Production of pullulanase and alfa-amylase enzymes from Bacillus sp. isolated from cow milk, determination of some optimization conditions of these enzymes

Use of enzymes has been attracted considerable interest specially inbiotechnological applications. -Amylase and pullulanase are important biotechnologicalenzymes that are used in industry such as in food, detergent, textile, paper and beer, etc.In the present study, extracellular enzymes that are secreted by a microorganism,which is isolated from cow milk and identified as G (+) Bacil, were determined bybiochemical methods. Bacteria was produced by Submerged Fermentation (SmF) process.-Amylase, pullulanase, -galactosidase and protease activity were done from supernatant.The best activity was observed in -amylase and pullulanase and therefore, optimization ofthese enzymes was carried out for further studies.Appropriate incubation time, optimum pH and optimum temperature were found tobe 48 h, 6.8 and 37 oC for -amylase and 44 h, 6.4 and 37 oC for pullulanase activity,respectively.The effect of different nitrogen and carbon containing substrates on the productionof these enzymes were tested in Bacillus sp. obtained results indicated that starch as acarbon containing substrate was caused to increase the -amylase production while starchand pullulan increased pullulanase production.Nitrogen containing substrates such aspepton, beef extract and trypton were caused to increament in -amylase production whilepepton only caused to increament in pullulanase production.Effect of various starches and flours on the production of enzymes were examinedand the result showed that both potato starch and wheat flour were caused to increase in theproduction of these two enzymes.Finally, the effect of metal ions on enzyme activity was tested. It was found that -amylase was activated by %103 with 10 mM Ca+2 and pullulanase was activated by %105with 50 mM Ca+2. -Amylase was inhibited by %85.89 and %71.25 with 50 mM Mn+2 and10 mM Zn+2, respectively. Pullulanase was inhibited by %97.68 and %97.14 with 10 mMand %50 mM Zn+2, respectively.Keywords: -amylase, pullulanase, optimization parameters, Bacillus sp.