Production and characterization of thermostable α-amylase from a newly isolated strain of Bacillus subtilis KIBGE-HAR

摘要

Investigation on the fermentation conditions for Alpha-amylase (1,4-α-D-glucan glucanohydrolase, E.C. 3.2.1.1) production was carried out with Bacillus subtilis KIBGE-HAR and a optimal synthetic medium for enzyme production was developed. Alpha-amylase production and cell population reached maximum after 24 hours of cultivation. The optimum temperature and pH for enzyme production were found to be 50°C and 7.0 respectively. Starch (15 g/l), which was used as a carbon source, supported the maximum production of enzyme. Peptone was used as a nitrogen source and the best concentration of peptone for α-amylase formation was found to be 5 g/l. High α-amylase titre was obtained in medium supplemented with 1.0 g/l yeast extract. Calcium chloride was added in the medium as a stabilizer and 0.2 mg/dl CaCl2 was found to be the most favorable concentration for ?-amylase production and stability. Among different reference media tested for ?-amylase production, medium 3 gave the maximum yield of enzyme and our own optimized medium 4 was proved to be optimal for ?-amylase production in contrast with the reference media. The optimum temperature and pH of the enzyme were found to be 60oC and 7.0 respectively. The most suitable buffer system for pH maintenance was proved to be Tris-HCl buffer (50 mM). Velocity of reaction reached to maximum in the presence of 2 % substrate i.e. starch. Gene Bank Accession: EU819144 (16S rRNA gene sequencing for Bacillus subtilis KIBGE- HAR) Introduction The α-amylase (E.C. 3.2.1.1) randomly hydrolyzes alpha 1,4 glucosidic linkages in starch, glycogen and related polysaccharides yielding dextrins, oligosaccharides, maltose and D-glucose (Takeshita et al., 1975). Bacterial α-amylases are extensively important in industrial processes such as production of ethanol and high fructose corn syrup, baking, in laundry washing powders and dish washing detergents, textile desizing, and paper recycling (Nigam and Singh, 1995).Thermostable enzymes are more versatile rather than thermolabile (Fogarth et al., 1974) as they have higher operational stability and a longer shelf life at elevated temperatures (Niehaus et al., 1999). Therefore the thermophilic microorganisms are of special interest for producing thermostable α- amylase, which can be use in a wide array of industrial processes (Chandra et al., 1980; McMohan et al., 1997). Bacteria belonging to the genus Bacillus have been widely used for the commercial production of thermostable α-amylase. These include B. coagulans, B. stearothermophilus, B. caldolyticus, B. brevis, B. acidocaldarius and B. thermoamyloliquefaciens (Campbell, 1954, 1955).Due to the increasing demand for thermostable α-amylase in various industries, it has been produced and characterized from different sources. The characteristics of α-amylase, such as its thermostability and pH profile should match its application.In this regard it is essential to work on the conditions that lead to the bulk production of thermostable amylase for industrial applications and to search for α-amylase with improved properties. Therefore, the present study was carried out to optimize the fermentation conditions for the production of α-amylase from Bacillus subtilis KIBGE-HAR and to characterize the enzyme produced in the culture supernatant as well as comparison of different reference media with that of our own optimized medium for the production of α-amylase. Materials And Methods OrganismThe strain was isolated from air and pure culture study was performed. A pure culture of Bacillus subtilis KIBGE-HAR was used and stock culture was maintained on nutrient agar slant and for alpha amylase production the strain was weekly revived in 5 ml starch broth. Media CompositionFor α-amylase production, a culture medium was prepared containing (g/l): 15.0 soluble starch, 1.0 Yeast extract, 5.0 Bacto Peptone, 0.5 MgSO4, 0.5 NaCl and 0.002 CaCl2. The pH of the medium was adjusted to 7.0 before sterilization (Aliya et al. 2007).Produ