Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation.

Dibyangana Raul,Suchita Mukhopadhyay,Tania Biswas,Suvroma Gupta,Shrayan Kumar Das

doi:10.1155/2014/568141

Dibyangana Raul, Suchita Mukhopadhyay + Show 3 more

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https://doi.org/10.1155/2014/568141

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Abstract

Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α-amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30–70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques.

Highlights

Amylase catalyses the breakdown of starch into sugars. α-Amylase can breakdown long-chain carbohydrates, yielding maltose from amylose, or maltose, glucose, and “limit dextrin” from amylopectin
After 48 hours of solid state fermentation at 37∘C using Bacillus subtilis, the production of α-amylase was detected by determining enzymatic activity using dinitrosalicylic acid (DNS) method at 40∘C
Alpha amylase activity of the extract was measured by DNS method [30]

Summary

Introduction

Amylase catalyses the breakdown of starch into sugars. α-Amylase can breakdown long-chain carbohydrates, yielding maltose from amylose, or maltose, glucose, and “limit dextrin” from amylopectin. Amylase catalyses the breakdown of starch into sugars. Α-Amylase can breakdown long-chain carbohydrates, yielding maltose from amylose, or maltose, glucose, and “limit dextrin” from amylopectin. Two major classes of amylases have been identified in microorganisms, namely, α-amylase and glucoamylase. Α-Amylases (endo-1,4-a-D-glucan glucohydrolase) are extracellular enzymes that randomly cleave the 1,4-a-D-glucosidic linkages between adjacent glucose units in the linear amylase chain. Glucoamylase (exo-1,4-a-D-glucan glucanohydrolase) hydrolyzes single glucose units from the nonreducing ends of amylose and amylopectin in a stepwise manner [1, 2]. These are calcium metalloenzymes, which are completely unable to function in the absence of calcium. Calcium stabilizes the interface between the central A domain (291 residues) with (β/α) barrel structure and the more variable B domain (104 to 206 residues) [3,4,5,6,7]

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Conclusion