Isolation, partial purification and characterization of α-amylase from Bacillus subtilis

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Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2014, 4 (1):49-54 (http://scholarsresearchlibrary.com/archive.

Sani *1, A. Abdulhamid 1, F. Bello 1, M. Yahaya 1 and A. I.

1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2014, 4 (1):49-54 ( ISSN : CODEN (USA) : JMBRB4 Isolation, partial purification and characterization of α-amylase from Bacillus subtilis I. Sani *1, A. Abdulhamid 1, F. Bello 1, M. Yahaya 1 and A. I. Bagudo 2 1 Department of Biochemistry, Kebbi State University of Science and Technology, Aliero, Birnin Kebbi, Kebbi State, Nigeria 2 Microbiology Unit, Department of Biological Sciences, Kebbi State University of Science and Technology, Aliero, Birnin Kebbi, Kebbi State, Nigeria ABSTRACT Nowadays, the use of α-amylase in some industries especially in food, beverage, textiles, leather and paper industries is increasing; there is a need for other source of the enzyme to be discovered as Nigeria is a tropical country which is rich of natural resources, particularly the microbes as enzyme producers. This research is aimed at isolation, partial purification and characterization of α-amylase from Bacillus subtillis. The partial purification of the enzyme was done by fractionation of raw extract with ammonium sulphate salt in a variety of saturated degree to get the partial purified enzyme. The partial characterization of partially purified enzyme was done by determination of the optimum ph and temperature at which the enzyme activity is maximum. The results showed that the partially purified enzyme has specific activity of ± u/mg, there was an increase of 33.5 times than the raw enzyme extract. The optimum ph of the purified enzyme was 6.0, but the enzyme can work in the ph range of The optimum temperature of the enzyme was 60 C. The B. subtilis can serve as other source of α-amylase especially for industrial purposes. Keywords: α-amylase; Bacillus subtillis; Partial Purification; Characterization; Ammonium sulphate; Industrial application INTRODUCTION Amylase is an enzyme which hydrolyses starch molecules to give diverse products including dextrins and progressively smaller polymers composed of glucose units [1]. This enzyme is of great significance in present day biotechnology with applications ranging from food, fermentation, textile to paper industries. Although amylase can be derived from several sources; including plants, animals and microorganisms, microbial enzymes generally meet industrial demands [2]. Today a large number of microbial amylases are available commercially and they have almost completely replaced chemical hydrolysis of starch in starch processing industries. Α-Amylase (1, 4-α-D-glucan-glucanhydrolase, EC ) is a widely distributed secretary enzyme and the most popular and important form of industrial amylases. Today a large number of enzymes are known which hydrolyze starch molecule into different products and a combined action of various enzymes is required to hydrolyze starch completely [1]. A number of reviews exist on amylases and their applications; however, none specifically covers α- amylases at length. 49

2 Α-Amylases are universally distributed throughout the animal, plant and microbial kingdoms. Over the past few decades, considerable researches have been undertaken with the extracellular α-amylase being produced by a wide variety of microorganisms. The major advantage of using microorganisms for the production of amylases is the economical bulk production capacity and microbes are easy to manipulate to obtain enzymes of desired characteristics [3]. Α-Amylases have been derived from several fungi, yeasts, bacteria and actinomycetes, however, the enzymes from fungal and bacterial sources have dominated applications in industrial sectors. Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis, and Bacillus amyloliquefaciens are known to be good producers of thermostable α-amylase, and these have been widely used for commercial production of the enzyme for various applications [4]. This research work is aimed at isolating, partially purify and characterize α-amylase from Bacillus subtillis. MATERIALS AND METHODS 2.1 Methods The research was conducted in four (4) phases; isolation of Bacillus subtilis, extraction of α-amylase from the B. subtilis, partial purification of the enzyme and characterization Isolation of Bacillus subtillis The bacterial cells were isolated from soil collected from University garden in Kebbi State University of Science and Technology Aliero, Nigeria. 1g of soil sample was added to a glass tube containing 9 ml sterilized distilled water, and 1ml of the mixture in the 1 st test tube were transfer to a 2 nd test tube containing 9ml of distilled water and same process was done continuously until the last 7 th test tube. 0.1ml of the sample from test tubes 4, 5, 6, and 7 were spread onto nutrient agar plates (prepared by dissolving 0.6 g peptone, 0.4 g casein hydrolyzate, 0.15 g beef extract, 0.3 g yeast extract and 0.2 g starch in 100 ml distilled water, ph was then adjusted to 7 and then 1.5 g agar was added in the 250 ml Erlenmeyer flasks, autoclaved at 121 C and 15 lb pressure for 15 min) and incubated at 37 C for 48 h. Gram staining was then conducted using standard procedure. The gram positive colonies were subjected to biochemical tests for identification of Bacillus subtilis Extraction of α-amylase The α-amylase was produced in fermentation media containing: NaCl 0.04%, peptone 0.20%, yeast extract 0.10% and starch 1.00%. ph was adjusted to 7 with NaOH. The media was sterilized by autoclaving at 121 C and 15 lb pressure for 15 min. After cooling, the media was transferred into test tubes and inoculated with the bacterial isolates and incubated at 37 C for 24 hours. The raw extract of the α-amylase in fermentation media was separated from the bacterial cells by cold centrifuge at speed of 6000 rpm for 30 minutes at 4 0 C Partial Purification of α-amylase Fractionation with Ammonium Sulphate In a variety of saturated degree of ammonium sulphate, a standard procedure was used to get the partially purified enzyme [5]. The raw extract was brought to 50% saturation of ammonium sulphate, the precipitates were removed by centrifuging at 20,000g for 15 minutes. The resulting supernatant was raised to 70% ammonium sulphate saturation and precipitate was collected by centrifuging at 20,000g for 15 minutes. The resulting supernatant was raised to 90% ammonium sulphate saturation and precipitate was also collected by centrifuging at 20,000g for 15mins. The resulting supernatants were subjected to enzyme assay Enzyme Assay Amylase activity was determined by measuring the release of reducing sugar from soluble starch. The reaction mixture contained 0.5 ml of crude enzyme and 1 ml of sodium phosphate buffer (ph 7.0). 1% soluble starch was 50

3 then added and incubated at 25 C for 10 min. The amount of reducing sugar released in the mixture was determined by the addition of 2 ml of 3,5-dinitrosalicylic acid method [6] followed by boiling for 10 min to develop color. The absorbance of the mixture was measured at 540 nm, and D-glucose was used to create a standard curve. One unit of enzyme activity was defined as the amount of enzyme releasing reducing sugar equivalent to 1 µmol glucose per minute under the assay condition Protein Content Estimation A standard method by [7] was used. Different dilutions of Bovine Serum Albumin (BSA) solutions were prepared by mixing stock BSA solution (1 mg/ ml) and distilled water in test tubes. The final volume in each of the test tubes was 5 ml. The BSA range was 0.2 to 0.8mg/ ml. From these different dilutions, 0.2 ml protein solutions were pipetted to different test tubes and 2 ml of alkaline copper sulphate reagent was added. The solutions were mixed well and incubated at room temperature for 10 min. 0.2 ml of Folin Ciocalteau solution was then added to each tube and incubated for 30 min. The absorbance of each mixture was then measured using spectrophometer at 600 nm. The absorbance was plotted against protein concentration to get a standard calibration curve. The concentration of the unknown samples was determined from the standard curve plotted Partial Characterization of Purified Enzyme The partial characterization of the purified enzyme was done by determining the optimum ph and optimum temperature at which the enzyme activity is high Determination of Optimum ph of the Purified Enzyme To 11 sets of test tubes in triplicates, 2ml of 0.1% starch solution was added, followed by the addition of 4ml of 0.1 M phosphate buffer with ph variations of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and ml of distilled water was added to the 11 th test tube and labeled as blank. All the tubes were incubated at 37 o C in a water bath for 5min to equilibrate. 0.1ml of the α-amylase was added and mixed in all the test tubes and returned into water bath for further incubation for 5min and 2ml of N\100 iodine solution was added immediately after incubation. The absorbance of each mixture was measured spectrophotometrically at 660 nm using reagent blank Determination of Optimum Temperature of the Purified Enzyme To 7 sets of test tubes in triplicates, 2ml of 0.1% starch solution was added, followed by addition of 4ml of 0.1 M phosphate buffer (with ph 7) and 1ml of distilled water. The variations of the temperature used were 45, 50, 55, 60, 65 and 70 C. The test tubes were left at these temperatures for 5 min. 0.1ml of the enzyme preparation was added to each tube. The tubes were incubated for another 5 min then 4ml of N\100 iodine solution was added after the incubation. The absorbance of each mixture was measured spectrophotometrically at 660 nm using reagent blank. RESULTS 3.1 Extraction and Partial Purification of α-amylase After inoculation of the production media with cells from plate 10-5 followed by incubation at 37 o C for 24 hours, the broth culture was observed to have cell growth and release of the enzyme. The culture was more turbid and bubbles were seen at the top of the liquid as presented in Figure 1. The results for the individual steps during the partial purification procedures are presented in Table 1. Table 1: Purification table for isolated α-amylase enzyme Purification Steps Total Activity (U) Total Protein (mg) Specific Activity (u/mg) Yield (%) Purification factor (X) Raw extract ± ± ± A.S. 50% ± ± ± ± ± A.S. 70% ± ± ± ± ± 0 A.S. 90% ± ± ± ± ± The values are presented as mean ± standard deviation of triplicates. A.S. means ammonium sulphate 51

4 Control media Production media Figure 1: Control and Production broth for α-amylase 3.2 Partial characterization The enzyme activities (unit) of the partially purified enzyme at the various ph values used are presented in Figure 2, while those of temperatures are shown in Figure 3. Figure 2: Optimum ph of partially purified α-amylase enzyme 52

5 Figure 3: Optimum temperature of partially purified α-amylase enzyme DISCUSSION 4.1 Partial Purification The phases of purification of α- amylase from B. subtilis can be seen in Table 1. It has been showed that there was high gradual specific activity in each step of purification. The highest activity was observed from ammonium sulphate fraction of 90% saturation. The specific activity increases from one purification step to another as follows; 3.5 times for ammonium sulphate fraction of 50%; 1.6 times for ammonium sulphate fraction of 70% and 6.0 times for ammonium sulphate fraction of 90%. The increase of specific activity for each purification process was an indication that the step of purification used was effective. The results obtained were then compared to those obtained by [8] who did purification on α- amylase from B. subtilis ITBCCB148. The α-amylase obtained showed increase of specific activity higher than α- amylase from wild B. subtilis, but the difference in enzyme activity may be as a result of the purification process (partial) undertaken. 4.2 Partial characterization Optimal ph The results obtained indicated that the optimum ph at which the α-amylase activity is high is 6.0. This observation agrees with what was earlier reported by [8, 9]. The α-amylase obtained showed the same optimal ph with α- amylase from wild B. subtilis (ph value 6) Optimal Temperature The results obtained showed optimal temperature value of 60 0 C at which the enzyme activity is high. This also agrees with what was reported earlier by [8, 9]. This is different when compared to those obtained by [10, 11]. The optimum temperatures at which α-amylase activity reaches maximum as reported by many researches were reviewed based on microbial sources by [12]. CONCLUSION The usefulness of an enzyme from any organism for starch hydrolysis depends upon its potential to degrade native starch to oligosaccharides, glucose and other products at high temperatures and over a wide range of ph. The ability of Bacillus sp. to degrade native starch, at a wide range of ph and the thermal stability of α-amylase are the attractive attributes which make this bacterial strain to be a potential source of this enzyme for starch hydrolysis, especially for many applications ranging from bread and baking industry, starch liquefaction and saccharification, textile desizing, paper industry, detergent application, analysis in medicinal and clinical chemistry. 53

6 REFERENCES [1] Windish W.W. and Mhatre N.S. (1965). Microbial amylases, In: Wayne W.U., editor. Advances in applied microbiology, Vol. 7. New York: Academic Press [2] Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. (2000). Biotechnol. Appl. Biochem.; 31: [3] Lonsane B, Ramesh M. (1990). Advances in applied microbiology, Vol. 35. San Diego: California Academic Press, 1-56 [4] Prakash, O.; Jaiswal, N. (2009). Appl. Biochem. Biotechnol. [5] Yandri, A.S., D. Herasari, T. Suhartati. (2007). Jurnal Sains MIPA, Edisi khusus, 13 (2): [6] Yang CH, Cheng KC, Liu WH. (2003). Food Sci. Agric. Chem., 5: [7] Lowry, O.H., N.J., Rosebrough, A.L., Farr, R.J. Randall (1951). J. Biol. Chem., [8] Yandri A.S., T. Suhartati, S. Hadi. (2010). European Journal of Scientific Research. 39, [9] Hayashida S, Teramoto Y, Inoue T. (1988). Appl. Environ. Microbiol; 54: [10] Schwermann B, Pfau K, Liliensiek B, Schleyer M, Fischer T, Bakker EP. (1994) [11] Folia Microbiol 1985;30:452 [12] Gupta R., Gigras P., Mohapatra H., Goswami V. K., Chauhan B. (2003). Process Biochemistry 38(11):

ISOLATION, CHARACTERIZATION AND PURIFICATION OF AMYLASE ENZYME

ISOLATION, CHARACTERIZATION AND PURIFICATION OF AMYLASE ENZYME 1 Rekhani Jyoti Pal, 2 Dr. Surendra Sarsaiya 1 Research Scholar, Sri Satya Sai University of Technology and Medical Sciences, (India) ABSTRACT