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 60oC
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 (Km) and maximum velocity (Vmax)
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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