtained by evaluating the difference in process performance caused by a change from the low ( 1) to the high (+1) level of the corresponding factor. Process performance was measured for enzyme production response, and both the t-test and p-value statistical parameters were used to confirm the significance of the factors studied (Table 3). The results showed that the BGL production was more significantly affected ( p < 0.05) by peptone concentration. The change in this variable from the low to the high level exhibited a positive influence that resulted in an increase in enzyme activity of 2.04 U on average. Grape waste concentration presented a positive influence at 94% confidence level ( p < 0.06). Despite the fact that the highest enzyme activity was obtained when grape waste and peptone were used at higher concentrations, it should be taken into account that peptone is an expensive substrate and, in this sense, a cost-benefit analysis is indispensable in view to obtain satisfying amounts of enzyme at lower or moderate costs. 4. Conclusions Among the various substrates tested, the best combination for extracellular BGL production by M. purpureus NRRL1992 on submerged cultivations was observed with grape waste and peptone. The utilization of inorganic nitrogen source (NH4Cl) in combination with agro-industrial residues resulted in lower production of BGL by M. purpureus, and the presence of peptone played a crucial role in the production of high levels of BGL. Peptone appeared to be more important than grape waste; however, the positive interaction observed between these components on BGL production is an interesting and attractive factor to obtain higher amounts of this enzyme. The production of extracellular BGL by M. purpureus seems to be inducible, and controlled by carbon (glucose) catabolite repression. To our awareness, this is the first report on extracellular BGL production by M. purpureus. Purification, characterization, and investigations on the potential applications of this enzyme are being conducted. Acknowledgement This work was supported by CNPq, Brazil.
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[24] Gomes I, Gomes J, Gomes DJ, Steiner W. Simultaneous production of high activities of thermostable endoglucanase and b-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol 2000;53:461–8. [25] Abdel-Fattah AF, Osman MY, Abdel-Naby MA. Production and immobilization of cellobiase from Aspergillus niger A20. Chem Eng J 1997;68:189–96.
[26] Thirumale S, Rani DS, Nand K. Control of cellulase formation by trehalose in Clostridium papyrosolvens CFR-703. Process Biochem 2001;37:241–5. [27] Suto M, Tomita F. Induction and catabolite repression mechanisms of cellulase in fungi. J Biosci Bioeng 2001;92:305–11. [28] Jørgensen H, Mørkeberg A, Krogh KBR, Olsson L. Growth and enzyme production by three Penicillium species on monosaccharides. J Biotechnol 2004;109:295–9.
Short communication
Production of extracellular b-glucosidase by Monascus purpureus on different growth substrates Daniel J. Daroit, Silvana T. Silveira, Plinho F. Hertz, Adriano Brandelli * Laborato´rio de Bioquı´mica e Microbiologia Aplicada, Departamento de Cieˆncia de Alimentos (ICTA), Universidade Federal do Rio Grande do Sul, Av. Bento Gonc¸alves 9500, 91501-970 Porto Alegre, RS, Brazil Received 8 November 2006; received in revised form 23 January 2007; accepted 24 January 2007
Abstract Various agro-industrial residues in combination with peptone, NH4Cl and/or soy bran were screened as substrates for extracellular bglucosidase (BGL) production by Monascus purpureus NRRL1992 on submerged fermentations (SmF). Higher BGL production was achieved when the agro-industrial residues were combined with peptone, and the utilization of NH4Cl (inorganic nitrogen source) had not supported high enzyme production. The combination between grape waste and peptone was the best for enzyme production, and was selected as the growth substrate for further investigations. The evaluation of the effects of the medium components on enzyme production showed that the influence of peptone was more important than grape waste. The production of extracellular BGL by M. purpureus was inducible and controlled by carbon (glucose) catabolite repression. # 2007 Elsevier Ltd. All rights reserved. Keywords: Agro-industrial wastes; Cellulase; Enzyme; b-Glucosidase; Monascus purpureus; Submerged fermentation
1. Introduction b-D-Glucosidases (E.C. 3.2.1.21) constitute a heterogeneous group of enzymes that occurs in several organisms, performing a variety of functions. The principal reaction catalyzed by this class of enzymes is the hydrolytic cleavage of b-glycosidic linkages in low-molecular-mass glycosides, and the affinity of BGL for a particular substrate is dependent upon the nature of the enzyme source, physiological function and the location of the enzyme [1,2]. BGLs have been exploited in a variety of biotechnological applications. In the enzymatic saccharification of cellulose, BGL produces glucose by cleaving cellobiose. Since cellobiose inhibits the action of endo- and exoglucanases, BGL also contributes to the efficiency of this process [3]. BGL have been also studied due to its potential to release flavour compounds such as terpenes from odorless non-volatile glycosidic precursors in fruit juices and wines [4], release phenolic compounds with antioxidant, nutraceutical and flavourant properties from their glycosilated forms in fruit and vegetable
* Corresponding author. Tel.: +55 51 3308 6249; fax: +55 51 3308 7048. E-mail address: [email protected] (A. Brandelli). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.01.012
residues [5], cassava detoxification [6], among other applications [1,2]. The filamentous fungi Monascus are used in Asia for centuries as a source of pigments for the coloring of traditional foods. This genus is subject of constant studies, mainly due to the growing interest for natural pigments to be used in the food industry. Although other metabolic products from Monascus species, like alcohols, organic acids, antimicrobial agents and substances with therapeutic activity have been described [7–9], little information about enzymes from Monascus is available [9]. Large amounts of agro-industrial residues are generated every year from diverse economic activities, increasing the biotechnological interest on the utilization of these residues as substrates (or raw-materials) in biotechnological processes [5,10–12]. The utilization of agro-industrial waste as growth substrates may represent an added value to the industry and meets the increasing consciousness for energy conservation. Submerged fermentations (SmF) are normally used in the fermentation industry because they are easier to handle and control in the fermentation process [13]. In this paper, several agro-industrial byproducts were tested as substrates for BGL production by M. purpureus in submerged fermentations (SmF). Then, the effects of selected substrates were evaluated to establish the best conditions for
D.J. Daroit et al. / Process Biochemistry 42 (2007) 904–908
BGL production. The repression of enzyme production was also investigated. 2. Materials and methods 2.1. Microorganism M. purpureus NRRL1992 was maintained on Sabouraud dextrose agar plates at 4 8C and subcultured periodically. Cultures reactivated by transferring onto fresh Sabouraud agar plates and cultured at 30 8C for 12–14 days were used for inoculum preparation.
905
Table 2 BGL activities on the different treatments for a 22 factorial experiment Run
Grape waste
Peptone
Enzyme activity (U/mL)
1 2 3 4 5 6 7
( 1) (10 g/L) ( 1) (10 g/L) (+1) (50 g/L) (+1) (50 g/L) (0) (30 g/L) (0) (30 g/L) (0) (30 g/L)
( 1) (1 g/L) (+1) (20 g/L) ( 1) (1 g/L) (+1) (20 g/L) (0) (10.5 g/L) (0) (10.5 g/L) (0) (10.5 g/L)
0.542 1.953 0.097 2.780 2.294 2.385 2.352
carried out adding the chosen substrates to the mineral medium, as indicated in Table 2.
2.2. Substrate selection Carboxymethylcellulose (CMC) and agro-industrial residues, including cheese whey powder, pinha˜o (seeds of Araucaria angustifolia) rind, grape waste of wine industry, and soy bran were tested as substrates (20 g/L) for BGL production by M. purpureus in combination with peptone (5 g/L), NH4Cl (2.5 g/ L) and/or soy bran (5 g/L), in mineral medium. The mineral medium contained: K2HPO4 (5.0 g/L), KH2PO4 (5.0 g/L), MgSO47H2O (0.1 g/L), CaCl2 (0.025 g/ L), ZnSO47H2O (0.01 g/L) and MnSO4 (0.01 g/L). The substrate combinations are specified in Table 1. The initial pH of the medium was adjusted to 6.0. Erlenmeyer flasks (125 mL) containing 25 mL of medium were inoculated with 250 mL (1%, v/v) of a conidial and mycelial suspension with OD620 of 0.4. For the preparation of this suspension, 12–14-day-old cultures were scraped from the surface of Sabouraud agar, added to a 0.85% NaCl saline sterile solution, and mixed until a homogeneous solution was obtained. The inoculated flasks were incubated at 27 8C on a rotatory shaker at 100 rpm for 9 days, and the substrate combination which gave the higher BGL activity in the screening tests was further employed in a first-order factorial design.
2.3. First-order factorial design The influence of the concentrations of the selected substrates was evaluated in a first-order factorial design (22) with three replicates in the central points, which means a total of five different treatment combinations [14]. Three levels of each independent variable were chosen, with the upper and lower limits of this set to be in the range described in the literature, and BGL activity was taken as the dependent variable. Table 2 shows the independent variables and their levels, as well as the response evaluated. All data were analyzed with the software Statistica 5.0 (Statsoft, Tulsa, OK, USA). The experiments were Table 1 BGL activity in Monascus purpureus culture supernatant after 9 days of submerged cultivation on different substrates Substrate (%, w/v)
Enzyme activity (U/mL)a
CMC (2.0%) + NH4Cl (0.25%) CMC (2.0%) + peptone (0.5%) Cheese whey powder (2.0%) + NH4Cl (0.25%) Cheese whey powder (2.0%) + peptone (0.5%) Cheese whey powder (2.0%) + soy bran (0.5%) Pinha˜o rind (2.0%) + NH4Cl (0.25%) Pinha˜o rind (2.0%) + peptone (0.5%) Soy bran (2.0%) Soy bran (2.0%) + NH4Cl (0.25%) Soy bran (2.0%) + peptone (0.5%) Grape waste (2.0%) + NH4Cl (0.25%) Grape waste (2.0%) + peptone (0.5%) Grape waste (2.0%) + soy bran (0.5%) Grape waste (2.0%) + NH4Cl (0.25%) + soy bran (0.5%)
0.085 0.014 0.363 0.027 0.202 0.018 1.043 0.029 0.398 0.024 0.066 0.020 0.638 0.044 0.827 0.034 0.779 0.018 1.096 0.039 0.085 0.009 1.682 0.038 0.664 0.024 0.223 0.016
a b ac d b a e f f d a g ef c
Different letters represents statistically different means at p < 0.05. a Means of duplicate cultivations and two enzymatic assays for each cultivation.
2.4. Enzymatic activity At the end of cultivation, or at defined intervals, samples of 500 mL were taken, centrifuged (12,000 g for 5 min), and the supernatant was used as enzyme source. BGL activity was assayed by a modified procedure, based on the method of Hang and Woodams [10]. The reaction mixture (200 mL) contained 90 mL of citrate buffer (250 mM, pH 4.5), 10 mL of p-nitrophenyl-b-D-glucopyranoside (pNPG; 4 mg/mL), and 100 mL of the culture supernatant. After incubation at 37 8C for 30 min, the reaction was stopped by adding 1 mL of cold sodium carbonate buffer (500 mM, pH 10). The activity of bglucosidase was estimated spectrophotometrically by reading the absorbance of the liberated p-nitrophenol at 405 nm (e = 18,700). One unit (U) of b-glucosidase activity was defined as the amount of enzyme required for the hydrolysis of 1 mmol of substrate (pNPG)/min, under the assay conditions.
2.5. Repression studies To evaluate the possible repression of enzyme production, the fungus was grown on mineral medium containing glucose (20 g/L) and peptone (5 g/L). The enzymatic activity was estimated as described above. The concentration of reducing sugars was determined by the 3,5-dinitrosalicylic acid (DNS) method, where 1 mL of DNS solution was added to 100 mL of culture supernatant and incubated at 100 8C for 10 min. After cooling, the absorbance was measured at 570 nm, using glucose as standard.
3. Results and discussion 3.1. Substrate screening for BGL production The results of BGL production by M. purpureus growing on different substrates are shown in Table 1. The combination between agro-industrial residues and peptone or soy bran resulted in higher BGL production when compared with the combination between CMC and peptone. This result is interesting due to the fact that CMC is a highly purified synthetic substrate, becoming expensive for large-scale enzyme production. The utilization of agro-industrial residues as potential substrates for the production of BGL and other cellulase, hemicellulase and pectinase components has attracted much attention [10,11,15,16], since it can contribute to lower the costs of enzyme production and also to reduce the environmental pollution caused by the accumulation of lignocellulosic wastes. It was demonstrated that the best substrate combination for BGL production was the grape waste supplemented with peptone (1.682 U/mL). Fig. 1 shows the pattern of BGL production within 14 days of cultivation and the productivity (U/mL day). Although the extracellular BGL activity continues to grow until
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Fig. 1. BGL production (~) by Monascus purpureus on mineral medium containing 20 g/L grape waste and 5 g/L peptone; and productivity (&). Each point is the mean S.E.M. of duplicate cultivations and two assays for each cultivation.
the 14th day of cultivation, a considerable fall in the productivity is observed at this point. In this sense, the cultivation period of 9 days was chosen for extracellular BGL production by M. purpureus. Grapes are the second world’s largest fruit crop, with more than 60 million tonnes produced annually. Grape pomace represents approximately 20% of the weight of processed grapes, which amounts to more than 9 million tonnes per year [17]. Thus, grape residues are abundant and can be employed in composting processes [18], as sources of antioxidant compounds [19], as substrates for enzyme [20], ethanol [21] and citric acid production [22], among other applications. Particularly, grape pomace represents a potentially valuable source of phenolic antioxidants [19], and the combination of fungal growth and BGL production could be explored to liberate free phenols from this substrate, as in the case of cranberry pomace [5]. In this work, inorganic nitrogen sources (NH4Cl) had not supported the production of high levels of BGL (Table 1), a fact also reported for other fungi [23]. On the other hand, some studies have found that inorganic nitrogen sources resulted in equal or higher BGL production than organic ones [16]. The results obtained indicate that the presence of peptone in the culture media played a critical role in the production of high levels of BGL by M. purpureus (Table 1). Organic nitrogen sources are usually used for pigment [8] and enzyme [7,9] production by Monascus species. Particularly, Liu et al. [7] observed that inorganic nitrogen sources resulted in much lower serine carboxypeptidase production by M. purpureus. Among the residues screened as substrates in combination with NH4Cl, soy bran showed higher results for BGL production (Table 1). Also, due to the high enzyme activity achieved when soy bran was utilized as the single substrate, this industrial byproduct was tested as an alternative to the utilization of peptone. Corn steep liquor [15] and soy meal [24] are examples of cheap substrates successfully employed as complex nitrogen sources in BGL production, contributing to lower the costs of enzyme production. However, BGL production on grape waste supplemented with soy bran was only 39% of that obtained on grape waste supplemented with peptone. Nevertheless, the use of soy bran in combination with grape waste showed similar results
to that obtained with peptone supplemented-pinha˜o rind. The lower recalcitrance, the presence of vitamins and minerals, and the essential amino acid content of soy bran, and also the higher lignin content of pinha˜o rind could be responsible for this result. Lignin, a highly insoluble complex branched polymer that surrounds and strengthens the cellulose microfibrils, is highly resistant to biodegradation and protects cellulose and hemicellulose against hydrolysis [3]. Cheese whey powder, in association with NH4Cl or soy bran, was not a good substrate for BGL production. However, the utilization of cheese whey powder and peptone has increased the enzyme production, being the second most effective substrate combination for this purpose (Table 1). Cheese whey is a lactoserich residue of the dairy industry, and lactose has been demonstrated to induce high production of enzymes of the cellulase complex, including BGL, in some fungi [25] and bacteria [26]. Cellulase genes are normally expressed at a low basal level, and the regulation mechanism of enzyme transcription known as carbon catabolite repression is common among fungi grown on easily attainable carbon sources [27]. The growth of fungal species on mineral medium containing glucose generally represses enzyme production, and only after glucose depletion the fungi begins the production of BGL [28]. In agreement with these results, M. purpureus produced a constitutive basal level of BGL in the presence of glucose, and the higher enzyme production started only after glucose depletion (Fig. 2). Similar results were found when M. purpureus was grown on mineral medium containing peptone as the sole carbon and nitrogen source (not shown). Thus, the production of extracellular BGL by M. purpureus seems to be inducible and controlled by carbon (glucose) catabolite repression. 3.2. Effects of medium components on BGL production The results obtained so far point to a positive interaction among the agro-industrial residues tested and peptone, producing higher BGL activity values. The extent of the contribution and the effects of grape waste and peptone on BGL production by M. purpureus in submerged cultures were studied in a 22 factorial
Fig. 2. Repression of BGL production by M. purpureus when grown on mineral medium containing 20 g/L glucose and 5 g/L peptone. (&) Reducing sugar (glucose) concentration, (~) enzyme activity. Each point is the mean S.E.M. of duplicate cultivations and two assays for each cultivation.
D.J. Daroit et al. / Process Biochemistry 42 (2007) 904–908 Table 3 Main effects and interactions analysis for enzyme activity in submerged culture by M. purpureus Factor
Effect (U/mL)
S.E.
t-Value
p-Value
Mean Grape waste (1) Peptone (2) 12
1.77 0.19 2.04 0.63
0.01 0.04 0.04 0.04
101.75 4.15 44.43 13.80
<0.000* 0.053** <0.000* 0.005*
* **
Significant factors ( p < 0.05). Significant factors ( p < 0.06).
design. Table 2 shows the independent variables (grape waste and peptone) and their levels, as well as the enzymatic activity (dependent variable) for each treatment combination. An estimation of main effects of variables on the response is obtained by evaluating the difference in process performance caused by a change from the low ( 1) to the high (+1) level of the corresponding factor. Process performance was measured for enzyme production response, and both the t-test and p-value statistical parameters were used to confirm the significance of the factors studied (Table 3). The results showed that the BGL production was more significantly affected ( p < 0.05) by peptone concentration. The change in this variable from the low to the high level exhibited a positive influence that resulted in an increase in enzyme activity of 2.04 U on average. Grape waste concentration presented a positive influence at 94% confidence level ( p < 0.06). Despite the fact that the highest enzyme activity was obtained when grape waste and peptone were used at higher concentrations, it should be taken into account that peptone is an expensive substrate and, in this sense, a cost-benefit analysis is indispensable in view to obtain satisfying amounts of enzyme at lower or moderate costs. 4. Conclusions Among the various substrates tested, the best combination for extracellular BGL production by M. purpureus NRRL1992 on submerged cultivations was observed with grape waste and peptone. The utilization of inorganic nitrogen source (NH4Cl) in combination with agro-industrial residues resulted in lower production of BGL by M. purpureus, and the presence of peptone played a crucial role in the production of high levels of BGL. Peptone appeared to be more important than grape waste; however, the positive interaction observed between these components on BGL production is an interesting and attractive factor to obtain higher amounts of this enzyme. The production of extracellular BGL by M. purpureus seems to be inducible, and controlled by carbon (glucose) catabolite repression. To our awareness, this is the first report on extracellular BGL production by M. purpureus. Purification, characterization, and investigations on the potential applications of this enzyme are being conducted. Acknowledgement This work was supported by CNPq, Brazil.
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