Problem Solving in Enzyme Biocatalysis

By Andrés Illanes, Lorena Wilson and Carlos Vera

Enzyme biocatalysis is a fast-growing area in process biotechnology that has expanded from the traditional fields of foods, detergents, and leather applications to more sophisticated uses in the pharmaceutical and fine-chemicals sectors and environmental management. Conventional applications of industrial enzymes are expected to grow, with major opportunities in the detergent and animal feed sectors, and new uses in biofuel production and human and animal therapy.

In order to design more efficient enzyme reactors and evaluate performance properly, sound mathematical expressions must be developed which consider enzyme kinetics, material balances, and eventual mass transfer limitations. With a focus on problem solving, each chapter provides abridged coverage of the subject, followed by a number of solved problems illustrating resolution procedures and the main concepts underlying them, plus supplementary questions and answers.

Based on more than 50 years of teaching experience, Problem Solving in Enzyme Biocatalysis is a unique reference for students of chemical and biochemical engineering, as well as biochemists and chemists dealing with bioprocesses.

Contains: Enzyme properties and applications; enzyme kinetics; enzyme reactor design and operation 146 worked problems and solutions in enzyme biocatalysis. 

• Language: English
• Publisher: Wiley
• Release date: Oct 2, 2013
• ISBN: 9781118341773

Andrés Illanes

Dr. Andrés Illanes is a Full Professor of the Biochemical Engineering School and Head of the Biocatalysis Group, Senior Researcher at the Biotechnology Nucleus of the Pontificia Universidad Católica de Valparaíso. Dr. Illanes’ fields of expertise are enzyme biocatalysis, enzyme immobilization and bioreactor design. He is author of over 150 publications in ISI WoS journals, several book chapters and editor of three books in enzyme biocatalysis for Wiley, Springer and Academic Press-Elsevier.

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Preface

You shall bring forth your work as a mother brings forth her child: out of the blood of your heart. Each act of creation shall leave you humble, for it is never as great as your dream and always inferior to that most marvelous dream which is Nature.

Decalogue of the Artist, Gabriela Mistral, Chilean Nobel Laureate

This book is primarily intended for chemical and biochemical engineering students, but also for biochemists, chemists, and biologists dealing with biocatalytic processes. It was purposely written in a format that resembles the Schaum's textbooks of my long gone college years. An abridged coverage of the subject is provided in each chapter, followed by a number of sample exercises in the form of solved problems illustrating resolution procedures and the main concepts underlying them, along with supplementary problems for the reader to solve, provided with the corresponding answers. Learning through problem solving is designed to be both challenging and exciting to students. There is no book on enzyme biocatalysis with this format and purpose, so we sincerely hope to have made a contribution to the toolbox of graduate and undergraduate students in applied biology, chemical, and biochemical engineering with formal training in college-level mathematics, organic chemistry, biochemistry, thermodynamics, and chemical reaction kinetics. The book pretends to be a complement of a previous book, Enzyme Biocatalysis (Springer, 2008), which I had the privilege and pleasure to edit.

Chapter 1 is an introduction, giving an updated vision of enzyme biocatalysis, its present status, and its potential development. Chapters 2 and 3 refer to enzyme kinetics in homogeneous and heterogeneous systems, respectively, considering both fundamental and practical aspects of the subject. Chapter 4 is devoted to enzyme reactor design and operation under ideal conditions, while Chapters 5 and 6 deal with the main causes of nonideal behavior (mass-transfer limitations and enzyme inactivation, respectively). Chapter 7 presents an overview of the optimization of enzyme reactor operation and different tools for optimization. The book is complemented by two documents. Appendix A refers to mathematical methods for those readers not sufficiently acquainted with the subject, while Epsilon Software Information presents a software program ( psilon) for solving and representing enzyme reactor performance under different scenarios.

Writing is certainly a most exciting endeavor. Undoubtedly, writing about enzymes is beyond being the subject of our expertise, because enzymes are the catalysts of life, so writing about enzymes lies in the boundaries of writing about life. And life, as the Iraqi poet Abdul Wahab said, is about standing straight, never bent or bow, about remaining glad, never sad or low. We hope our readers will keep straight and glad while getting a little more acquainted with these catalysts of life.

A book is a journey from expectation to consolidation, an act of love from conception to birth. It has been a most rewarding experience to travel and conceive in the company of my co-authors, two brilliant young colleagues of mine, Dr. Carlos Vera and Dr. Lorena Wilson, who were formerly my students and now fly well over my shoulders. My gratitude also to my colleague Dr. Raúl Conejeros, who authored Chapter 7 and beyond that contributed to spice this book throughout with mathematical modeling and elaborated thinking. Do not blame him too much; he is a good mix of wisdom and kindness. Our gratitude also to Dr. Felipe Scott for developing, together with Dr. Carlos Vera, the software Epsilon, presented in Epsilon Software Information to the development of the Epsilon Software.

A significant part of this book was written in Spain, at the Chemical Engineering Department of the Universitat Autònoma de Barcelona, where I was (again) warmly hosted while undertaking a sabbatical (my third one there) during the second semester of 2012. My personal gratitude to my colleagues there, Dr. Josep López and Gregorio Álvaro, who made me feel at home and who shared the ecstasy and the agony of this pregnancy.

My gratitude to the people at my institution, the Pontificia Universidad Católica de Valparaíso, who supported and encouraged this project, particularly to the rector, Professor Claudio Elórtegui, and the Director of my School of Biochemical Engineering, Dr. Paola Poirrier. Special thanks to Dr. Atilio Bustos, Head of the Library System, for his enthusiastic support and advice. My deepest appreciation also to the people at Wiley: Rebecca Stubbs and Sarah Tilley, and Shikha Pahuja at Thomson Digital, who were always helpful, warm, and supportive.

Last but not least, my gratitude to my life partner Dr. Fanny GuzmÁn, expert in peptide synthesis, loving care, and savoir-vivre.

Engineering is about products and processes. A book is both. We hope you will enjoy the product as much as we have enjoyed the process.

Andrés Illanes

Valparaíso, July 2013

Nomenclature

Epsilon Software Information

Available alongside this book is a copy of the software psilon which allows the simulation of the operation of enzymatic reactors. psilon has been designed to illustrate the main topics included in this book, offering an additional tool to improve the understanding of the design and operation of enzymatic reactors (specifically Chapters 4 to 6). Guidelines for software installation are given below:

i. Program Installing

Two situations may occur during installation:

– Matlab® is already installed in user's computer

The program was built using Matlab®'s compiler toolbox. Hence, if Matlab® is installed in the user's computer, open the distrib folder and run the file Epsilon_32.exe or Epsilon_64.ex, depending on the system's architecture.

– Matlab® is not installed in user's computer

First, open the Matlab® package (Epsilon_64_pkg or Epsilon_32_pkg, depending on the system's architecture). Two files will be created in the current directory. Second, open MCRInstaller.exe and follow the installer instructions. Once the installation is complete, open the newly created Epsilon_64.exe or Epsilon_32.exe.

ii. Program Description

This software was designed with a simple interface, with the purpose of allowing the comparison of reactor behavior under different scenarios (reaction kinetics and operation modes). The program considers reactor performance for biocatalysts having the most common enzyme kinetics (Michaelis-Menten, competitive and non-competitive inhibition by product and uncompetitive inhibition by substrate) and operating in usual reactor configurations (BSTR, CPBR and CSTR). The program also allows incorporating the effect of external diffusional restrictions (EDR) or catalyst inactivation during reactor operation. Figure 1 shows the interface of psilon.

Figure 1 psilon interface.

iii. Using Epsilon

The use of psilon is straightforward. First, select reaction kinetics; a scheme showing the selected kinetic mechanism should appear. Then input the value of the model parameters into the bottom table. After that, choose the reactor configuration and the plot type desired to show the reactor performance. Furthermore, it is possible to include the effect of EDR and enzyme inactivation in the simulation.

If EDR is selected, introduce the value of the Damkoehler number for substrate, αs, and product, αp, (in the case of product inhibition). The former is defined by Equation (3.15); the latter is defined as:

(1) equation

where KP is the inhibition constant by product and hP is the film volumetric mass transfer coefficient for product. If enzyme inactivation is taken into account, input the value of the corresponding inactivation parameters (see Chapter 6). A scheme of the inactivation mechanism will appear if this option is selected.

To plot reactor performance under the determined conditions, press the update button. To compare this condition with another, you will need to repeat the process choosing the new operational conditions, and then press the update button again. Press the clear plots button to reset the program.

To simulate the reactor operation under ideal conditions, psilon will solve the mathematical expressions presented in Table 4.1 for the corresponding kinetics and reactor mode of operation. The effect of EDR on reactor behavior is simulated using the corresponding Equations (5.7) to (5.9). To calculate the effectiveness factor, it is assumed that, at steady-state, mass transfer rate and reaction rate should be equal at the catalyst surface. So, when product inhibition is considered, the corresponding system of two equations will be solved using Newton's Method (see Appendix A). For the simulation of biocatalyst deactivation, the program considers a two-stage series mechanism (see Equations (6.2) and (6.3)). In the case of continuous reactors, psilon will solve Equation (6.16) for a set of given times ranging from zero to final reaction time. Again, the former Equation will be solved using Newton's Method (see Appendix A). For a batch reactor, inactivation kinetics is incorporated into Equation (4.4), similarly as shown by Equation (6.7). The resulting differential equation will be solved by numerical integration.

This software has been developed using Matlab and Matlab Compiler which can be found at the Mathworks website http://www.mathworks.co.uk/products/compiler/mcr/

Acknowledgement

With respect to Dr. Felipe Scott contribution to Epsilon Software Information referred to the Epsilon Software, a special acknowledgement should be done since he made the most significant contribution to the development of that software.

1

Facts and Figures in Enzyme Biocatalysis

1.1 Introduction

1.1.1 Enzyme Properties

Enzymes are the catalysts of life. Each of the chemical reactions that make up the complex metabolic networks found in all forms of life is catalyzed by an enzyme, which is the phenotypic expression of a specific gene. Thus the chemical potential of an organism is dictated by its genomic patrimony and enzymes are the biological entities that convert information into action. They are tightly regulated both by controls at the genomic level and by environmental signals that condition their mode of action once synthesized.

Enzymes are highly evolved complex molecular structures tailored to perform a specific task with efficiency and precision. They can be conjugated with other molecules or not, but their catalytic condition resides in their protein structure. The active site—the molecular niche in which catalysis takes place—represents a very small portion of the enzyme structure (only a few amino acid residues), while the remainder of the molecule acts as a scaffold and provides necessary structural stability. Many enzymes are conjugated proteins associated with other molecules that may or may not play a role in the catalytic process. Those that do are quite important as they will determine to a great extent an enzyme's technological potential.

Enzyme biocatalysis refers to the use of enzymes as biological catalysts dissociated from the cell from which they derive; the major challenge in this process is building up robust enzyme catalysts capable of performing under usually very nonphysiological conditions. The goal is to preserve the outstanding properties of enzymes as catalysts (specificity, reactivity under mild conditions) while overcoming their constraints (mostly their poor configurational stability). The pros and cons of enzymes as catalysts are thus the consequence of their complex molecular structure. Enzymes are labile catalysts, with enzyme stabilization being a major issue in biocatalysis and a prerequisite for most of their applications. Several enzyme stabilization strategies have been proposed, including: searching for new enzymes in the biota and metagenomic pools [1], improving natural enzymes via site-directed mutagenesis [2] and directed evolution [3], catalyst engineering (chemical modification [4], immobilization to solid matrices [5], and auto-aggregation [6]), medium engineering (use of nonconventional reaction media) [7], and, most recently, reactivating enzymes following activity exhaustion [8]. Enzyme immobilization has been a major breakthrough in biocatalysis and has widened its field of application considerably [9].

1.1.2 Enzyme Applications

Enzymes have found a wide spectrum of applications, from a very large number of enzyme-catalyzed industrial processes to use within the toolbox of molecular biology. Besides their use as process catalysts in industrial processes, which will be analyzed in Section 1.2, enzymes have found important applications in:

Chemical and clinical analysis, due to their high specificity and sensibility, which allow the quantification of various analytes with high precision. Worth mentioning are their uses in flow injection analysis [10], robust electrodes for process control [11], and nanosensors [12]. Enzymes are also widely used in various diagnostic kits and as detectors in immunoassays [13].

Therapy, due to their high specificity and activity, which allow the precise and efficient removal of unwanted metabolites. Many therapeutic applications have been envisaged, including for such enzymes as asparaginase, billirubin oxidase, carboxypeptidase, α-glucosidase, α-galactosidase, phenylalanine ammonia lyase, streptokinase, urease, uricase, and urokinase. The US Food and Drug Administration (FDA) has already approved applications in cardiovascular disorders, pancreatic insufficiency, several types of cancer, the replacement therapy for genetic deficiencies, the debridement of wounds, and the removal of various toxic metabolites from the bloodstream [14].

Environmental management (waste treatment and bioremediation). Enzyme specificity allows the removal of particularly recalcitrant pollutants from hard industrial wastes and highly diluted effluents. Enzymes are also used in the final polishing of municipal and industrial effluents following conventional treatment and as enhancers of the hydrolytic potential of the microbial consortia in the first step of anaerobic digestion [15]. In addition, they are increasingly being used in bioremediation of polluted soils and waters with recalcitrant compounds [16], although the high cost of the enzymes is still prohibitive in most such cases [17].

Biotechnological research and development. Enzymes are fundamental components of the toolbox for biotechnology research, especially in the areas of molecular biology and genetic engineering. Thermostable DNA polymerases are the basis of the polymerase chain reaction (PCR) [18], which is fundamental to gene amplification, while restriction endonucleases and ligases are fundamental tools in recombinant DNA technology [19].

Highly purified enzymes are required for most of these applications, which are sold at very high unitary prices. Therefore, despite their low volume of production, the market size is significant.

1.2 Enzymes as Process Catalysts

Enzymes used as catalysts for industrial processes are generally rather impure preparations sold as commodities at low unitary prices, although this tendency is changing to some extent, with increasing usage in an immobilized form and in organic synthesis. It can be estimated that industrial applications make up roughly 70% of the enzyme market size.

Enzyme transformations can be used in industrial applications to create the desired product (e.g. high-fructose syrup is produced from starch by the sequential operations of liquefaction, saccharification, and isomerization, catalyzed by α-amylase, amyloglucosidase, and glucose (xylose) isomerase, respectively). Enzymes can also be used as additives, in order to confer certain functional properties on the product, as illustrated by many applications in the food sector (amylases and proteases in bread making, phytases and β-glucosidases in animal feed upgrading, pectinases in fruit juice and wine making, β-galactosidases in low-lactose milk and dairy products, and so on).

Enzymes have been used systematically in industry since the beginning of the 20th century. Originally they were mostly crude preparations extracted from plant and animal tissues and fluids, but with the development of fermentation technology and industrial microbiology in the middle of that century, microbial enzymes began to take over. These had the advantages of intensive and reliable production and a wide spectrum of activities. In recent decades, advances in recombinant DNA technology have allowed genes of any origin to be expressed into a suitable microbial host [20], as well as in protein engineering techniques that allow enzyme properties to be improved through progressively rational approaches [21]. While plant and animal enzymes still have some application niches, most of the enzymes used industrially today are produced by microbial strains.

Until recently, most of the enzymes used as industrial catalysts were hydrolases, which are particularly robust and are frequently extracellular proteins that do not require coenzymes, making them well suited to use under harsh process conditions. Most industrial enzymatic processes are catalyzed by carbohydrases, proteases, and lipases, which are mostly used in their hydrolytic capacity to degrade substrates (frequently polymers) into products of lower molecular complexity. Some traditional industrial applications are listed in Table 1.1.

Table 1.1 Enzymes traditionally used as industrial catalysts.

Most of the just-mentioned applications can be considered mature technologies, although significant improvements are ongoing. Major breakthroughs in detersive enzymes have been made rather recently, with the incorporation of highly robust lipases and α-amylases into the already sophisticated proteases currently in use. Phytases have recently been developed and successfully introduced into the fast-growing animal feed market. Major technological breakthroughs are being made in the use of enzymes in biofuel production. α-amylases used in the first-generation production of bioethanol from starchy feedstocks are continuously being improved, although land competition for food production has turned attention to the production of second-generation bioethanol from cellulosic raw materials through the use of cellulase preparations. There are now highly active enzyme cocktails with well-dosed activities that can efficiently hydrolyze cellulose from pretreated woods at very high solids concentrations, delivering high-glucose syrups for ethanol fermentation. High-tonnage plants for second-generation bioethanol are going into operation and their impact on the energy bill is forecasted to be significant by 2020.

1.3 Evolution of Enzyme Biocatalysis: From Hydrolysis to Synthesis

Enzymes were long considered catalysts for the molecular degradation of natural substrates in aqueous media, and most of their traditional industrial uses accord with this, as illustrated in Table 1.1. However, a new paradigm for enzyme biocatalysis that challenges this view has appeared in recent decades and a myriad of technologically relevant reactions of organic synthesis (in which the high selectivity of enzymes is well appreciated), frequently conducted in nonaqueous media and on non-natural substrates, have been developed [22]. Here the goal is molecular construction rather than degradation, which is quite meaningful from an industrial perspective, since the potential added value is certainly much higher than in conventional degradation processes.

Technological breakthroughs in both medium engineering and catalyst engineering are the basis of this change of paradigm. Enzyme catalysis in nonconventional (nonaqueous) media has undergone impressive development since the pioneering work of Klibanov and others in the 1980s [23], so that enzymatic reactions can now be efficiently conducted in several nonaqueous media, such as organic solvents, ionic liquids, supercritical fluids, and semisolid systems. Screening for new enzymes and genetic improvement, along with developments in enzyme immobilization, have been driving forces for novel applications of enzymes, providing robust and finely tuned enzyme catalysts for the performance of organic synthesis reactions. The introduction of catalysts of biological origin in a strictly chemical industry has not been easy, but their enormous potential, stemming from their exquisite selectivity, activity under mild conditions, and compliance with the green chemistry concepts [24], has provided them a firm position [25]. It is not presumptuous to say then that enzyme biocatalysis is there to stay.

Enzymes that perform the metabolic reactions of synthesis are usually complex, unstable coenzyme-requiring proteins, so their use as process biocatalysts involves major technological challenges [26]. However, readily available hydrolases can, under certain conditions, catalyze the reverse reactions of synthesis; that is, formation of a chemical bond instead of its cleavage by water. In this way, robust and readily available carbohydrases can catalyze the formation of glycosidic bonds in order to synthesize oligosaccharides and glycosides; proteases can catalyze the formation of peptide bonds to allow the synthesis of peptides; acylases can catalyze peptide-type bond formation in the synthesis of antibiotics and other bioactive compounds; and lipases can catalyze esterification, transesterification, and interesterification reactions. In order for them to do so, the water activity must be low enough to depress the competing hydrolytic reactions, which means that nonconventional media will be required in most cases [27]. Lipases are particularly well endowed for performance in poorly aqueous media and are therefore the most commonly used class of enzyme in organic synthesis [28].

Examples of the use of hydrolases in industrial synthesis reactions include the following:

Carbohydrases: Several hydrolases are in current use in the synthesis of different types of oligosaccharide and glycoside. Worth mentioning are the industrial production of prebiotic galacto-oligosaccharides with β-glactosidases [29] and fructo-oligosaccharides [30] with β-fructofuranosidases, whose application in the design of functional healthy foods has grown considerably.

Proteases: Several proteases, such as papain, thermolysin, α-carboxypeptidase, alcalase, neutrase, and subtilisin, have been used in the synthesis of functional peptides of industrial interest for therapeutic applications, nutritional supplementation, and flavor enhancement [31,32]. Of particular note is the use of thermolysin in the large-scale synthesis of the leading noncaloric sweetener aspartame (L-α-aspartyl-L-phenylalanine-1-methyl ester) [33].

Acylases: Penicillin acylases, traditionally used for the production of β-lactam nuclei as an intermediate stage in the production of semisynthetic β-lactam antibiotics, are now being used in their synthetic capacity to conduct the side-chain derivatization of such nuclei in order to yield the corresponding antibiotics [34]. The environmentally offensive chemical synthesis is being gradually replaced by enzymatic synthesis with immobilized penicillin acylases, which works on green chemistry principles. Certainly, amoxicillin and cephalexin are currently being produced by biocatalysis at an industrial level. Penicillin acylases are quite versatile enzymes and can be used in the synthesis of a variety of building blocks for the production of bioactive compounds and as catalysts for side reactions in