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
CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
L-asparaginase as a bacterial enzyme reduces the availability of asparagine (an amino acid), that is necessary for the growth of some tumor cells. L-asparaginase selectively targets the metabolism of cancer cells by exploiting deficiencies in metabolic pathways and catalyzing the degradation of L-asparagine into L-aspartic acids and ammonia, causing nutrient starvation of cancer cells and bringing about their demise.
L-asparaginase which catalysis the conversion of L- asparagine to L-aspartate and ammonia has been identified in various organisms, including bacteria, yeasts, molds, archaea and animals. In clinical applications, L-asparaginase is used to treat acute lymphoblastic leukaemia, lymphoma and other cancer cells, as it removes the L-asparaginate required for the survival of lymphoblastic leukemia cells. Two types of commercial L-asparaginase are currently in clinical use for chemotherapy, these enzymes are Escherichia coli and Erwinia chrysanthemi although they have some drawbacks in that they exhibit low substrates specificity and high glutaminase activity, which can cause liver dysfunction, pancreatitis, leucopenia, neurological seizures, and coagulation abnormalities that can lead to intracranial thrombosis or hemorrhages. Therefore, it is important to identify sources and methods of producing greater amounts of glutaminase-free ASNases and exhibit high substrates affinity and therapeutic activity. L-asparaginase is given as a chemotherapeutic treatment (drugs) by injection into the vein, muscles or under the skin. L-asparaginase also has high commercial value due to its vast applications in the food and pharmaceutical industries. In the food industry, L-asparaginase is used to reduce L-asparagine, which is a precursor of carcinogenic acrylamide. During this process, L-asparaginase is treated at a high temperature together with raw food materials or is sometimes blanched in hot water, therefore the enzyme should be capable of withstanding high temperatures. However, the low activity and instability of L-asparaginase at high temperatures have restricted its application in the food industry.