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ISOLATION, PURIFICATION and PROPERTIES of a Mammlian

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ISOLATION, PURIFICATION and PROPERTIES of a Mammlian ISOLATION, PURIFICATION AND PROPERTIES OF A MAMmLIAN GLUCOSIMSE AND A BACTERIAL FRUCTOSIDASE BY • ' ' KEITH WILLIAM DAWES A Thesis presented to the Faculty of Science of the University of London in '4 partial fulfilment of the requirements for the Degree of Doctor of Philosophy The Bourne Laboratory October, 1978 Royal Holloway College Egham Surrey ProQuest Number: 10097444 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10097444 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 TO MY PARENTS Acknowledgements The author would like to thank the late Professor E.J, Bourne for the opportunity to carry out this research in the Bourne Laboratory, Gratitude is due to Dr H, Weigel for his guidance throughout and to Dr P. Dey and Dr. P. Finch for showing interest in this work. The author would also like to thank Dr E. Detyna for deriving the theoretical rate equations, and to the Science Research Council and Royal Holloway College for financial assistance. ABSTRACT Isolation,Purification and Properties of a Mammalian Glucosidase and a Bacterial Fructosidase BY KEITH WILLIAM DAWES PART 1 The enzyme acid a-g-glucosidase has been isolated from three batches of pig's liver, and purified by molecular gel filtration. Disc gel electrophoresis of the purified enzyme established homogeneity. An isoenzyme of acid a-D-glucosidase, purified to homogeneity, was isolated from one batch. ~ Sodium dodecyl sulphate electrophoresis indicated that the enzyme existed in subunits with a molecular weight of 126,000, 101,500, and 87,500 for each. A study of the enzymic properties of the enzyme was carried out. The enzyme liberated D-glucose in a exo-manner from cibrachon blue amylose. The enzymic hydrolysis of phenyl a-D-glucopyranoside yielded the a-anomer of D-glucose, as confirmed by nmr spectroscopy. A series of rate equations were derived and applied to describe the reaction. The enzyme hydrolysed a variety of u-linked glucosyl oligosaccharides, glycosides and polysaccharides. ' Maltose was the preferred substrate with a K of 4*0 "to 4*4 mM. Glycogen was completely hydrolysed to D-gîucose. ~ A comparison of the properties of the two isoenzymes showed that they both had similar pH optima, thermal stabilities, and subunit structures. The isoenzymes could be separated by cellulose acetate electrophoresis. One isoenzyme had a diminished affinity for glycogen and dextran T^^. PART 2 The extracellular levanase of Streptococcus salivarius strain '51' was isolated from a culture grown on a levan containing medium,gel filtration, ion-exchange and affinity chromatography were used in an attempt to purify the enzyme to homogeneity. A partially purified enzyme preparation was used to study the properties of the enzyme. Heat dénaturation studies indicated that two enzymes were present in the preparation, one of which was thermally unstable. Both enzymes had the ability to hydrolyse both levan and inulin. The preparation was also able to hydrolyse raffinose, sucrose, inulin- and levan- oligosaccharides and methyl p-D-fructofuranoside. CONTENTS Page CHAPTER I PART I ACID a-D-GLUCOSIDASE OF SWINE LIVER 1.1. INTRODUCTION I.A. Classification of Acid oc-D- 10 Glucosidase I.B. Mammalian Acid ct-D-Glucosidases. 11 I.e. Use of Inhibitors to probe the 24 Active Site. I.D. Mechanism of Action of Glycoside 31 Hydrolases. I.E. The role of Acid a-D-glucosidase 46 in Mammalian metabolism and its significance in Type 11 Glycogenesis. 1.2. RESULTS AND DISCUSSION 2,A, Isolation and Purification of the enzyme a. General aspects of purification 54 b. Preliminary purification of the 61 enzyme c. Separation of high molecular 64 weight impurities by gel filtration d. Separation of low molecular 66 weight impurities by gel filtration e. Final purification of the enzyme to 68 homogeneity by gel filtration f. An attempt to purify the enzyme to 70 homogeneity by the use of one gel filtration column eluted with a competitive inhibitor of acid a-D-glucosidase. 76 g. Separation and isolation of an isoenzyme of acid a-D-glucosidase h. Homogeneity of the Glucosidase 86 preparation Page 2.B. Properties of Isoenzyme I a. General Aspects 89 b. Subunit structure and molecular 89 weight of the purified enzyme c. Mode of Action i) Exo-nature of the acid a-glucosidase ii) The configuration of the product 97 released during enzymic hydrolysis iii) Effect of pH on the rate of 122 enzyme hydrolysis iv) Thermal stability of the enzyme ' 123 v) pH stability of the enzyme 123 vi) Action on Glycosides and 127 Disaccharides to show its broad specificity vii) Dextranase properties of the enzyme viii) Studies on the Degradation of 142 linear and branched Polysaccharides 2.C. Properties of Isoenzyme II a. General Aspects 152 b. The relationship between Isoenzyme I 152 and Isoenzyme II c. The molecular weight and subunit 158 structure of isoenzyme II d. Effect of pH on the rate of enzyme 1&0 hydrolysis e. Thermal stability of isoenzyme II 160 f. Activity of isoenzyme II towards 163 maltose,dextran T^^ and glycogen Page CHAPTER II PART II LEVANASE OF STREPTOCOCCUS SALIVARIUS '51' II.1. INTRODUCTION I.A. Naming of the enzymes associated 167 with levan synthesis and degradation I.B. Definition of the term levan 167 I.e. Brief survey of thebacterial 168 fructans and their role in dental caries I.D. Levansucrase 172 1.E. p-fructosidases igQ II.2 RESULTS AND DISCUSSION 2.A. Isolation and Purification a. General aspects of purification ^ b. Preparation of the enzyme 194 c. Preliminary purification of the ^ enzyme preparation d. The partial purification of the 1^7 enzyme by gel permeation chromatography using Sephadex G-200 e. The instability of the enzyme 201 to purification by ion-exchange chromatography f. Purification of the enzyme by 218 gel permeation chromatography on Ultrogel AcA.22. g. Attempt at the purification of 223 the enzyme by affinity chromatography h. Removal of small and larger molecular 23 0 weight proteins by gel permeation chromatography on Biogel A-5m and by use of a molecular filtration membrane i. Use of adsorption chromatography on 234 hydroxyl&P&tite for the purifi­ cation of ^ salivarius levanase Summary 237 Page 2.B. Properties of S. salivarius strain '51' Levanase a. Determination of the pH at 238 maximum levanase activity h. Effect of variation of the 238 reaction temperature on the enzyme activity c. Heat inactivation of the levanase 243 and inulinase activity d. Levanase properties of the partially purified enzyme e. Activity of the preparation 247 towards p-(2— ^1 ) and P~(2— &»6) links in polysaccharides f. Hydrolysis of oligosaccharides 250 containing a terminal p-linked fructosyl unit g. The coupling of an enzymic method 256 for the determination of glucose and fructose with the levanase system: activity of the preparation towards p-linked fructosyl oligosaccharides and other substrates 267 h. Inhibition studies on the inulinase and levanase properties of the enzyme CHAPTER III GENERAL METHODS A. Common Procedures 270 B*. Paper chromatographic methods 270 C. Paper electrophoresis 272 D. Terms used in chromatography 272 E. Buffers 272 F. Gel filtration chromatography 273 G. Ion-exchange chromatography 275 H. Hydroxylapatite chromatography ’ 275 Page I. Affinity chromatography on a modified 276 Biogel P-300 packed column J, Con A-Sepharose chromatography 278 K. Charcoal celite chromatography 278 L, Gas liquid chromatography 279 M. Analytical techniques 279 N. Electrophoretic techniques 283 CHAPTER IV EXPERIMENTAL A. Experiments relating to Chapter I-2-A 289 Experiments 1 to 11 B. Experiments relating to Chapter I-2-B 297 Experiments 12 to 23 C. Experiments relating to Chapter I-2-C 309 Experiments 24 to^O B. Experiments relating to Chapter II-2-A 311 Experiments 31 to 49 E. Experiments relating to Chapter II-2-B 326 Experiments 50 to 59 BIBLIOGRAPHY 333 APPENDIX I An attempted study to show the mode 353 of action of acid a-D-glucosidase at branch points in amylopectin APPENDIX II Further studies of the configuration 359 of the product released during enzymic hydrolysis 10 Chapter I Part I Acid a-D-glncosidase of Swine liver 1-1. Introduction I.A. Classification of Acid a-D-glucosidase The enzyme a-D-glucosidase catalyses the following reaction: CH,OH enzyme (► OH H + ROH HO OH 1 Its catalogue number according to "The Enzyme Handbook" is 3.2.1.20. However, in the early literature enzyme preparations which have catalysed the above reaction have been given a variety of names which are often based on the substrates they hydrolysed. Some examples are given in Table 1. Table 1 Variation in the naming of a-D-glucosidase Hame Reason for Haraing Glucoamylase Property of producing solely glucose from amylose Y-Amylase Y-amylolysis of glycogen Maltase Hydrolysis of maltose Dextranase Hydrolysis of dextrans a-Glucosidase Stresses broad specificity of action This variation clearly resulted from the lack of the detailed knowledge of the substrate specificity of the enzyme. This is amplified by the 11 p definition given by Gottschalk in 1950» He defined an a-D-glucosidase as an enzyme which cleaned the a-D-glucosidic linlcage in oligosaccharides and in a-D-glucosides• It was therefore popular to terra the enzyme as a maltase because of its ability to hydrolyse 4'Q-a-D-glucopyranosyl-a-D-glucopyranose (maltose) .
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