Partial Characterization of α-amylase Produced from Aspergillus niger using Potato Peel as Substrate

Alpha amylase (endo-1,4-α-D-glucan glucanohydrolyase E.C 3.2.1.1) is an extra cellular enzyme. It produces ultimately a large number of products i.e. maltose and glucose sub units by splitting up 1,4-α-D-glucosidic bonds randomly which are present among consecutive molecules of glucose in straight amylose chain (Saleem and Ebrahim, 2014; Mathew et al., 2016; Kanti, 2016). Alpha amylases are widely used in various industries such as baking, brewing, confectionary, sugar (i.e. fructose and glucose), paper coating, alcohol, pharmaceutical, textile, syrup industries, starch, detergents, digestive aids, and for treatment of sewage (Sundarram and Murthy, 2014; Elaiyaraja et al., 2016; Veerapagu et al., 2016). Alpha amylases are wide spread in occurrence which can be obtained by different resources e.g. microorganisms, animals and plants etc. However, fungi and bacteria are used for commercial production of amylases (Mathew et al., 2016; Singh et al., 2016), because of a few advantages i.e. reliability, less time and space, low cost required for enzyme production, ease of manipulation and economical bulk production capacity (Khan and Yadav, 2011; Mahmood et al., 2016).

Aspergillus niger is a fungus which is prevalent in nature and belongs to genus Aspergillus. It is usually found as saprophytes growing on stored grain, dead leaves, and other decaying vegetation. It looks as very dark brown patches or carbon black. Aspergillus niger, dominates in solid state fermentation, is a very vital group of microorganisms. It posses some very special properties such as good tolerance to less availability of water and capacity to spread over and to enter inside the solid substrate, therefore, it is extensively grown and used in food industry for making many enzymes such as α-amylases, amyloglucosidases, cellulases, lactase and acid proteases (Singh et al., 2016; Manpreet et al., 2005). In recent years solid state fermentation (SSF), where the fungus is grown on moist solid substrate, has been utilized increasingly for the production of α-amylase (Xu et al., 2008) because of numerous benefits e.g. less capital investment, simple technique, marginal end product inhibition, superior and high volumetric productivity, less catabolite repression, low energy requirement, requirement of simple equipment for fermentation, better product recovery and less water output (Singh et al., 2016; Gangadharan et al., 2006).

The cost of α-amylase production is dependent on the cost of the substrate used during SSF. For the cost effective production of alpha amylases, several researchers considered the use of easily available and inexpensive food and agro wastes such as, wheat bran, potato peel, wheat straw, rice straw, rice husk, and sugarcane bagasse, banana waste and waste of coffee as substrate for α-amylase production (Murthy et al., 2009; Simair et al., 2017). Potatoes are peeled in various processed food industries and are used for the production of different foodstuffs such as chips, fries and mashed potatoes etc. (Shukla and Kar, 2006; Schieber et al., 2009). The potato peel is considered as waste, discarded and allowed to rot, so it creates many pollution and disposal problems. Therefore, for cost effective production of enzyme, potato peel should be used as a cheap source of substrate because it also include adequate amount of nutrients like protein and carbohydrates, which are essential for the growth of microorganisms (Ajao et al., 2009). The objective of present work is to characterize crude α-amylase from Aspergillus niger using potato peel as substrate.

In the present study, potato peel was selected as substrate for α-amylase production. It was collected from Lays, Pepsi-cola International (Pvt) Ltd, Lahore, Pakistan.

Aspergillus niger was obtained from the Microbiology Laboratory, Food & Biotechnology Research Center, Pakistan Council of Scientific & Industrial Research Laboratories Complex, Lahore. The fungus was grown on slants of potato dextrose agar (PDA) for five days before storage, and maintained at 4°C on PDA.

Five days old PDA slant culture full of fungal spores were taken and ten ml of sterilized distilled water was added. Under sterilized conditions, spore clusters were broken with the help of an inoculum needle and homogenized suspension of spores were prepared and used as an inoculum source.

Twenty gram of raw potato peel was weighed in 250 ml Erlenmeyer flask and hydrated with 2 ml of salt solution comprising (g/l) MgSO4 2, KH2PO4 10, MnSO4 0.5 and NaCl 2. The material was mixed thoroughly, cotton plugged and sterilized at 121°C, 15lb psi for 15 min. After sterilization, the cooled media was inoculated with one milliliter spore suspension of Aspergillus niger and incubated at 30°C for 5 days.

In each of above flasks, 50 ml of citrate buffer (pH 5) was poured and shaken vigorously in a rotary shaker for 1 hour at 200 rpm. Then the fermentation mixture was filtered and centrifugation was taken place for 15 min at 4°C at 8,000 rpm. The supernatant (crude enzyme) was filtered and used to measure activity of crude enzyme.

Amylase activity was measured by method as described by Okolo et al. (1995). Reaction mixture containing 1 ml of enzyme extract and 1 ml of substrate (i.e. 1% soluble starch solution) was taken in test tube and incubated for 30 min at 50°C. After that reaction was stopped by adding 3ml of DNS reagent and boiled for 10 min. The reaction mixture was allowed to cool at room temperature and absorbance was measured by spectrophotometer at 540nm (Miller, 1959). One unit (IU) α-amylase activity was defined as the amount of enzyme that releases 1 µg of maltose per minute under the standard reaction conditions.

Characterization of the crude enzyme was conducted by studying the effect of different parameters such as incubation time (10-100 min), incubation temperature (20-100°C), substrate concentration (0.25-2%), pH (4-9) of reaction mixture and metal ions (Na+1, Ca2+, Mg2+, Mn2+, Zn2+, Fe2+, Cu2+, Ag1+, Cs1+ & Co2+) on α-amylase activity.

All the data obtained from different experiments were analyzed statistically by SPSS software. ANOVA test was used at p < 0.05 significance level.

Alpha amylase activity was performed at different incubation periods (10, 20, 30, 40, 50, 60, 70, 80, 90 & 100 min) and it was observed that enzyme was optimally active (2779.49 U/g) for 10 minute of incubation but subsequently, there was gradual decline in enzyme activity (Figure 1). Similar results were reported by Kanwal et al. (2004). Maximum activity for α-amylase at five minutes incubation of reaction mixture has been reported earlier (Ramachandran et al., 2004; Alva et al., 2007).

Effect of different temperatures i.e. 20, 30, 40, 50, 60, 70, 80, 90 & 100°C of reaction mixture on activity of α-amylase was evaluated. Maximum enzyme activity (2947.08 U/g) was gained at 40°C, whereas, the activity of enzyme showed a declining trend with the increase or decrease in temperature (Figure 2). Thermostability of the enzyme was checked by incubating crude enzyme solution at various temperature for different time period. Results showed that enzyme was stable at temperature 40oC for 70min attaining 96 % enzyme activity. As the temperature gradually increased, the enzyme activity become decreased with the passage of time. Ayansina and Owoseni (2010) showed same results for Aspergillus flavus. Maximum α-amylase activity was reported for Aspergillus spp. and Aspergillus niger at 45°C (Avwioroko et al., 2015; Wang et al., 2016). Greatest activity for amylase was obtained at incubation temperature of 30°C (Obafemi et al., 2018; Varalakshmi et al., 2009; Nouadri et al., 2010), 50°C (Patel et al., 2005; Mahmood and Rahman, 2008) and even 60°C (Yahya et al., 2016).

The incubation of enzyme-substrate reaction mixture was carried out at various pH values such as 4, 5, 6, 7, 8 & 9. At pH 6, maximum enzyme activity (2895.02 U/g) was noted and the activity of enzyme was decreased, due to increase or decrease of pH from the optimum value (Figure 3). Enzyme stability was also assessed by pre-incubating enzyme at various pH buffers and enzyme was found stable in pH range of 4-6. Avwioroko et al. (2015) reported that Aspergillus spp. related with cassava spoilage produced α-amylase which showed optimum activity within pH range of 4-5. Alpha amylase indicated excellent activity at about pH 6 (Obafemi et al., 2018; Nouadri et al., 2010; El-Safey and Ammar, 2004). It was studied in previous literature that α-amylase was best active at pH 6.8 (Kanwal et al., 2004), pH 5 (Patel et al., 2005), pH 7 (Tiwari et al., 2007), pH 7 (Wang et al., 2016) and pH 5.6 (Yahya et al., 2016), respectively.

Enzyme activity was investigated with different concentrations of substrate (soluble starch) i.e. 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 & 20.0 mg/ml. It was noted that enzyme was optimally active (3014.30 U/g) at 10.0mg/ml soluble starch, subsequently it remained nearly constant (fig 4). Kinetic constants like Km and Vmax was calculated through Line-Weaver Burk plot. The crude enzyme had Km value of 3.00 mg/ml and Vmax of 1000.0 µM/g using soluble starch as substrate. Banerjee and Gosh (2017) reported Km and Vmax of 0.387 mg.ml-1 and 35.03 U µl-1 min-1, for glucoamylase using soluble starch respectively. Crude α-amylase produced from Aspergillus sp. exhibit maximum Vmax of 10 U/mg protein and Km in range of 0.37 -1.25%w/v (Avwioroko et al., 2015). α-amylase produced from Aspergillus oryzae had Km and Vmax of 1.4 mg ml-1 and 37.037 U ml-1 respectively (Shah et al., 2014).

The study of different metal ions exhibited that enzyme activity was enhanced by Ca2+, Zn2+, Cs1+, Co2+, Mn2+and Fe2+, whereas, inhibited by Na+1, Mg2+, Ag1+, Cu2+ (Figure 5). α-amylase activity was improved due to Mn2+, Ca2+, and Fe2+ ions and inhibited due to Mg2+, Na+1, Cu2+, Hg2+, Ag1+ ions has been observed by many workers (Patel et al., 2005; Tiwari et al., 2007; Varalakshmi et al., 2009; Nouadri et al., 2010). Wang et al. (2018) reported that most of the cloned α-amylase were Ca2+ independent. α-amylases produced from Aspergillus spp. grown on degrading cassava were slightly stimulated by Mg2+ and Na+, moderately activated by Fe3+ and Ca2+, whereas strongly activated by potassium ion K+ (Avwioroko et al., 2015).

In the current research work, the results presented that potato peel could be used as a good substrate in fermentation media for microbial growth as it contains all the essential nutrients. A significant enzyme activity can be obtained by utilizing potato peel, an agro-residue, as a substrate for SSF. Maximum α-amylase activity was obtained at pH 6, 40°C substrate concentration 1% incubation period 10 minutes, the enzyme activity was enhanced by Ca2+, Cs1+, Mn2+ and Co2+, whereas inhibited by Na1+, Mg2+, Ag1+ and Cu2+.