Abstract —Microbial α-amylase is a highly demanded industrial enzyme with extensive commercial applications in various sectors. Studies were carried out with a bacterial strain producing extracellular α-amylase, isolated from rhizospheric soil of Euphorbia hirta. The isolate was gram positive, motile rod, bearing terminal endospore. It exhibited >98% similarity with the reference strains in the GenBank. The phylogenetic tree constructed on the basis of 16S rRNA gene sequences revealed that it clustered with the closest members of Bacillus subtilis and identified as Bacillus subtilis KC3. Furthermore, the effects of incubation period, temperature, pH, different carbon and nitrogen sources, metal salts and different substrate concentrations of the medium were optimized. The maximum enzyme production was found after 48 h (22.92 U/ml) of incubation at temperature 40°C and pH 7. The optimal temperature and pH for enzyme activity were 50°C and 6.5 respectively. Barley starch (27.27 U/ml) was observed to be the best inducer followed by corn starch (24.30 U/ml) and maltose (19.10 U/ml). The addition of glucose to the culture medium greatly reduced the synthesis of α-amylase (5.45 U/ml) which demonstrates that a classical glucose effect is operative in this organism. The effects of different metal ions (Ca 2+, Fe 2+ , Mg 2+ , Zn 2+ and Cu 2+ ) on amylolytic activity were investigated and it was found that 0.1% of Ca 2+ increased enzyme production (28.83U/ml), whereas other metal ions exhibited inhibitory effects. The enzyme production was maximum at 2% substrate (starch) concentration, which declined beyond it. These characteristics of Bacillus subtilis KC3, suggest that this is a promising isolate which merits further investigations for potential applications in various biotechnological processes. Index Terms—α-amylase, bacillus subtilis KC3, euphorbia hirta, rhizospheric soil I. INTRODUCTION Amylases constitute a group of industrial enzymes, which alone covers approximately 30% of the enzyme market. They have opened new frontiers of many commercial biotechnological processes including renewable energy, pharmaceuticals, saccharification or liquefication of starch, detergent industries, warp sizing of textiles, fibres, paper industries, foodstuffs, baking, clarification of haze formed in beer or fruit juices and for pretreatment of animal feed to improve digestibility [1]- [6]. Amylases are known to be produced by a variety of bacteria and fungi and their applications at industrial level have stimulated interest to explore their amylolytic activity in several microbes to be used as bioresources [2], [7]-[9]. The horizon got further Manuscript received August 10, 2012; revised September 20, 2012. The authors are with the Department of Botany, P. U., Patna- 800005, Bihar, India (e-mail: vijayalakshminishant@gmail.com; ksushmabt@gmail.com; Tel. +91-9470412424, singh.abhabt@gmail.com; Tel. +91-9430458247, cp_ptc@yahoo.co.in). magnified with the discovery of new strains of microorganisms and development of more efficient production strategies because microbes have substantial potential to contribute to several commercial purposes. Moreover, microbial amylases have a broad spectrum of industrial applications as they are more stable with great genetic diversity, high enzymatic activity in a wide range of conditions (extreme pH, temperature, osmolarity, pressure etc.), simple and cost effective production and easy manipulation to obtain enzymes of desired characteristics [10] -[12]. The production of microbial amylases from bacteria is dependent on the type of strain, composition of medium, method of cultivation, cell growth, nutrient requirements, incubation period, pH, temperature, metal ions and thermostability [8]. In fact, such industrially important microorganisms found within the genus Bacillus, can be exploited commercially due to their rapid growth rate leading to short fermentation cycles, capacity to secrete proteins into the extracellular medium and safe handling [8]. Bacillus is endowed to produce thermostable α-amylase and also large quantities of other enzymes. Indeed, 60% of commercially available enzymes are obtained from different species of Bacillus i.e. B. subtilis, B. stearothermophilus, B. licheniformis and B. amyloliquefaciens [11]. Some Bacillus strains produce enzyme in the exponential phase, whereas some others in the mid stationary phase. Though, different Bacillus species have similar growth patterns and enzyme profiles, but their optimized conditions vary, depending upon the strain. The objectives of the present study are to screen Bacillus species isolated from Euphorbia hirta rhizosphere in order to study their suitability with regard to α-amylase production. II. MATERIAL AND METHODS A. Sampling The samples of rhizospheric soil of rooted Euphorbia hirta were collected from the Research Garden, Department of Botany, Patna University, Patna (25°36ƍ39.6ƎN 85°08ƍ38.4ƎE) and stored for further study. B. Isolation, Screening and Characterization Bacteria were isolated by serial dilution and streak plate methods. The aliquots (0.1 ml) were plated in triplicates on Nutrient Agar (NA) medium [(w/v) 0.5% peptone; 0.3% beef extract; 0.5% NaCl; 1.5% agar, pH 7] and incubated at 30±2°C for 72 h. The nutrient agar plates containing 1% starch (Starch Agar plates) were inoculated with test bacterial isolates and incubated at 30±2°C. The α-amylase producing Isolation and Characterization of Bacillus Subtilis KC3 for Amylolytic Activity Vijayalakshmi, K. Sushma, S. Abha, and P. Chander International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 2, No. 5, September 2012 336