Partial Purification and Characterisation of â-Amylase Isolated From Malted Pearl Millet (Pennisetum glaucum)

The industrial use of enzymes in the degradation of starch has become predominant over conventional methods of acid starch hydrolysis. As such, cheap sources of starch hydrolysing enzymes are being sought for. â-amylase, an exo-hydrolase which releases maltose by hydrolysing starch from the non-reducing end, is one of such important enzymes found to be common to the seeds of higher plants such as cereal grains. In this research, â-amylase was extracted from malted Pearl Millet grains and partially purified by ammonium sulphate precipitation (0-60%). The activity of â-amylase peaked at 72h of malting (7.40 ìmol/min/mL) with specific activity of 1.84 ìmol/min/mg. The enzyme extract at 72h was purified for characterization assays, giving a 1.33 purification fold and a yield of 9.28% after extensive dialysis against extraction buffer. Enzyme characterisation assays showed pearl millet â-amylase has optimal activity at pH 5.0 and 50 °C while a K value of 10 mg/mL and V value of 11.11 ìmol/min/mL were obtained m max when corn starch was used as substrate. The enzyme also showed significant activity over a wide temperature and pH range, indicating its potential as an industrial enzyme. The results from this study show that malted Pearl Millet is a cheap source of â-amylase with favourable kinetics and the desired characteristics of activity at low pH and elevated temperatures.


Introduction
However, amylases as biocatalysts are now being Starch is a carbohydrate food reserve in employed for efficient starch liquefaction and plants and known to be one of the most saccharification against the crude acid hydrolysis abundant biopolymers on earth (Anto et al., method (Pervez et al., 2014;Ayodeji et al., 2006). This polysaccharide, is abundant in the 2017). endosperm of several small seeded cereals Pearl Millet (Pennisetum glaucum) is an (millets) and are converted to fermentable annual, warm-season crop widely grown usually sugars during biotechnological and industrial for grazing, hay, cover crop and wildlife. It yields processes of malting (Kolawole and Kolawole, higher than foxtail millet and regrows after 2015). Several indigenous foods and drinks are harvest if sufficient stubble is left (Chad and made from the flour and malt of these millets Jimmy, 2014). The grass grows 4-8 ft tall, on across South Asia and Africa (Kolawole and smooth ½-1inch diameter stems, with more Ebiloma, 2017). Depolymerization of starch may upright side shoots (tillers) compared to be accomplished by various chemical and sorghum (Kajuna and Lewis, 2001). The seeds of physical treatments or by enzymatic treatment.
Millets are cylindrical, typically white, or yellow, but there are varieties with colours ranging from This paper therefore presents a âbrown to purple. Pearl Millet's deep root system amylase from malted pearl millet and describes grows relatively fast (Hannaway and Larson, some of its properties with a view to exploiting 2004), and though, widely distributed across the enzyme for industrial purposes. Africa, India, and Asia, Pearl Millet, can be grown throughout the continental United States and Materials and Methods Puerto Rico. Despite its wide distribution Collection of Sample however, pearl millet is chiefly used as animal Newly cultivated and harvested grains of fodder material (Chad and Jimmy, 2014) and pearl millet were obtained from the Oja Oba therefore, discovery of alternative uses for the market, Akure, Ondo State, South-western hardy cereal does not threaten human food Nigeria and were authenticated at the production.
Department of Crop, Soil and Pest Management, Amylases are among the most important the Federal University of Technology, Akure, enzymes used in several biotechnological Nigeria. applications particularly in starch processing industries to hydrolyse the polysaccharides such Malting of Pearl Millet Grains as starch into simpler sugars. This is the basis for The malting of the pearl millet grains was various industrial processes like preparation of done as described by Kharkrang and Ambasht glucose syrups (Prakash et al., 2011). â amylases (2012) with some modifications. Dried grains (EC 3.2.1.2) are exo-hydrolases which release â-Pearl Millet (300 g) was weighed into a plastic -maltose from the non-reducing ends of alpha-1, vessel. The grains were carefully rinsed and then 4 linked poly and oligoglucans until the first soaked with distilled water for 6 h. The soaked alpha-1, 6 branching point along the substrate grains were sieved and spread in an incubator set molecule is encountered (Saini et al, 2017). It is a at 25 °C where they were left to grow and starch hydrolyzing enzyme used in foods, germinate. An aliquot sample from the soaked pharmaceuticals and brewing industries to grains was obtained to represent the grain at 0 h. convert starch into maltose (Aiyer, 2005). High The germinating grains were sprinkled with little value is placed on the thermal stability and quantity of distilled water every day until the 5th activity at low pH in industrial processes. day and also sampled daily for analysis (Macnicol However, most  The extraction of crude â-amylase from the particularly Bacillus species such as Bacillus germinated grains was done as described by cereus, Bacillus megaterium and Bacillus Kharkrang and Ambasht (2012) with some polymyxa (Femi-Ola and Ibikunle, 2013; modifications. The vegetative parts of the Niziolek, 1997;Hensley et al, 1980), as source of samples were removed by rubbing them the enzyme, with minimal work being done on between the palms, followed by suspension in plant â-amylases. â -amylases from soybean, distilled water and decantation. 30 g of each barley, and wheat have been used industrially sample was weighed and homogenized using an (Hesam et al, 2015). However, soybean âelectric blender with 7.5 mL of 50 mM sodium amylase is relatively expensive and barley and phosphate buffer pH 6.9. The homogenized wheat â-amylases are lacking in thermostability.
samples were then centrifuged at 6000 rpm x g The Pearl Millet is thought to be a promising at 4 °C for 25 min. The obtained supernatant was source of â-amylase since â-amylase is one of used as the crude enzyme solution. the major proteins in the cereal, which is responsible for it's usability as a brewer's grain.
Beta Amylase Activity Assay and Determination of In addition, the crop is hardy to environmental Total Protein conditions, pests, and diseases and it's use in Amylase activity assay was carried out enzyme production poses minimal threat to according to the method of Bernfeld (1955). human and animal food production. Briefly, 0.5 mL of enzyme solution was added Kinetic Parameters of â-amylase from Pearl Millet into a test tube containing 0.5 mL 1% starch Kinetic parameters (K and Vmax) were m solution in 50 mM Sodium acetate buffer pH 4.8. determined at best conditions by measuring the The reaction was allowed to proceed for 5 min at initial reaction rates with soluble starch at 40°C before termination. The amount of various concentrations (0.5 -4 %) for âreducing sugar released was estimated using amylase. The accurate values of the apparent dinitro salicylic acid (DNSA) reagent as described kinetic parameters were obtained from double by Miller (1959). One unit of enzyme is defined reciprocal plot by Lineweaver and Burk (1934). as the amount of enzyme that is required to liberate 1 ìmol of maltose per min under the Results and Discussion described assay condition.
Total Crude Protein Content The total protein content in the sample A protein standard curve was obtained using was determined using the method of Bradford the method of Bradford (1976) using Bovine (1976) using Bovine Serum Albumin (BSA) as the Serum Albumin (BSA) as the standard protein standard protein.
( Figure 1). At 0h, the total protein content was 1.36 mg/mL which increased slightly to 1.46 th Partial Purification of â-amylase from Crude mg/mL after 24 h and 2.27 mg/mL at 48 h. A Extract gradual increase in the protein concentration was noticed from the beginning of the malting Precipitation of â-amylase from the crude nd process up till 72 h with a value of 4.02 mg/mL enzyme was done stepwise until it attained 60% th before a sharp drop to 2.54 mg/mL at 96 h of ammonium sulphate saturation and the solution malting ( Figure 2). The protein concentration was incubated overnight in a cold laboratory nd reaching maximum at 72 h in this study agrees system. The resulting suspension was with the reports of Egwin and Oloyede (2006) centrifuged at 10,000 rpm x g for 15 min at 4 °C.
and El Nour et al. (2013), with both studies The pellet was obtained as the precipitated âplacing the maximum protein concentration of amylase and was suspended in 2 mL of 50 mM nd th malted millet between 72 h and 96 h of sodium phosphate buffer pH 6.9. The precipitate germination. This increase in protein content can was dialysed extensively, as described by as well be correlated with the activity of the Sattayasai (2012), with some modifications, nd enzyme which was observed to peak at the 72 h against 50 mM sodium phosphate buffer pH 6.9 of malting. .

Characterization of Partially Purified âamylase
Activity of â-amylase in Crude Extract Effect of Temperature on the Activity of â-amylase Cereal amylases play a very important role in the metabolism of germinating cereals (Agbo from Pearl Millet et al., 2017). These enzymes are synthesized The effect of temperature on â-amylase under the influence of plant growth hormones activity was determined at different such as gibberellic acid upon activation by water temperatures between 25°C and 80°C in 50 mM imbibition and they exist in multiple forms sodium acetate buffer solution, pH 4.8 under (Muralikrishna and Mirmala, 2005). Germinated standard assay conditions previously described.
pearl millet grains were investigated as a possible source for industrially viable â-amylase.

Effect of pH on the Activity of â-amylase from
The results of the study showed promising Pearl Millet amylase activity from the germinated grain The effect of pH on the activity of the partially extract. A â-amylase activity standard curve was purified â-amylase was determined by obtained using the method of Bernfeld (1955) performing the enzyme activity assay earlier (Figure 3). The activity of â-amylase at 0h of described at different buffer solution pH using malting was 5.23 ìmol/min/mL while the specific the following buffers (50 mM): Sodium acetate activity was 3.86 ìmol/min/mg protein. After 24 buffer (pH 4.0 -5.0), potassium phosphate h of malting, â-amylase activity as well as the buffer (pH 6.0 -7.0) and Tris-HCl buffer (pH 8.0specific activity showed a slight increase, being 9.0) at 25°C. 5.65 ìmol/min/mL and 3.89 ìmol/min/mg phenomenon in which the starch content of the protein respectively. At 48h, a more marked grains, as well as other metabolic resources increase was noted with â-amylase activity being begin to run out, therefore impeding the 6.25 ìmol/min/mL and specific activity being germinating grain's ability to produce more 4.61 ìmol/min/mg. â-amylase activity peaked at enzymes and other proteins (Kolawole and 72 h being 7.65 ìmol/min/mL in agreement with Ebiloma, 2017). reports on maximum â-amylase activity of malted millet between 72h and 96h (Egwin and â-amylase Partial Purification Process The 72 h extract which showed maximum 2013) but the specific activity plummeted â-amylase activity was chosen for partial significantly to 1.85 ìmol/min/mg, a purification. Purification was carried out by 60% phenomenon which can be correlated with the ammonium sulphate precipitation and dialysis. noted increase in protein content of the Pearl The clear solution remaining after the Millet grains at this time. â-amylase activity at ammonium sulphate precipitation showed no â-96h showed a marked decrease from the peak amylase activity. The summary of purification down to 6.13 ìmol/min/mL while the specific procedure is shown in Table 1. The two step activity increased slightly to 2.41 ìmol/min/mg purification process revealed a specific activity of ( Figure 4). Degradation of the starch endosperm 2.44 ìmol/min /mg yield of 9.28% and of the malted pearl millet grains is thought to be purification fold of 1.33. responsible for this. This is explained by the Lineweaver-Burk plot for the Pearl Millet â-The partially purified 72 h extract was amylase is rather high, 10.00 mg/mL (Figure 6), subjected to characterisation assays to indicating a low affinity of the enzyme for the determine the usefulness of the enzyme in the corn starch substrate while the V value (11.11 industry. max ìmol/min/mL) is comparable to the results reported (12.50 ìmol/min/mL) by Tapan et al. (2006) for Heliodiaptomus viduus (Gurney) â-The reaction between the partially amylase. purified â-amylase and corn starch obeyed the Michelis-Menten rule (Figure 5), reaching condition increased between 25°C, 30 and 40°C Effect of pH on â-amylase Activity with relative activities of 42.94%, 46.86% and At pH 4, the relative activity was 55.66% 71.166%, respectively. Maximum activity was while at pH 5.0 the relative activity peaked at recorded at 50 °C while temperature upwards of 100% after which a successive decline was 60°C showed a decline in the relative activity of recorded at pH 6 (74.83%), pH 7 (52.12%), ph 8 the amylase (Figure 8). The optimal activity (35.66%) and pH 9 (29.07%) (Figure 7). The temperature at 50 °C is comparable with partially purified Pearl Millet â-amylase revealed reported values 50°C to 60°C for â-amylase from optimum activity at pH 5.0, showing significant microbial sources including Actinomycetes and preference for acidic pH than most bacterial âamylases which have their pH optimum at Bacillus subtillis (El Nouret al., 2013; Femi-Ola around pH 6.0 (Femi-Ola et al, 2013;Madi et al, and Ibikunle, 2013;Obi and Odibo, 1984). At o 1987) while exhibiting similarity to the optimum 60 C is the enzyme still shows appreciable pH 5.5 reported by Oyefuga et al. (2011) for âactivity, comparable with the enzyme's relative o amylase isolated from Saccharium offinacium activity at room temperature, 25 C (41.72% to nodes. Most commonly described â-amylase lack 42.94%respectively). Relative activity at 70 and significant activity at extreme pH (2-3) (Eke and 80 was recorded at 16.02% and 7.33% Oguntimehin, 1992; Hyun and Zeikus, 1985).

Kinetic analysis of â-amylase from Pearl Millet
respectively. The decline in activity below or Extreme pH can initiate chemical reactions that above the optimum temperature results from the can destroy amino acid residues of the protein changes in the protein structure due to the molecules of the enzyme, thus resulting in changes in the temperature of the environment irreversible deactivation of the enzyme (Agbo et al, 2017). Loss of activity at (Creighton, 1990). Thus, Pearl Millet â-amylase temperatures far exceeding the optimum shows potential for industrial processes such as temperature is usually irreversible (Prescott et al, o starch liquefaction which require low pH 2008). An optimum temperature of 50 C places environments (Richardson, 2002) this amylase as a fairly thermostable enzyme, sharing it's optimum temperature (and pH) with acid amylase, an industrially used amylase Effect of Temperature on â-Amylase Activity isolated from Aspergillus niger (Vengadaramana, The thermal studies showed that there was a gradual increase in the activity of the partially 2013). purified â-amylase as the assay temperature h intervals at 25ºC at near neutral pH (50mM Sodium phosphate buffer pH 6.9). A steady rise in total protein content is observed from 0h till 48h. Maximum protein concentration is indicated by the peak at 72h of germination, followed by a decline in total protein content at 96h. Each point represents the mean ± SD values of independent experiments carried out in triplicate. . Activity peaks at 72h while specific activity drops, showing that while amylase activity is maximum at 72h, protein content also exhibits a massive increase at that point. This is then followed immediately by a drop in â-amy lase activity and a slight rise in specific activity due to a reduction in the total protein content of the germinating grain. Each point represents the mean ± SD values of independent experiments carried out in triplicate. Beta Amylase Specific Activity Figure 6: The Lineweaver-Burk plot of the effect of substrate concentration (1% -4% Starch) on the activity of partially purified pearl millet â-amylase at 25ºC in acidic pH (16mM Sodium acetate buffer pH 4.8). Vmax was determined to be 11.11ìmol/mL/min while Km was 10 mg/mL. Each point represents mean ± SD values of independent experiments carried out in triplicate.

Figure 7:
The effect of pH on partially purified pearl millet â-amylase at 25ºC within the range of pH 4-9, using 16mM Sodium acetate buffer from pH 4-5, 16mM Sodium phosphate buffer from pH 6-7 and 16mM Tris-HCl buffer from pH 8-9. Each point represents the mean ± SD values of independent experiments carried out in triplicate.