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 Directory of

THERAPEUTIC
ENZYMES
Edited by

Barry M. McGrath
National University of Ireland
Galway, Ireland

Gary Walsh
University of Limerick
Limerick, Ireland

Boca Raton London New York

A CRC title, part of the Taylor & Francis imprint, a member of the
Taylor & Francis Group, the academic division of T&F Informa plc.

Copyright © 2006 Taylor & Francis Group, LLC

2714_Discl.fm Page 1 Thursday, June 9, 2005 9:09 AM

Published in 2006 by
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

© 2006 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group
No claim to original U.S. Government works
Printed in the United States of America on acid-free paper
10 9 8 7 6 5 4 3 2 1
International Standard Book Number-10: 0-8493-2714-8 (Hardcover)
International Standard Book Number-13: 978-0-8493-2714-8 (Hardcover)
Library of Congress Card Number 2005012902

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with
permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish
reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials
or for the consequences of their use.

No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or
other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information
storage or retrieval system, without written permission from the publishers.

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Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for
identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data

Directory of therapeutic enzymes / [edited by] Barry M. McGrath and Gary Walsh.
p. ; cm.
Includes bibliographical references and index.
ISBN-13: 978-0-8493-2714-8 (alk. paper)
ISBN-10: 0-8493-2714-8 (alk. paper)
1. Enzymes-Therapeutic use Catalogs. [DNLM: 1. Enzymes-therapeutic use. QU 135 D5985 2006]
I. McGrath, Barry M. II. Walsh, Gary, Dr.

RM666.E55D57 2006
615'.35--dc22 2005012902

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Copyright © 2006 Taylor & Francis Group, LLC

 Preface

Today myriad enzymes are used for various industrial, analytical, and therapeutic pur-
poses. In the context of this publication, they form a significant subcategory of modern
biopharmaceuticals. Thus, we aim to overview these therapeutic enzymes, focusing in
particular on more recently approved products produced by recombinant means.
This book is primarily aimed at those in the biopharmaceutical/biotechnology industries
who wish to gain a comprehensive understanding of enzyme-based therapeutic products.
The publication also will be of direct relevance to students undertaking advanced under-
graduate/postgraduate programs in relevant aspects of the biological sciences, as well as
to researchers in these areas.
Chapter 1 provides a summary overview of applied enzymology, while Chapter 2
focuses upon the theory and applications of enzyme engineering. Between them, these
chapters set an appropriate backdrop for the remaining 11 chapters, which focus upon
actual enzyme products that have now gained regulatory approval for general medical
use. Chapters 3 through 13 highlight the manifold applications of approved therapeutic
enzymes, including use in the treatment of blood-clotting disorders, certain cancers, and
a variety of genetic disorders.
As editors, we wish to extend our sincere gratitude to all contributing authors and to
our publisher, Taylor & Francis. Finally, we wish to dedicate this book to our parents,
Hugh and Deirdre McGrath and Bina and the late John Walsh.

Copyright © 2006 Taylor & Francis Group, LLC

2714_C000.indd v 7/5/2005 11:13:57 AM

 Contributors

Barngrover, Debra Therapeutics Operation Management, Genzyme Corporation,

Cambridge, Massachusetts

Bayol, Alain Analytical Department, Sanofi-Synthelabo Recherche, Labège, France

Bras, Jean-Marc Analytical Department, Sanofi-Synthelabo Recherche, Labège, France

Brun, Nikolai Novo Nordisk A/S, Bagsvaerd, Denmark

Couderc, René Functional Genomic Department, Sanofi-Synthelabo Recherche, Labège,

France

Demeester, Joseph Laboratory of General Biochemistry and Physical Pharmacy, Faculty

of Pharmacy, Ghent University, Ghent, Belgium

De Smedt, Stefaan C. Laboratory of General Biochemistry and Physical Pharmacy,

Faculty of Pharmacy, Ghent University, Ghent, Belgium

Edmunds, Tim Therapeutic Protein Research, Genzyme Corporation, Framingham,

Massachusetts

Erhardtsen, Elisabeth Novo Nordisk A/S, Bagsvaerd, Denmark

Ferrara, Pascual Functional Genomic Department, Sanofi-Synthelabo Recherche, Labège,

France

Grinnell, Brian W. Biotechnology Discovery Research, Lilly Research Laboratories, Eli

Lilly and Co., Indianapolis, Indiana

Kakkis, Emil BioMarin Pharmaceutical Inc., Novato, California

Klausen, Niels Kristian Novo Nordisk A/S, Bagsvaerd, Denmark

Körholz, Dieter Department of Pediatrics, University of Leipzig Clinic and Policlinic for
Children and Adolescents, Leipzig, Germany

Lascombes, Françoise Clinical and Exploratory Pharmacology, Sanofi-Synthelabo

Recherche, Nancy, France

Loison, Gérard Functional Genomic Department, Sanofi-Synthelabo Recherche, Labège,

France

Loyaux, Denis Functional Genomic Department, Sanofi-Synthelabo Recherche, Labège,

France

Copyright © 2006 Taylor & Francis Group, LLC

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 Macias, William L. Lilly Laboratory for Clinical Reseach, Lilly Research Laboratories,
Eli Lilly and Co., Indianapolis, Indiana

Mauz-Körholz, Christine Division of Pediatric Hematology and Oncology, Clinic and

Policlinic for Children and Adolescents, University of Leipzig Medical Center, Leipzig,
Germany

McGrath, Barry M. Gene Therapy Group, Regenerative Medicine Institute, National

University of Ireland, Galway, Ireland

O’Connell, Shane Chemical and Environmental Sciences Department, University of

Limerick, Limerick, Ireland

Persson, Egon Novo Nordisk A/S, Bagsvaerd, Denmark

Rexen, Per Novo Nordisk A/S, Bagsvaerd, Denmark

Sanders, Niek N. Laboratory of General Biochemistry and Physical Pharmacy, Faculty

of Pharmacy, Ghent University, Ghent, Belgium

Shanley, Nancy Department of Applied Science, Limerick Institute of Technology,

Moylish, Limerick City, Ireland

Wahn, Volker Clinic for Children and Adolescents, Uckermark Medical Center,
Schwedt/Oder, Germany

Walsh, Gary Industrial Biochemistry Program, University of Limerick, Limerick, Ireland

Yan, S. Betty Lilly Laboratory for Clinical Research, Lilly Research Laboratories, Eli Lilly
and Co., Indianapolis, Indiana

Copyright © 2006 Taylor & Francis Group, LLC

2714_C000.indd viii 7/5/2005 11:13:58 AM

 Contents

1 Applied Enzymology: An Overview ............................................................................... 1

Nancy Shanley and Gary Walsh

2 Enzyme Engineering ........................................................................................................ 17

Barry M. McGrath

3 Tissue Plasminogen Activator-Based Thrombolytic Agents .................................... 55

Gary Walsh

4 Activated Protein C ........................................................................................................... 69

Brian W. Grinnell, S. Betty Yan, and William L. Macias

5 Deoxyribonuclease I ......................................................................................................... 97
Niek N. Sanders, Stefaan C. De Smedt, and Joseph Demeester

6 β-Glucocerebrosidase Ceredase® and Cerezyme® .................................................... 117

Tim Edmunds

7 α-Galactosidase................................................................................................................135
Debra Barngrover

8 Urate Oxidase ................................................................................................................... 159

Alain Bayol, Françoise Lascombes, Denis Loyaux, Jean-Marc Bras,
Gérard Loison, René Couderc, and Pascual Ferrara

9 L-Asparaginase Review of Pharmacology, Drug Resistance,

and Clinical Applications .............................................................................................. 179
Christine Mauz-Körholz, Volker Wahn, and Dieter Körholz

10 Recombinant Factor VIIa ............................................................................................... 189

Elisabeth Erhardtsen, Nikolai Brun, Niels Kristian Klausen,
Egon Persson, and Per Rexen

11 Factor IX (Protease Zymogen) ....................................................................................... 209

Barry M. McGrath

12 α-L-Iduronidase: The Development of Aldurazyme (Laronidase) ........................ 239

Emil Kakkis

13 Additional Therapeutic Enzymes ................................................................................ 261

Shane O’Connell

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 1
Applied Enzymology: An Overview

Nancy Shanley and Gary Walsh

CONTENTS
1.1 Introduction............................................................................................................................ 1
1.1.1 History of Enzymology ........................................................................................... 2
1.1.2 Classification of Enzymes ....................................................................................... 2
1.1.3 Applied Enzymology ............................................................................................... 3
1.2 Enzymes: Sources and Production ..................................................................................... 3
1.2.1 Immobilized Enzymes ............................................................................................ 5
1.3 Enzymes Used for Analytical Purposes ............................................................................. 6
1.3.1 Enzymes in Clinical Chemistry .............................................................................. 6
1.3.2 Enzymes Used in Immunoassay Systems ............................................................ 8
1.3.3 Enzyme-Based Biosensors ....................................................................................... 8
1.4 Enzymes Used for Industrial Purposes ........................................................................... 10
1.4.1 Detergent Proteases ................................................................................................ 10
1.4.2 The Application of Proteases in the Food and Drink Industry ....................... 11
1.4.3 Additional Applications of Proteases .................................................................. 12
1.4.4 Carbohydrases and Additional Industrial Enzymes ........................................ 12
1.5 The Future of Industrial Enzymes .................................................................................... 14
References....................................................................................................................................... 14

1.1 Introduction
Several thousand enzymes have been identified to date. This group of biomolecules first
found industrial application in the early 1900s. Today, a myriad enzymes are employed for
industrial, analytical, and therapeutic purposes [1–3]. While the following sections of this
book focus specifically upon therapeutic uses of enzymes, this chapter provides a sum-
mary overview of their additional applied uses, offering a framework for the interested
reader to better understand the entire application range of these versatile biomolecules.
Enzymes are biological catalysts. They speed up the rate of biochemical reactions
by several orders of magnitude (see Table 1.1) and function under relatively mild condi-
tions of pH and temperature [4]. With the exception of a small group of catalytic RNA
molecules [5], all enzymes are protein-based. They are globular proteins, typically soluble

1
Copyright © 2006 Taylor & Francis Group, LLC

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 2 Directory of Therapeutic Enzymes

TABLE 1.1
Rate Enhancement Produced by Selected Enzymes

Enzyme Rate Enhancement

Carbonic anhydrase 106

Chorismate mutase 106
Triose phosphate isomerase 109
Carboxypeptidase A 1011
AMP nucleosidase 1012
Phosphoglucomutase 1012
Succinyl CoA transferase 1013
Staphylococcal nuclease 1014
Orotidine monophosphate decarboxylase 1017

in aqueous-based solutions, and sensitive to environmental conditions such as changes

in temperature and pH. Enzymes are high-molecular mass molecules, ranging from 13 to
500 kDa, although many fall within the 30 to 100 kDa range. They generally display a
high degree of specificity with respect to the substrate(s) with which they interact. The
region of the enzyme where the substrate is transformed to form the product is known as
the active site. Some enzymes also require the presence of a nonprotein cofactor at the
active site in order to maintain catalytic activity.

1.1.1 History of Enzymology

The study of enzymes began in earnest toward the end of the 18th century. Much of the
initial work upon these molecules stemmed from digestive physiological research that
focusing upon the ability of stomach secretions to digest meat and saliva to convert starch
into sugars. The scientist Frederick Kuhne [28] proposed the term “enzyme” in 1878. In
1884, Jokichi Takamine [28] was awarded a patent for a product termed “Taka-diastase,”
the active component of which was an amylolytic enzyme derived from Aspergillus oryzae
grown on rice. Purification of enzymes began in the 1920s. Urease, for example, was first
purified and crystallized in 1926, while pepsin, trypsin, and several other digestive
enzymes were crystallized in the 1930s (Figure 1.1). By then, scientists were also attempting
to use crude enzyme preparations for a variety of industrial and medical purposes. But in
many instances, such early attempts met with limited success, largely due to an incomplete
understanding of the nature of enzymes themselves in terms of substrate specificity and the
influence of environmental parameters upon enzyme activity.

1.1.2 Classification of Enzymes

Enzymes may be grouped into families on the basis of a number of different criteria. They
may be classified according to the type of substrate they transform. They are often named
simply by adding “ase” to the end of the substrate name, hence the general classifications
of “protease,” “carbohydrase,” “lipase,” “pectinase,” etc. Polymer-degrading enzymes are
also often classified as “endo-” or “exo-” acting, depending upon the position of the bonds
within the substrate attacked by the enzyme.
The most comprehensive and internationally recognized method of enzyme classifica-
tion is based upon the type of reaction catalyzed [6]. Using this system, enzymes are
assigned to one of six basic categories: oxidoreductases, transferases, hydrolases, lyases,
isomerases, and ligases (see Table 1.2).

Copyright © 2006 Taylor & Francis Group, LLC

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 Applied Enzymology: An Overview 3

FIGURE 1.1
The three-dimensional structure of (a) porcine pepsin and (b) porcine trypsin. (Courtesy of the protein databank,
http://www.pdb.org. Entry ID numbers 3PEP and 1S81, respectively. With permission.)

TABLE 1.2
Classification of Enzymes According to the Reaction Catalyzed

Grouping Reaction Catalyzed

Oxidoreductases Catalyze oxidation–reduction reactions such as CH→C–OH, or overall removal or addition

of hydrogen atom equivalents, for example: CH (OH)→C⫽O
Transferases Mediate the transfer of various chemical groups, such as aldehyde, ketone, acyl, sugar,
phosphoryl, etc. from one molecule to another
Hydrolases Catalyze hydrolysis reactions, i.e., the transfer of various functional groups to water
Lyases Catalyze additions to, or formation of, double bonds such as C⫽C, C⫽O, and C⫽N
Isomerases Catalyze various types of isomerations, including racemization reactions
Ligases Mediate the formation of C–C, C–O, C–S, and C–N bonds, usually via condensation
reactions coupled to ATP cleavage. Often termed synthetases

1.1.3 Applied Enzymology

Today, enzymes find a broad range of applications within industry, medicine, and as ana-
lytical reagents [1–3]. The major enzyme preparations used for therapeutic purposes are
summarized in Table 1.3 and are discussed further in the forthcoming chapters. The
enzymes used for analytical or industrial purposes are summarized below. But, before
specific enzymes and their applications are considered, the more general issues of enzyme
sources, methods of manufacture, and enzyme immobilization should be discussed first.

1.2 Enzymes: Sources and Production

Enzymes that have found applied use have traditionally been sourced from a range of
microbial, plant, and animal species [3,7]. Traditionally, most therapeutic enzymes are
extracted directly from the natural source, i.e., from animal or human tissue. In most
instances, enzymes used for diagnostic and in particular for industrial purposes have been
sourced from microorganisms. This producer microorganism is classified as generally

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 4 Directory of Therapeutic Enzymes

TABLE 1.3
Major Enzymes Used for Therapeutic Purposes

Enzyme Therapeutic Application

Tissue plasminogen activator (tPA) Thrombolytic agent

Urokinase Thrombolytic agent
(Activated) protein C Severe sepsis
DNase Cystic fibrosis
Glucocerebrosidase Gaucher’s disease
α-galactosidase Fabry’s disease
Urate oxidase Hyperuricemia
Asparaginase Cancer treatment, most notable childhood leukemia
Factor VIIa Hemophilia
Factor IX (protease zymogen) Hemophilia
α-iduronidase Mucopolysaccharidosis I (MPS I)
Pancreatin/lactase/α-amylase/proteases Used as digestive aids
Hyaluronidase Various potential uses for myocardial infarction, cancer
therapy, and in anesthesia for eye surgery
Superoxide dismutase Oxygen toxicity, anti-inflammatory agent
Various proteases, including papain, Debriding agents (cleansing of wounds)
collagenase, and trypsin

recognized as safe (GRAS)). GRAS microorganisms are nonpathogenic, nontoxic, and gene-
rally do not produce antibiotics. Microorganisms represent an attractive source of enzymes
(and indeed, many other proteins) because they can be cultured on a large scale over a
short time-span by well-established methods of fermentation. Accordingly, they can pro-
duce an abundant, regular supply of the desired enzyme product.
The advent of genetic engineering has facilitated the production of virtually any pro-
tein in recombinant production systems, with associated potential benefits in terms of
production levels and, potentially, product safety [7]. A range of enzyme products are now
produced by recombinant means. Recombinant enzymes used for industrial and diagnos-
tic purposes are generally produced in engineered microbial systems, whereas many
therapeutic enzymes are produced in animal cell lines, a consequence of the need for post-
translational modifications.
Most enzymes (both recombinant and nonrecombinant) produced by fermentation and
cell culture are secreted directly by the producing cells into the culture medium.
Extracellular production displays process advantages, by obviating the necessity of lysing
(disrupting) the cells in order to bring about the release of the protein (enzyme) of interest,
thereby simplifying downstream processing. Whole cells can be removed by simple filtra-
tion or centrifugation of the extracellular medium.
In a few instances, the enzyme of interest is produced intracellularly. Accordingly, it is
necessary to disrupt the cells upon completion of fermentation and cell harvesting. This
releases not only the enzyme of interest, but also the entire intracellular content of the
cell. Subsequent purification procedures to obtain the final enzyme product are thereby
rendered more complicated. Examples of intracellular enzymes that have found industrial
application include asparaginase, penicillin acylase, and glucose isomerase.
The complexity of downstream processing undertaken after initial enzyme recovery or
extraction depends upon the required specification of the final product. Bulk industrial
enzymes generally require minimal or no further purification, and the final product is
generated by concentration of crude extract, excipient addition, and drying (if required).
Therapeutic enzymes, on the other hand, are invariably purified to homogeneity before
final formulation, a process requiring multiple high-resolution chromatographic steps.
Processing of enzymes used for analytical purposes generally falls somewhere between

Copyright © 2006 Taylor & Francis Group, LLC

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 Applied Enzymology: An Overview 5

Collection/culture of enzyme source

Enzyme extraction from source

Clarification (centrifugation or filtration)

Concentration, Concentration Concentration

excipient addition, (e.g. ultrafiltration) (e.g. ultrafiltration)
potency adjustment

Drying, if required Partial purification Full purification

(drum drying, spray drying) (1 to 2 chromatographic steps) (3 to 5 chromatographic steps)

(a)
Excipient addition, Excipient addition,
potency adjustment potency adjustment

Drying, if required Drying, if required

(lyophilization) (lyophilization)

(b) (c)

FIGURE 1.2
An overview of a generalized manufacturing scheme for (a) industrial grade, (b) analytical, and (c) therapeutic
enzymes.

these two extremes, with most such enzymes being partially purified. An overview of
downstream processing of these enzyme products is provided in Figure 1.2.

1.2.1 Immobilized Enzymes

Enzymes are expensive to produce. It therefore makes economic sense, where practicable,
to recover the enzyme after it carries out its intended function, so that it can be reused or
recycled. Enzyme immobilization technology makes this possible. Immobilized enzymes
often display increased stability due to the fact that their support material can provide
protection from pH and temperature changes in the surrounding environment.
Enzymes can be immobilized by a variety of techniques, for example, adsorption, encap-
sulation, covalent attachment, and entrapment of the enzyme [8,9] (Figure 1.3). Entrapment
is achieved by incubating the enzyme along with gel monomers (e.g., acrylamide or meth-
acrylic acid) and then promoting gel polymerization. The gel pore size must be large
enough to allow free passage of enzyme reactants and products but small enough to retain
the entrapped enzyme molecules. Industrially, polymeric gel beads containing the immo-
bilized enzyme are usually packed into columns. As they pass down the column, the sub-
strate molecules diffuse into the beads and are converted into the product by the trapped
enzyme. Penicillin acylase and glucose isomerase are examples of two enzymes that are
used industrially in an immobilized state. Glucose isomerase catalyzes the interconversion

Copyright © 2006 Taylor & Francis Group, LLC

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 6 Directory of Therapeutic Enzymes

E
E
E E E E
E
E
E

(a) (b)
(c) (d)

FIGURE 1.3
Enzymes may be immobilized by methods of (a) adsorption, (b) covalent attachment, (c) encapsulation, and
(d) entrapment.

of glucose to fructose, both of which are widely used in the production of sweets, jams, soft
drinks, etc. Fructose is twice as sweet as glucose, so smaller quantities of fructose can be
used to achieve the same degree of sweetness in food products.

1.3 Enzymes Used for Analytical Purposes

Almost 100 enzymes now find routine analytical application in the detection and quanti-
fication of analytes of medical, environmental, or industrial significance [10,11]. These
enzymes are obtained from a variety of animal, plant, and microbial sources. Most of these
enzymes are produced by direct extraction from native producer sources, although some
(e.g., alkaline phosphatase) are produced mainly by genetic engineering. The annual
world market for analytical enzymes has well surpassed the € 100 million mark. In the
context of therapeutic enzymes, this may seem modest. Enzymes used for analytical (and
industrial) purposes, however, have a much lower cost of production and sales value per
unit of activity than therapeutic proteins.
It is not usually necessary to purify enzyme-based diagnostic reagents to homogeneity.
However, it is essential that the purification procedure involved removes any proteins or
other molecules present in the initial preparation that might interfere with the assay.

1.3.1 Enzymes in Clinical Chemistry

The substrate specificity of enzymes and their catalytic efficiency render these biomole-
cules attractive as analytical reagents. In most instances, the enzyme employed is one that
uses the target analyte directly as a substrate. Changes in the concentration of substrate (or
appearance of easily quantifiable product) are then monitored, usually spectrophotometri-
cally. The magnitude of the signal generated is directly proportional to the concentration
of the target analyte.
Biochemical research continues to increase our understanding of normal and abnormal
metabolic activity. A variety of enzyme preparations have been used as diagnostic reagents
in the in vitro clinical diagnostics sector for many years (see Table 1.4). Clinical chemistry
is the detection, monitoring, and quantitation of a broad variety of marker substances
present in clinical biological samples. Substances of diagnostic value include metabolic
products such as urea, glucose, cholesterol, or steroid hormones. Other diagnostic markers
of higher molecular mass include specific proteins that may be released from damaged
tissue or whose normal concentration is altered due to abnormal metabolism. Diagnostic
tests that detect viruses and microorganisms have also been developed.

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 Applied Enzymology: An Overview 7

TABLE 1.4
Some Enzymes Used Directly or Indirectly as Diagnostic Reagents — Their Sources and Likely
Applications

Enzyme Source Application

Acetyl cholinesterase Bovine erythrocytes Analysis of organophosphorous

compounds such as pesticides
Alcohol dehydrogenase Yeast Determination of alcohol levels in
biological fluids
Alkaline phosphatase Calf intestine and kidney, Conjugation to antibodies allows its
Recombinant (Picca sp.) use as an indicator in ELISA systems
Arginase Beef liver Determination of L-arginine levels in
plasma and urine
Ascorbate oxidase Cucurbita sp. Determination of ascorbic acid levels;
eliminates interference by
ascorbic acid
Cholesterol esterase Pig/beef pancreas, Pseudomonas sp., Determination of serum cholesterol
Recombinant (Streptomyces sp.) levels
Creatine kinase Rabbit muscle, beef heart, pig heart Diagnosis of cardiac and skeletal
malfunction
Glucose-6-phosphate Yeast, Leuconostoc mesenteroides Determination of glucose and ATP in
dehydrogenase conjunction with hexokinase
Glucose oxidase Aspergillus niger Determination of glucose in biological
samples in conjunction with
peroxidase; a marker for
ELISA systems
Glutamate Beef liver Determination of blood urea nitrogen
dehydrogenase in conjunction with urease
Glycerol kinase Candida mycoderma, Arthrobacter sp. Determination of triglyceride levels
in blood in conjunction with lipase
Glycerol-3-phosphate Rabbit muscle Determination of serum triglycerides
dehydrogenase
Hexokinase Yeast Determination of glucose in
body fluids
Peroxidase Horseradish Indicator enzyme for reactions in
which peroxide is produced
Phosphoenolpyruvate Maize leaves Determination of CO2 in body fluids
carboxylase
Urease Jack bean Determination of blood urea
nitrogen; marker enzyme for
ELISA systems
Uricase Porcine liver Determination of uric acid
Xanthine oxidase Buttermilk Determination of xanthine and
hypoxanthine in biological fluids

Reproduced from Walsh, G., Proteins: Biochemistry and Biotechnology, John Wiley & Sons, Chichester, UK, 2001.
With permission from John Wiley & Sons.

Dehydrogenase and oxidase enzymes are the most common substances used for ana-
lytical purposes. Dehydrogenases catalyze the following general reaction type:

S ⫹ NAD⫹ → P ⫹ NADH ⫹ H⫹

NADH absorbs at 340 nm whereas NAD+ does not, thereby allowing spectrophotometric
monitoring of the generation of NADH. The amount of NADH produced bears a direct
stoichiometric relationship with the concentration of the substrate (the analyte whose
concentration is to be determined) that is present.

Copyright © 2006 Taylor & Francis Group, LLC

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 8 Directory of Therapeutic Enzymes

Oxidases represent a second class of enzyme often utilized for analytical purposes.
However, unlike dehydrogenases, none of the oxidase reaction products is easily measur-
able. This difficulty may be overcome by the inclusion of a second “linker” enzyme in the
assay system. The linker enzyme is chosen on the basis of its ability to utilize one of the
products of the primary reaction as a substrate and, in turn, to generate an easily detect-
able product. A specific example of such a diagnostic system is that of the glucose oxi-
dase/peroxidase system, widely used to determine blood glucose concentrations. In this
reaction sequence, the first reaction is catalyzed by glucose oxidase, while the second is
catalyzed by peroxidase:

Glucose ⫹ O2 ⫹ H2O → gluconic acid ⫹ H2O2

2H2O2 ⫹ 4-aminophenazone ⫹ phenol → quinoneimine ⫹ 4H2O

The dye quinoneimine, a reaction product, absorbs at 520 nm, allowing spectrophotometric
determination of its concentration produced. This same reaction strategy can be used to
detect and quantify additional analytes. For example, a combination of cholesterol oxidase
and peroxidase may be used to determine cholesterol concentrations.

1.3.2 Enzymes Used in Immunoassay Systems

A selection of enzymes also find analytical application as reporter groups for immunoas-
says. Owing to the specificity of antibody–antigen interaction, antibodies are often used to
detect and quantify target analytes against which they have been produced [12]. Such
immunoassay-based systems now find routine use for a variety of clinical, industrial, and
environmental purposes (e.g., the detection and quantification of specific drugs or other
analytes in blood, or the detection and quantification of pesticides in food or environmen-
tal samples).
The binding of an antibody to an antigen is an event that is itself not readily detectable.
One common strategy to overcome this difficulty is to conjugate an easily detectable
reporter group to the antibody. Initially, radioactive tags were commonly used for this pur-
pose (radioimmunoassay). More recently, the use of enzyme-based reporter groups (enzyme
immunoassay) has become more common. Enzymes may be covalently coupled to antibod-
ies by a variety of relatively straightforward chemical procedures, for example, by incuba-
tion with bifunctional cross-linking reagents such as glutaraldehyde. Enzymes capable of
converting a chromogenic substrate into a readily detectable colored product (which is
therefore easily detected and quantified by spectrophotometry) are most commonly used,
for example, alkaline phosphatase isolated from calf stomach. This enzyme can catalytically
dephosphorylate p-nitrophenylphosphate (PNPP), yielding inorganic phosphate and
p-nitrophenol. The latter product is yellow, absorbing maximally at 405 nm. Additional
enzymes for which chromogenic substrates are available and which, therefore, have been
used as reporter groups in immunoassays include β-galactosidase (lactase), urease, and
glucose oxidase. Enzyme immunoassays of various different formats have been designed
and commercialized. The principle of one such assay type is illustrated in Figure 1.4.

1.3.3 Enzyme-Based Biosensors

In an immobilized format, enzymes can be used as the biological-sensing element of bio-
sensors (Figure 1.5). Biosensors are analytical devices capable of detecting and quantifying
specific target analytes often present in complex samples [13,14]. They consist of a bio-
logical component, most often an enzyme, immobilized onto a transducer. A transducer is

Copyright © 2006 Taylor & Francis Group, LLC

2714_C001.indd 8 7/5/2005 9:13:05 AM

 Applied Enzymology: An Overview 9

E
E
E

Wash & Assay E

= Antigen, = Immobilized antibody, E = Antibody−enzyme conjugate

FIGURE 1.4
An overview of the principles and operation of a noncompetitive enzyme-linked immunosorbant assay (ELISA).

Target analyte

Quantitative
Transducer Electronics read out

Additional
Biological sensing
analytes in
elements immobilized
sample
in close proximity to
transducer

FIGURE 1.5
Basic biosensor design. (Reproduced from Walsh, G., Proteins: Biochemistry and Biotechnology, John Wiley & Sons,
Chichester, UK, 2001., With permission from J. Wiley & Sons.)

a device that senses one form of energy and converts it to another. The transducer can, for
example, be an electrode that converts the biochemical signal generated by the enzyme
reaction (e.g., changes in concentration of substrate or product, absorbance, etc.) into a
quantifiable electrical signal. The electrical signal generated by the transducer is related to
the concentration of the analyte. A standard curve can be constructed from readings
obtained when the electrode is immersed in standard solutions containing known concen-
trations of the analyte.
Most enzyme biosensors use either membrane-based enzyme entrapment, or the
enzyme is covalently bound to the inside of a nylon tube. The stability of the electrode is
dependent on the stability of the enzyme, which is partially dependent on the method of
immobilization. Many enzyme electrodes are available to detect, for example, glucose,
urea, creatine, and pyruvate in clinical samples. However, only the glucose biosensor has
been widely commercialized.

Copyright © 2006 Taylor & Francis Group, LLC

2714_C001.indd 9 7/5/2005 9:13:05 AM

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