# Biotechnological Potential of Agro-Industrial Wastes as a Carbon Source to Thermostable Polygalacturonase Production inAspergillus niveus

Authors: Alexandre Maller; André Ricardo Lima Damásio; Tony Marcio da Silva; João Atílio Jorge; Héctor Francisco Terenzi; Maria de Lourdes Teixeira de Moraes Polizeli
Journal: Enzyme Research
 (2011)

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## Abstract

Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as nutrients for microbial growth and production of enzymes. The aim of this work was to study polygalacturonase (PG) production byAspergillus niveus cultured on liquid or solid media supplemented with agro-industrial wastes. Submerged fermentation (SbmF) was tested using Czapeck media supplemented with 28 different carbon sources.  Among these, orange peel was the best PG inducer. On the other hand, for solid state fermentation (SSF), lemon peel was the best inducer. By comparing SbmF with SSF, both supplemented with lemon peel, it was observed that PG levels were 4.4-fold higher under SSF. Maximum PG activity was observed at 55∘C and pH 4.0. The enzyme was stable at 60∘C for 90 min and at pH 3.0–5.0. The properties of this enzyme, produced on inexpensive fermentation substrates, were interesting and suggested several biotechnological applications.


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## Body

## 1. Introduction

Pectolytic enzymes are involved in the degradation of pectin, a structural component of the middle lamella and the primary cell walls of plants. Pectins are complex colloidal acidic polysaccharides that show a backbone of galacturonic acid residues withα-1,4-glycosidic linkages [1]. These molecules possess L-rhamnose, arabinose, galactose, and xylose in the side chains. Also, the carboxylic groups in the galacturonic acid chain are neutralized by different ions, as Na+, K+, and NH4 + [2]. Pectins comprise a family of oligosaccharides and polysaccharides that have common features, but are extremely diverse in their fine structures. However, all pectins are rich in galacturonic acid (GalA) and they have at least 65% GalA.Pectinolytic enzymes break down pectin or pectate by the hydrolysis ofα-1,4-glycosidic linkages and they have varied biotechnological applications. The acidophilic pectinases have extensive applications in the manufacture of fruit juices and wine. They are used in apple juice preparation and clarification, to facilitate pressing and juice extraction. Moreover, pectic enzymes are used to reduce haze or gelling of grape juice in wine manufacture and to enhance the quality of cider apple varieties that are bitter, sweet, or sour [3, 4]. The alkaline pectinase also has various industrial applications, such as wastewater treatment, paper manufacturing, oil extraction, coffee and tea fermentation, processing and degumming of many plant fibers [3].Several fungal species are effective degraders of pectic substances, being able to produce high amounts of pectinolytic enzymes [1]. A novel strain of A. niveus was isolated from Brazilian soil, which produces high levels of several hydrolytic enzymes, such as xylanase [5, 6] and amylases [7, 8]. In this work, we demonstrated that this fungus also produced high polygalacturonase levels when grown on agricultural wastes, such as orange peel and passion fruit peel. This work leads to future biotechnological applications, and it also contributes to diminish the environmental pollution consequent of the accumulation of citric residues that are discarded in the environment.


## 2. Materials and Methods

### 2.1. Organism and Growth Conditions

Aspergillus niveus was isolated from Mangifera indica in our laboratory. The microorganism was identified and deposited in the culture collection of Pernambuco Federal University (PE, Brazil). The organism was maintained on slants of potato dextrose agar (PDA) medium covered with mineral oil, at 4°C. The fungus was incubated on PDA medium, at 30°C for 15 days previous to the cultivation and optimization experiments. After that, 5×106 conidia from these cultures were inoculated into 125-mL Erlenmeyer flasks containing 25 mL of liquid Czapeck medium [9] with 1.0% citric pectin Sigma (w/v) or other carbon sources as described in Results. The cultures were incubated at 40°C, under agitation (100 rpm) or under static conditions, for different periods, depending on the experiment. Other media were used to standardize the pectinolytic production, such as M-5 [10], Adams [11], Khanna [12], SR-Segato Rizzatti et al. [13] and Czapeck medium [9]. Cultures were filtered through Whatman no. 1 in a Buchner funnel. The filtrate was saved as a source of crude extracellular polygalacturonase. Micelial pads were ground with sea sand, at 4°C with ten vol. of cold 100 mM sodium acetate buffer, pH 6.0. After centrifugation (15,000 xg, 15 min, 4°C), the supernatant fraction was the source of crude intracellular enzyme.


### 2.2. Culture Condition under SSF

The fungus was inoculated (5×106 conidia/mL) on SSF medium, composed by 2 g of different agro-industrial residues plus 4 mL of sterile distilled water. After the incubation period, the cultures were added of 50 mL of distilled water, maintained on ice and agitated for 30 min, after that, the extract fluid was separated from the solid residues as described in Section 2.1, and the filtrate was the source of crude extracellular polygalacturonase.


### 2.3. Enzymatic Assays and Protein Determination

Polygalacturonase activity was assayed according to Miller [14]. The enzymatic assays were carried out with 50 μL of enzyme and 1.0% polygalacturonic acid sodium salt from Sigma-Aldrich in 100 mM acetate buffer pH 4.0, as substrate. The reactions occurred at 60°C, for 5 min. A unit was defined as the amount of enzyme that releases 1 μmol of reducing sugar per min under the assay conditions. Protein was assayed according to Lowry et al. [15], using bovine serum albumin as the standard. Total activity and total protein represent U/mL or mg/mL multiplied by total volume of culture filtrate.


### 2.4. Reproducibility of the Results

All data are the mean of at least three independent experiments showing consistent results.


## 2.1. Organism and Growth Conditions

Aspergillus niveus was isolated from Mangifera indica in our laboratory. The microorganism was identified and deposited in the culture collection of Pernambuco Federal University (PE, Brazil). The organism was maintained on slants of potato dextrose agar (PDA) medium covered with mineral oil, at 4°C. The fungus was incubated on PDA medium, at 30°C for 15 days previous to the cultivation and optimization experiments. After that, 5×106 conidia from these cultures were inoculated into 125-mL Erlenmeyer flasks containing 25 mL of liquid Czapeck medium [9] with 1.0% citric pectin Sigma (w/v) or other carbon sources as described in Results. The cultures were incubated at 40°C, under agitation (100 rpm) or under static conditions, for different periods, depending on the experiment. Other media were used to standardize the pectinolytic production, such as M-5 [10], Adams [11], Khanna [12], SR-Segato Rizzatti et al. [13] and Czapeck medium [9]. Cultures were filtered through Whatman no. 1 in a Buchner funnel. The filtrate was saved as a source of crude extracellular polygalacturonase. Micelial pads were ground with sea sand, at 4°C with ten vol. of cold 100 mM sodium acetate buffer, pH 6.0. After centrifugation (15,000 xg, 15 min, 4°C), the supernatant fraction was the source of crude intracellular enzyme.


## 2.2. Culture Condition under SSF

The fungus was inoculated (5×106 conidia/mL) on SSF medium, composed by 2 g of different agro-industrial residues plus 4 mL of sterile distilled water. After the incubation period, the cultures were added of 50 mL of distilled water, maintained on ice and agitated for 30 min, after that, the extract fluid was separated from the solid residues as described in Section 2.1, and the filtrate was the source of crude extracellular polygalacturonase.


## 2.3. Enzymatic Assays and Protein Determination

Polygalacturonase activity was assayed according to Miller [14]. The enzymatic assays were carried out with 50 μL of enzyme and 1.0% polygalacturonic acid sodium salt from Sigma-Aldrich in 100 mM acetate buffer pH 4.0, as substrate. The reactions occurred at 60°C, for 5 min. A unit was defined as the amount of enzyme that releases 1 μmol of reducing sugar per min under the assay conditions. Protein was assayed according to Lowry et al. [15], using bovine serum albumin as the standard. Total activity and total protein represent U/mL or mg/mL multiplied by total volume of culture filtrate.


## 2.4. Reproducibility of the Results

All data are the mean of at least three independent experiments showing consistent results.


## 3. Results

### 3.1. Time-Course of Polygalacturonase Production

Regarding the nutritional composition of the culture medium, an experiment was carried out according to Cereia et al. [16]. A. niveus was preliminarily grown on a variety of liquid media (Table 1). Among them, Czapeck medium was the best inducer for the PG production.Table 1
Effect of liquid media on growth and PG activity ofA. niveus.
MediaRelative growth (%)Relative activity (%)M-524±0.432±0.8Adams100±0.362±0.4Khanna6±0.118±1.7SR27±0.433±1.3Czapeck34±0.4100±1.7A. niveus was grown in 1% citric pectin media, for 3 days at 40°C.Then, the time-course of PG production was followed only with Czapeck medium added of 1% citrus pectin Sigma-Aldrich (w/v). The incubation occurred without agitation, up to 9 days, at 40°C, or under agitation for up to 5 days, at 40°C. Maximum growth occurred after four days without agitation (Figure1(a)) and the PG production occurred after five days (Figure 1(b)).Time course ofA. niveus cultivation. (a) Growth, (b) PG production, (■) static condition, (●) agitation condition.
(a)(b)


### 3.2. Effect of Carbon Sources on Growth and Enzymatic Production on SbmF and SSF

The effect of the carbon sources on SbmF was studied by supplementing the Czapeck medium with 28 carbohydrates and/or agro-industrial wastes (Table2). The cultures were incubated under agitation for 5 days and the fungal growth was expressed as total protein. The best source for PG production activity was orange peel, which was 21-fold higher than the basal activity in medium supplemented by glucose. Another agro-industrial residue tested was passion fruit peel, which resulted in PG levels 19-fold higher than the one in glucose-medium. Furthermore, lemon peel, apple peel, gum guar, commercial mate herb (Illex paraguariensis), and corncob were also tested and contributed to produce high PG activity from A. niveus. Sigma-Aldrich, CPKelco 8003, and Vetec citrus pectins led to about 19-fold increase in PG activity in relation to that attained on glucose-containing media. Pectins from different origins induced lower PG levels as well as polypectate acid salt sodium and monogalacturonic acid that were poor inducers.Table 2
Effect of different carbon sources on the production of PG fromA. niveusunder SbmF.
SourceGrowth(mg total of protein)Activity(U total)Sigma-Aldrich Citrus Pectin1.9±0.07319±3None0.1±0.0636±3Glucose0.1±0.0416±2Monogalacturonic acid0.1±0.05102±5Polygalacturonic acid0.2±0.08124±47128 Citrus Pectin*2.5±0.06286±2USP-B Citrus Pectin*2.5±0.07274±4JMH6 Citrus Pectin*2.5±0.09236±4GENU 8001 Citrus Pectin*2.2±0.08268±3GEN  8003 Citrus Pectin*2.6±0.04315±3Sucrose3.8±0.05197±4Vetec Citrus Pectin1.7±0.09307±5Galactose3.1±0.1026±2Trehalose2.6±0.0567±3Raffinose2.7±0.0460±2Arabinose3.8±0.04172±4Penetrose5.2±0.07170±3Gum guar3.7±0.11244±2Apple peel (Malus domestica)4.5±0.09248±7Orange peel (Citrus sinensis)2.4±0.10335±9Lemon peel (Citrus latifolia)3.1±0.12300±8Passion fruit peel (Passiflora edulis)3.6±0.11313±6commercial mate herb (Illex paraguariensis)4.9±0.09136±7Rice straw (Oryza sativa)1.0±0.0435±4Sugar cane bagasse (Saccharum officinarum)1.6±0.0795±6Corn cob (Zea mays)1.2±0.03105±8Wheat bran (Triticum aestivum)3.1±0.1392±5Soya bran (Glycine max)3.3±0.1073±4* FROM CPKelco Brasil S/A.Polygalacturonase production on SSF was studied by incubating the fungus with 8 different agro-industrial residues (Table3) for 7 days. Under this condition, lemon peel was the best inducer of PG activity.  Besides, passion fruit peel was an interesting inducer showing the second best level. The other residues tested showed about half of the activity of the carbon sources previously mentioned.Table 3
Effect of different carbon sources on the production of PG fromA. niveusunder SSF.
SourceGrowth(mg total of protein)Activity(U total)Orange peel (Citrus sinensis)46±2.9495±33Lemon peel (Citrus latifolia)39±1.51324±57Passion fruit peel (Passiflora edulis)42±7.3960±15Apple peel (Malus domestica)42±2.4311±36Rice straw (Oryza sativa)5±0.1318±27Wheat bran (Triticum aestivum)26±1.4407±25Corn cob (Zea mays)14±0.5416±1Sugar cane bagasse (Saccharum officinarum)5±0.1272±10


### 3.3. Biochemical Characterization of Polygalacturonase Activity

The enzyme used to biochemical characterization was the extracellular PG produced in medium supplemented with Sigma-Aldrich citrus pectin, because it showed elevated activity and few contaminants. The optima of temperature and pH were 55°C and the pH range of 3.0–4.5 (Figures2(a) and 2(b)). The enzyme retained 91% of the activity after 90 min at 60°C; higher temperatures severely inactivated the enzyme (Figure 2(c)). PG activity remained stable after 24 h at 4–6°C at the pH range of 3.0–5.0, with a decrease of 15% at pH 5.5 and 91% at pH 8.0 (Figure 2(d)).Biochemical characterization of the PGs produced byA. niveus. (a) Effect of the temperature; (b) pH influence, (c) thermal stability, (d) pH stability,  (■) 60°C; (●) 65°C; (▲) 70°C.
(a)(b)(c)(d)The effect of different salts and EDTA (1 and 10 mM, final concentration) on PG activity is shown in Table4. EDTA and Mn2+ generated a slight increase in the enzymatic activity. However, 10 mM Hg2+, Ba2+, and Cu2+ inhibited 96, 61, and 52% of the PG activity, respectively.Table 4
Effect of metal ions and EDTA on the activity of the PG produced byA. niveus.
CompoundRelative activity (%)1 mM10 mMNone100±2.1100±1.3Hg2+59±2.94±1.0NH4 +87±3.087±2.1Ca2+97±2.878±2.5Zn2+87±2.675±2.2Ba2+101±1.339±2.8Cu2+60±1.948±2.4Na+95±2.893±2.0Mn2+117±1.4113±3.2Mg2+72±2.992±2.9Al2+98±2.393±2.1EDTA110±3.294±3.3


## 3.1. Time-Course of Polygalacturonase Production

Regarding the nutritional composition of the culture medium, an experiment was carried out according to Cereia et al. [16]. A. niveus was preliminarily grown on a variety of liquid media (Table 1). Among them, Czapeck medium was the best inducer for the PG production.Table 1
Effect of liquid media on growth and PG activity ofA. niveus.
MediaRelative growth (%)Relative activity (%)M-524±0.432±0.8Adams100±0.362±0.4Khanna6±0.118±1.7SR27±0.433±1.3Czapeck34±0.4100±1.7A. niveus was grown in 1% citric pectin media, for 3 days at 40°C.Then, the time-course of PG production was followed only with Czapeck medium added of 1% citrus pectin Sigma-Aldrich (w/v). The incubation occurred without agitation, up to 9 days, at 40°C, or under agitation for up to 5 days, at 40°C. Maximum growth occurred after four days without agitation (Figure1(a)) and the PG production occurred after five days (Figure 1(b)).Time course ofA. niveus cultivation. (a) Growth, (b) PG production, (■) static condition, (●) agitation condition.
(a)(b)


## 3.2. Effect of Carbon Sources on Growth and Enzymatic Production on SbmF and SSF

The effect of the carbon sources on SbmF was studied by supplementing the Czapeck medium with 28 carbohydrates and/or agro-industrial wastes (Table2). The cultures were incubated under agitation for 5 days and the fungal growth was expressed as total protein. The best source for PG production activity was orange peel, which was 21-fold higher than the basal activity in medium supplemented by glucose. Another agro-industrial residue tested was passion fruit peel, which resulted in PG levels 19-fold higher than the one in glucose-medium. Furthermore, lemon peel, apple peel, gum guar, commercial mate herb (Illex paraguariensis), and corncob were also tested and contributed to produce high PG activity from A. niveus. Sigma-Aldrich, CPKelco 8003, and Vetec citrus pectins led to about 19-fold increase in PG activity in relation to that attained on glucose-containing media. Pectins from different origins induced lower PG levels as well as polypectate acid salt sodium and monogalacturonic acid that were poor inducers.Table 2
Effect of different carbon sources on the production of PG fromA. niveusunder SbmF.
SourceGrowth(mg total of protein)Activity(U total)Sigma-Aldrich Citrus Pectin1.9±0.07319±3None0.1±0.0636±3Glucose0.1±0.0416±2Monogalacturonic acid0.1±0.05102±5Polygalacturonic acid0.2±0.08124±47128 Citrus Pectin*2.5±0.06286±2USP-B Citrus Pectin*2.5±0.07274±4JMH6 Citrus Pectin*2.5±0.09236±4GENU 8001 Citrus Pectin*2.2±0.08268±3GEN  8003 Citrus Pectin*2.6±0.04315±3Sucrose3.8±0.05197±4Vetec Citrus Pectin1.7±0.09307±5Galactose3.1±0.1026±2Trehalose2.6±0.0567±3Raffinose2.7±0.0460±2Arabinose3.8±0.04172±4Penetrose5.2±0.07170±3Gum guar3.7±0.11244±2Apple peel (Malus domestica)4.5±0.09248±7Orange peel (Citrus sinensis)2.4±0.10335±9Lemon peel (Citrus latifolia)3.1±0.12300±8Passion fruit peel (Passiflora edulis)3.6±0.11313±6commercial mate herb (Illex paraguariensis)4.9±0.09136±7Rice straw (Oryza sativa)1.0±0.0435±4Sugar cane bagasse (Saccharum officinarum)1.6±0.0795±6Corn cob (Zea mays)1.2±0.03105±8Wheat bran (Triticum aestivum)3.1±0.1392±5Soya bran (Glycine max)3.3±0.1073±4* FROM CPKelco Brasil S/A.Polygalacturonase production on SSF was studied by incubating the fungus with 8 different agro-industrial residues (Table3) for 7 days. Under this condition, lemon peel was the best inducer of PG activity.  Besides, passion fruit peel was an interesting inducer showing the second best level. The other residues tested showed about half of the activity of the carbon sources previously mentioned.Table 3
Effect of different carbon sources on the production of PG fromA. niveusunder SSF.
SourceGrowth(mg total of protein)Activity(U total)Orange peel (Citrus sinensis)46±2.9495±33Lemon peel (Citrus latifolia)39±1.51324±57Passion fruit peel (Passiflora edulis)42±7.3960±15Apple peel (Malus domestica)42±2.4311±36Rice straw (Oryza sativa)5±0.1318±27Wheat bran (Triticum aestivum)26±1.4407±25Corn cob (Zea mays)14±0.5416±1Sugar cane bagasse (Saccharum officinarum)5±0.1272±10


## 3.3. Biochemical Characterization of Polygalacturonase Activity

The enzyme used to biochemical characterization was the extracellular PG produced in medium supplemented with Sigma-Aldrich citrus pectin, because it showed elevated activity and few contaminants. The optima of temperature and pH were 55°C and the pH range of 3.0–4.5 (Figures2(a) and 2(b)). The enzyme retained 91% of the activity after 90 min at 60°C; higher temperatures severely inactivated the enzyme (Figure 2(c)). PG activity remained stable after 24 h at 4–6°C at the pH range of 3.0–5.0, with a decrease of 15% at pH 5.5 and 91% at pH 8.0 (Figure 2(d)).Biochemical characterization of the PGs produced byA. niveus. (a) Effect of the temperature; (b) pH influence, (c) thermal stability, (d) pH stability,  (■) 60°C; (●) 65°C; (▲) 70°C.
(a)(b)(c)(d)The effect of different salts and EDTA (1 and 10 mM, final concentration) on PG activity is shown in Table4. EDTA and Mn2+ generated a slight increase in the enzymatic activity. However, 10 mM Hg2+, Ba2+, and Cu2+ inhibited 96, 61, and 52% of the PG activity, respectively.Table 4
Effect of metal ions and EDTA on the activity of the PG produced byA. niveus.
CompoundRelative activity (%)1 mM10 mMNone100±2.1100±1.3Hg2+59±2.94±1.0NH4 +87±3.087±2.1Ca2+97±2.878±2.5Zn2+87±2.675±2.2Ba2+101±1.339±2.8Cu2+60±1.948±2.4Na+95±2.893±2.0Mn2+117±1.4113±3.2Mg2+72±2.992±2.9Al2+98±2.393±2.1EDTA110±3.294±3.3


## 4. Discussion

Aspergillus niveus turned out to be a good pectinase producer in cultures grown with agro-industrial residues. It is very convenient to use such residues to produce the enzyme in industrial scale, once their use reduces costs and aggregates value to the organic material, bringing benefits to the environment as well as the industry. When grown in Czapeck medium, the fungus had maximum PG production after 5 days.  Patil and Dayanand [17] and Friedrich et al. [18] described similar period of time for pectinase production by different strains of Aspergillus niger in submerged cultures. The present study demonstrated that fruit peels, especially orange peels, considerably stimulated the production of PG under SbmF. However, Niture and Pant [19] described that orange peel induced elevated levels of PG II in Fusarium moniliforme grown in semisolid medium. Besides, our results showed that PG production by A. niveus was better under SSF than under SbmF, considering the same carbon source.  The PG production with rice straw was 9.1-fold higher under SSF than in SbmF, followed by lemon peel and wheat bran (4.4-fold), corn cob (3.9-fold), passion fruit (3.1-fold), sugar cane bagasse (2.9-fold), orange peel (1.5-fold), and apple peel (1.3-fold). These results suggested that the high enzymatic production is due to a close contact of the microorganism with the carbon source, observed in SSF.A. niveus is a thermotolerant fungus [20]; so, the PG secreted was quite active and stable at 60°C. Mohamed et al. [21] described a PG with optimum activity at 40°C. Kashyap et al. [22] and Moyo et al. [23] describe the maximum temperature of 50°C for the PG ofA. nigerand Kluyveromyces wickerhamii,respectively. The optimum temperature for PG from Bacillus sp. [4], Trichoderma harzianum [24], T. reesei[21], and A. niger [25] was about 40 and 50°C.TheA. niveus PG was stable for 90 min at 60°C. Mohamed et al. [24] describe that the PG of Trichoderma harzianum was stable for 30 min at 60°C. Thus, the PG from A. niveusmay be advantageous for the industrial processes of candy, syrups, juice, and drink production.PG activity was predominantly acidic, presenting two plateaus (pH range of 3–4.5 and 5–6.5, suggesting more than one enzymatic form). ForA. niger [24] and Fusarium moniliforme[18], the maximum PG activity occurred at pH 5.0, and for T. reeseiPGs at pH 4.5 and 4.2 [24]. The enzyme from A. niveuswas stable in a pH range of 3.0–5.0, for 24 h at 4–6°C. The PG from T.harzianum [24] was stable at pH 5.0, and PG from A. fumigatus [3] was stable in a pH range of 3.0–9.0. Kobayashi et al. [26] demonstrated that PG from Bacillus sp. was stable at pH 6.0 and 12.0 at 30°C for 1 h.EDTA and Mn2+ increased the enzymatic activity of the PG fromA. niveus, and Hg2+, Ba2+, and Cu2+ inhibited it. The effect of Hg2+ suggested the presence of SH groups on the protein, which was a covalent bound with this metal turning the three-dimensional structure unstable and decreasing its enzymatic activity. PGII from T. harzianum [24] was totality inhibited by 1mM Mn2+ and Co2+. The activity of Sporotrichum thermophile Apinis PG was stimulated by Fe2+ and Mn2+ both at 1 and 5 mM, while Ca2+ and Cu2+ stimulated only at 1 mM and 5 mM. Mg2+ strongly inhibited enzyme activity [27]. The PG of Bacillus MG-cp-2 was stimulated by Ca2+ [4].In conclusion, agro-industrial residues, such as orange and lemon peel, induce high levels of a thermostable acid PG byA. niveus. Finally, the use of these residues on industrial enzymatic production would aggregate value to waste and would reduce the environmental pollution.



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*Source: 289206-2011-06-20.xml*