Isolation, Identification and characterisation of Amylase enzyme produced by different Microorganisms isolated from soil

Abstract

Amylase is one of the most widely used enzyme in industries from decades. Its main function is to hydrolyze the starch molecule into glucose unit and oligosaccharides. Among different types of amylases, α-amylase is in high demand due its wide spread use in food, textile, baking and detergent industries. It can be acquired from microorganisms, plants and animals. This study was conducted with the aim to isolate, identify and to characterize the amylase enzyme produced by microorganisms from soil. The soil sample were collected from the different places of Dharan. The screening of amylase producing microorganisms were carried out by starch hydrolysis test on starch agar plates. The organisms producing highest zone of clearance were identified and further used for amylase production and characterization. Based on colonial characteristics, cultural characteristics, staining and biochemical test the organisms were identified as Bacillus subtilis whereas the isolated fungi was identified as Aspergillus niger on the basis of colonial characteristics and LPCB staining. The identified organisms were further used for the production of amylase on submerged fermentation. The amylase activity was assayed by DNS method. The produced enzyme was centrifuged and partially purified using ammonium sulphate. The amylase production was found to be maximum at 40ºC and 6.5 pH for Bacillus amylase and 30 ºC and pH 7.5 for Aspergillus niger. Further, Characterization of the enzyme was done for the industrial application of the enzyme. Crude amylase showed its maximal activity at pH 7 and 45 ºC For Bacillus subtilis and 30 ºC and pH 6.5 for Aspergillus niger. Crude amylase showed great potential for its application in detergent industry.

Keywords –amylase, enzyme, submerged fermentation, DNS method, ammonium sulphate, characterization.