ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS

ISOLATION, PURIFICATION AND CHARACTERIZATION OF FREE AND IMMOBILIZED ALPHA-AMYLASE FROM BACILLUS LICHENIFORMIS

ABSTRACT

Bacillus licheniformis was grown in nutrient agar and incubated for 15 hours at 35 degrees Celsius. Following incubation, the bacteria cells were collected using centrifugation. The cell-free supernatant was utilised to calculate alpha-amylase activity. The alpha-amylase was extracted and purified via ammonium sulphate precipitation, gel filtration, and ion exchange chromatography. It was purified up to 15.5 times and yielded 20.2% on a DEAE- Sephadex column, having a final specific activity of 12.14 u/mg. The alpha-amylase was immobilised by entangling in calcium alginate beads. The free and immobilised enzymes had broad temperature ranges of 20oC to 70oC, with optimum temperatures of 60oC and 70oC, and optimum pH levels of 7.0 and 8.0, respectively. Initial velocity experiments using maltose as a substrate indicated a KM value of 2.5 mg/ml and 1.0 mg/ml for the free and immobilised enzymes, respectively, as well as a Vmax value of 0.4unit/mg/min and 0.95unit/mg/min for the free and immobilised enzymes. Ca2+, Mn2+, and Na+ all increased the activity of both free and immobilised enzymes, whilst Hg2+ and Zn2+ were found to be severe inhibitors of both.

Chapter one

INTRODUCTION

1.1.Background of the Study

Amylase is a digestive enzyme classed as a saccharidase (an enzyme that cleaved poly-saccharides). It is largely a constituent of pancreatic juice and saliva, needed for the breakdown of long-chain carbohydrate (such as starch) into smaller units like disaccharides and trisaccharides.

Alpha-amylase is the primary form of amylase present in humans and other mammals. It is also present in seeds storing starch as food reserve and it is secreted by several fungi. Although found in numerous organs, alpha-amylase is particularly abundant in pancreatic juice and saliva.

Saliva contains alpha-amylase, which breaks down starch into maltose and dextrin. It converts large insoluble starch molecules into soluble ones, such as amylodextrin, erythrodextrin, and achrodextrin, resulting in gradually smaller starches and, finally, maltose.

The pancreas produces alpha-amylase, which hydrolyses dietary starch into disaccharides and trisaccharides, which are then transformed to glucose by other enzymes to provide energy for the body (Alistair et al., 2006).

Although amylase can be generated from plants, animals, and bacteria, microbial amylase is more suitable for industrial use. Large amounts of microbial amylase have fully replaced chemical starch hydrolysis in starch processing companies (Pandey et al. 2000).