ISOLATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF α-AMYLASE FROM Bacillus alcalophilus

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ABSTRACT

The aim of this study was to isolate, partially purify and characterize α-Amylase from Bacillus alcalophilus. The enzyme (α-amylase) was isolated from Bacillus alcalophilus using cassava as only carbon source. 20 g of soil sample were weighed out and dissolved in 40 ml of distilled water in a clean conical flask, mixed vigorously and heated at 60^oC for 60 min in water bath and taken as the stock culture. From the stock preparation, ten folds serial dilution were carried out and the 10^-4 to 10^-6 dilutions were plated out in a media plate. Percentage ammonium sulphate saturation, ammonium sulphate precipitation and gel filtrations were carried out to partially purify α-amylase from Bacillus alcalophilus. The α-amylase was then characterised by studying the effect of pH, change in temperature, substrate concentration and metal ion on the enzyme`s activity. The specific activity of the crude enzyme was 160.26 U/mg proteins. After ammonium sulphate precipitation and gel filtration, the specific activity were found to be 88.9 and

285.9U/mg protein, respectively. The optimum pH was found to be 7.5 and 70°C respectively. The α-amylase activity was found to be enhanced by Ca²⁺, Mg²⁺, Mn²⁺and Co²⁺, whereas Fe²⁺

was found to have inhibitory effect. The enzyme retained more than 80% of its activity at 60 min in the presence of Ca²⁺, Mg²⁺ , Mn²⁺and Co²⁺, and lost up to 90% of its activity in the presence

of Fe²⁺. In this study, Ca²⁺ maintained more stability of the enzyme than all other metal ions. The Michaelis-Mentens constant (K_m) and maximum velocity (V_max) obtained from the Line Weaver Burk plot of initial velocity data of different substrate concentrations were 1.159 mg/mL and 16.24 μmol/min respectively. In conclusion, this study revealed the potentials Bacillus alcalophilus to serve as other source of α-amylase, especially for industrial purposes.

TABLE OF CONTENTS

Title Page

Abstract

Table of Contents

List of Figures

List of Tables

List of Abbreviations

CHAPTER ONE: INTRODUCTION

1.1       Amylase Family

1.1.1    α-Amylases

1.1.2    β-Amylase

1.1.3    γ-Amylase

1.1.1.4 The α-Amylase Family

1.1.1.4.1Endo Amylase

1.1.1.4.2Exo Amylases

1.1.1.4.3Debranching Amylase

1.1.1.4.4The Transferases

1.2       Sources of α- Amylase

1.3       Uses of α-Amylase

1.3.1    Fermentation

1.3.2    Flour additive

1.3.3    Molecular biology

1.3.4    Medical Uses

1.3.5    Other uses

1.3.6    Hyperamylasemia

1.4       Methods of Production of α- Amylase from Microbial Sources

1.4.1    Solid State Fermentation

1.4.2    Submerge Fermentation

1.5       Bacillus alcalophilus

1.6       Process Optimization for Production of α- Amylase

1.6.1    pH

1.6.2    Temperature

1.6.3    Metalions

1.6.4    Moisture

1.6.5    Particle Size of Substrate

1.6.6    Oxidative Stress

1.6.7    Purification

1.6.7.1 Affinity Adsorption Chromatography

1.6.7.2 Countercurrent Chromatography

1.6.7.3 Substitute Western Affinity Purification Assay (SWAP)

1.6.7.4 Magnetic Affinity Adsorption

1.6.7.5 Expanded Bed Adsorption

1.7       Alpha Amylase Characterizations

1.7.1    pH Stability

1.7.2    Effects of Temperature

1.7.3    Effect of Metal Ions

1.7.4    Effect of Inhibitors on α-Amylase Activity

1.7.5    Effect of Substrate Concentrations

1.7.5    Digesting Property of Raw Starch

1.8       Stabilization of Alpha Amylase

1.8.1    Immobilized Amylases

1.8.2    Cloning of Alpha Amylase Genes

1.8.3    Enzyme Engineering

1.9       Aim and Objectives of the Study

1.9.1    Aim of the Study

1.9.2    Specific Objectives of the Study

CHAPTER TWO: MATERIALS AND METHODS

2.1       Materials

2.1.1    Soil samples

2.1.2    Cassava tubers

2.1.3    Apparatus and Instruments

2.1.4    Chemical / Reagents

2.2       Methods

2.2.1    Processing of Starch from Cassava

2.2.2    Preparation of Buffer Solutions

2.2.2.1 Sodium Acetate Buffer (stock solution)

2.2.2.2 Preparation of Working Acetate Buffer Solution

2.2.2.3 Sodium Phosphate Buffer (stock solution)

2.2.2.4 Tris-HCl Buffer (stock solution)

2.2.3    Isolation of Bacillus alcalophilus from Soil

2.2.3.1 Preparation of Media and Plate Pouring

2.2.3.2 Inoculations of Folds of Diluted Soil Solution on the Prepared Plate and Sub Culturing

2.2.3.3 Storage of Pure Bacterial Isolates

2.2.3.4 Microscopic Features of the Isolated Bacteria

2.2.3.5 Bacterial Identification

2.2.4    Fermentation Experiment

2.2.4.1 Constitution of the Fermentation Broth

2.2.4.2 Inoculation of the Broth

2.2.4.3 Harvesting of the Fermented Broth

2.2.4.4 Mass Production of Enzyme

2.2.5    Assay of Alpha Amylase Activity

2.2.6    Protein Determination

2.2.6.1 Preparation of Reagents/Solutions

2.2.6.2 Procedure for Protein Determination

2.7       Purification of α-amylase from Bacillus Alcalophilus

2.2.7.1 Determination of Percentage Ammonium Sulphate Saturation

            Suitable for α- Amylase Precipitation

2.2.7.2 Ammonium Sulphate Precipitation of α-Amylase

2.2.7.3 Gel Filtrations

2.2.8    Studies on Partially Purified Enzyme

2.2.8.1 Effect of pH Changes on α-amylase Activity

2.2.8.2 Effect of Temperature Change on α-amylase Activity

2.2.8.3 Effect of Metal Salts Concentrations on

            α-Amylase Isolated from Bacillus alcalophilus

2.2.9    Effect of Substrate Concentration on α-amylase Activity

CHAPTER THREE: RESULTS

3.1       Examination of Pure Sub-cultured Bacterial Species

3.1.2    Percentage yield of Starch from Cassava

3.2       Effect of Incubation days on α-amylase production

3.3       Purification of Crude α-amylase

3.3.1    Ammonium Sulphate Precipitation

3.3.2    Gel filteration

3.3.3    α-Amylase Purification Table

3.4       Characterization of Partially Purified Enzyme

3.4.1    Effect of pH

3.4.2    Effect of Temperature

3.5       Effects of Metal ions Concentrations on pH 7.5 activity of α-amylase

3.6.      Determination of Kinetic Parameters

CHAPTER FOUR: DISCUSSION

4.1       Discussion

4.2       Conclusions

4.3       Suggestion for Further Studies

            References

            Appendices

CHAPTER ONE

INTRODUCTION

Enzymes are produced by plants, animals and microorganisms. Microbial enzyme production is of great importance as they are more economical to produce, calculable, tractable and stable (Burhan et al., 2003). Amylases are very important family of enzymes that hydrolyze starch into dextrins and small polymers of glucose. Two major classes of amylase, mostly identified amongst microorganisms are α-amylase and glucoamylase (Vijayabaskar et al., 2012). The β-Amylase, mostly from plant origin has also being recorded from microbial source (Pandey et al., 2000). The use of α-amylase in some industries especially in food, beverage, textiles, leather and paper industries is increasing. There is a need for other source of the enzyme to be discovered as Nigeria is a country which is rich in natural resources, particularly the microbes as enzyme producers. Over the years, amylases have been isolated from bacteria, fungi and actinomycetes. Bacterial amylases are mostly reported from thermophilic, acidophilic and alkalophilic bacteria (Kim et al., 1995). They are commercially available and have replaced chemical hydrolysis of starch to a great extent in industries (Pandey et al., 2000). Bacillus alcalophilus, B. substilis, B. licheniformis, B. amyloliquifaciens and B. stearothermophilis are most prominent among the bacterial sources of amylase (Carlos et al., 2002).

α-Amylase is a ubiquitous enzyme produced by plants, animals and microbes, and they play an important role in carbohydrate metabolism (Swetha et al., 2006). Amylase (1, 4-α-D-glucano hydrolase; E.C 3.2.1.1) is applied in food, paper and clothing industries. They are also applied idustrially in fermentation such as brewing, baking, digestive acid production, fruit juice, starch syrups and chocolate cake’s production (Pandey et al., 2000). Several reports on starch degrading microorganisms from different source and respective amylase activity have been reported (Nguyan et al., 2002; Balkan and Ertan, 2005). The soil is one of the richest sources of starch degrading microorganisms as it contains starchy substances required for the continuous microbial growth and reproductive life. pH and thermal stability are very important factors considered in industrial enzymatic bio-reactions, most limitations in the applications of enzymes industrially could be attributed to these factors. Most industrial application of α-amylase (e.g. in starch liquefaction industries) takes place at high temperature ranges and during the liquefaction-saccharification process, bye-products are given off which can lower the pH of the reaction medium. Stabilization of these parameters (pH and temperature) in α-amylase is of great importance due to the high industrial utilization of the enzyme. Most of the α-amylases reported till dates are metal ion-dependent enzymes and these metal ions are known to be stabilizers for amylases isolated from various microorganisms (Sudha, 2013). By various ways, these metal ions affect enzyme catalysis. They can act by modifying the electron flow in the enzyme substrate reaction or by changing the orientation of the substrate with reference to specific functional groups at active site (Singh et al., 2014). Also, these metal ions can accept or donate electrons and act as electrophiles, mask nucleophiles to prevent unwanted side reactions, bind enzyme and substrate by coordinate bonds, hold the reacting groups in the required orientation, and simply stabilize a catalytically active conformation of the enzyme (Sudha, 2013; Singh et al., 2014). These stabilizing effects generally simplify industrial procedures during the downstream processing and help reduce the production of compounds that increase pH of the reaction medium......

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