Isolation and characterization of a thermostable-amylase from Bacillus subtilis

The present study is aimed at assessing the ability of Bacillus subtilis to convert starch into reducing sugars. Maximum enzyme activity showed by B. subtilis was 236 Uml. B. subtilis showed optimum growth at pH 8 and optimum temperature for the growth of bacterial isolate was found to be 45°C. The optimal pH and temperature of the amylase activity were 7.0 and 37°C, respectively. The enzyme was found to be stable in the pH range of 5 to 10. Maximum -amylase activity was determined in 1% starch. The enzyme was found to be in two forms with relative molecular mass of 43 and 18 kDa in SDSpolyacrylamide gel electrophoresis. B. subtilis can be exploited for starch conversion biotechnologies.


INTRODUCTION
For years microorganisms have been the principal source of many different enzymes, which were identified after extensive research and currently find their main uses in industrial applications (Bon, 1995).The majority of industrial enzymes used belong to the hydrolase group, which are active on many natural substrates.Amylase (EC 3.2.1.1,-1,4-D-glucanohydrolyase) is capable of catalyzing the production of high yields of specific maltooligosaccharides on degrading starch and is of considerable commercial interest (Collins et al., 1993a).Thermostable -amylases have extensive commercial applications in starch processing, brewing and sugar production, desizing and textile industries and in detergent manufacturing process (Leveque et al., 2000).
Amylases can be obtained from several sources (Van der Maarel et al., 2002;Aquino et al., 2003).Thermophilic microorganisms have gained a great deal of attention recently (Becker et al., 1997;Beg et al., 2000).Enzymes from these microorganisms are of special interest since they are not usually denatured by high temperatures and are even active at elevated temperatures (Adams and Kelly, 1998;Fitter and Heberle, 2000).They are usually stearothermophilus (Boyer and Ingle, 1972;Sajedi et al., 2005).The properties of each -amylase such as *Corresponding author.E-mail: rehman_mmg@yahoo.com.Tel: 92-42-9231249.industrial applications such as Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus thermostability, pH profile, and Ca-independency must be matched to its application.For example, -amylases used in starch industry must be active and stable at low pH but in detergent industry at high pH values (Nielsen and Borchert, 2000).
The present study is concerned with the isolation and characterization of the -amylase from B. subtilis.Some properties of the amylase are also determined.

Sample collection and screening of -amylase producing bacteria
Bacteria for the production of -amylase were isolated from 20 soil samples collected from different industries, gardens and fruit markets (Pakistan).One gram of soil sample was added to a glass tube containing 10 ml sterilized distilled water, shaked and placed in a water bath at 80°C for 15 min.After cooling, 50 µl of the sample was spread on 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 adjusted at 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.The colonies forming clear zones around them were picked up and streaked on nutrient agar plates to get pure culture and to confirm zone formation.

Determination of optimum growth conditions
For optimum growth of the bacterial isolate, two parameters that are temperature and pH were considered.For determination of optimum temperature, 5 ml LB broth was added in 4 sets, each of three test tubes, autoclaved and inoculated with 20 µl of freshly prepared culture of bacterial isolate by overnight growth at 37°C in LB broth.The four sets of tubes were incubated at 20°C, 30°C, 37°C and 45°C.After an incubation period of 12 h, their absorbance was taken at 600 nm using a LAMBDA 650 UV/Vis Spectrophotometer (PerkinElmer, USA).For determination of optimum pH, test tubes having 5 ml LB broth were prepared in 6 sets, each containing 3 test tubes and their pH was adjusted at 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 then autoclaved.These tubes were inoculated with 20 µl freshly prepared culture of bacterial isolate.After an incubation period of 12 h, their absorbance was taken at 600 nm.

Effect of starch on bacterial growth
Growth curves of B. subtilis were determined in 1% starch and in LB broth medium.For bacterial isolate 100 ml medium was taken in one set consisting of 3 flasks, autoclaved and then inoculated with 100 µl of the freshly prepared inoculums.The cultures were incubated at 45°C in an incubator shaker at 150 rpm.An aliquot of culture was taken at regular intervals (0, 4, 8, 12, 16, 20, 24 and 28 h) to measure absorbance at 600 nm.

Physical, biochemical and molecular characterization
The isolate was Gram stained.For biochemical characterization the isolate was tested for Voges-Proskauer test, sporulation test and utilization of different sugars.The procedures adopted for all the above physical and biochemical tests were taken from Cappuccino and Sherman (2001).For molecular characterization genomic DNA was extracted as described by Carozzi et al. (1991) and the 16S rRNA gene was amplified by PCR using 16S rRNA primers (RS-1; 5 -AAACTC-AAATGAATTGACGG-3 , and RS-3; 5 -ACGGGCGGTGTGTAC-3 ) (Rehman et al. 2007).PCR was performed by initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and a final extension at 72°C for 5 min.The PCR product of 0.5 kb was removed from the gel and cloned in pTZ57R/T vector.The amplified 16S rRNA gene was purified with a Fermentas purification kit (#K0513) and the amplified products were electrophoresed on 1% agarose gel.Sequencing was carried out by Genetic analysis system model CEQ-800 (Beckman) Coulter Inc. Fullerton, CA, USA.The 16S rRNA gene sequences were compared with known sequences in the GenBank database to identify the most similar sequence alignment.

Amylase activity 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 phos-phate buffer (pH 7.0) containing 1% soluble starch and incu-bated 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 (Yang et al., 2003) followed by Shafaat et al. 3335 boiling for 10 min and to develop colour.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 mol glucose per minute under the assay condition.All analytical measurements were performed at least in triplicates.

Effect of pH, temperature and various substrates on amylase activity
The effect of pH on the activity of -amylase was measured by incubating 0.5 ml of the diluted enzyme and 0.5 ml of phosphate buffer ranging pH from 5 to 10, containing 1% soluble starch for 5 min at 100ºC.The effect of temperature on the enzyme activity was determined by performing the standard assay procedure for 10 min at pH 8.0 with in a temperature range of 30 to 100ºC.After treatment the residual enzyme activity was assayed.
The amylase activity was also assayed by measuring the reducing sugar released during the reaction, using complex polysaccharide substrates (starch, carboxymethylcellulose, molasses and sugarcane bagasse).The reaction mixture contained 50 µl of 1% solution of the substrates separately prepared in 2 mM imidazole-HCl buffer of pH (7.0) and 250 µl of enzyme solution.The reaction was stopped by adding 100 µl dinitrosalicylic acid solution after incubation at 37°C for 1 h.The reaction mixture was then heated in boiling water for 5 min and the absorbance was taken at 540 nm after cooling at room temperature.

SDS-polyacrylamide gel electrophoresis
Protein profile of bacterial cells, grown with 1% starch and without starch, was done to compare the inducibility of amylase.The crude enzyme was precipitated with ammonium sulphate (60%) and allowed to stand over night at 4°C with constant stirring.The mixture was centrifuged at 10,000 g for 20 min at 4°C.The precipitate was collected, re-dissolved in distilled water.The proteins of B.subtilis were isolated according to Rehman et al. (2009), estimated according to Lowry et al. (1951), and electrophoresed according to Laemmli (1970).Aliquots of 15 to 20 µl were loaded onto a 12% SDS polyacrylamide linear resolving gel overlaid with a 6% stacking gel.Gels were stained with Coomassie Brilliant blue R-250.

Statistical analysis
Observations were made and all the experiments run in triplicate.At least three separate flasks were usually maintained for one treatment.Each time three readings were taken, their mean, and standard error of the mean were calculated.

Isolation and identification of the bacterial isolate
A total of 20 soil samples collected from different ecological environments were analyzed for bacteria producing amylase.About 28 cultures of Bacillus species were isolated, purified and screened for the production of -amylase in 1% starch.Of all the cultures tested, bacterial isolate SA-J3 gave the maximum hollow zone around the agar plates containing 1% starch and was selected for further studies.Table 1 shows some physical and biochemical characteristics of the bacterial isolate.
On the basis of this characterization the bacterial isolate has been identified as Bacillus sp.
The partially amplified (500 bp) and sequenced 16S rRNA gene from local isolates (SA-J3) was uploaded to the NCBI (National Center for Biotechnology Information) website to search for similarity to known DNA sequences and to confirm the species of the local isolate.The nucleotide sequences coding for the 16S rRNA gene after BLAST query revealed that this gene is 94% homologous to Bacillus subtilis (SA-J3).The nucleotide sequences coding for the 16S rRNA gene of B. subtilis have been submitted to the GenBank database under accession numbers JF804982.

Optimum growth conditions
B. subtilis showed optimum growth at pH 8 and the most suitable temperature for the growth of bacterium was found to be 45°C.The growth curve pattern was studied by growing the bacterium in 1% starch and comparing with the control culture in which no starch was added.The growth pattern of B. subtilis was significantly different from those of control and the growth rate of bacterial isolate was higher in the presence of 1% starch as compared to growth in the presence of LB medium.The maximum growth (O.D) was determined after 16 and 20 h of growth in 1% starch and LB medium, respectively (Figure 1).This shows that the bacterium has efficient ability to convert starch into its sub-products and eventually into glucose.

Effect of pH, temperature and various substrates on amylase activity
The effect of pH on -amylase activity as a function of pH  is shown in Figure 2. Optimum pH was found to be 7 where the enzyme activity was 199 Uml -1 . The enzyme activity at pH 5.0 and 10.0 were 151 and 152% of that at pH 7, respectively.The crude enzyme was found to be stable in the pH range of 5.0 to 10.0.These results suggest that the activity of enzyme is fairly higher in alkaline pH, making this enzyme attractive for the detergent industry.The optimum temperature, pH and incubation period for amylase production in B. subtilis were 50 to 70ºC, 5.0 to 9.0 and 36 h, respectively (Swain et al., 2006).lpha-amylases produced by other Bacillus sp. have shown optimum activities at pH values as low as 3.5 or as high as 12 (Burhan et al., 2003;Konsula and Liakopoulou-Kyriakides, 2004).Figure 3 shows the activity of the enzyme assayed at temperatures ranging from 30 to 100°C at pH 7 and starch concentration of 1%.A slow decrease in enzyme activity was observed at values above 70°C.The optimum temperature of this -amylase was 37°C (236%), which was higher or similar to that described for other Bacillus -amylases (Sidhu et al., 1997;Burhan et al., 2003;Konsula and Liakopoulou-Kyriakides, 2004).The residual activities of crude amylase at 90°C was 169%, maximum enzyme activity lost as compared to optimum temperature (37°C) was 67%.
Figure 4 shows the substrate specificity of -amylase.Different polysaccharides at concentration of 1% were used and it was found that maximum -amylase activity was observed in 1% starch.

SDS-PAGE analysis
In the present study two alkaline -amylases with molecular weights of 45 and 18 kDa were observed in SDS-PAGE (Figure 5).Swain et al. (2006) reported the purified -amylases from B. subtilis were in two forms with molecular mass of 18.0 ± 1 and 43.0 ±+ 1 kDa in native SDS-PAGE.Shaw et al. (1995) reported the protein of 59 kDa in Thermus sp.Yang and Liu (2007) reported amylase as a single band of about 65 kDa by SDSpolyacrylamide gel electrophoresis.Murakami et al. (2007) reported two alkaline thermotolerant -amylases having molecular masses of 105 and 75 kDa, respectively.A protein with molecular weight of 53 kDa was found in B. subtilis WB600 (Liu et al., 2008).Another protein with molecular weight of 51.4 kDa was reported by Wang et al. (2008).

Conclusion
In the present study we have isolated -amylase, from locally isolated B. subtilis, with maximum enzyme activity of 236 Uml -1 . The optimal pH and temperature of the enzyme were 7.0 and 37°C.Two forms with molecular masses of 45 and 18 kDa were determined by SDS-PAGE and the enzyme was stable at 30 to 100°C and at pH 5.0 to 10.0.The highest -amylase activity was determined in 1% starch.B. subtilis can be exploited for industrial biotechnologies.

Figure 1 .
Figure 1.Effect of 1% starch on the cell growth of B. subtilis in LB broth medium after incubation at 37°C.

Figure 2 .
Figure 2. Effect of pH on the -amylase activity.

Figure 5 .
Figure 5. SDS-PAGE pattern of extracellular ammonium sulfate precipitated proteins of B. subtilis.M, protein marker; T, treated (with 1% starch) and C, control (without starch).The gel is 12% stained with Coomassie blue.

Table 1 .
Morphological and biochemical characteristics of bacterial isolate.