B. stearothermophilus and B. amyloliquefaciens were also considered as good producers for the thermostable α-amylase. Regarding the optimization, production and partial purification of extracellular α-amylase from Bacillus sp. marini, Ashwini et al. (2011) found that the maximum enzyme production was recorded in presence of starch as carbon source, yeast extract as nitrogen source, 6.5% NaCl concentration, temperature 40°C and pH 7.0, finally Bacillus sp. marini produced 8000 U of amylase at these optimum conditions. Further, Deb et al. (2013) studied the production and partial characterization of extracellular amylase enzyme from B. amyloliquefaciens P-001. They found that maximum enzyme production was obtained after 48 h of incubation in a fermentation medium with initial pH 9.0 at 42°C under continuous agitation at 15 rpm. The size of inoculum was also optimized which was found to be 1% (v/v). Enzyme production was 2.43 times higher after optimizing the production conditions as compared to the basal media. Studies on crude amylase revealed that optimum pH, temperature and reaction time of enzyme activity were 6.5, 60°C and 40 min, respectively. Meanwhile, Saha et al., (2014) found that the specific activity of amylase produced by B. amyloliquefaciens was 13.5 U/mg. Onofre et al., (2016) evaluated α-amylase produced by the endophytic strain of Penicillium digitatum in solid state fermentation (SSF) and submerged fermentation (SMF). They found that the maximum yield of the enzyme was observed with SSF, using rice bran as substrate after 72 h of fermentation, with 1,625 U/mL. The α-amylase had an optimal pH at 6.5 and optimal temperature at 37°C. All the ions resulted in a decrease in the activity of α- amylase in the concentration of 5 mM. Rasmy (2018) studied the kinetic properties of α-amylase produced by B. megaterium RAS103 under optimum conditions in submerged fermentation. He found that amylase activity was maximized to 106.39±2.36U/ml under the optimized culture conditions of a fermentation medium of 2% starch supplemented by 3g/L yeast extract, adjusted at pH 8.0, inoculated with 1% bacterial inoculum and incubated at 35°C for 24h. The Vmax and Km values of the produced amylase were 81.30U/ml and 0.878mg/ml, respectively for hydrolysis of starch in a reaction mixture of pH 6.0 at 45°C for 20min. The purpose of this work was to study the optimization of cultural conditions such as temperature, pH, carbon sources, nitrogen sources for α-amylase production by B. amyloliquefaciens as well the evaluation of kinetic properties for the produced α-amylase.

Introduction Enzymes are an important class of proteins produced by living cells of microorganisms, plants and animals to catalyze specific biochemical reactions of the metabolic pathways of the cells. Among the produced enzymes, amylases are the most important group for biotechnology and account approximately 65% of enzyme market in the world (Balkan & Figen, 2007 and Abd-Elhalem et al., 2015). Among them, α-amylase (E.C.3.2.1.1) is a calcium metalloenzymatic that hydrolysis the internal α-1,4-glycosidic linkages in starch and leads to the fermentation of low molecules weight oligosaccharides as glucose, maltose and maltotriose units (Aiyer, 2005). Microbial enzymes such as amylases are widely used in industrial processes due to their low cost, large productivity, chemical stability, environmental protection, plasticity and vast availability (Mishra and Behera, 2008). Today, amylases find potential widespread application in different industrial processes especially in food industry for liquefaction and saccharification of starch into fructose and glucose syrups (Khusro et al., 2017). Karnwal and Nigam (2013) reported that the heat stable α-amylase produced from B. amyloliquefaciens was the first liquefying enzyme used on a large commercial scale. Several bacterial species like Bacillus cereus and B. subtilis have been explored for production of amylases enzyme (Konsoula and Liakopoulou-Kyriakides, 2007). As well B. licheniformis

Materials and Methods Bacterial strain Bacillus amyloliquefaciens was obtained from Agric. Microbiology Department, Faculty of Agric., Ain Shams Univ., Egypt. B. amyloliquefaciens strain was sub-cultured on nutrient agar medium pH 7.0 (Taha, 1964) for purification, then maintained as a stock culture at 4-5°C in refrigerator for subsequent studies. Qualitative detection of α-amylase B. amyloliquefaciens was tested for α-amylase production using plate assay method by employing clear zone technique (Atlas et al., 1995) on starch agar medium (Harrigan and McCance, 1976). Assay of α-amylase activity Amylase activity was assayed by the method described by Miller (1959). Inoculum preparation Cells of B. amyloliquefaciens were transferred to a 100 ml conical flask containing 25 ml nutrient broth medium (Taha, 1964). The flask was kept on shaker (150 rpm) at 37oC for 24 h. One ml from homogenous bacterial suspension contains about 8×106 CFU/ml was used as a stock inoculum. Production medium of α-amylase Production of α-amylase by B. amyloliquefaciens was carried out using basal medium according to Bose and Das (1996). Optimization of cultural conditions for α-amylase productivity and activity Effect of pH on enzyme production and enzyme activity was estimated by adjust pH at different values (6.0, 6.5, 7.0, 7.5 and 8.0), production medium was inoculated by B. amyloliquefaciens with different inoculum sizes namely 100, 500, 1000, 1500 and 2000 μl/ml. Similarly, the effect of fermentation period was studied by adjusted different incubation periods (24, 48, 72, 96 and 120 h.). The inoculated medium was incubated at different incubation temperatures (33, 35, 37 and 39°C). Also, the effect of carbon, nitrogen sources was studied by replacing the basic sources in the production medium with different carbon sources (glucose, lactose, sucrose, dextrin and starch) and nitrogen sources (beef extract, yeast extract, urea, tryptone, potassium nitrate and sodium nitrate). Effect of fermentation medium on α-amylase production by B. amyloliquefaciens Two media, basal broth medium pH 7.0 (Bose and Das, 1996) and starch broth medium pH 7.0 (Harrigan and McCance, 1976) were used in this experiment at static incubator at 30°C for 72 h. Effect of static and shaking incubation on α-amylase production Basal broth medium (pH 7.0) was inoculated by 24 h old B. amyloliquefaciens (1500 μl/ml). The inoculated medium was divided into two groups, the first was incubated using shaking incubator (150 rpm) while, the other was incubated using static incubator at 30°C for 72 h (Sundarram and Murthy, 2014). Effect of optimal conditions on amylase production Basal broth medium (pH 7.0) amended with the best carbon and nitrogen sources and inoculated by 24 h old B. amyloliquefaciens (1500 μl / ml) was incubated at 37 oC using shaking incubator (150 rpm) for 72 h. At the end of incubation period in all previous experiments, the cultures were centrifuged at 5000 rpm for 15 min at 4°C to remove the bacterial cells. Then, the supernatant was collected, and α-amylase enzyme was assayed using the method described by Miller (1959) as abovementioned. Determination of protein content and specific activity of the produced α-amylase Protein content and specific activity of the produced α-amylase were determined in extracted crude enzyme according to the method described by Bradford (1976) using bovine serum albumin (BSA) as a standard. Characterization of the produced α-amylase Produced α-amylase from the optimal conditions experiment was used to evaluate the factors affecting the enzyme activity such as temperature, pH, substrate concentration, sodium chloride and metal ions. Optimum temperature for enzyme activity was determined by incubating crude enzyme substrate reaction mixture at different temperatures i.e: 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 and 80°C for 15 min using shaking incubator and assaying the enzyme activity as mentioned before (Miller, 1959). The effect of pH on amylase activity was determined by incubating the reaction mixture with different buffers of 0.1 M (pH 4.0-5.5, acetate buffer), (pH 6.0-7.5, phosphate buffer) and (pH 8.0- 9.0, Tris-HCl buffer). The relative activities of the produced α-amylase were estimated. Concerning the effect of substrate concentrations on the produced α-amylase activity, different concentrations of soluble starch (0.4, 0.8, 1.2,1.6, 2.0, 2.4, 2.8, 3.2, 3.6, 4.0% w/v) were investigated for detection of amylase kinetics properties. The reaction mixture contains extracted amylase was incubated at optimum temperature 65oC, pH 6.0 for 15 min using shaking incubator. The reaction velocity and activity of the produced α-amylase were estimated by Michael’s-Menten equation following by using Lineweaver-Burk plots for calculate Km and Vmax. Effect of sodium chloride concentrations on the produced α-amylase activity was investigated. The extracted enzyme was mixed with different concentrations of sodium chloride (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6 % w/v), pH (6.0), soluble starch at a concentration of 2.8% was added as a substrate. Then, the reaction mixture was incubated at optimum temperature 65oC for 15 min using shaking incubator. Effect of metal ions concentration on α-amylase activity was investigated, the reaction mixture contains the extracted enzyme was mixed with different metal ions i.e: Fe2+, Cu2+, Ca2+, Mn2+ and Ni2+ at two concentrations (1 and 5 mM from each ion), pH 6.0 and soluble starch (2.8%) a substrate. Then, the reaction mixture was incubated at optimal conditions as mentioned above. At the end of incubation period in all above experiments for evaluation of enzyme activity, the produced α-amylase was assayed using the method described by Miller (1959) as abovementioned.

Sundar et al., 2012

Gupta et al., 2008). Table 5. α-Amylase production by B. amyloliquefaciens using different carbon sources Carbon sources Obtained glucose (μg/ml) Amylase activity (IU/ml/min.) Glucose 109.74 60.97 Lactose 115.90 64.39 Sucrose 77.77 43.21 Dextrin 115.90 64.39 Starch 122.94 68.30 Nitrogen sources Generally, the obtained data in Table (6) clearly indicate that the organic nitrogen sources (beef extract, urea, yeast extract and tryptone) gave higher records of the produced glucose and amylase activity compared to the inorganic nitrogen sources (potassium nitrate and sodium nitrate). The highest values of produced glucose and amylase activity were 125.57 µg/ml and 69.76 IU/ml/min, respectively when tryptone was used as a nitrogen source in fermentation medium. Whereas, the lowest values of abovementioned parameters were observed with sodium nitrate. Different inorganic and organic nitrogen sources were applied for α-amylase production by various bacterial strains (Haq et al., 2010

Demirkan, 2011

Results and discussion Qualitative detection of α-amylase production Figure (1) show that the bacterial strain Bacillus amyloliquefaciens was able to hydrolyze starch as a result of its ability for amylases production showing zone of hydrolysis around the colonies on starch agar medium supplemented with soluble starch. In this concern, some species of genus Bacillus produces a large variety of extracellular enzymes such as amylases, which are considerable for industrial importance (Swain et al., 2006, Deb et al., 2013). Figure 1. Zone of clearance due to the hydrolysis of starch by B. amyloliquefaciens. Effect of cultural conditions on α-amylase production by B. amyloliquefaciens Initial pH Data in Table (1) indicate that obtained glucose and amylase activity were higher when the pH of fermentation medium ranged from 6.5-7.0 compared to other investigated pH values. The highest values of produced glucose and amylase activity were found to be 55.81 mg/ml and 31.01 IU/ml/min, respectively when the initial pH of fermentation medium was 7.0. Whereas, the lowest records of abovementioned parameters were observed when the initial pH of fermentation medium was 6.0. α-Amylase production by bacteria significantly depends on the medium pH because it effects on the growth and many metabolic reactions as well as the movement of molecules across cell membrane. The obtained results are in accordance with Sundarram and Murthy (2014). Many studies revealed an optimum pH range between 6.0 and 7.0 for the growth of bacterial strains and amylases production because bacteria required neutral pH for optimum growth (Gangadharan et al., 2006 and Gupta et al., 2008). Table 1. α-Amylase production by B. amyloliquefaciens at different pH values pH values Obtained glucose (μg/ml) Amylase activity (IU/ml/min.) 6.0 34.55 19.19 6.5 53.55 29.75 7.0 55.81 31.01 7.5 48.10 26.72 8.0 47.4 26.35 Inoculum size Table (2) show that both the produced glucose and amylase activity were affected by the inoculum size of B. amyloliquefaciens. The two parameters were increased gradually with the increasing of inoculum size and reach their maximum values 57.16 mg/ml and 31.75 IU/ml/min respectively when the fermentation medium was inoculated with 1500 μl/ml. Meanwhile, the increasing of inoculum size to 2000 (μl/ml) was useless. Similar results were observed by Deb et al., (2013). Table 2. α-Amylase production by B. amyloliquefaciens using different inoculum sizes Inoculum size (μl/ml) Obtained glucose (μg/ml) Amylase activity (IU/ml/min.) 100 34.81 19.34 500 36.04 20.02 1000 39.56 21.98 1500 57.16 31.75 2000 54.70 30.39 Incubation period Results in Table (3) show that the records of produced glucose and amylase activity were increased with the increasing of incubation period to reach their maximum values (98.51 mg/ml and 54.73 IU/ml/min, respectively after 72 h of incubation. Further, the increase of incubation time significantly decreases the enzyme production. This may be due to nutriented efficiency of the culture medium and accumulation of toxic metabolites. These results are in accordance with those observed by Asgher et al., (2007) and Haq et al., (2010) who found that the highest activity of α-amylase production by B. subtilis and B. amyloliquefaciens was recorded at 37°C after 48 h of incubation. Table 3. α-Amylases production by B. amyloliquefaciens at different fermentation periods. Incubation period (h) Obtained glucose (μg/ml) Amylase activity (IU/ml/min.) 24 42.76 23.76 48 45.25 25.14 72 98.51 54.73 96 72.06 40.03 120 69.56 38.65 Nutritional medium Obtained results in Figure (2) clearly indicate that, produced glucose and amylase activity were higher (92.50 μg/ml and 51.38 IU/ml/min) when the basal broth medium (77.80 μg/ml and 43.20 IU/ml/min) was used as a fermentation medium rather than the using of starch broth medium. The decrease in amylase biosynthesis when used starch for fermentation medium might be due to inaccessibility of dissolved oxygen to the bacterial cells because of the high viscosity of carbon source in the fermentation medium. Figure 2. α-Amylase production by B. amyloliquefaciens at two nutritional media Static and shaking incubation Data in Figure (3) clearly indicate that the shaking method gave higher values 116.95 μg/ml and 64.97 IU/ml/min for produced glucose and amylase activity, respectively by bacterial strain B. amyloliquefaciens as compared to static method. This may be because shaking increase the dissolved oxygen which enhance bacterial growth. Therefore, the shaking method was used in all subsequent experiments that performed in the current study. Similar results were observed by Sundarram and Murthy (2014). Figure 3. α-Amylase production by B. amyloliquefaciens under shaking and static incubation. Incubation temperature Data in Table (4) show that the produced glucose and amylase activity were increased with the increasing of incubation temperature and reached their maximum records at 37oC. The highest values of produced glucose and amylase activity were 119.59 μg/ml and 66.44 IU/ml/min, respectively. These results are in agreement with reported by Nusrat and Rahman (2007) and Haq et al., (2010). The decrease in amylase activity that obtained at high temperature (39oC) might be due to inhibition of cell division and growth as well as protein denaturation of bacterial cells (Oyeleke et al., 2011). Table 4. α-Amylase production by B. amyloliquefaciens at different incubation temperatures Incubation temperature (˚C) Obtained glucose (μg/ml) Amylase activity (IU/ml/min.) 33 84.52 46.96 35 102.47 56.93 37 119.59 66.44 39 53.38 29.65 Carbon sources Data in Table (5) show that the highest records of produced glucose and amylase activity (122.94 μg/ml and 68.30 IU/ml/min, respectively) were observed when starch was used as a carbon source. Whereas, the lowest values of abovementioned parameters were observed when lactose was used as a carbon source in fermentation medium. In general, obtained data in Table (5) clearly indicated that the oligosaccharides such as dextrin and polysaccharides such as starch gave higher records of abovementioned criteria compared with either monosaccharides or disaccharides (glucose, lactose and sucrose). In this concern, starch was known to increase amylases production by B. subtilis (Sodhi et al., 2005