Characterization of Alkaliphilic, Surfactant Stable and Raw Starch Digesting Α-Amylase from Bacillus subtilis Strain JS-16

J Microb Biochem Technol ISSN:1948-5948 JMBT, an open access journal Biomaterials: Down Stream Processing *Corresponding author: Dr. Kalpana Mody, Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute, Council of Scientific and Industrial Research (CSIR), Gijubhai Badheka Marg, Bhavnagar 364 002, Gujarat, India, Tel: +91-278-2561354; Fax: +91-278-2567562; E-mail: khmody@csmcri.org


Introduction
α-Amylases (E.C. 3.2.1.1) produced by plants, animals and microbes, hydrolyze α-1-4 glycosidic linkages in starch to dextrin, maltotriose, maltose and glucose. Amylases have been extensively used in food, fermentation, textile and paper industries with 30% production worldwide and 25% share in the enzyme market. Microbial amylases are preferred for industrial production considering the economics, faster production, wide range of operation parameters and minimum space requirements [1][2][3][4]. Demand for extremozymes with a blend of unique catalytic properties for specific biochemical processes is ever increasing. Extremozymes from alkaliphiles have found their applications in tanneries, paper and pulp, laundry, detergents and waste water treatment Bacillus sp. (B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens), a dominant and omnipresent representative of Phylum Firmicutes has been widely used source of microbial amylase [5,6]. Alkaline amylases are best suited as detergent additives and for starch saccharification in food and textile industries [7].
Present study deals with production of alkaline amylase by an alkaliphilic Bacillus subtilis strain JS-16 isolated from a soda ash industry located in west coast of Gujarat, India. Further, it was purified and characterized for its unique properties that make it a potential biochemical catalyst for various industries.

Bacterial identification and phylogenetic analysis
Morphological, physiological and biochemical characteristics of JS-16 was studied according to Bergey's Manual of Determinative Bacteriology [8]. Fatty Acid Methyl Ester (FAME) analysis was performed (MIDI Sherlock Microbial Identification System). For molecular identification genomic DNA was extracted by standard chloroform-isoamyl alcohol method [9]. PCR amplification of 16S rRNA was performed using forward and reverse primers as 8f (fD1) 5′-AGA GTT TGA TCC TGG CTC AG-3′ and 1495r (rP2) 5′-ACG GCT ACC TTG TTA CGA CTT -3′ respectively [10]. Reaction mixture for PCR amplification contained 10X PCR buffer 5 µl, 200 mM dNTPs 5 µl, 2.5 U Taq DNA polymerase, 20 pM of each primers (Sigma, India) and 50 ng of bulk DNA. Amplification was performed in a thermal cycler (Bio-Rad MyCycler, Thermal cycler, California, USA) for an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 58°C for 1 min and 72°C for 2 min and a final extension at 72°C for 5 min. Purified PCR product was sequenced for BLAST analysis [11]. 16S rRNA gene sequence was deposited in GenBank with accession number GQ280086. Phylogenetic analysis was done by MEGA 4.1 [12] software and tree was constructed using neighbor-joining method [13].

Effect of carbon sources
Amylase production was carried out using soluble starch, maltose, dextrin and sugar cane bagasse. M9 medium supplemented with 0.5% of the above mentioned carbon source was inoculated with the culture and incubated at 37°C for 96 h.

Amylase assay
Amylase activity was determined at 50°C for 15 min in 20 mM Tris-HCl buffer (pH-9.0) by measuring the release of reducing sugar from starch using Dinitrosalicylic acid (DNS) method [14]. In blank, enzyme was added after addition of DNS reagent. The absorbance was taken at 540 nm. One unit of amylase activity was defined as the amount of enzyme that produced 1 µmol of maltose equivalent per minute under specified conditions.

Protein estimation
Protein concentration was determined by Folin phenol method [15] with bovine serum albumin as standard. Protein content of the chromatographic fractions was measured at 280 nm.

Purification of Amylase
Ammonium sulfate precipitation and dialysis: Crude amylase was precipitated with 70% saturation of ammonium sulfate and kept overnight at 4°C. Precipitate was centrifuged and dissolved in minimum volume of 20 mM Tris-HCl buffer (pH 9.0). Dialysis was done using dialysis tubing (Sigma, D-0655).
Anion exchange chromatography: Prior to equilibration, the DEAE-cellulose (SRL, Mumbai, India) was activated by suspending in 0.5 M HCl, degassed for 20 min, and washed with distilled water till it was acid free followed by treatment with 0.5 M NaOH and finally washed with distilled water. This process was repeated thrice to activate the support. The support is loaded on the column (2 cm×18 cm) and was equilibrated with 20 mM Tris-HCl buffer of pH 9.0. The ammonium sulfate precipitated and dialyzed enzyme solution was loaded on the column and eluted with a stepwise concentration gradient of sodium chloride (0, 0.25, 0.5, 0.75 and 1 M NaCl) in the same buffer. The fractions, each 3 ml, were collected at a flow rate of 0.6 ml/min. Protein concentration and amylase activity of each fraction was determined. Active fractions were pooled.
Size exclusion chromatography: Pooled fraction (5 ml) from ion exchange chromatography was loaded to a Sephadex-G-100 (GE Healthcare, Uppsala, Sweden) column (1.25 cm×25 cm) preequilibrated with 20 mM of Tris-HCl buffer (pH 9.0) and then eluted with the same buffer. Fractions of 2 ml each were collected at a flow rate of 19 ml/h. Protein concentration and amylase activity of each fraction was determined. Active fractions were pooled, concentrated by lyophilization and checked for purity by SDS-PAGE.
Zymogram analysis was done by running native PAGE (10%). A 3% agarose plate was prepared into which 0.5% soluble starch was incorporated. After electrophoresis, the native PAGE gel was laid over the agarose gel and incubated at 40°C for 3 h. The agarose gel after incubation was overlaid with Lugol's Iodine solution to visualize the band.

Effect of pH and temperature on amylase activity
Optimum pH for amylase activity was determined at 50°C for 15 min using different buffers viz. citrate buffer (6.0), Mc Levine buffer (7.0), Tris-HCl buffer (8.0-9.0) and glycine-NaOH buffer (10.0-11.0). Stability of the purified enzyme with respect to varying pH (6.0-10.0) was determined by incubating in respective buffer for 30 min.
The optimum temperature for amylase activity was determined by incubating the assay system from 30°C to 80°C. Stability of the purified enzyme with respect to temperature was also determined by incubating the enzyme from 30°C to 80°C at pH 9.0 for 30 min.

Kinetic studies
The kinetic properties of amylase were determined using varying concentration of soluble starch. K m and V max values were calculated by Lineweaver Burk double reciprocal plot.

Raw starch hydrolysis by crude amylase
Raw corn, wheat and potato starch granules (10 mg) were separately mixed with 5 ml of the crude enzyme and final volume made up to 10 ml with 20 mM Tris-HCl buffer (pH 9.0). The reactions were incubated at 50°C in a shaking (100 rpm) water bath. Sample aliquots were collected after 4, 8, 12, 16, 20 and 24 h for the estimation of reducing sugars [14]. Degree of hydrolysis of raw starch (Rh) was defined by the formula: Rh (%)=(A1/A0)×100, where A1 was -amount of sugar in the supernatant after hydrolysis and A0 was the initial amount of raw starch [17].

Amylase production and purification
Soluble starch was the best carbon source for amylase production followed by dextrin and maltose (Figure 3). Sugar cane bagasse was not a good inducer. Bacillus sp. strain TSCVKK produced optimal amylase with 1% dextrin [18]. The three-step purification yielded 15.16 fold purification of amylase with 4.13% yield and specific activity of 13.5 Units/mg of protein (Table 2). 1.7 fold purification and 74% yield was reported from Bacillus subtilis WB600 recombinant amylase [19].

SDS-PAGE and zymogram analysis
The purification homogeneity assayed from native page revealed two bands of equal intensities corresponding to molecular mass of about 99 and 87 kDa. Two zones of clearance in zymogram supported the trends obtained in native PAGE. Correspondingly, four bands of molecular mass of about 35, 42, 48 and 58 kDa were obtained in the SDS-PAGE (Figure 4). Two bands with amylase activity in zymogram might be a possible explanation for the hetero-dimeric forms. Bacillus sp. A 3-15 amylase also reported two forms with molecular mass of 86 and 60.5 kDa after partial purification [20]. Similar results were

Effect of pH and temperature on amylase activity
Bacillus subtilis strain JS-16 amylase exhibited good activity from pH 8.0 (76.0% relative activity) to 9.0 with optimum at pH 9.0 ( Figure  5A). An optimum pH of 6.0 was reported by amylase from Bacillus subtilis AX20 [22]. After 30 min incubation at pH 9.0, approximately 48% residual activity was observed, which was drastically reduced after 60 min incubation ( Figure 5B). JS-16 amylase exhibited good activity from 20°C-80°C with optimum at 50°C. Moreover, 70% relative activity was seen at 80°C ( Figure 5C). Thermo stability test showed that about 80% residual activity was observed at 50°C which was drastically decreased at 60°C ( Figure 5D). This trend is in accordance to that of Nesterenkonia sp. strain F amylase [23].

Effect of additives and surfactants on amylase activity
Fe 3+ ions enhanced amylase activity while Hg 2+ strongly inhibited it. 55% residual activity was seen with Ca 2+ ions ( Figure 6A). JS-16 amylase was Ca 2+ ions independent similar to that from Bacillus subtilis AX20 [22] and in contrast to Bacillus sp. strain TSCVKK amylase [18]. Considerable activity was observed with PMSF and β-mercaptoethanol ( Figure 6B). Enhanced activity in PMSF was contradictory to a thermostable alkaline α-amylase from Bacillus sp. A 3-15 that was inhibited by 3 mM PMSF [20]. EDTA did not affect amylase activity indicating it as metal independent. SDS enhanced amylase activity by two-fold. In Triton X-100 and Tween 80, residual activity of 62% and 72% respectively was retained. Nesterenkonia sp. strain F amylase retained 90% activity on incubation with 0.1-0.5% SDS [23]. Similarly 82% and 80% activity was exhibited by Bacillus sp. A 3-15 and Bacillus sp. PN5 respectively, with 1% SDS [20,24]. 90% activity was retained by Bacillus sp. strain TSCVKK amylase with 0.1% Triton X-100 and Tween 80 [18]. Present study reports massive enhancement of amylase activity with SDS and greater stability in surfactants (Tween 80 and Triton X-100) making it a potential candidate for detergent market. However, when surf and tide were used as additives, amylase retained a very low residual activity of 27 and 13% respectively. Sensitivity to oxidants present in the detergents should be a plausible explanation for reduction in amylase activity. Increase of α-amylase activity in the presence of SDS might be the first of its kind to be reported.

Kinetic studies
Km value was 10 mg/ml and the Vmax was 0.2 g μmol/min/ml (11. 56 μmol/min/mg protein). Values of kinetic parameters differ with different substrates or assay conditions, and thus, the Km value of amylase from JS-16 was well within the range of other α-amylases (0.35-11.66 mg/ml) [25].

Conclusion
The Bacillus subtilis strain JS-16 isolated from the sludge samples of a soda ash industry in Gujarat produced α-amylase with promising properties. Activity of amylase at an alkaline pH, over a broad range of temperature and stimulation in the presence of SDS, are the key properties which could make this isolate a potential candidate for its use as a source of amylase suitable especially for the liquid detergent industry where SDS is largely used as surface active agent.