b ¡\il i: ilq.5l¡Tt-!iÈ' ta. S.S LIBRARY STUDIES OF BARLEY LIMIT DEXTRINASE I I I I by I l I ¡ I Michael John Sissons ì B.Ag.Sc. (University of Adelaide)' I I M.Ag.Sc. (La Trobe UniversitY) i l A thesis submitted to the University of Adelaide for the degree of Doctor of Philosophy Departnent of Plant Science, Waite Agricultural Resea¡ch Institute, Glen Osmond, South Australia October, 1991 LIST OF CONTENTS Contents Page No. Summary i-ü Statement of originality and consent for photocopy or loan lU Acknowledgements iv List of publications v Chapter 1: LITERATURE REVIEW 1.1 Introduction I 1.2 Properties of limit dextrinase 2 t.2.r Purification 2 1.2.2 Enzyme properties 3 t.2.3 Effect of inhibitors 3 r.2.4 Polymorphism 7 t.2.5 Substrate specificity 7 1.2.5.1 Action of limit dextrinase on oligosaccharides 7 t.2.5.2 Polysaccharides 7 1.3 Methods of Assay 9 1.3.1 Detection of Limit Dextrinase Activity in Electrophoretic Gels 10 t.4 Synthesis of limit dextrinase t2 1.4.1 Mechanism of Increase in Limit Dextrinase Activity during Germination 13 1.5 Effect of Barley Genotype and Environment on Limit Dextrinase ActivitY 15 1.6 Limit dextrinase - Role in Malting and Brewing 16 1.6.1 Effect of Kilning on Limit Dextrinase Activity 18 r.6.2 Effect of Mashing on Limit Dextrinase Activity 18 1.6.3 Limit 0extrinase-Role in Speciality Brewing, Distiling and Related Iridustries 2l 1.6.4 Relationship beween Limit Dextrinase Activity, Wort Fermentability and Alcohol Production 22 r.7 Role of Limit Dextrinase in Starch Degradation 23 1.8 Conclusions 24 Chapter 2t MATERIALS AND METHODS 2.1 Protein Assay 25 2.2 Enzyme Assays 25 2.2.1 Column Fractions and Purifred Enzyme 25 2.2.2 Extracts of BarleY and Malt Flour 25 2.3 Electrophoretic Methods 26 2.3.1 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis 26 2.3.2 Isoelectic Focusing 27 2.3.3 Silver Staining 28 2.4 Preparation of CYclohexaamYlose Sepharose 68 28 2.5 Red Pultulan AssaY 30 2.6 Micromalting and Methods of Malt Analysis 31 2.6.1 Micromalting Procedure 31 2.6.2 Mashin g and Determination of Extract 3T 2.6.3 Measurement of Apparent Attenuation Limit 34 2.6.4 Moisture Determination 35 2.6.5 Preparation of Malt Flour for Extraction of Limit Dextrinase 35 Chapter 3 PURIFICATION AND CHARACTBRIZATION OF BARLEY LIMIT DEXTRINASE 3.1 INTRODUCTION 36 3.2 MATERIAIS AND METHODS 36 3.2.r Plant Material 36 3.2.2 Purif,rcation of Limit Dexuinase from Malt Extract 37 3.2.2.1 Ammonium sulPhate fractionation 37 3.2.2.2 Desalting 3l 3.2.2.3 Anion-exchan ge chromatograPhY 37 3.2.2.4 Gel frltration chromatograPhY 38 3.2.2.5 Affi nity chromatograPhY 38 3.2.3 Characterization of Ba¡ley Limit Dextrinase 38 3.2.3.r Molecular wei ght determination 38 39 3.2.3.2 Isoelectric Point determi n ation 3.2.3.3 Effect of temPerature on activity and stabilitY 39 3.2.3.4 Effect of pH on activitY 39 3.3 RESULTS 39 3.3.1 Enzyme Purification 39 3.3.2 Characterization 43 3.3.2.1 Molecula¡ wei ght determination 43 3.3.2.2 Isoelectric point 45 3.3.2.3 Effects of temPerature on activitY and stabilitY 45 3.3.2.4 Effect of PH on activitY 46 3.4 DISCUSSION 46 3.4.t Purihcation of Barley Limit Dexuinase 46 3.4.2 Characterization of Limit Dextrinase 48 Chapter 4 DEVELOPMENT OF IMMUNOCHEMICAL METHODS 4.1 INTRODUgTION 49 4.2 MATEzuAIJ AND METT{ODS 50 4.2.1 Immunization Schedule 50 4.2.2 Assessment of AntibodY Titre 50 4.2.3 Separation of IgG from other Serum Proteins 52 4.2.4 Conjugation of Polyclonal IgG Antibodies to Horseradish Peroxidase 52 4.2.5 Immunobloning of SDS-PAGE Gels for Determining the Specificity of the Antiserum 53 4.2.6 Immunoblotting of IEF Gels 54 4.2.6.r Method usingHRP 54 4.2.6.2 Method using alkaline phosphatase conjugate 54 4.2.7 Cro ssed Immu noelectroPhoresis 55 4.2.8 Crossed Immuno-Isoelectric Focusing 56 4.3 RESULTS AND DISCUSSION 58 4.3.1, Separation of IgG from other Serum Proteins 58 4.3.2 Antibody Tire and SPecificitY 60 4.3.3 Development of an ELISA for Measuring Limit Dextrinase in Crude Extracts 1l 4.3.3.r ELISA protocol for measuring limit dextrinase activiry in malt extracts 75 4.3.4 Evaluation of the ELISA 77 4.3.4.1 SpeciftcitY 77 4.3.4.2 Assay linearity 78 4.3.4.3 Potential interference from malt extract 79 4.3.4.4 Evidence that the specihc anti-limit dextrinase antibody recognises active enryme 80 4.3.4.5 Assay reproducibilitY 83 4.3.4.6 Comparison of the ELISA with other enzyme assays 83 4.3.5 Extraction of Limit Dextrinase 89 4.3.6 Crossed Immuno-Isoelectric Focusing 94 4.3.7 Development of an Immunobloting Procedure for Isoelectric Focusing Gels 94 Chapter 5 STUDIES OF THE GENETIC VARIABILITY OF LIMIT DEXTRINASE 5.1 INTRODUCTION tt2 5.2 MATERIALS AND METHODS 113 5.2.1 Cultiva¡ Differences in Limit Dextrinase Activity and Isoenzymes Detected by IEF-Immunoblotting 113 5.2.r.t Plant material 113 5.2.1.2 Analyses 113 5.2.1,.3 Statistical methods tr4 5.3 RESULTS AND DISCUSSION 114 5.3.1 Cultiva¡ Differences in Limit Dextrinase Activity tt4 5.3.2.r Cultivarl)ifferences in LD detected by IEF-Immunoblotting t2t 5.3.2.2 Isozyme variation in Hordeum spontaneum C. Koch r25 5.3.3 Attempts to l-ocate the Chromosome carrying the Structural Gene for Limit Dextrinase 131 5.3.3.1 Studies with wheat-barley addition lines 131 5.3.3.1.1 Introduction t3l s.3.3.r.2 Methods 131 5.3.3.1.3 Results and Discussion t32 5.3.3.2 studies with wheat nullisomic-tetrasomic lines 135 5.3.3.2.1 Introduction 135 s.3.3.2.2 Method 135 5.3.3.2.3 Results and discussion 136 Chapter 6 CHANGES IN LIMIT DEXTRINASE DURING KERNEL DEVELOPMENT 6.1 INTRODUCTION 140 6.2 MATERIAI.S AND METHODS t4r 6.2.1 Barley Material t4l 6_2.2 Preparation of Samples for Analysis r4t 6.2.3 Analyses t4t 6.2.4 Immunoblottin g Analysis t42 6.2.5 Dissection Studies t42 6.3 RESULTS AND DISCUSSION t42 6.3.r Changes in Limit Dextrinase During Kernel Development t42 6.3.2 Analysis of Isoenzyme Va¡iation during Development 149 6.3.3 Location of Limit Dextrinase in Different Tissues of the Developing Kernel 15r Chapter 7 STUDIES OF THE CHANGES IN LIMIT DEXTRINASE DURING GERMINATION, MALTING, KILNING AND MASHING AND 'BOUND' AND 'FREE' FORMS 7.1 INTRODUCTTON r54 7.2 MATERIAI-S AND METFIODS t54 7.2.r Germination Studies r54 7.2.2 Malting, Kilning and Mashing Studies 155 7.2.2.1 Malting 155 7.2.2.2 Kilning 156 7.2.2.3 Mashing 156 7.2.3 Studies of 'Bound' and 'Free' Forms of LimitDexninase 156 7.2.3.r Effect of reducing agents and papain upon the release of 'bound' and 'free'enzyme 156 7.2.3.2 Determination of the proportion of 'bound' and'free' enzyme r57 7.3 RESULTS AND DISCUSSION 157 7.3.1 Changes in Limit Dexrinase Activity during Germination t57 7.3.2 Changes in Limit Dexhinase Activity during Malting 160 7.3.3 Changes in Limit Dextrinase Activity during Kilning r63 7.3.4 Changes in Limit Dexrinase Activity during Mashing t67 7.4 Studies on 'bound' and 'free' forms of limit dextrinase 170 7.4.1 Effect of Reducing Agents and Papain upon'Bound' and'Free' EnzYme r70 7.4.r.1 Introduction r70 7.4.1.2 Methods 17l 7.4.r.3 Results t7r 7.4.r.4 Discussion 175 7.4.2 Determination of the Proportion of 'Bound' and'Free'Enzyme 176 7.4.2.1 Methods r76 7.4.2.2 Results 176 7.4.2.3 Discussion 177 Chapter 8 RELATIONSHIP BETWEEN LIMIT DEXTRINASE AND TIIB APPARENT ATTENUATION LIMIT 8.1 INTRODI.JCTION 179 8.2 MATEzuALS AND METFIODS 180 8.2.t Relationship between Limit Dextrinase and Apparent Attenuation in Genetically Diverse Cultivars 180 8.2.2 Relationship between Limit Dexüinase and Apparent Attenuation in Selected Cultivars Malted for 4,7 and 10 daYs 180 8.3 RESULTS AND DISCUSSION 181 8.3.1 Rel ati on ship bet'ween Limit Dextrin ase Activity and the Apparent Attenuation Limit in Genetically Diverse Cultivars 181 8.3.2 Relationship between Limit Dextrinase and Apparent Attenuation in Selected Cultivars Malted for 4,7 and 10 days 184 190-197 Chapter 9¿ GENERAL DISCUSSION APPENDIX 5.1 198 APPENDIX 5.2 t99-202 APPENDIX fi. I 203 APPENDIX 8.2 204-205 APPENDIX 8.3 206 APPENDIX 8.4 207-210 APPENDIX 8.5 2tt-2t2 BIBLIOGRAPHY I SUMMARY Limit dextrinase (LD) is potentially an important sta¡ch degrading enryme in barley because a-and p-amylase cannot hydrolyse the a-l,Gbonds in amylopectin and limit dextrins. The methods used to date to measure the activity of this enryme in crude extracts of germinated seed or malt are subject to interference from other sta¡ch degrading enzymes present in the extract.