SELECTION OF FRESHWATER BACTERIA WITH POTENTIALS FOR COMMERCIAL L-ASPARAGINASE PRODUCTION

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ABSTRACT

L- asparaginase is an important enzyme that hydrolyzes the amino acid L-asparagine to produce L-aspartate and ammonia which leads to cell death by starvation. The enzyme has been used as an anti-cancer or anti-leukemic agent to treat lymphoblastic leukemia. However, a number of commercially available enzymes are from bacterial sources that cause allergic reactions during applications. This research was aimed at finding alternative non-toxic sources of L-asparaginases. Five sediment samples were collected from Great Kwa River and analyzed by the pour plate technique for isolation of distinct bacterial strains. A total of 133 bacterial strains were obtained and screened for L-aspraginase production by the rapid plate technique and 35 isolates were positive. Some of the strains were found to produce the enzyme slowly which made them not suitable for fermentative productions. However, 21 strains could produce the enzyme within 72 h and were considered for secondary screening in liquid medium. Only five of the isolates were successful at this stage and were subjected to tertiary screening under shake flask fermentation conditions.  Results showed that only one strain could produce detectable and quantifiable amounts of L-asparaginase under study conditions. The bacterium produced 54 µg/mL of L-asparaginase. However, when extraneous nitrogen sources were introduced into fermentation flasks, only NH4Cl was found capable to significantly enhance production. The organic forms of nitrogen were found to repress enzyme biosynthesis. The bacterium was identified as Bacillus sp. strain GKRS-D7 and is recommended for further investigations into production of clinically-benign L-asparaginases.

TABLE OF CONTENTS

TITLE PAGE              –           –           –           –           –           –           –           i

CERTIFICATION                  –           –           –           –           –           –           ii

DEDICATION           –           –           –           –           –           –           –           iii

ACKNOWLEDGEMENT     –           –           –           –           –           –           iv

ABSTRACT   –           –           –           –           –           –           –           –           v

TABLE OF CONTENTS       –           –           –           –           –           –           vi

LIST OF TABLES     –           –           –           –           –           –           –           x

LIST OF PLATES      –           –           –           –           –           –           –           xi

LIST OF FIGURES   –           –           –           –           –           –           –           xii

CHAPTER ONE: INTRODUCTION

1.1.Background of the Study –           –           –           –           –           –           1

1.2. Aims and Objectives of the Study           –           –           –           –           4

1.3. Justification of the Project           –           –           –           –           –           4

CHAPTER TWO: LITERATURE REVIEW

2.1. Historical view of L-asparaginase            –           –           –           –           6

2.2. Functions and structure of L-asparaginase          –           –           –           8

2.2.1. Functions of L-asparaginase     –           –           –           –           –           8

2.2.2    Structure of L-asparaginase                –           –           –           –           8

2.3.      Side effects and adverse reaction of L-asparaginase chemotherapy.    9

2.3.1    Side effects of L-asparaginase            –           –           –           –           9

2.3.2    Adverse reaction of Erwinia L-asparaginase                         –           9

2.4       Types of microbial L-asparaginase production           –           –           9

2.4.1    Bacterial L-asparaginase         –           –           –           –           –           10

2.4.2.   Fungi L-asparaginase –           –           –           –           –           –           11

2.5.      Medical and Industrial application of L-asparaginase

by enzymes production           –           –           –           –           –           11

2.5.1. Enzymes as therapeutic agent in the medical application.        –           11

2.5.2. Enzymes as pharmaceutical and food processing in the

industrial application   –           –           –           –           –                       12

CHAPTER THREE: MATERIALS AND METHODS

3.1       Materials         –           –           –           –           –           –           –           14

3.2                   Methodology –           –           –           –           –           –           –           14

3.2.1    Sampling         –           –           –           –           –           –           –           14

3..2.2   Sample Preparation and plating          –           –           –           –           14

3.2.3.   Isolation of morphologically- distinct bacteria           –           –           15

3.2.4.   Purification and maintenance of isolates        –           –           –           15

3.2.5    Primary screening of bacterial isolates for L-asparaginase    

            production potential.   –           –           –           –           –           –           15

3.2.6    Secondary screening of primary positive isolates for

                        L-asparaginase production in Liquid medium.           –           –           16                   

3.2.7  Tertiary screening of secondary isolates for L-asparaginase

                        productionin shake flask.        –           –           –           –           –           17

3.2.8    Characterization of selected L-asparaginase producing bacterium     17

3.2.9    Evaluation of effect of Carbon sources in L-asparaginase

                        production by Selected bacterium isolate A1 –           –           –           17

CHAPTER FOUR: RESULTS

4.1. Distribution of Morphologically- Distinct Bacterial isolates in

            Sample            –           –           –           –           –           –           –           –           20

4.2       Primary Screening of Bacteria for L-asparaginase Production

            Potential          –           –           –           –           –           –           –           –           20

4.3.      Secondary screening of primary Positive bacteria for L-asparaginase Production     –           –           –           –     –           –           –           26

4.4.      Tertiary Screening of Bacteria for L-asparaginase production           –           26

4.5.      Preliminary identification of Selected L-asparaginase producing

            bacterium                    –           –           –           –           –           –           26

4.6 Evaluation of the effect of extraneous carbon on L-asparaginase

production by Bacillus sp. GKRS-D7      –           –           –           –           27

CHAPTER FIVE: DISCUSSION, CONCLUSION, RECOMMENDATION

5.1. Discussion            –           –           –           –           –           –           –           34

5.2. Conclusion           –           –           –           –           –           –           –           35

5.3. Recommendation             –           –           –           –           –           –           35

REFERENCES        –           –           –           –           –           –           –           36