Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001
© Deb et al.; licensee Springer. 2013
Received: 12 February 2013
Accepted: 4 April 2013
Published: 10 April 2013
Amylases are one of the most important enzymes in present-day biotechnology. The present study was concerned with the production and partial characterization of extracellular amylase from Bacillus amyloliquefaciens P-001. The effect of various fermentation conditions on amylase production through shake-flask culture was investigated. Enzyme production was induced by a variety of starchy substrate but corn flour was found to be a suitable natural source for maximum production. Tryptone and ammonium nitrate (0.2%) as nitrogen sources gave higher yield compared to other nitrogen sources. Maximum enzyme production was obtained after 48 hrs of incubation in a fermentation medium with initial pH 9.0 at 42°C under continuous agitation at 150 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 was 6.5, 60°C and 40 minutes respectively. About 73% of the activity retained after heating the crude enzyme solution at 50°C for 30 min. The enzyme was activated by Ca2+ (relative activity 146.25%). It was strongly inhibited by Mn2+, Zn2+ and Cu2+, but less affected by Mg2+ and Fe2+.
Microbial enzymes are widely used in industrial processes due to their low cost, large productivity, chemical stability, environmental protection, plasticity and vast availability (Burhan et al.2003; Mishra &Behera 2008). Bacillus species such as Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis are used as bacterial workhorses in industrial microbial cultivations for the production of a variety of enzymes as well as fine biochemicals for decades. A large quantity (20-25g/l) of extracellular enzymes has been produced and secreted by the various Bacillus strains which have placed them among the most significant industrial enzyme producers. The estimated value of world market is presently about US$ 2.7 billion and is estimated to increase by 4% annually through 2012. Detergents (37%), textiles (12%), starch (11%), baking (8%) and animal feed (6%) are the main industries, which use about 75% of industrially produced enzymes (Harwood 1992; Ferrari et al.1993; Schallmey et al.2004; Das et al.2011). Amylases are among the most important enzymes and are of great significance for biotechnology, constituting a class of industrial enzymes having approximately 25-30% of the world enzyme market (Azad et al.2009; Rajagopalan &Krishnan 2008). Initially the term amylase was used originally to designate an extracellular enzymes capable of hydrolyzing α-1,4-glucosidic linkages in polysaccharides containing three or more 1,4-α-linked glucose units. The enzyme acts on starches, glycogen and oligosaccharides in a random manner, liberating reducing groups. These enzymes are found in prokaryote as well as in eukaryotic organisms. They are widely distributed in microbial, plant and animal kingdoms. In the present day scenario, amylases have a great commercial value in biotechnological applications ranging from food, fermentation, textile to paper industries. These uses have placed greater stress on increasing amylase production and search for more efficient processes (Aehle &Misset 1999; Lin &Hsu 1997;Wolfgang 2007). For the maximum enzyme production, medium optimization is a prime step for its commercial usage.
The present work describes the effects of culture conditions on amylase production in batch experiments in shake flasks and under controlled conditions in a laboratory incubator. In this study, we show that enzyme synthesis is affected by carbon and nitrogen sources and maximal activity is attained with inorganic than organic nitrogen sources. The optimum enzyme production by the bacterial isolate was found at 42°C, whereas maximum enzyme activity was observed at 60°C. The enzyme was activated by Ca2+ (relative activity 146.25%). It was strongly inhibited by Mn2+, Zn2+ and Cu2+, but less affected by Mg2+ and Fe2+.
Results and Discussion
Therefore, further studies on enzyme production in shake-flask cultures were carried out using Bacillus amyloliquefaciens P-001. The organism was used for extracellular amylase production in shake-flask culture using basal medium (0.1% KH2PO4, 0.25% Na2 HPO4, 0.1% NaCl, 0.2% (NH4)2SO4, 0.005% MgSO4 .7H2O, 0.005% CaCl2, 0.2% tryptone and 1% soluble starch, pH 6.5) (Bose &Das 1996) for 48 hrs hours of incubation at 37°C and enzyme activity was obtained 35.0 U/ml. To enhance the production of enzyme various parameters associated with the production of amylase were studied in the medium used for the enzyme production. Optimization of culture conditions is very important for maximum microbial growth and enzyme production by microorganisms (Kathiresan &Manivannan 2006). Among the physical and chemical parameters, optimum temperature, pH range, carbon and nitrogen sources are the most important for enzyme production by microbes (Bose &Das 1996; Gupta et al.2003).
Effect of culture conditions for extracellular amylase production from Bacillus amyloliquefaciens P-001 in shake-flask cultivations
Amylase activity (U/ml) (Mean ± SE)
Relative activity (%)
Total soluble protein (mg/ml) (Mean ± SE)
Incubation temperature (°C)
Incubation period (hr)
Inoculums volume (%)
Temperature is a vital environmental factor which controls the growth and production of metabolites by microorganisms and this is usually varied from one organism to another (Banargee &Bhattacharya 1992; Kumar &Takagi 1999). Bacterial amylases are produced at a much wider range of temperature. Bacillus amyloliquefaciens, B.subtilis, B. licheniformis and B. stearothermophilus are among the most commonly used Bacillus sp. reported to produce α-amylase at temperatures 37–60°C (Mendu et al.2005;Mielenz 1983; Syu &Chen 1997; Mishra et al.2005). A wide range of temperature (35-80°C) has been reported for optimum growth and α-amylase production in bacteria (Burhan et al.2003; Castro et al.1992; Prakash et al.2009; Lin et al.1998). In present study, for the determination of optimum temperature for enzyme production, the fermentation was carried out at different temperatures (32 to 45°C). Enzyme production was gradually increased with increasing temperature and maximum enzyme production was observed at 42°C (Table 1). The optimum range for enzyme production was 40-42°C. Nusrat &Rahman (2007) reported that, α-amylase production was maximum at temperature 37°C by the Bacillus amyloliquefaciens. Haq et al. (2010) reported that, the better activity of α-amylase in stirred fermentor with working volume of 4.5 L was at 37°C in 48 h by using randomly induced mutant strain of Bacillus amyloliquefaciens EMS 6.
From the time course study in shake culture, it was found that the rate of enzyme production was increased with the increase in the fermentation period and reached its maximum activity after 48 hour incubation (Table 1). The total protein content obtained was 3.97 mg/ml at that time. A prolonged incubation time beyond 48 hour did not increase the enzyme production. These findings are similar to the result reported by Haq et al. (2010). A similar result was also found by Asgher et al. (2007) studied on Bacillus subtilis, Kaur &Vyas (2012) in case of Bacillus sp. DLB 9 and Riaz (et al.2003) in case of Bacillus subtilis. It might be due to the accumulation of other by products in the medium Riaz et al. (2003). Efficient induction might not occur until the stationary phase has been reached and the available carbon source was reduced (Huang et al.2003; Wanderley et al.2004). But, Abate et al. (1999) reported that the production of α-amylase by Bacillus amyloliquefaciens starts at the beginning of the exponential growth phase reaching the maximum level after 24 hour and after that, α-amylase level decreased drastically probably due to the accumulation of high level of protease activities concomitant with the sporulation process at the end of the exponential growth phase. Similar findings have been reported on Bacillus amyloliquefaciens Hillier et al. (1997) and Bacillus sp. ANT-6 Burhan et al. (2003).
The volume of inoculum plays an important role in the fermentation of enzymes Lin et al. (1998). In our study, 1% inoculum induced the maximum amylase production (Table 1). As the inoculum level was further increased, the production of enzyme was gradually decreased. It may be due to the fact that at high concentration of inoculum level, the bacteria grow rapidly and the nutrients present in the media were insufficient to overcome the growth of bacteria. Thus, the production of amylase was affected at higher concentration of inoculum. Our findings are in a good agreement with Riaz et al. (2003).
The cultural conditions and composition of media for optimal production of amylase by B. amyloliquefaciens P-001 has been developed in this study. Enzyme synthesis was affected by carbon and nitrogen sources and maximal activity was attained with inorganic than organic nitrogen sources. The optimum enzyme production by the bacterial isolate was found at 42°C, whereas maximum enzyme activity was observed at 60°C which could make the enzyme from B. amyloliquefaciens P-001 more suitable for future use in various industries. It can be concluded that, B. amyloliquefaciens P-001 can be a potential producer of extracellular amylase which could find applications in industry. Due to the importance of these findings, further studies need be carried on in order to commercialize the production process.
The bacterial culture Bacillus amyloliquefacience P-001 was obtained from the Microbial Biotechnology Division, National Institute of Biotechnology, Ganakbari, Savar, Dhaka. It was maintained on nutrient agar medium. The organism was maintained at 4°C in refrigerator for routine laboratory use. For the long term preservation, the log phage growth bacteria were maintained in 15% glycerol broth at -20°C.
Plate assay method
The Bacillus isolates were tested for amylase activity by employing zone clearing technique Atlas et al. (1995) using starch agar medium. The inoculated plates were incubated at 37°C for two days. After incubation, the zone of hydrolysis of starch was detected by flooding the plates with iodine solution. The development of blue colour indicated the presence of starch, while the areas around the hydrolytic bacteria appeared clear.
Preparation of seed culture
Vegetative inoculums were used in the present studies. Fifty millilitre of inoculums medium containing nutrient broth 13 g/l, pH 7.4 ± 0.2 was transferred to a 100 ml conical flask and cotton plugged. It was sterilized in an autoclave at 15 lbs/inch2 pressure at 121°C for 20 min. After cooling to room temperature, a loopful of freshly grown culture was aseptically transferred to it. The flask was incubated overnight at 37°C and 150 rpm in a rotary shaking incubator.
Enzyme production in shake flask cultures
The enzyme production was carried out in the basal Asgher et al. (2007) medium containing 0.1% KH2PO4, 0.25% Na2 HPO4, 0.1% NaCl, 0.2% (NH4)2SO4,0.005% MgSO4 .7H2O, 0.005% CaCl2, 0.2% tryptone and 1% soluble starch. 1 ml of 24 hours grown inoculums were cultivated in 250-ml Erlenmeyer flasks containing 100 ml of medium with an initial pH 7.0. The cultures were shaken at 150 rpm in a orbital shaker incubator at 37°C for at least 48 h unless otherwise stated. After the incubation, the fermented broth was centrifuged in a refrigerated centrifuge machine at 8000 rpm for 15 minutes at 4°C.
Amylase was determined by using soluble starch, 1% (w/v), as substrate in 0.05 M Sodium phosphate buffer (pH 6.5) essentially according to Gomes et al. (2001). The reaction mixture containing 1.8 ml substrate solution and 0.2 ml suitably diluted enzyme solution was incubated at 50°C for 10 min. The reaction was stopped by adding 3 ml dinitrosalicylic acid (DNS). The reducing sugar released was determined by the method ofMiller (1959). The absorbance was measured at 540 nm with spectrophotometer (Jenway 6305, USA). One unit (U) of enzyme activity is defined in all cases as the amount of enzyme releasing 1 μg of reducing sugar as maltose per minute, under assay conditions.
Soluble protein estimation
Extracellular soluble protein in culture filtrate was estimated by the Lowry’s method using bovine serum albumin (BSA) used as Standard Lowry et al. (1951). 2 ml of analytical reagent was added to 0.2 ml suitably diluted test samples (enzyme solution). The mixture was mixed well and allowed to stand for 10 min at 50°C. Then 0.2 ml of the folin-ciocalteau reagent was added and shaken to mix well and incubated at room temperature for about 30 min. Optical density of the reaction mixture was measured at 600 nm, against a blank prepared with 0.2 ml buffer. A standard curve was constructed with each experiment using bovine serum albumin as a known protein. The amount of the soluble protein was calculated from the standard curve of as mg protein per ml of test samples.
The research was supported by National Institute of Biotechnology, Savar, Dhaka, Bangladesh.
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