Extraction , partial purification and characterization of pectinases isolated from Aspergillus species cultured on mango ( Mangifera indica ) peels

Pectinase was produced from a culture of Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus. Pectinase synthesis was achieved using mango (Mangifera indica) pectin extract as an inducer during pectinolytic fungi isolation while submerged fermentation process was carried out using ground mango peels as the sole carbon source. Substrate fermentation was evaluated within seven days by monitoring the pectinase activity every 24 h. The highest pectinase secretion was obtained from A. niger and A. fumigatus after 92 h (day 4) of incubation, while in A. flavus, it was after 120 h (day 5). Crude enzyme extracts from the three organisms were partially purified by a combination of ammonium sulphate precipitation and dialysis with an approximately two-fold purification of the pectinase and a yield of 5.4, 7.66 and 5.99% for A. niger, A. fumigatus and A. flavus, respectively after dialysis. The specific activities of 1.62, 1.79 and 1.86 U/mg for A. niger, A. fumigatus and A. flavus enzymes were calculated, respectively. Pectinase from A. niger and A. fumigatus had pH and temperature optima of 5.0 and 40°C, respectively, while that from A. flavus had pH and temperature optima of 5.0 and 45°C. The Michealis constant, Km and the maximum velocity, Vmax determined from Lineweaver-Burk plots of initial velocity data at different concentrations of the mango pectin extract were 0.357 mg/ml and 35.34 U; 0.156 mg/ml and 68.0 U; and 0.261 mg/ml and 60.61 U; for the enzymes from A. niger, A. fumigatus and A. flavus, respectively. The results suggest that mango peels can be used for value added synthesis of pectinase, an important enzyme with numerous biotechnological applications.


INTRODUCTION
In the processing and utilization of mango fruit in products such as juices, nectars concentrates, jams, jelly powders and flakes, wastes are generated in the form of peels and kernels.According to Larrauri et al. (1996), byproducts of industrial mango processing may amount to 35 to 60% of the total fruit weight.These by-products represent a serious disposal problem and ways for a sustainable agricultural production have to be discovered.Mango kernels have been utilized as source of oil (Moharram and Moustafa, 1982), natural antioxidants (Puravankara et al., 2000), starch (Kaur et al., 2004), flour (Arogba, 2002) and feed (Ravindran and Sivakanesan, 1996).Mango peels can be utilized in the production of value added products such as biogas, pectin peel oil, dietary fiber and predominantly pectinases that can be easily harnessed.Among these products, pectin and pectinases have a wide global market (Bali, 2003).Pectins are complex and structural polysaccharides found in the primary cell wall and middle lamella of fruits and vegetables where they function as hydrating agent and cementing material of the cellulosic network (Jarvis et al., 2003;Favela-Torres et al., 2006).Pectic polysaccharides such as homogalacturonan (HGA), xylogalacturonan (XGA), apiogalacturonan, rhamnogalacturonan I (RGI) and rhamnogalacturonan II (RGII) have been isolated from plant primary cell wall (Harholt et al., 2010).Pectinases are a group of enzymes that catalyze the breakdown of pectins.They are classified into protopectinases, esterases and depolymerases based on their preferred substrate, the degradation mechanism and the type of cleavage (Jayani et al., 2005).Submerged (SMF) and solid state fermentation (SSF) processes have been widely used for pectinase production by different organisms.However, microbial pectinases are produced mostly by SMF in a process that is influenced by the type and concentration of the carbon source, the culture pH, the incubation temperature and the oxygen concentration (Rashmi et al., 2008).

MATERIALS AND METHODS
3,5-Dinitrosalicylic acid (DNS) was a product of Sigma chemical company (USA).All other chemicals used in this work were of analytical grade and were products of Merck (Germany), BDH chemical limited (England), May and Baker limited (England).

Collection of mango fruits
Mature mango fruits (Mangifera indica) were collected from the major markets in Amechi Awkunanaw in Enugu State, Nigeria.

Ground mango peels
The fruit peels were peeled, cut into pieces and washed with 96% ethanol to disinfect the peels.The washed peels were then sun dried for seven days and ground into powder.

Extraction of pectin
Pectin was extracted using the method described by McCready (1970) and the yield determined by the equation:

Isolation of microorganism
Three Aspergillus strains were isolated from soil of decaying mango fruits and vegetables located in the University of Nigeria Nsukka Campus Enugu State, Nigeria using the method described by Martin et al. (2004).The soil samples were collected in clean dry plastic containers and transported to the laboratory.

Fungal identification
Three days old pure culture of Aspergillus niger, Aspergillus fumigatus and Aspergillus flavus, were used in preparing microscopic slides.A clump of the mycelia was dropped on the slide and a drop of lactophenol blue was added to it.Identification was carried out using 400 magnifications according to the method of Barnett and Hunter (1972).Species identification was performed by examining both macroscopic and microscopic features of a three day old pure culture.Colour, texture, nature of mycelia and/or spores produced, growth pattern in addition to microscopic features such as separation, spore shapes and so on were examined.The spores of the three Aspergillus strains were grown and maintained on potato dextrose agar.

Pectinase production
Pectinase was produced by submerged fermentation according to the method of Martin et al. (2004).Submerged fermentation (SmF) technique was employed using 21 250 ml Erlenmeyer flask containing 100 ml of sterile cultivation medium optimized for pectinase with 0.1% NH 4 NO 3, 0.1% NH 4 H 2 PO 4, 0.1% MgS0 4 × 7H 2 O and 1% of ground mango peel.The flask was stoppered with aluminium foil and autoclaved at 121°C for 15 min to sterilize the culture medium.Three days old cultures were used to inoculate the flasks.In every sterile flask, two discs of the respective fungal isolates were added using a cork borer of diameter 10 mm.The culture was incubated for seven days at room temperature (30°C).The fermentation media was agitated at 250 rpm on rotary shaker.At each day of harvest, flasks were selected from the respective groups and mycelia biomass separated by filtration through Whatman filter paper No. 1.The filtrate for each day was assayed for pectinase activity till the 7th day of fermentation.After the seven days pilot SmF studies, the day of peak pectinase activity was chosen for mass production of enzyme from the respective fungal isolates.Several (21) 250 ml Erlenmeyer flasks were used to produce 3.0 L of the enzyme.Harvesting was carried out on the respective peak days of enzyme activity.The harvested 3.0 L of the enzyme was used as crude enzyme.

Pectinase assay
Pectinase activity was evaluated by assaying for polygalacturonase *Corresponding author.E-mail: daniel.udenwobele@unn.edu.ng.Tel: +2347031524415.(Pg) activity of the enzyme.This was achieved by measuring the release of reducing groups from mango pectin using a modification of the 3,5-dinitrosalicylic acid (DNS) reagent assay method described by Miller (1959) as described in Wang et al. (1997) with little modifications.The reaction mixture containing 0.5 ml of 0.5% mango pectin in 0.05 M sodium acetate buffer of pH 5.0 and 0.5 ml of enzyme solution were incubated for 1 h. 1 ml of DNS reagent was added and the reaction was stopped by boiling the mixture in a boiling water bath for 10 min.The mixture volume was made up to 4 ml with 1 ml of Rochelle salt solution and 1 ml of distilled water.The reaction mixture was allowed to cool and then absorbance was read at 575 nm.The standard curve was prepared for reducing groups with galacturonic acid.One enzyme unit of pectinase is the number of reducing groups measured in terms of galacturonic acid, produced as a result of the action of 0.5 ml of enzyme extract in 1 min at 30°C.Protein was determined by the method of Lowry et al. (1951).Specific activity is the ratio of the total activity to total protein.

Enzyme purification
The crude enzyme was brought to 80% ammonium sulphate saturation and then dialysed.Dialysis tubes preserved in 90% ethanol were rinsed several times with distilled water and then buffered till they were clean of the ethanol.0.05 M sodium acetate buffer pH 5.0 was used for enzyme dialysis.Dialysis was carried out for 14 h with continuous stirring and buffer changed every 6 h aiming to remove low molecular weight substances and other ions that may interfere with enzyme activity.After dialysis was complete, the partially purified enzyme was stored frozen at -24°C.

Enzyme characterization
The effects of pH and temperature were determined according the method described by Miller (1959) as described in Wang et al. (1997) with little modifications as described previously under pectinase assay.The kinetic parameters were calculated from Lineweaver-Burk plots of initial velocity data at different concentrations of mango pectin extract (0.1, 0.5, 1.0, 5.0, 10.0 and 20.0 mg/ml).

RESULTS
All the experiments were conducted in triplicate and the result is the mean of the data derived.Three strains of pectinolytic fungi (A.niger, A. fumigatus and A. flavus) were isolated from soil of decaying fruits and vegetables.Pectin was extracted from mango peel with a yield of 15.2% at pH of 2.2, temperature of 70°C and extraction time of 60 min.The three Aspergillus species: A. niger, A. fumigatus and A. flavus, grown in media containing mango peel, produced significant quantities of pectinases.Crude enzymes from A. niger and A. fumigatus had their highest activities on day 4, while A. flavus had its highest activity on day 5 (Figure 1). Figure 2 shows the pectinase activities of mass-produced crude extract.The polygalacturonase activities of A. niger, A. fumigatus and A. flavus obtained from ammonium sulphate precipitation of pectinases were 45.92, 41.69 and 51.19 µmol/min, respectively, at 80% ammonium sulphate saturation (Figure 10).Tables 1, 2 and 3 show the percentage yield and purification fold of pectinases from the three pectinolytic microorganisms after dialysis, while the specific activity of the partially purified enzyme is presented in Figure 3.The effect of pH on the activity of pectinases from the three microorganisms is shown in Figure 4.     achieved, indicating that pectinases from A. flavus that have the greatest enzyme specific activity, were the most purified.They were followed by pectinases produced by A. niger and then A. fumigatus, after partial purification (dialysis).

Pectinase characterization
The optimal pH of 5.0 was obtained for the pectinases from the three Aspergillus species (Figure 4) while an optimal temperature of 40°C was obtained for A. niger and A. fumigatus and 45°C for A. flavus, respectively (Figure 5).These results are in consonance with the reports of Favela-Torres et al. ( 2006), Niture and Pant, (2001) and Manachini et al., (1987).Jayani et al., 2005).

Kinetic studies
Kinetic parameters, V max and K m, of the enzymes were calculated from Lineweaver-Burk plot (Figures 7,8 and 9).Pectinases from A. niger, A. fumigatus and A. flavus Ammonium sulphate saturation (%) had V max and K m values of 35.34 U and 0.367 mg/ml; 68.03 U and 0.156 mg/ml and 60.61 U and 0.261 mg/ml, respectively (Table 4).Thus, the increasing order of V max for the three organisms is A. niger>A.flavus>A.fumigatus.K m values less than 0.15 and up to 5.0 mg/ml (<0.15 to 5.0 mg/ml) and specific activities 8.8 to 7000 U/mg were reported for some fungal pectinases by Lucie (2000).The K m value obtained in this study is not only comparable but also in agreement with the report by Rombouts and Pilnik (1980) that the K m for most fungal pectinases is less than 1 mg/ml.

Conclusion
Most work on pectinase microbial production has been oriented in the direction of using pure pectin extract, especially apple pectin extract, as the sole carbon source, while in this work we used mango peels for pectinase production.Our findings provide alternative and cheaper source of substrates for microbial pectinase production.Thus, mango peel could be an attractive and promising substrate especially in submerged fermentation for the production of pectinases by Aspergillus species.Pectinase from A. fumigatus with the highest yield during the purification process, indicated highest V max and the lowest K m (highest catalytic efficiency) value at acidic pH of 5.0 and optimum temperature of 40°C.Therefore, it can be employed in industries for hydrolysis of pectic biomass to utilizable bio-products.

Figure 1 .
Figure 1.Determination of duration for peak pectinase production from Aspergillus species.
Figure 4 also shows minor pH peaks for pectinases from A. fumigatus, A. flavus and A. niger.The effect of temperature on pectinase activities of the three isolates is presented in Figure 5.The effect of temperature Figu spec

Figure 10 .
Figure 10.Ammonium sulphate precipitation profile of pectinases from the three Aspergillus strains.
Figure 4 also shows minor peak activities for A. fumigatus and A. flavus at pH 8.0 and A. niger at pH 8.5.At pH of 8.0, pectinases from A. fumigatus and A. flavus indicated an optimal temperature of 40 and 45°C respectively, while that from A. niger indicated a temperature optimum of 40°C at pH of 8.5 as shown in Figure 6.The minor peaks at pH 8.0 for A. fumigatus and A. flavus and 8.5 for A. niger in Figure 4 may represent optimal pH for an isoform of pectinase, possibly pectin lyase.Pectin lyases have pH optima in the alkaline range 7.5-10.0and temperature optima of 40-50°C (