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Molecular cloning, overexpression and biochemical characterization of bacterial amylase for biotechnological processes
Amylases are the enzymes that act on glycosidic bond of starch and related polysaccarides. They comprise 25% of enzyme utilised in a variety of industry. It is used to obtain maltose, glucose and maltodextrins in various lenghts during industrial processes. Amylases are widely distributed enzymes in bacteria, fungi, higher plants and animals. Thermophilic enzymes are widely demanded in order to be stable at harsh process conditions. Isolating these enzymes from thermophilic microorganism is increasing trend because of ease of enzyme production. In this study Î±-amylase gene region from a thermophilic Bacillus sp. isolated from Balçova Geotermal region in İzmir was cloned to compotent E. coli BL 21 cells. Additionally protein expression was reinforced with pKJE7 chaperone plasmid. Cloned gene was sequenced and found as 1542 bp in length. Thermophilic amylase that has a 59.9 kD molecular weight was expressed and purified from this recombinant strain. Mass spectrometric analysis were performed and the enzyme was matched with Î±-amylase family protein of Geobacillus thermodenitrificans NG80-2 using NCBInr database. The aminoacid sequence of this enzyme was seen to be similar 92% with our obtained enzyme. According to the results of characterization studies, the amylase enzyme was seen to have highest activity at pH 8.0 and 60Â°C. The enzyme was also showed to have resonable activity between pH5 and 9. 85% of the enzyme activity was retained at 70Â°C. Furthermore, amylase activities at 65 and 85Â°C were observed to remain stable for 5 and 2 hours, respectively. It was also showed that the activity was stable and pH7 and 9 for 6 hours. The effects of some metal ions, chemical agents and organic solvents on enzyme activity were examined so, Co+2, Mg+2,Ca+2 was determined to be as inducer for the enzyme activity. Conversely the activity was inhibited by Cu+2. Furthermore methanol, DDT and Triton X-100 was found to have no effect on the enzyme activity.