GELATINASE PRODUCTION POTENTIAL OF BACTERIA ISOLATED FROM ATIMBO AND KWA RIVER SEDIMENTS

ABSTRACT

            The industrial importance of gelatinase cannot be overemphasized as more jaw dropping discoveries have uncovered the various undisputable applications of the enzyme in several aspects of science. In this study, gelatinase producing bacteria were probed from sediment samples of Kwa River and Atimbo River, Cross River State. Primary screening uncovered 3 isolates (B5, A7 and B6) that could elaborate gelatinase activity. This exposed the quantity of gelatinolytic bacteria to be 5.4% of the entire sediment sample population. The isolates that showed positive gelatinolytic activity were then quantitatively screened using the Bradford method which displayed high production in 2 isolates (B6 and B5).  The isolate that showed maximum gelatinolytic activity was then centrifuged and evaluated to determine the extracellular production of the enzyme using cell free supernatants. The cell free supernatants of positive isolates were used as crude gelatinase. Determination of carbon source required by the bacteria for optimal production of the gelatinase showed high rates of enzyme activity in sucrose which was selected. The successful organism was identified to be of Bacillus species using identification tests.

TABLE OF CONTENTS

Title page                                                                                                       i

Certification                                                                                                   ii

Dedication                                                                                                      iii

Acknowledgements                                                                                       iv

Abstract                                                                                                         v

Table of Contents                                                                                          vi

List of Figures                                                                                               vii

List of Plates                                                                                                  viii

List of Tables                                                                                                 ix

CHAPTER ONE: INTRODUCTION                                                        1

1. Aim and objectives of study                                                               3
   1. Justification of study                                                                          3

CHAPTER TWO: LITERATURE REVIEW                                          4

• Gelatins                                                                                               4
• Sources of gelatin                                                                               7
• Production of gelatin                                                                          9
• Gelatin structure                                                                                 12
• Mechanism of gelatin gelation                                                            14
• Properties of gelatin                                                                            15
• Industrial applications of gelatin                                                        17
• Proteases                                                                                             21
  • Microbial proteases                                                                             22
  • Applications of microbial proteases                                                    24
  • Matrix Metalloproteases (MMPs)                                                       27
• Gelatinase                                                                                           29
• Types of gelatinases                                                                            30
• Structure of gelatinases                                                                       31
• Activation of gelatinases                                                                    32
• Initiation of gelatinases                                                                       33

CHAPTER THREE: MATERIALS AND METHODS                          34

• Materials                                                                                             34
• Methods                                                                                              34
• Sampling                                                                                             34
• Sample collection                                                                                  34
• Sample preparation                                                                               35
• Microbial analysis                                                                                 35
  • Enumeration                                                                                          36
  • Isolation                                                                                                36
  • Primary screening for gelatinase-liquefying bacteria                 36
  • Secondary screening for gelatinase production in submerged culture  37
  • Evaluation of carbon sources for optimal gelatinase production           39
  • Characterization and identification of selected

gelatinase-producing bacterium                                                             41

CHAPTER FOUR: RESULTS                                                                      42

• Microbial analysis                                                                                  42
• Primary screening for gelatinase-liquefying bacteria                            42
• Secondary screening for gelatinase production in submerged culture  43
• Evaluation of carbon sources for optimal gelatinase production          43
• Characterization and identification of selected

gelatinase-producing bacterium                                                              44

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATION                                                                                                                     49

• Discussion                                                                                           49
• Conclusion                                                                                          50
• Recommendation                                                                                51

References