Hand Out Enzyme

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Enzymatic Hydrolysis of CelluloseExperimental and Modeling Studies

Natalija Andersen Ph.D. Thesis October 2007



The present work represents the result of my PhD study carried out as a part of the research school Novozymes Bioprocess Academy, which is collaboration between the industrial partner, Novozymes A/S, and two departments at the Technical University of Denmark (DTU), BioCentrum and Department of Chemical Engineering. Experimental part of this study was conducted at Center for Microbial Biotechnology (CMB), BioCentrum - DTU, while modeling study was conducted at IVC-SEP group, Chemical Engineering department. The study was carried out in the period January 2004 to December 2006. During my PhD study I received grants from Otto Mnsteds Fond to cover the expenses for participation at conferences in the USA and Chile. I would very much like to thank my four supervisors, Professor Lisbeth Olsson (CMB), Docent Michael L. Michelsen (IVC-SEP), Professor Erling H. Stenby (IVC-SEP) and Senior Scientist Katja S. Johansen (Novozymes) for their valuable guidance, inspiration, and support throughout the whole period. Lisbeth, thank you for your enormous enthusiasm, always positive thinking, and encouragement when the things got rough. Michael, you helped me a lot with modeling and all my small and big questions about it, and I am very grateful for that. Erling, you contributed a lot to our discussions by your holistic view and by ability to see things from a different angle, and Katja, I thank you for valuable discussions on enzymes and for providing the industrial point of view. During my PhD study I co-supervised Cleo C.W. Chang who conducted her master project on enzymatic hydrolysis of cellulose. She did a great work and I wish her all the best in her PhD and future career. I would also like to thank all my former and present colleagues at CMB and Chemical Engineering department, for three very good and pleasant years. It has been a pleasure to be a part of this open-minded, international environment. However, a special thank to Kristian Krogh for interesting and inspiring brain-stormings and lots of help in the lab, and to Kianoush Hansen, Tina Johansen and Jette Mortensen for technical support. Susan, Maya and Gianni, it was great shearing an office with you. i

Finally, I would like to thank my family and friends for their enormous support during the last three years. I would especially like to thank my parents Milica and Zvonimir Popovic for both moral and economical support during my studies, and to my wonderful daughter, Sara for being so loving, nice and easy.

Natalija Andersen August, 2007 Copenhagen, Denmark


CONTENTS1. Introduction1 1.1 References3 2. Cellulosic material.....5 2.1 Lignocellulose (plant cell wall polysaccharides).5 2.1.1 Cellulose 2.1.2 Hemicellulose 2.1.3 Lignin 2.1.4 Pretreatment of lignocellulose 2.2 Cellulose in model substrates.12 2.3 References..15 3. Analytical methods for quantification of enzymatic hydrolysis..19 3.1 Traditional enzyme assays.....19 3.1.1 Nielson-Somogyi assay 3.1.2 PAHBAH assay 3.1.3 DNS assay 3.1.4 2-cyanoacetamide assay 3.1.5 Ferricyanide assay 3.1.6. Summary of the results reducing saccharide assays 3.2 Chromatographic techniques.24 3.3 Other (novel) techniques....25 3.3.1 Optimization of PACE for cellulose hydrolysis studies 3.4 Summary....33 3.5 References..34 4. Cellulolytic enzymes.37 4.1 Molecular structure of cellulolytic enzymes..38 4.2 Mechanisms of cellulase activity...41 4.3 Classification of cellulases.42 4.3.1 The complete cellulolytic system (multiple cellulases) 4.3.2 Endoglucanases 4.3.3 Cellobiohydrolases 4.3.4 -glucosidases 4.3.5 Summary of the enzymes used in this study 4.4 References..50 5. Synergism between the enzymes.55 5.1 References..56 5.2 Article A: Enzymatic hydrolysis of cellulose using mono-component enzymes show synergy during hydrolysis of Phosphoric Acid Swollen Cellulose (PASC), but competition on Avicel...........................57 6. Factors affecting enzymatic hydrolysis of cellulose..69 6.1 Enzyme related factors..70 6.2 Physical properties of the substrate affecting the hydrolysis.73 6.3 References..76


Article B: Enzymatic degradation of cellulose - Investigation of declining hydrolysis rate79 7. Mathematical modeling of enzymatic degradation of cellulose...91 7.1 Major aspects and challenges during modeling of hydrolysis process .94 7.1.1 Specific surface area (SSA) and crystallinity index (CrI) 7.1.2 Available/accessible surface area 7.1.3 Deactivation of enzymes 7.2 De-polymerization type of model..99 7.2.1 Individual enzyme kinetics for E1, E2, and E3 7.2.2 Enzyme kinetic parameters used in the model 7.2.3 Comparison of model predictions and experimental results 7.3 Summary..111 7.4 References112 7.5 Nomenclature...115 8. Application of Metabolic Control Analysis (MCA) theory to the enzymatic hydrolysis of cellulose....117 8.1 MCA theory.118 8.1.1 Control coefficients and the summation theorem 8.1.2 Elasticity coefficient and the connectivity theorem 8.2 MCA and enzymatic hydrolysis of cellulose...121 8.2.1 Experimental procedure 8.2.2 Construction of the kinetic model on Gepasi 8.2.3 Enzymatic hydrolysis of PASC Results 8.2.4 Kinetic model of enzymatic hydrolysis of cellulose Results 8.2.5 MCA of the kinetic models - Results 8.2.6 Summary and discussion of the results 8.2.7 Conclusions 8.3 References....132 9. Conclusions and future perspectives....133 9.1 References136 10. Appendix....137 10.1 Nelson-Somogyi assay.137 10.2 4-Hydroxybenzoic acid hydrazide (PAHBAH) assay.139 10.3 Dinitrosalicylic acid (DNS) assay140 10.4 2-Cyanoacetamide assay..141 10.5 Ferricyanide assay142 10.6 8-Aminonaphtalene-1,3,6-trisulfonic acid (ANTS) derivatization..143 10.7 Polyacrylamide gel preparation ......144 10.8 Gel imaging .146 10.9 Glucose oligomer ladder preparation ..147 10.10 Electrophoresis 148 10.11 Enzymatic hydrolysis ..149 10.12 Glucose oxidase-peroxidase assay ..150 11. Sammenfatning p dansk151



LIST OF ABBREVIATIONSANTS BET BG BMCC BSA C1 C2 C3 C4 C5 C6 CBH CBM CD CMC CrI DMSO DNS DP EG FPA HPAEC HPLC IUPAC PACE PAD PAHBAH PASC Rxn SSA TEMED 8-aminonaphthalene-1,3,6-trisulfonic acid Bennet-Emmit-Teller -glucosidase Bacterial micro-crystalline cellulose Bovine serum albumin Glucose Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose Cellobiohydrolase Carbohydrate binding module Catalytic domain Carboxymethyl cellulose Crystallinity index Dimethyl sulfoxide Dinitrosalicylic acid Degree of polymerization Endoglucanase Filter paper activity High performance anion-exchange chromatography High pressure liquid chromatography International Union of Pure and Applied Chemistry Polysaccharide analysis using carbohydrate gel electrophoresis Pulsed amperometric detection 4-Hydroxybenzoic acid hydrazide Phosphoric Acid Swollen Cellulose Reaction Specific surface area N,N,N',N'-Tetramethylethylenediamine




IntroductionCellulose is the major polymeric component of plant material and is the most abundant polysaccharide on Earth. In nature, a variety of microorganisms are known for producing a set of enzymes capable of degrading this insoluble polymer to soluble sugars, primarily cellobiose and glucose. Enzymes involved in these processes are called cellulases and are consisting of at least three classes of enzymes, namely, endogluganases (EG), cellobiohydrolases (CBH) and -glucosidases (BG). Cellulases can be used in the variety of applications within food, vine, animal feed, textile and pulp and paper industry (Bhat, 2000). The application and interest in cellulases has particularly increased in recent years with the utilization of the enzymes in the production of bioethanol from lignocellulose (Sun and Cheng, 2002). Bioethanol can be blended at low concentrations with petrol (gasoline) or diesel for use in todays vehicles, and is considered to be a sustainable transportation fuel. Alternatively, if bioethanol is used in higher, or 100 % concentrations, adopted vehicles are typically needed. The main motivation for investments in research and process development concerning bioethanol production is environmental concern related to global warming. The focus is, in particular, turned towards the reduction of CO2 emissions and other so-called green house gases. Moreover, sustainable bioethanol production would decrease the dependency on the traditional, natural oil, reserves, which can due to their restricted geographical localization cause political tension and economical instability. Under EU proposal 0547 from November 7, 2001, a series of goals were set for member states to introduce biofuels for diesel and gasoline. By 2005, 2 % of transport fuel should be accounted for by biofuels; by 2020, the goal is 20 %. First-generation biofuels are made from food crops, such as sugar cane and corn. This can offer some CO2 benefits and can help to improve domestic energy security. Nevertheless, concerns exist about the sourcing of feedstocks, including the impact it may have on biodiversity and land use, and competition with food crops. Second1

Chapter 1: Introduction

generation biofuels are made from non-food feedstocks, such as waste from agriculture and forestry. Second-generation biofuels could significantly reduce CO2 production, do not compete with food crops, and, some types can offer better engine performance (www.shell.com, August 2007). Third-generation biofuels technology is directed towards, so called, synthetic biology, e.g. discovery, development and commercialization of engineered cellulase enzymes that are incorporated into the corn plants themselves, or development of crops whose lignin-content (the hard, woody part of plants' cell walls) has been artificially weakened and reduced, and