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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
10.13 Some early achievements of protein engineering 10-13
10.14 Computational approaches in protein engineering 10-14
10.14.1 Molecular dynamics simulations 10-15
10.14.2 Quantum mechanical calculations 10-15
10.14.3 Docking and ligand optimization 10-16
10.14.4 Machine learning algorithms in protein design 10-17
10.15 Directed evolution techniques 10-18
10.15.1 Error-prone PCR 10-18
10.15.2 DNA shuffling 10-18
10.15.3 Saturation mutagenesis 10-19
10.15.4 Phage display 10-19
10.16 Post-translational modifications 10-19
10.16.1 Glycosylation engineering 10-19
10.16.2 Phosphorylation engineering 10-20
10.16.3 Methylation and acetylation 10-20
10.16.4 PEGylation for enzyme stability 10-20
10.17 Structural flexibility and allosteric regulation 10-20
10.17.1 Intraprotein communication pathways 10-21
10.17.2 Allosteric site identification 10-21
10.17.3 Modulator design 10-21
10.17.4 Coupling allosteric regulation with catalytic function 10-21
10.18 Protein–protein and protein–ligand interactions 10-22
10.18.1 Characterizing binding sites 10-22
10.18.2 Fine-tuning affinity and specificity 10-22
10.18.3 Interaction networks 10-22
10.18.4 Biophysical methods for interaction studies 10-23
10.19 Applications in synthetic biology 10-23
10.19.1 Metabolic pathway engineering 10-23
10.19.2 Genetically encoded sensors 10-24
10.19.3 Protein-based logic gates 10-24
10.19.4 Gene circuits for dynamic control 10-24
10.20 Engineering multi-functional proteins 10-25
10.20.1 Fusion proteins 10-25
10.20.2 Protein scaffolds 10-25
10.20.3 Modular protein design 10-25
10.20.4 Dual-enzyme systems 10-26
xx
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
10.21 Ethical and safety considerations 10-26
10.21.1 Bioethics in protein engineering 10-26
10.21.2 Biosafety and environmental concerns 10-26
10.21.3 Intellectual property rights 10-27
10.21.4 Regulatory frameworks 10-27
10.22 Studies in protein engineering 10-27
10.22.1 Therapeutic proteins 10-28
10.22.2 Industrial enzymes 10-28
10.22.3 Diagnostic proteins 10-29
10.23 Single-molecule techniques in protein engineering 10-29
10.23.1 Atomic force microscopy 10-30
10.23.2 Single-molecule FRET 10-30
10.23.3 Optical tweezers 10-30
10.23.4 Patch-clamp technique 10-31
10.24 High throughput screening methods 10-31
10.24.1 Fluorescence-activated cell sorting (FACS) 10-32
10.24.2 Microfluidics-based assays 10-32
10.24.3 Yeast surface display 10-32
10.24.4 Mass spectrometry-based methods 10-33
10.25 Protein engineering for nanotechnology 10-33
10.25.1 Protein-based nanocarriers 10-34
10.25.2 Biosensors 10-34
10.25.3 Protein nanowires and nanotubes 10-35
10.25.4 DNA–protein hybrid structures 10-35
References 10-36
xxi
Preface
There has been a long-felt requirement for a textbook representing current and
recent developments in the field of pharmaceutical biotechnology. The few available
books have become out of date, in particular due to the development of new
biotechnology from advances in concepts and techniques. This volume has been
written with a view to provide background knowledge on the state of the art of the
subject and provide a practical view of the developments to date in genes, enzymes
and proteins. The book covers several different facets of evolutional progress and
achievements in each line of inquiry. Attention is focused on newer perspectives on
the roles of genes, enzymes and proteins in the field of pharmaceutical biotechnology. The current volume is organized into three important areas—enzymes,
genomics and proteomics—and is comprised of ten chapters. The book begins
with the general properties, mechanisms, applications, production, immobilization
and purification of enzymes, with some applications in the form of biosensors and
biotransformation reactions. This information is followed by characterizations in
genomics, genomes sequencing, comparative genomics and genomic evolution.
Other highlights pertain to protein engineering. This book will be of interest to
biochemists, biologists, microbiologists, biotechnologists, food technologists and
others involved in research on the biotechnological applications of proteins and
enzymes.
xxii
Acknowledgement
We would like to dedicate this book to all students, researchers, academicians, and
all the scholars. who have sincerely contributed to the area of Pharmaceutical
Biotechnology, depicted in the book, at national as well as international level. We
would also like to acknowledge the University of Nizwa, Sultanate of Oman for
extending its support in accomplishing this book project successfully. We are
grateful to the Natural and Medical Sciences Research Center (NMSRC) housed
at the University of Nizwa for providing central resources of advanced analytical
instruments for our research work and for promoting interdisciplinary research
studies. This centre has given a valuable base to this book project. Thus, authors are
thankful to NMSRC for offering excellent facilities required for the completion of
this book.
Natural and Medical Sciences Research Center, University of Nizwa
Last, but not least, we show our sincere gratitude to the whole team of IOP
Publishing for furnishing their active cooperation and support.
xxiii
Author biographies
Professor Ahmed Al-Harrasi
Ahmed Al-Harrasi is a professor of organic chemistry and the vice
chancellor for graduate studies, research and external relations at
the University of Nizwa. He obtained his BSc in Chemistry from
SQU and his MSc and PhD in Organic Chemistry from Free
University of Berlin as a DAAD-fellow. Then he received the
Fulbright award in 2008 for postdoctoral research in Chemical
Biology from Cornell University. He is a founder and chair of the
Natural and Medical Sciences Research Center. He is a member of
the Scientific Council of UNESCO. He was named on the list of top 2% scientists for
the last three years. He has authored over 800 scientific papers and more than
20 books and book chapters. He received the Order of Royal Commendation from
His Majesty, The Sultan of Oman as an outstanding Omani individual for his
remarkable contribution and active role in research.
Dr Saurabh Bhatia
Saurabh Bhatia is an Associate Professor within the Natural and
Medical Sciences Research Centre at the University of Nizwa,
Oman. He has published 127 referred journal articles and written
94 book chapters. Dr Bhatia has also authored 10 books and is the
Associate Editor on several international journals.
Dr Ajmal Khan
Ajmal Khan is currently working as an Associate Professor in
Natural and Medical Sciences Research Centre at the University
of Nizwa, Oman. Dr Khan published more than 450 articles in
international peer review journals with impact factor of more than
2000 and citation more than 9000. Besides this, he published two
US patents, and 3 books and 13 books chapters. Dr Khan is
Associate Editor for four journals and in editorial board of three
international journals.
xxiv
IOP Publishing
Introduction to Pharmaceutical Biotechnology, Volume 2
(Second Edition)
Enzymes, proteins and bioinformatics
Ahmed Al-Harrasi, Saurabh Bhatia and Ajmal Khan
Chapter 1
Introduction to enzymes and their applications
1.1 Introduction
The cell is the structural and functional unit of life—the basic building block of
living systems. Cells effectively utilize biocatalysts, known as enzymes, which are
notable for their high catalytic efficiency and specificity for substrates and reactions.
Enzymes have amazing catalytic power and their high level of specificity for their
substrate makes them suitable for biological reactions. They are crucial for cellular
metabolism. Each and every chemical reaction that takes place in plants, microorganisms and animals proceeds at a quantifiable rate as a direct result of enzymatic
catalysis. Most of the history of biochemistry is directly or indirectly related to the
history of enzyme research. Catalysis in biological systems was initially reported in
the early 1800s based on research into the digestion of meat. In this report the
catalytic activity of secretions from the stomach, the conversion of starch into sugar
by saliva, and various plant extracts were reported.
In 1837, Berzelius documented the catalytic nature of fermentation. In the 1850s
Louis Pasteur reported that fermentation was a process initiated by living organisms. During this study it was reported that the fermentation of sugar into alcohol by
yeast was catalyzed by ferments. He also hypothesized that these ferments are close
to the structure of yeast. These ferments were later called enzymes (in yeast). The key
breakthrough in the history of enzymes came in 1897 when Edward Buchner
isolated, from yeast cells, the soluble active form of the set of enzymes that catalyzes
the fermentation of sugar to alcohol. Emul Fischer reported the first systematic
studies on enzyme specificity in the early twentieth century [1]. Later, in 1926, James
Sumner extracted urease in pure crystalline form from jack beans [2]. He also
recognized the protein nature of urease. In 1930, John Northrop and his co-workers
crystallized pepsin and trypsin and established them as proteins [3]. In subsequent
years enzymology developed rapidly (table 1.1). The important developments during
doi:10.1088/978-0-7503-5387-8ch1 1-1 ª IOP Publishing Ltd 2024. All rights,
including for text and data mining (TDM), artificial intelligence (AI) training, and similar technologies, are reserved.
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 1.1. Chronology of enzyme studies.
Name Year Work
Anfinsen 1956–8 The sequence of an amino acid regulates the folding pattern
and activity of a ribonuclease.
Beatle and tatum 1940 ‘One gene one enzyme’ hypothesis.
Bertrand 1896–7 Co-enzyme or co-ferment (currently known as co-factors).
Berzelius 1835 Concept of catalysis.
Berzelius 1837 Exploration of biological catalysis.
Briggs and haldane 1925 Derivation of enzyme rate equations using the steady-state
approximation.
Buchner 1897 Isolation of the soluble active form of enzymes from yeast
cells.
Chances 1943 Application of spectroscopic techniques for studying
enzymes.
Cori and cori 1937–9 Muscle phosphorylase.
Duclaux henri 1898 Nomenclature: substrate plus suffix ‘ ase’.
Fischer 1894–5 ‘Lock and key’ hypothesis of enzyme specificity.
Harden and young 1901–3 Methods for the derivation of kinetic rate laws; principle of
enzyme–substrate complex. 1906 Co-ezymase (NAD).
Jacob, Monod and
changeux
Koshland 1953 ‘Induced fit’ hypothesis.
Kuhne 1878 Explored trypsin catalyzed reactions; introduction of word
Michaelis and menten 1913 Extension of the kinetic theory of enzyme catalysis.
Northrop and kunitz 1930–3 Crystallization of proteolytic enzymes.
Pasteur 1850 Fermentation of sugar into alcohol by yeast.
Payen and persoz 1833 Alcohol precipitation of thermolabile ‘diastase’ from malt.
Phillips, johnson and
north
Sumner 1926 Crystallization of urease.
Sutherland 1956 Cyclic AMP adenyl cyclase.
Umbarger, yates and
pardee
Wilhelmy 1850 Quantitative evaluation of the rates of sucrose inversion.
1961 Allosterism.
‘enzyme’.
1965 Three-dimensional structure of lysozyme obtained at 1.5 A
resolution.
1956 Regulation of enzyme activity via feedback inhibition.
this period are: the elucidation of major metabolic pathways, such as the glycolysis
and tricarboxylic acid cycle; the detection of numerous biochemical events of
digestion, coagulation, muscular contraction and endocrine function, and their
roles in the maintenance, control and integration of complex metabolic processes;
the kinetic backgrounds to explain the observations of enzyme action and inhibition;
and the development of protocols for examining the structures of functionally
sensitive proteins. There has been exhaustive research on enzyme-catalyzed reactions
1-2
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
and enzymes involved in cell metabolism. At present, 2000 different enzymes have
been recognized, each of which catalyzes a different chemical reaction. Currently,
more focus is being directed towards the application of enzymes. The high efficiency
of enzymes makes them commercially valuable and their specificity of action is
offering diverse advantages in clinical medicine.
1.2 Properties of enzymes
Enzymes are the complex protein molecules, often called biocatalysts, which are
produced by living cells. They are highly specific both in the reactions that they
catalyze and in their choice of reactants, which are known as substrates. An enzyme
typically catalyzes a single chemical reaction or a set of closely related reactions [4].
Side reactions resulting in the wasteful formation of by-products are rare in enzymecatalyzed reactions, in comparison to uncatalyzed ones. Enzymes can also be defined
as soluble, colloidal and organic catalysts that are produced by living cells, but are
capable of acting independently of the cells [4]. Enzymes are currently being used in
diverse areas in the food, feed, paper, leather, agriculture and textiles industries,
resulting in major cost reductions. Simultaneously, rapid scientific progress is now
encouraging the chemistry and pharmacological industries to embrace enzyme
technology, a trend supported by concerns regarding energy, raw materials, health
and the environment. One of the most common advantages of enzymes is their
ability to function continuously even after their removal or separation from the cells.
This means that even after the separation of cells from in vivo environments, they
continue to work efficiently under in vitro conditions; we can conclude that these
biocatalysts remain in an active state even after their isolation. Enzymes are
primarily non-toxic, biodegradable, and can be produced in large quantities by
microorganisms for various industrial applications. In this chapter, the isolation,
production, purification, utilization and application of enzymes (in soluble and
immobilized or insoluble form) are discussed in detail. Procedures such as recombinant DNA technology and protein engineering are frequently used to produce more
efficient and beneficial enzymes. The industrial production and utilization of
enzymes is an important part of industry. Interdisciplinary collaboration between
areas such as chemistry, process engineering, microbiology and biochemistry is
required to develop the best possible enzyme technology, and eventually to achieve
increased production and maintain the enzyme’s physico-chemical properties under
in vitro environments.
For catalytic action, small quantities of an enzyme are sufficient, where this
quantity of enzyme is much smaller in comparison to its substrates. The overall
concentration of substrate transformed per mass of enzyme is often very large.
Without exception, all enzymes are proteinaceous and exhibit all the properties of a
protein. The treatment of enzymes by extreme temperature or extreme pH, or by
treatment with other denaturing agents, results in the complete loss of catalytic
activity. Structural configurations such as the primary, secondary, tertiary and
quaternary structures of enzyme proteins are essential for their catalytic activity. The
degree of catalytic activity chiefly depends on the integrity of the enzyme’s structure
1-3
H
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
as a protein. As per reports, enzymes have molecular weights ranging from about
12 000 to over 1 million Da. A number of enzymes consist only of polypeptides and
contain no chemical groups other than amino acid residues, e.g. pancreatic
ribonuclease. Numerous enzymes require a specific, heat stable, low molecular
weight organic molecule, known as a co-enzyme. Moreover, a number of enzymes
require both a co-enzyme and one or more metal ions for activity. A complete
biochemically active compound is formed by the combination of a catalytically
active enzyme (also called the protein part) with a co-enzyme or a metal ion—this is
called a holoenzyme. The protein part of a holoenzyme is called an apoenzyme. In
this arrangement a co-enzyme may bind covalently or noncovalently to the
apoenzyme. In certain enzymes the co-enzyme or metal ion is only loosely and
transiently bound to the protein. However, in others it is tightly and permanently
bound, in which case it is known as a prosthetic group. A prosthetic group signifies a
covalently bound co-enzyme. According to reports, co-enzymes and metal ions are
stable under heating, while the protein part of an enzyme (the apoenzyme), is
denatured by heat.
oloenzyme Apoenzyme Prost hetic group
Total enzyme Protein Non protein()() ( )
=+
−
Prosthetic groups may be classified functionally into two major classes: coenzymes and co-factors. Co-enzymes may be considered to be biosynthetically
related to the vitamins, such as the co-enzyme nicotinamide adenine dinucleotide
(NAD) which is vital for cellular energy metabolism and integrates the vitamin
niacin into its chemical makeup. Moreover, a co-enzyme may be considered as a cosubstrate, experiencing a chemical transformation throughout the enzyme reaction
(NAD is reduced to NADH), the reversal of which requires a separate enzyme,
perhaps from a different cellular site. Co-enzymes might thus travel intra-cellularly
between apo-enzymes and, by transferring chemical groupings, integrate several
metabolic processes. Table 1.2 shows a list of the more common co-enzymes and
their functions. In contrast to co-enzymes, co-factors, such as pyridoxal phosphate
or hem groups, remain with one enzyme molecule and in conjunction complete a
cycle of a chemical change brought about by one enzyme turnover [5]. Other
enzymes, such as carboxypeptidase, require metal ions as co-factors, the divalent
Table 1.2. Several co-enzymes employed in the transfer of specific atoms or functional groups.
Co-enzyme Entity transferred
Thyamin pyrophosphate Aldehydes
Tetrahydrofolate Other one-carbon groups
Pyridoxal phosphate Amino groups
Nicotinamide adenine dinucleotide Hydrogen atoms (electrons)
Flavin adenine dinucleotide Hydrogen atoms (electrons)
Co-enzyme A Acyl groups
Biocytin CO
3′-Deoxyadenorylcohalamine (co-enzyme B12) H atoms and alkyl groups
2
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 1.3. Several enzymes and their co-factors.
Enzyme Co-factor(s) Enzyme Co-factor(s)
Pyruvate kinase K
Nitrate reductase Mo Peroxidase Fe++or Fe
Glucose 6-phosphatase Hexokinase Mg
DNA polymerase Zn Glutathione peroxidase Se
Cytochrome oxidase Cu** Cytochrome oxidase Fe
Carbonic anhydrase Zn
Arginase Mn Alcohol dehydrogenase Zn
+
and Mg
++
+
Urease Ni
Catalase Fe++or Fe
++
++
++
+
or Fe
+++
+++
+++
cations Mg2+,Zn2+and Mn2+being the most common; these are often called
enzyme activators [6]. Table 1.3 lists several enzymes and their respective co-factors.
1.3 Catalysis
The role of a catalyst is to increase the speed of a chemical reaction. When the rate of
a chemical reaction is governed by a soluble catalyst, which may result in a further
increase in the rate of chemical reaction, it is called homogeneous catalysis. In this
case catalysis occurs in a solution. When the catalyst is in a separate phase from the
reactants, or when catalysis occurs on a insoluble surface or an immobilized matrix, it
is known as heterogeneous catalysis. Enzymes are also called biological catalysts. These
biological catalysts generally have the properties of homogeneous catalysts, however, a
number of enzymes present in membranes are insoluble, and thus are called heterogeneous catalysts. Enzyme specificity is the absolute specificity of protein catalysts to
identify and bind to only one or a few molecules. In this process the enzyme carries a
defined arrangement of atoms in their active site to bind with the substrate. This active
site on the enzyme should have a shape that accurately matches the substrates. Thus
specificity is achieved when an enzyme with an active site binds with the chemical
reactants (the substrates) at their active sites via weak bond interactions. To undergo a
chemical reaction, this active site carries certain residues that form a temporary bond
with the chemical reactants, termed the binding site, whereas the catalytic site carries
the residues that are responsible for catalysis. Specificity is achieved when a substrate
binds to an enzyme that has a defined arrangement of atoms in the active site. An
enzyme always catalyzes a single type of chemical reaction, which involves the
formation and breakdown of covalent bonds. Since they are specific to one particular
reaction, this feature of enzymes is called reaction specificity, also known as absolute
reaction specificity, i.e. no by-products are formed.
1.4 The structure of enzymes
Enzymes always act as catalysts and small quantities compared to their substrate are
required to considerably increase the rate of chemical reactions, wherein the
enzymes themselves experience no overall change [7, 8]. In contrast to all true
catalysts, an enzyme does not alter the ultimate equilibrium position of a reaction,
1-5