RELATIONSHIP BETWEEN DIGESTIVE ENZYMES, PROTEINS AND ANTI-NUTRITIVE FACTORS IN MONOGASTRIC DIGESTION By: Theofilos Kempapidis A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy Department of Chemical and Biological Engineering Faculty of Engineering The University of Sheffield December 2019 ‘To my beloved mother, whom I couldn’t have done this without and to the memory of my beloved father that passed away during my PhD.’ i ABSTRACT Monogastrics’ lack of some digestive enzymes is compensated by using exogenous enzymes in the animal feed. Those interact with feed ingredients causing enzyme inhibition. Two known inhibiting substances are phytic acid and polyphenolics. Phytase is an important exogenous enzyme that is breaking down phytic acid. The effect of sorghum polyphenolic-rich extracts on the 6-phytase activity was investigated using an ITC by calculating its relative activity. Results show inhibition of the phytase, from all three extracts. For a better understanding of this assay, a real-time mass spectroscopic analysis was performed, that showed the pattern in which the phytase is hydrolysing phytic acid. Consecutively, the effect of phytic acid on the activity of three proteases exogenous and endogenous chymotrypsin and endogenous trypsin was studied. A colorimetric assay using casein as a substrate was used. The results showed different levels of inhibition of all three enzymes by phytic acid. Finally, the interaction between the exogenous chymotrypsin and the phytase was studied by using the ITC enzyme assay and an SDS-Page gel analysis. ITC results showed partial inhibition of the phytase activity, while SDS-Page results showed complete inhibition. Literature supports the results of this study which can have a negative nutritional and economic effect. This information could be used to create a conceptual model, that would be a useful tool to understand the fundamental interactions of enzymes, substrates and anti-nutritive factors in order to improve monogastrics’ nutrition. ii ACKNOWLEDGMENTS I would like to thank Dr Falconer who always had his door open and Dr Cameron who has always been there for me. I would also like to thank DSM Nutritional Products who funded this PhD project. I would like to thank everyone in the department of Chemical and Biological Engineering, as well as the Department of Animal and Plant Sciences who offered me help, support and training. I am grateful to Niall Bradshaw and Hayden Hodges for their valuable contributions to my work. I would also like to thank Dr Eleni Routoula & Dr Kristina Schiavone who were always there for me both as friends and colleagues. I am forever grateful to my mother and sister, who I love unconditionally, my aunt and grandparents who have always supported me and tried to help me in any way they could. I would like to thank my thank my dad for being there for me and providing me with his knowledge in animal nutrition. Finally, I would like to thank a long list of friends for the emotional support and help, as they are my other family. My biggest thank you to: Nantia, Elpida, Antigone, Jules, Manolis, Jonny, Erman, Remi, Myree, Eralp, Lukas, Spyri, Katerina, Polyvios, Georgia, Stavroula, Eleni, Katerina and Marianthi. iii PUBLICATIONS Kempapidis, T., Bradshaw, N.J., Hodges, H.E., Cowieson, A.J., Cameron, D.D. and Falconer, R.J., 2020. Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of- flight mass spectroscopy. Analytical Biochemistry, 606, p.113859. iv CONTENTS ABSTRACT II ACKNOWLEDGMENTS III PUBLICATIONS IV CONTENTS V LIST OF FIGURES X LIST OF TABLES XIII CHAPTER 1 ‘GENERAL INTRODUCTION’ 2 1.1 SUMMARY 3 1.2 INTRODUCTION 4 1.3 LITERATURE REVIEW 8 1.3.1 Nutrition in monogastric animals 8 1.3.2 Digestion in Monogastrics 9 1.3.3 Digestive systems in monogastrics 9 1.3.4 Digestion in pigs 10 1.3.5 Digestion in poultry (chicken) 11 1.3.6 Digestive/endogenous enzymes 12 1.3.7 Monogastric Animal Feed 14 1.3.7.1 Grains 14 1.3.7.2 Protein meals 14 1.3.7.3 Vegetable protein sources 14 1.3.7.4 Animal protein sources 15 1.3.7.5 Oils and fats 15 1.3.7.6 Minerals and vitamins 15 v 1.3.8 Feed enzymes 16 1.3.8.1 Proteases 18 1.3.8.2 Phytase 19 1.3.9 Anti-nutritional factors in animal feed 20 1.3.10 Isothermal Titration Calorimetry 22 1.3.10.1 ITC Theory and Operation 23 1.3.10.2 ITC for enzyme kinetics 23 1.3.10.3 Kinetic ITC Experiments 25 1.4 AIMS AND OBJECTIVES 27 1.5 HYPOTHESES 28 CHAPTER 2 ‘THE EFFECT OF SORGHUM POLYPHENOLS ON THE ACTIVITY OF A 6-PHYTASE’ 30 2.1 SUMMARY 31 2.2 Introduction 32 2.3 MATERIALS AND METHODS 45 2.3.1 Chemicals and Materials 45 2.3.2 Extraction of polyphenols 45 2.3.3 Analysis of Polyphenolics 46 2.3.4 Total Phenolic Content Assay 46 2.3.5 ITC 47 2.3.5.1 ITC Optimization 48 2.3.5.2 ITC Enzyme Activity Assay 48 2.3.6 Anion Exchange Chromatography 49 2.3.7 Mass spectrometry 49 2.3.8 Software 50 2.3.9 Mathematical calculations 51 2.4 RESULTS AND DISCUSSION 52 vi 2.4.1 Polyphenol Extraction and Analysis 52 2.4.2 ITC Enzymatic activity assay 61 2.4.2.1 Optimization 61 2.4.2.2 Ion Exchange Chromatography and Mass Spectrometry Analysis 65 2.4.2.3 Interpretation of the phytase-phytic acid thermograms 68 2.4.2.4. Inhibition of phytase by Sorghum Polyphenols 73 2.4.3. Application of results in monogastric animal nutrition 80 CHAPTER 3 ‘THE EFFECT OF PHYTIC ACID ON THE ACTIVITY OF PROTEASES’ 87 3.1 SUMMARY 88 3.2 INTRODUCTION 89 3.3 MATERIALS AND METHODS 102 3.3.1 Chemicals and Materials 102 3.3.2 ITC Protease activity assay 103 3.3.3. SDS-Page Gels 105 3.3.3.1 Sample Preparation 105 3.3.3.2 General procedure 105 3.3.4 Pierce Protease Assay Kit 106 3.3.5 Sigma's Non-specific Protease Activity Assay - Casein as a Substrate 107 3.3.5.1 Assay Validation 107 3.3.5.2. Analysis 108 3.4 RESULTS AND DISCUSSION 112 3.4.1 ITC Enzyme activity assay 112 3.4.2 SDS Page analysis 112 3.4.3 Pierce Protease Assay Kit 115 3.4.4 Protease activity assay 117 3.4.4.1 Validation 117 3.4.4.2. Analysis 120 vii 3.4.5 General Discussion 133 3.5 CONCLUSIONS 137 CHAPTER 4 ‘INTERACTIONS BETWEEN PHYTASE AND PROTEASES’ 140 4.1 SUMMARY 141 4.2 INTRODUCTION 142 4.3 MATERIALS AND METHODS 147 4.3.1 Chemicals and Materials 147 4.3.2 ITC 148 4.3.2.1 ITC sample preparation 148 4.3.2.2 ITC Enzyme Activity Assay 148 4.3.3 SDS-Page 149 4.3.3.1 SDS-Page gel analysis 149 4.3.3.2 SDS-Page gel data analysis 150 4.4 RESULTS AND DISCUSSION 151 4.4.1 ITC Enzyme Activity Assay for 6-phytase in the presence of serine chymotrypsin 151 4.4.2 SDS-Page gel analysis for the interaction between the 6-phytase and the serine chymotrypsin 154 4.5 CONCLUSIONS 161 CHAPTER 5 ‘GENERAL DISCUSSION’ 163 5.1. GENERAL CHEMISTRY 164 5.1.1 Mapping the phytase, protease and anti-nutritive factors system 166 5.1.2 Comparison of different enzymatic activity techniques 167 5.2. GUT COMPLEXITY ON ENZYME INTERACTIONS 169 5.2.1. Anti-nutritive factors 169 viii 5.2.2 The effect of pH, temperature, salts and water 170 5.2.3 A model that outlines the interactions between feed ingredients 171 5.3 IMPLICATIONS OF INTERACTIONS IN NUTRITION 173 5.3.1 How can these interactions affect the feed composition 173 5.3.2 How to design a better animal feed 174 5.4 Conclusions Limitations and future work 175 REFERENCES 179 APPENDIX 195 A1 Abstract for oral presentation at the International Society of Enzymology Abstract 195 A2 Abstract for poster presentation at the 3rd European Sustainable Phosphorus Conference (ESPC3) 196 A3 Figures showing the different stages of the ITC enzyme activity assay for Liberty, Cracka and MR-Buster sorghum varieties 197 A4 Tables for detailed inhibition of proteases using the colorimetric assay (Chapter 3) 200 ix LIST OF FIGURES Figure 1.1 ........................................................................................................................................ 24 Figure 1.2 ........................................................................................................................................ 27 Figure 1.3 ........................................................................................................................................ 27 Figure 2.1 ........................................................................................................................................ 32 Figure 2.2 ........................................................................................................................................ 34 Figure 2.3 ........................................................................................................................................ 36 Figure 2.4 ........................................................................................................................................ 38 Figure 2.5 ........................................................................................................................................ 40 Figure 2.6 ................................................................................................................................... 4241 Figure 2.7 ........................................................................................................................................ 44 Figure 2.8 ........................................................................................................................................ 55 Figure 2.9 .......................................................................................................................................