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STUDIA UNIVERSITATIS BABEŞ – BOLYAI, BIOLOGIA, LV, 1, 2010, p. 119-130 OBTAINING AND CHARACTERIZATION OF AN α-AMYLASE BY A STRAIN OF BACILLUS SUBTILIS MIHAIL DRĂGAN-BULARDA 1 , RAHELA CARPA 1 , MIHAELA MIŞCA 1 and VICENłIU CĂLEAN 1 SUMMARY. This paper presents the results of obtaining a Bacillus subtilis strain, by UV irradiation, a strain that should produce alpha-amylase. We had in view the increase of biomass on a mineral medium with yeast extract, to which soluble starch 2% was added, and we used therefore, an installation with fermentation pot. The production of alpha-amylase was monitored, determining the maximum specific increase rate, respectively the relation between the increase of the cell mass and the enzyme production. We had also in view to obtain a raw enzymatic preparation and a partial purified one, for which K m and V max of reaction were determined. The enzyme production proved to be closely correlated with the quantity of biomass. The bacteria culture reached constant production efficiency after the lag phase. The raw enzymatic preparation presented a slightly higher alpha-amylase activity than the partial purified one, probably because of the starch traces present in the raw enzymatic extract. Keywords: α-amylase, Bacillus subtilis, characterization, enzyme kinetics, fermentation, purification Introduction Amylases are among the most important starch-converting enzymes, and they can be derived from several sources. However, bacterial and fungal amylases have dominated applications in the industrial sector (breweries, alcohol production, bakeries, juice production from various fruits) (Vasilescu, 1963; Pandely, 2003; Drăgan-Bularda and Samuel, 2008). Also, the amylolytic enzymes find a wide spectrum of applications in food industry for production of glucose syrups, high fructose corn syrups, maltose syrups, reduction of viscosity of sugar syrups, reduction of haze formation in juices, and find a wide range of application in baking, paper, textile and detergent industry (Sivaramakrishnan et al., 2006). The alpha-amylase hydrolyses the α-1.4 bonds within the amylose and amylopectin chains (dextrinogenous amylases), resulting many maltodextrins, some maltose and 1 Babeş-Bolyai University / 1 Kogălniceanu St., 400084 Cluj-Napoca, Romania e-mail: mihai.dragan@ubbcluj.ro e-mail: k_hella@yahoo.com e-mail: vicentiu_84@yahoo.com
M.. DRĂGAN-BULARDA,. R. CARPA, M. MIŞCA, V.-CĂLEAN glucose from amylose, respectively, many maltodextrins and limit dextrin (with ramifications through α-1.6 bonds), some maltose and glucose from amylopectin (Baig et al., 1984; Banu et al., 1987; Dan, 2000; Jurcoane, 2000; Malhontra et al., 2000; Iorga and Câmpeanu, 2004; Drăgan-Bularda and Samuel, 2008). The α-amylase produced by bacteria is a metalloenzyme with high molecular mass of about 50,000 Da, characterized by the presence in its structure of some inorganic ions, related to the polypeptide chains (Zarnea et al.,1988). These are Ca 2+ (1-3) ions for stabilizing the structure and 1-2 Zn 2+ ions that determine the dimer formation of the enzyme (www.resb.org). Due to the fact that it is a protein, the biosynthesis of the alpha-amylase takes place at ribosome level and is secreted by the bacteria in the culture medium (Drăgan-Bularda and Samuel, 2008). In the last decades, more and more microorganisms were used as potential sources for various biotechnologies (Zarnea and Dumitru, 1994). Bacterial α-amylase, which are usually produced by bacteria belonging to Bacillus genus (Nigam and Singh, 1995) are widely used in the starch-processing industry for the hydrolysis of starch (Lonsane and Ramesh, 1990). There were tested conditions for optimizing the biosynthesis of some enzymatic complexes (protease-amylase complex) by using Bacillus subtilis strains (Bahrim and Dan, 1994; Dan et al., 1994), respectively, there was studied the influence of the input of aminoacids on the biosynthesis of some enzymes, among these also the alpha-amylase on Bacillus subtilis strains, or the effect of some water-soluble vitamins on the biosynthesis in case of fermentation with Bacillus subtilis strains. In most cases the enzymatic process is inhibited by high substrate and product concentration and also instability of the enzyme repetitive or prolonged use. Immobilization is an important technique for continuous and repeated use of enzymes in industrial application and also rapid separation of the enzyme from the reaction medium. The general methods employed for immobilization are entrapment, microencapsulation, copolymerization, cross-linking, physical adsorption, chemical attachment and covalent binding (Gangadharan et al., 2009). Tests were conducted for immobilizing some microbial hydrolases with protease and amylase activity on collagen support (Tcacenco et al., 1994). α-amylase produced by Bacillus amyloliquefaciens ATCC 23842 was immobilized in calcium alginates. Also, alginate beads have been successfully used for entrapment of α- amylase of Bacillus subtilis and effectively used for starch hydrolysis (Rajagopalan and Krishnan, 2008). Immobilization is a very effective alternative in overcoming problem of instability and repetitive use of enzymes. Entrapment method of immobilization is advantageous over other methods that do not involve chemical modification of the enzyme (Gangadharan et al., 2009). It should be emphasized that the application of α-amylase in different industrial processes demands specific characteristics including thermostability, particular pH activity profiles, pH stability and Ca-independency, consequently, demand for novel amylases is increasing worldwide, as the variety of enzyme applications is increasing in various industrial sectors (Pandely et al., 2000). Industrially 120
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