Production of alpha Amylase from Soil Bacteria and characterization of the enzyme activity and Kinetic Study

Author Name: Bhargavi Basavaraju, *Avantika Gurung, Manisha Singh
Author Email: mishusin.bri@gmail.com

Abstract

Enzymes obtained from micro organism are more stable and economic. Among the commercially valuable microbial enzymes, Amylases are most important and are of great significance in present day industry. Amylases are used for various purposes such as in Brewing industry, Food additive, also used in Clothing and Dishwasher Detergents to dissolve starches. In the present study, bacterial strain was isolated from garden soil and screened for the production of the enzyme α-amylase. The bacteria that showed positive test for enzyme production was subjected for Biochemical tests for its identification. The organism was identified as bacillus species which is gram positive. In further investigation, the bacteria showed a positive response to citrate test. Kinetic study also showed that the optimum pH for enzyme activity was pH 8 and optimum temperature was 65°C at 575nm. It was observed that salt tolerance of α-amylase activity was obtained at 4M NaCl concentration at 575nm. The effect of the substrate concentration in the enzymes was observed at 2% starch concentration at 540nm. The activity of purified enzyme was 100μl α-amylase. Thus the optimum conditions for enzyme activity were standardized in the current work.

Keywords

Amylases, BACILLUS, Kinetic Study, Screening test, Starch hydrolysis.

Introduction

Amylases are the most important industrial enzymes in the present day biotechnology [1]. The amylase family of enzymes is of great significance due to its wide area of potential application’s Biodin and Effront were the first to use bacillus species for the production of alpha amylase on commercial scale [2]. These contribute a major class of industrial enzymes constituting 25% of the enzyme market.
Amylase is an enzyme that catalyses the hydrolysis of starch into sugars. It is used for the various purposes such as in brewing industry, food additive and also in clothing and dishwasher detergents to dissolve starches [3]. For the preparation of sizing agents and removal of the starch sizing from the woven cloth, preparation of starch sizing plate’s Liquefaction of heavy starch pastes formed during heating steps in the manufacture of corn and syrups, in bakery industry. The microbial source of amylase is preferred to other sources because of its plasticity and vast availability [4]. The major advantages of using microorganisms for production of amylases are the ability to produce in bulk and ease at which it can be manipulates for desired products [5]. In the present study, the production of the amylase is carried out from the Screening of Bacillus sp in soil, purified, immobilized and the various enzymatic parameters like pH, temperature ,salt tolerance ,substrate concentration ,enzyme activity was checked.

Conclusion

The current work was an attempt to synthesize the Amylase enzyme from commonly available soil bacteria and to analyze the optimum conditions for the maximum enzyme activity. The bacteria that was isolated from soil and gave a positive result for amylase production was found to be gram positive Bacillus coagulans. The organism was further used for the synthesis of the amylase enzyme by providing appropriate production medium. The enzyme thus produced was purified using various techniques to obtain maximum purity. The pure enzyme was later used for the kinetic study. The optimum conditions for the maximum enzyme activity were found to be 650C Temperature, pH 8, Substrate concentration was 2gms and salt tolerance was 4M NaCl.The work can further be extended for the standardization of the other parameters also.

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