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

ABSTRACT

The bacteria Bacillus licheniformis was cultured in nutrient agar and then incubated for 15h at 35^oC. The bacteria cells were harvested by centrifugation after incubation. The cell free supernatant was used to estimate alpha-amylase activity. The alpha-amylase obtained was isolated and purified using ammonium sulfate precipitation, gel filtration and ion exchange chromatography. It was purified up to 15.5 fold and a yield of 20.2% on DEAE- Sephadex column with a final specific activity of 12.14 u/mg. The alpha-amylase was immobilized by entrapment in calcium alginate beads. The free and immobilized enzyme had broad temperature ranges from 20^oC to 70^oC with optima of 60^oC and 70^oC respectively and optimum pH of 7.0 and 8.0 respectively. Initial velocity studies for the determination of kinetic constants with maltose as substrate revealed a K_M value of 2.5 mg/ml and 1.0 mg/ml for the free and immobilized enzyme respectively and a V_max value of 0.4unit/mg/min and 0.95unit/mg/min for the free and immobilized enzyme respectively. Both the free and immobilized enzyme activity were enhanced by Ca²⁺ , Mn²⁺ , and Na⁺ while Hg²⁺ and Zn²⁺ were found to be strong inhibitors of both the free and immobilized enzyme.

CHAPTER ONE

1.0. INTRODUCTION

1.1. Background of Study

Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaved poly-saccharides). It is mainly 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 major form of amylase found in humans and other mammals. It is also present in seeds containing starch as food reserve and it is secreted by many fungi. Although found in many tissues, alpha-amylase is most prominent in pancreatic juice and saliva. Alpha-amylase found in saliva breaks starch down to maltose and dextrin. It breaks large insoluble starch molecules into soluble forms e.g. amylodextrin, erythrodextrin and achrodextrin producing successively smaller starches and ultimately maltose. The pancreas produces alpha-amylase which hydrolyses dietary starch into disaccharides and trisaccharides which are converted by other enzyme to glucose to supply the body with energy (Alistair et al., 2006).

Although amylase can be derived from several sources such as plants, animals and microbes, the microbial amylase meet industrial needs and demands. Large numbers of microbial amylase have completely replaced chemical hydrolysis of starch in starch processing industries (Pandey et al., 2000).

Nowadays the use of enzyme in industrial sector is increasing due to the increase of industries, especially in food, beverages, textile, leather and paper industries. Besides its uses in industry, it can also be used in treatment of industrial waste such as cellulase which is able to convert cellulose of wood and paper wastes to ethanol (Vielle and Zeikus., 1996). One of the enzymes widely used in industrial sectors is alpha-amylase. Alpha-amylase from Bacillus species has found application in many industries such as pharmaceutical, textile, paper, detergent and chemical industries. Therefore, these enzymes account for about 39% of the world‟s enzymes production (Gomes and Steiner, 2004).

A biocatalyst is termed immobilized, if its mobility has been restricted by chemical means. Immobilized enzymes are used in food technology, biotechnology, biomedicine and analytical chemistry. Immobilized enzymes offers variety of advantages over free enzyme catalysis including increased stability of enzyme, easy recovery of enzyme, easy separation of reactant and product, repeated or continuous used of a single batch of enzyme which will ultimately save the enzyme, labor and overhead costs (Gerhatz, 1990).

Enzymes can be immobilized to a multitude of different carriers by entrapment, adsorption, ionic binding and covalent binding (Varavinit et al., 2002). Entrapment is taken as the most preferable method because it prevents excessive loss of enzyme activity after immobilization, increases enzyme stability and protects enzyme from microbial contamination (Kennedy and Cabral, 1987).

Physical entrapment of alpha-amylase in calcium alginate beads has shown to be a relatively easy, rapid and safe technique (Dey et al., 2003) in comparison with other immobilization methods.

1.2 Statement of Research Problem

a. Amylases possess important applications in the production of syrup with high glucose content, sweetener manufacture, detergent and ethanol (Pandey et al., 2000).

b. The annual sale of alpha-amylase in global market is estimated to be eleven million dollars (Kilara and Desai, 2002).

c. There is a need to discover more bacterial sources of alpha-amylase that will produce alpha-amylase with better properties e.g. thermostability that will be of greater use to the industries.

d. There is a need to discover more ways of producing alpha-amylase in bulk and that will be economically viable.

e. Most of the enzymes used in the industrial sector in Nigeria including food industries are still imported enzymes and economically, this is not favorable to the nation because Nigeria is rich in natural resources especially the microbial which can be use as enzymes producer for example alpha-amylase enzyme.

f. There is a need to immobilize alpha-amylase in order to explore the various benefits that can come from the immobilization process.

1.3 Justification

a. Bacterial alpha-amylase is preferred for the application in starch processing and textile industries due to its stability at higher temperature (75-105ºC) and its neutral to alkaline pH (Shah and Kothari, 1991).

b. B. licheniformis, B. coagulans, B. polymyxa, B. vulgaris have been used for alpha amylase production in solid state fermentation (Babu and Satyanarayana, 1995).

c. Due to the increase in the demand for these enzymes in various industries, there is therefore a need to discover more strains of bacteria that can produce alpha-amylase with better properties in terms of thermo-stability, mass production of the enzyme and consistency.

d. There is also a need to compare the kinetics and physico-chemical properties of the free and immobilized alpha amylase to discover various benefits that immobilization of alpha amylase can offer to the industries.

e. The strain of B. licheniformis used for this research work is a local strain isolated from Kaduna metropolis soil in Kaduna State, Nigeria and it is different from imported strains of the bacteria that are usually used for other research work.

f. Also there is no documented work on the kinetic studies and effect of metal ions on immobilized alpha amylase.

1.4 Aim

To isolate, purify and characterize free and immobilized alpha-amylase from Bacillus licheniformis.

1.4.1 Objectives

a. Isolation of the enzyme alpha-amylase in fermentation media from the cell of the Bacteria isolate of Bacillus licheniformis.

b. Purification of alpha-amylase obtained from the Bacillus licheniformis

c. Immobilization of the enzyme by entrapment in calcium alginate beads.

d. Characterization of the free and immobilized alpha amylase.

1.5 Research Hypothesis (Null)

Immobilization of alpha-amylase does not affect the stability, kinetics and physico-chemical properties of the enzyme.

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