Production and Characterization of a Thermostable and Ca++ Independent Amylase Enzyme from Soil Bacteria

Aims: Present study was done with the aim of isolation of efficient amylase producing bacterial isolates from soil. Amylase enzyme production, purification, SDS-PAGE and characterization of different important parameters as pH, temperature, substrate concentration etc. was done for obtaining maximum enzyme activity.
Study Design: An experimental study.
Methodology: Isolation was done by serial dilution and spread plate method on nutrient agar at 50ºC and 11 pH. Then screening of bacterial isolates for amylase production was done using starch agar media and on addition of iodine solution bacterial colonies confirmed the amylase enzyme production by decolourization of media from purple to white due to starch hydrolysis. Bacterial isolates with highest zone of starch hydrolysis were selected for enzyme production. Amylase production was done using amylase screening broth at 50ºC, at 250 rpm for 72 h in incubator shaker. Enzyme activity was determined after centrifugation of the culture broth at 10,000 rpm for 10 min. Identification of bacterial isolate was done by performing different morphological and biochemical tests. Then enzyme purification was done using acetone and ammonium sulphate precipitation methods. Molecular weight determination was done by SDS-PAGE. For obtaining maximum enzyme activity evaluation of optimum values for important parameters such as pH, substrate concentration, temperature, reaction time, thermostability, effect of cations, effect of chelating agents and different raw starches was done.
Results: Bacterial isolate 16A showed the highest zone of starch hydrolysis and maximum amylase production (48.86 UmL-1) was obtained on 48 h of incubation at 50ºC. On the basis of morphological and biochemical characterization 16A was identified as Bacillus sp. Enzyme showed 1.6 fold purification with acetone precipitation and 1.54 fold with ammonium sulphate precipitation. Molecular weight of amylase enzyme determined was 97.4 kD approximately, by SDS-PAGE. Enzyme characterization showed that maximum enzyme activity was obtained at 15 minutes reaction time, 75% substrate concentration, 7 pH and 50ºC temperature. A good thermostability was showed by amylase enzyme for 24 h at 50ºC and 15 h at 100ºC. The enzyme activity was enhanced by Fe++ (2 X), Mn++ (3 X) and Triton X-100 (2 X) while was completely inhibited Zn++, Hg++, Cu++, Fe++, Co++, SDS, tween-20 and EDTA. On enzyme characterization maximum enzyme activity of 138 UmL-1 has been observed that is a very good activity.
Conclusion: On the basis of results during present study this is visualized that the enzyme is very suitable for many industrial applications because of its desirable qualities for its industrial applicability as Ca++independent nature and thermostability at 80-100ºC. The broad range of pH and moderate thermostability makes this amylase enzyme a useful additive to liquid detergents that can function in hard and warm water. Calcium independent amylase is suitable for manufacturing of fructose syrup, where Ca++ is an inhibitor of glucose isomerase. So this enzyme can be very useful in different industrial applications.