ISOLATION AND CHARACTERIZATION OF AMYLASE ENZYME FROM LICHEN PARMELIA SP COLLECTED FROM ARUNACHAL PRADESH

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ISOLATION AND CHARACTERIZATION OF AMYLASE ENZYME FROM LICHEN PARMELIA SP COLLECTED FROM ARUNACHAL PRADESH

Journal Title: Journal of Drug Discovery and Therapeutics - Year 2013, Vol 1, Issue 2

Abstract

The plant material (lichen) was collected from the hills of the Arunachal Pradesh and it was identified as the species of the genus Parmelia. The lichen was crushed in the extraction buffer in order to isolate the enzyme. The extracted material was first tested for the presence of the protein by Lowry’s method which showed positive result. The extracted material was now tested for the presence of the enzyme amylase. 1% starch solution was used as substrate in order to test the activity of amylase. In our first experiment we wished to optimize the amount of the crude extracted enzyme, amylase from the plant material. Here we used different volumes of the extract i.e 1.0 ml, 2.5 ml, 5.0 ml, 7.0 ml and 10.0 ml [Table 1]. From the observation it could be inferred that the optimum volume of crude extract was 5ml. This is because as we increased the volume above 5ml the activity of the enzymes didn’t showed any significant increase. Therefore we used 5ml of crude extract in our further experiments. We tried to find out the time of incubation for the maximum activity of the enzyme.The time of incubation was varied from 1min to 20 min. [Table 2]. The activity increased from 1min to 10 min. But we found that from 15mins onwards the amount of starch hydrolyzed started to decrease. However we were not able to cover the short time intervals between 10 min to 15mins which may show the maximum activity. Therefore, we optimized the incubation period of 10 min only for our further experiments.The use of any enzyme for the commercial purpose depends on the temperature in which it shows its maximum activity. Our next aim was to optimize the temperature for the maximum activity of the crude extracted amylase from the foliose lichen. In our experiment a temperature range of 50C to 55 0C was maintained [Table 3].It was observed that at pH 6 and at incubation time of 10 min. the enzyme showed its maximum activity at a very low temperature of 150C. A drastic fall of enzyme activity was recorded as we increase the temperature from 250C onwards. However, a slight increase in activity was seen at 550C. This observation can be compared with the occurrence of the foliose lichen that was collected from the hills of Arunachal Pradesh at a low temperature of 10-15 0C.This low temperature activity of the amylase therefore can be used for the industrial purpose. For an enzyme activity, the pH is an important factor. So our next aim was to optimize the pH for the enzyme activity. The different pH solutions were used from pH 3.0 to pH 10 [Table 4]. It was found that the enzyme showed maximum activity at pH 7.0 while the activity is less at acidic and basic pH compared the neutral pH.Further we tried to purify the enzyme amylase by giving the ammonium sulphate cut at different concentrations, which ranged from 1.5 M to 4 M [Table 5]. It was found that the amylase showed maximum activity at an ammonium sulphate concentration of 1.5 M and 2 M. To remove the ammonium sulphate dialysis [Table 6] was done for further purification. It was observed that 0.1ml of the enzyme showed the activity comparable to the crude enzyme extract keeping all the parameters constant such as pH 7.0, incubation period of 10mins and temperature of 15°C. For the purpose of purification salting out was done by ammonium sulphate.The trace of ammonium sulphate was removed from the protein by dialysis. The activity of the enzyme both in precipitated state in ammonium sulphate and after dialysis were measured and found that the activity has increased significantly [Table 7], with respect to the crude enzyme. Lastly the partially purified enzyme amylase from the foliose lichen, Parmelia was studied to observe the effect of metals on enzyme activity. This was done to see the role of metals as metals behave as the cofactors for the enzyme activity. The different metals used were calcium, potassium, sodium, copper and iron [Table 8]. With Cu, we found that at very low concentration it increased the activity however it inhibited the activity at higher concentration. So, Cu is not desirable in the production process. Ca and Fe, however showed increase in the enzyme activity and are therefore desirable metals for the large scale production of the enzyme.Therefore all these results interpret that the enzyme amylase isolated from the foliose lichen showed its optimum activity at the low temperature of 15°C, at an incubation period of 10mins with Ca and Fe as its metal inducers. This low temperature nature of the enzyme amylase can be exploited in future for its commercial application.

Authors and Affiliations

Aniket Mukherjeee| M.Sc. in Botany, Barasat Govt. College, Calcutta University, West Bengal, India-700124

Keywords

Amylase enzyme Lichen Parmelia SP Arunachal Pradesh

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