Alkaline active α-amylases from alkaliphilic bacillus sp.: screening and enzyme prouction
The term alkaliphiles defines microorganisms that grow optimally at pH values above 9, often between 10 and 12, but are unable to grow or grow slowly at the near neutral pH value of 6.5. Consequently, their extremozymes such as amylases also have high catalytic efficiency and stability at an alkaline pH range of pH9- 11. These alkaline active amylases have potential applications for hydolysing starch under high pH conditions in the starch-based industries and also as ingredients in enzyme-based detergents. Potential application of alkaliphilic amylases has led to their bioprospecting from the microorganisms found thriving in naturally occurring alkaline environments such as soda lakes found along the East African Rift valley. In this study, five alkaliphilic bacteria designated Bacilus sp. LBW 213, Bacilus sp LBW 2.719, Bacilus sp LBW 33, Bacilus sp LBW 35 and Bacilus sp LBW 313 previously isolated from Lake Bogoria, a soda lake found in the east African Rift Valley in Kenya were screened for the production of alkaline-tolerant amylases. The five Bacilli sp were grown on solid Horikoshi medium pH (10.5) for 72 h, after which the plates were flooded with Gram’s iodine solution in order to identify amylase-producers. All 5 bacteria exhibited extracellular starch hydrolysing activities, as depicted by the presence of clearance zones or ‘halos’ around their colonies after staining with Gram’s iodine solution. The ratios of the diameter of halos to that of colonies were then used as a semi-quantitative method for classifying the bacteria. Bacillus sp. LBW 33 and LBW 35 were considered as excellent producers while the rest were very good producers. Based on this analysis, all the bacteria were considered for further experiments. The five Bacilli sp. from Lake Bogoria in the presence of soluble starch were cultivated in liquid broth at 37oC, 100rpm in a shaker incubator for up to 48h, during which samples were withdrawn after every 12h to determine the cell growth by measuring their optical density at 600nm in order to generate characteristic bacterial growth curves. During each withdrawal, cells were harvested by centrifugation and the cell free culture supernatants used as crude enzyme source for extracellular amylase assay using a glucose standard. All the bacteria exhibited both growth and α-amylase enzyme production. During the cultivation, all five bacteria exhibited a general growth profile, reaching optimal growth at 24 h with maximum OD at 600nm of 4.370(Bacillus sp.LBW 213), 4.670 (Bacillus sp. 27 19), 5.206(Bacillus sp.LBW 33), 6.10(Bacillus sp.LBW 35) and 6.310(Bacillus sp.LBW 313). Page | 8 There was a general increase in α-amylase production by most of the bacteria, reaching optimum levels during late exponential phases of bacterial growth (24 h) for most of the bacteria except for Bacillus sp.LBW313, during cell death phase (36 h). Therefore, in this study the range of enzymatic activity for all the five bacteria was 5.5 x 10-3 – 38 x 10-3U/ml. Although production levels by the bacteria were low compared to those of other alkaliphilic bacteria studied, Bacillus sp. LBW 33 was the most promising candidate for enzyme production (highest producer), thus making it ideal for further studies.