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Reversion Reactions Catalyzed by Glucoamylases I and II From Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1986 Reversion reactions catalyzed by glucoamylases I and II from Aspergillus niger: product analysis, kinetics equilibria, and modeling Zivko Nikolov Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Chemical Engineering Commons Recommended Citation Nikolov, Zivko, "Reversion reactions catalyzed by glucoamylases I and II from Aspergillus niger: product analysis, kinetics equilibria, and modeling " (1986). Retrospective Theses and Dissertations. 8282. https://lib.dr.iastate.edu/rtd/8282 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS While the most advanced technology has been used to photograph and reproduce this manuscript, the quality of the repr^uction is heavily dependent upon the quality of the material submitted. For example: • Manuscript pages may have indistinct print. In such cases, the best available copy has been filmed. • Manuscripts may not always be complete. In such cases, a note will indicate that it is not possible to obtain missing pages. • Copyrighted material may have been removed from the manuscript. In such cases, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, and charts) are photographed by sectioning the original, beginning at the upper leA-hand comer and continuing from left to right in equal sections with small overlaps. Each oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or as a 17"x 23" black and white photographic print. Most photographs reproduce acceptably on positive microfiîm or microfiche but lack the clarity on xerographic copies made from the microfilm. For an additional charge, 35mm slides of 6"x 9" black and white photographic prints are available for any photographs or illustrations that cannot be reproduced satisfactorily by xerography. 8703741 Nikolov, Zivko REVERSION REACTIONS CATALYZED BY GLUCOAMYLASES I AND II FROM ASPERGILLUS NIGER: PRODUCT ANALYSIS, KINETICS, EQUILIBRIA, AND MODELING Iowa Slate University PH.D. 1986 University Microfilms I nt6rn&ti On si 300 N. zeeb Road, Ann Arbor, Ml 48106 Reversion reactions catalyzed by glucoamylases I and II from Aspergillus niger; product analysis, kinetics equilibria, and modeling by Zivko Nikolov A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major: Chemical Engineering Signature was redacted for privacy. Signature was redacted for privacy. For the M^or Department Signature was redacted for privacy. For the Graduate College Iowa State University Ames, Iowa 1986 il TABLE OF CONTENTS Page DEDICATION v GENERAL INTRODUCTION 1 Explanation of Dissertation Format 2 REFERENCES 4 SECTION I. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF DISACCHARIDES ON AMINE-BONDED SILICA COLUMNS 5 SUMMARY 6 INTRODUCTION 7 EXPERIMENTAL 9 Apparatus 9 Materials 9 Chromatographic Conditions and Measurements 10 RESULTS AND DISCUSSION 11 ACKNOWLEDGEMENTS 20 REFERENCES 21 SECTION II. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF TRISACCHARIDES ON AMINE-BONDED SILICA COLUMNS 22 SUMMARY 23 INTRODUCTION 24 EXPERIMENTAL 26 Chromatographic Apparatus and Procedures 26 Trisaccharides 26 RESULTS AND DISCUSSION 28 ACKNOWLEDGEMENTS 40 REFERENCES 41 ill SECTION III. RETENTION OF CARBOHYDRATES ON SILICA AND AMINE-BONDED SILICA STATIONARY PHASES: APPLICATION OF THE HYDRATION MODEL 42 SUMMARY 43 INTRODUCTION 44 EXPERIMENTAL 46 RESULTS AND DISCUSSION 49 ACKNOWLEDGEMENTS 60 REFERENCES 61 SECTION IV. KINETICS, EQUILIBRIA, AND MODELING OF THE GLUCOAMYLASE-CATALYZED FORMATION OF DI- AND TRISACCHARIDES FROM GLUCOSE 62 SUMMARY 63 INTRODUCTION 65 MATERIALS AND I4ETH0DS 75 Enzyme Source 75 Substrates 75 Enzyme Assays 75 Reversion Reaction Experiments 76 Gas Chromatography 77 Initial Rate Determination 77 KINETIC MODEL 79 Estimation of Kinetic Parameters 84 RESULTS AND DISCUSSION 88 Comparisons of Model Predictions with Experimental Results 101 CONCLUSIONS 120 REFERENCES 122 ACKNOWLEDGEMENTS 124 iv APPENDIX 125 Glucoamylase Purification 125 REFERENCES 132 V DEDICATION I dedicate this dissertation to Mother, with love. 1 GENERAL INTRODUCTION Glucoamylase (a-l,4-glucan glucohydrolase, EC 3.2.1.3) is a typical exo-glucanase in its production of D-glucose from the nonreducing ends of amylose, amylopectin, and maltooligosaccharide molecules with inversion of configuration of the anomeric carbon (1). It is an important industrial enzyme used in the hydrolysis of liquefied starch to syrups of high glucose concentration. Although glucoamylase is capable of hydrolyzing a-1,6- as well as a-l,4-glucosidic linkages (2), glucose yields higher than 95% of theoretical are not achieved in concentrated starch solutions because of the occurrence of glucose condensation reactions, commonly called reversion reactions (3). Formation of reversion product, mainly di- and tri- saccharides, was often attributed to the catalytic action of transgluco- sylase contamination present in the glucoamylase preparations. However, a number of studies published in the last twenty years indicate that glucoamylase itself is capable of synthesizing various a-linked di- and trisaccharides in concentrated glucose solutions (4-10), The reported reversion products include isomaltose (6-0-a-D-glucopyranosyl-D-glucose), maltose (4-0-a-D-glucopyranosyl-D-glucose), nigerose (3-0-a-D-gluco- pyranosyl-D-glucose), kojibiose (2-0-a-D-glucopyranosyl-D-glucose), a,3-trehalose (a-O-D-glucopyranosyl-3-D-glucopyranoside), isomaltotriose 2 2 (6 -0-ct-D-glucopyranosyl-D-isomaltose), panose (6 -0-a-D-glucopyranosyl-D- 2 maltose), isopanose (4 -0-a-D-glucopyranosyl-D-isomaltose), and maltotriose 2 (4 -O-cx-D-glucopyranosyl-D-maltose). Two disaccharides, a,3-trehalose and kojibiose, were observed for the first time in our previous work (10). 2 Recently research Interest has been directed towards the kinetics and modeling of reversion reactions (9, 11-13). The common features of these studies are that maltose and isomaltose were the only condensation products detected and modeled, except for Beschkov et al. (11) who also included maltotriose, and that high performance liquid chromatography (HPLC) was used as an analytical method. Critical examination of the literature data shows significant dis­ agreement in the number and the type of reversion products formed. Some of the discrepancies may be caused by differences in experimental condi­ tions, like pH, temperature, and initial substrate concentration, or use of glucoamylase preparations with a different degree of purity and from a different origin. In addition, the applied chromatographic methods were probably not efficient enough to separate and identify all reaction products. Although evidence exists that glucoamylase is capable of synthesizing a-glucosidic bonds other than a-1,4 and a-1,6, fundamental understanding of condensation reactions is still lacking. The objectives of the kinetic studies (Section IV) were to examine the effect of reaction conditions on the rates and equilibria of reversion reactions as well as to compare the catalytic activities of A. niger glucoamylase forms. Attention has been focused on testing the validity and generality of the derived model which describes the simultaneous formation of reversion products. Explanation of Dissertation Format The dissertation contains four sections written in a form suitable for publication. The first three sections, which deal with HPLC of 3 carbohydrates, have already been published in the Journal of Chromatography (14-16), The last section presents the work on glucose reversion reac­ tions. The purification procedure of glucoamylase forms is described in the Appendix. Each section, as well as the General Introduction and Appendix, contains a list of references cited within. 4 REFERENCES 1. F. W. Parrish and E. T. Reese, Carbohydr. Res., 3 (1967) 424. 2. J. H. Pazur and T. Ando, Biol. Chem., 235 (1960) 297. 3. L. A. Underkofler, L. J. Denault, and E. F. Hou, St'ârke, 17 (1965) 179. 4. J. H. Pazur and S. Okada, Carbohydr. Res., 4 (1967) 371. 5. T. Watanabe, S. Kawamura, H. Sasaki, and K. Matsuda, Starke, 21 (1969) 18. 6. T. Watanabe, S. Kawamura, H. Sasaki, and K. Matsuda, Starke, 21 (1969) 44. 7. E. J. Hehre, G. Okada, and D. S. Genghof, Arch. Biochem. Blophys., 135 (1969) 75. 8. J. H. Pazur, A. Cepure, S. Okada, and L. S. Forsberg, Carbohydr. Res., 58 (1977) 193. 9. J. A. Roels and R. van Tilburg, Starch/Starke, 31 (1979) 338. 10. Z. L. Nikolov, M.S. Thesis, Iowa State University, Ames, 1983. 11. V. Beschkov, A. Marc, and J. M. Engasser, Biotechnol. Bioeng., 26 (1984) 22. 12. S. Adachi, Y. Ueda, and K. Hashimoto, Biotechnol. Bioeng., 26 (1984) 121. 13. F. Shiraishi, K. Kawakami, and K. Kusunoki, Biotechnol. Bioeng., 27 (1985) 498. 14. Z. L. Nikolov, M. M. Meagher, and P. J. Reilly, J. Chromatogr., 319 (1985) 51. 15. Z. L. Nikolov, M. M. Meagher, and P. J. Reilly, ^J. Chromatogr., 321 (1985) 393. 16. Z. L. Nikolov and P. J. Reilly, Chromatogr., 325 (1985) 287. 5 SECTION I. HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY OF DISACCHARIDES ON AMINE-BONDED SILICA COLUMNS 6 SUI'IMARY
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