Readings in Biochemical Sciences 1 – Extra Credit Opportunity

1. What five lessons were learned from studying the structure and function of lysozyme?2. What types of biochemical experiments were used to provide evidence for these five lessons? 3. Industrial grade enzymes are a multibillion-dollar business.

3A. Describe the primary difference between classical enzyme development, and present enzyme development, with regard to isolating desirable enzymes with improved biochemical properties.

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3B. What three steps are required in both development processes to scale up from mutant isolation to a commercialized product ready for the market? Briefly describe each of the three steps.

4. Briefly describe the role of enzymes in each of the three biochemical process listed below and include the name of at least one enzyme used in that process.

4A. The detergent industry 4B. Starch conversion4C. Textile applications Roger L. Miesfeld, PhD
Distinguished Professor
Chemistry & Biochemistry
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Fifty years of research have led to a detailed
understanding of the mechanisms of enzymatic
How Enzymes Work
Dagmar Ringe and Gregory A. Petsko
azing at the three-dimeneven if almost every other facsional structures of enzytor were eliminated by mutating
mes that regularly grace
the enzyme, the protein would
the covers of scientific publicastill be a respectable catalyst.
tions, it is hard to imagine that
Second, Koshland was right:
there are still people alive who
The active-site residues usually
remember when many biochemadjust to permit the binding
ists thought that enzymes had no
of the specific substrate. Inordered structure. But that was the
duced-fit changes involving
case until James Sumner crystalthe movement of entire protein
lized urease in 1926 (1)—a develdomains by several nanometers
opment so revolutionary that he
have been observed (6). Third,
was taken into custody as a danthe protein structure can create
gerous lunatic when he tried to
specialized microenvironments
explain what he had done to a
that dramatically alter the reac35
famous European scientist. When
tivity of key catalytic groups, in
biochemists realized that enzymes
some cases by shielding the
had persistent structure and that
catalytic site from contact with
Elucidating the active site. In the crystal structure of a lysozyme mutant bound to
destruction of that structure could a synthetic sugar substrate, the sugar ring in the active site is distorted, and the scis- bulk solvent. Fourth, enzymes
abolish enzyme activity, they rap- sile bond is close to the acid-base residues Asp52 (left) and Glu35 (lower right; can distort the substrate, causidly adopted the view that enzymes mutated to Gln in this structure) (5). All these features were deduced by Phillips and ing it to adopt a high-energy
were rigid scaffolds whose speci- co-workers more than 40 years ago (4). Unexpectedly, the structure also shows that conformation with increased
ficity and catalytic power came lysozyme can form a covalent intermediate with its substrates (5).
reactivity (7). Finally, enzymes
from the inflexible fit of the right
provide extra stabilizing intersubstrate onto the preformed enzyme surface, and biophysical experiments. The induced fit actions for the transition state (or unstable interthe way a key fits a lock. Fifty years ago, Daniel hypothesis was still controversial, and most mediates) in the reaction mechanism. Specific
Koshland challenged this view, proposing that models of enzyme function postulated a fairly stabilization of the transition state, particularly
the enzyme surface was flexible and that only rigid catalyst. Proximity—the holding of sub- electrostatically, is thought to be so important
the specific substrate would induce the proper strate molecules and catalytic groups on the that an entire industry—the development of
interactions that led to catalysis (2).
enzyme in close approximation and in orien- catalytic antibodies—has been based on this
Studies of enzyme mechanisms were driven tations favoring the appropriate bond-break- single principle (8–10).
by a wish to understand the ability of enzymes ing and bond-making steps—was generally
Most, if not all, enzymes derive the bulk of
to accelerate the rate of a chemical reaction by held to have an important role in catalysis, but their catalytic power from varying combinastaggering amounts—up to 1020 times the rate other details were murky.
tions of these simple factors. Confirming eviof the uncatalyzed reaction in water (3)—while
The fog lifted, brilliantly, over the course dence has come from a wide range of elegant
displaying a specificity so tight that some of a single weekend, when Phillips took the experiments, notably site-directed mutagenesis,
enzymes can discriminate between sulfate and atomic model of his newly determined which allows specific groups on the enzyme
phosphate. As we celebrate not only the lysozyme structure, built into its active site a to be changed or removed (11–13), and high50th anniversary of Koshland’s “induced fit” model of the oligosaccharide substrate, and resolution x-ray crystallography, especially of
hypothesis but also ~50 years of high-resolu- deduced a set of structural factors that he enzyme-substrate and enzyme-intermediate
tion protein structure determination by x-ray believed could explain the ability of this complexes (14).
crystallography, it is instructive to look back on enzyme to digest the peptidoglycan cell walls
What was missing in this picture? Three
the history of attempts to explain enzymatic of many bacteria. Forty years of follow-up relatively recent discoveries stand out. One is
catalysis and to summarize what we understand experiments proved his inspired reasoning the contribution of quantum mechanical tuntoday about how these remarkable macromole- correct in almost every detail, although a neling to the rates of enzyme-catalyzed reaccules function.
recent study provides a new wrinkle (see the tions whose mechanisms involve the transfer
Before the first crystal structure of an figure) (5). Moreover, the factors he enumer- of hydrogen ions (15). Another is the precise
enzyme was determined, that of lysozyme by ated turned out to be applicable to almost all matching of the pKa’s (a logarithmic measure
David Phillips and his team in 1965 (4), spec- other enzymes.
of the proton affinity of a weak acid) of the
ulations about how enzymes worked were
What are the lessons from lysozyme? First, donor and acceptor atoms in hydrogen bonds
based on deductions from indirect biochemical proximity and orientation are critical. Much of that stabilize the transition state. Such matchwhat an enzyme does is to bring the reacting ing can lead to short, symmetrical hydrogen
species together in a geometry that favors reac- bonds of greater-than-normal strength (16, 17).
Department of Biochemistry, Brandeis University, Waltham,
MA 02454, USA. E-mail:
tion. This is so important that in some cases, But perhaps the most active area of current
13 JUNE 2008
VOL 320
Published by AAAS
Downloaded from on September 21, 2015
research is the possible role of protein
dynamics in aiding the reacting species in
crossing the transition-state barrier to the
reaction. As originally formulated, the structure of the enzyme was proposed to favor
atomic vibrations along the reaction coordinate while disfavoring those that would not
lead to productive bond-making or bondbreaking steps (18). Recent evidence from
different enzyme systems suggests that this
factor may indeed contribute to catalytic efficiency (19, 20).
Given that we now have a good understanding of the principles underlying enzyme
catalytic proficiency and specificity, it seems
appropriate to ask where the field is likely to
go next. Practical applications, such as the
creation of enzymes catalyzing novel reactions, are under way. Further investigations
into the role of protein dynamics in enzymatic catalysis are still needed. But we
believe that a crucial next step will be to go
beyond the milieu of dilute aqueous solution
and individual purified enzymes that has
defined enzymology for the past 100 years.
Most enzymes function in the interior of the
cell, where the substrate concentration is typically very low and the protein concentration
may exceed 100 mM. How do enzymes function in a crowded medium of low water activity, where there may be no such thing as a
freely diffusing, isolated protein molecule? In
vivo enzymology is the logical next step
along the road that Phillips, Koshland, and
their predecessors and successors have traveled so brilliantly so far.
References and Notes
1. J. B. Sumner, J. Biol. Chem. 69, 435 (1926).
2. D. E. Koshland Jr., Nature 432, 447 (2004).
3. C. Lad, N. H. Williams, R. V. Wolfenden, Proc. Natl. Acad.
Sci. U.S.A. 100, 5607 (2003).
4. C. C. Blake et al., Proc. R. Soc. London B 167, 378
5. D. J. Vocadlo, G. J. Davies, R. Laine, S. G. Withers, Nature
412, 835 (2001).
6. T. A. Steitz, R. Harrison, I. T. Weber, M. Leahy, Ciba
Found. Symp. 93, 25 (1983).
7. D. L. Pompliano, A. Peyman, J. R. Knowles, Biochemistry
29, 3186 (1990).
8. S. D. Lahiri, G. Zhang, D. Dunaway-Mariano, K. N. Allen,
Science 299, 2067 (2003).
9. A. Warshel et al., Chem. Rev. 106, 3210 (2006).
10. R. A. Lerner, C. F. Barbas III, K. D. Janda, Harvey Lect. 92,
1 (1996–1997).
11. J. R. Knowles, Nature 350, 121 (1991).
12. T. C. Bruice, S. J. Benkovic, Biochemistry 39, 6267
13. D. A. Kraut, K. S. Carroll, D. Herschlag, Annu. Rev.
Biochem. 72, 517 (2003).
14. I. Schlichting et al., Science 287, 1615 (2000).
15. Z. D. Nagel, J. P. Klinman, Chem. Rev. 106, 3095 (2006).
16. W. W. Cleland, P. A. Frey, J. A. Gerlt, J. Biol. Chem. 273,
25529 (1998).
17. D. A. Kraut et al., PLoS Biol. 4, e99, (2006).
18. T. Alber et al., CIBA Found. Symp. 93, 4 (1982).
19. S. Hammes-Schiffer, S. J. Benkovic, Annu. Rev. Biochem.
75, 519 (2006).
20. K. A. Henzler-Wildman et al., Nature 450, 838 (2008).
21. We dedicate this paper to the memory of our good friend
and long-time collaborator Jeremy R. Knowles.
New results provide support for the hypothesis
that interactions between proteins involve
selection from an ensemble of different
How Do Proteins Interact?
David D. Boehr and Peter E. Wright
nteractions between proteins are central
to biology and are becoming increasingly
important targets for drug design. Upon
forming complexes, protein conformations
usually change substantially compared to the
unbound protein. Two main hypotheses have
been advanced to explain these changes (see
the figure). According to the “induced fit”
hypothesis, the initial interaction between a
protein and a binding partner induces a conformational change in the protein through a
stepwise process (1). In the “conformational
selection” model, it is assumed that, prior to
the binding interaction, the unliganded protein exists as an ensemble of conformations
in dynamic equilibrium. The binding partner
interacts preferentially with a weakly populated, higher-energy conformation-causing
the equilibrium to shift in favor of the
selected conformation. This conformation
then becomes the major conformation in the
complex (2). Although biochemistry textbooks have championed the induced fit
mechanism for more than 50 years, there is
now growing support for the additional bind-
Department of Molecular Biology and Skaggs Institute for
Chemical Biology, The Scripps Research Institute, La Jolla,
CA 92037, USA. E-mail:; wright@
ing mechanism, including the seminal work
by Lange, Lakomek, and co-workers on page
1471 of this issue (3).
A major stumbling block for the conformational selection hypothesis has been the
inability to characterize the structures of the
predicted multiple conformations (or conformational substates) of a protein. The structural
models resulting from x-ray crystallography
tend to identify only a single dominant conformation, although different crystal forms of the
same protein can provide insights into the
range of conformations accessible to the protein (4). Help comes from nuclear magnetic
resonance (NMR), a powerful method for
characterizing protein dynamics and the protein conformational ensemble at the atomic
level. Various NMR observables (5, 6) give
structural information about lowly populated,
higher-energy conformations that are invisible to other techniques.
In a previous report, Vendruscolo and coworkers (7) combined data from NMR relaxation experiments with molecular dynamics simulations to characterize a structural
ensemble of the protein ubiquitin. However,
the experimental data only covered nanosecond time-scale dynamics and thus failed to
capture the slower time scales that are important for molecular recognition.
VOL 320
Published by AAAS
Lange et al. have now extended the
methodology to slower time scales by using
residual dipolar couplings (RDCs) (3), which
serve as restraints for structural determination
by NMR and also provide dynamic information over a wide range of time scales (8). By
analyzing RDCs measured for a large range of
solution conditions, Lange et al. construct a
structural ensemble for ubiquitin that describes its dynamic behavior up to the
microsecond time scale.
The most striking feature of the ensemble
is the presence of conformations that are
nearly identical to the 46 known bound forms
of ubiquitin observed in x-ray crystal structures. The results provide very strong evidence that complex formation by ubiquitin
involves conformational selection processes. Gsponer et al. recently reported a similar
result for calmodulin. Using the methodology
of Vendruscolo and co-workers, they showed
that the nanosecond ensemble for apocalmodulin contains conformations similar to
calmodulin bound to myosin light chain
kinase (9).
The structural ensemble reported by Lange
et al. is consistent with the energy landscape
theory of protein folding and function (2, 10,
11). This theory posits that there are multiple
protein conformations in dynamic equilib-
13 JUNE 2008
Industrial enzyme applications
Ole Kirk*, Torben Vedel Borchert and Claus Crone Fuglsang
The effective catalytic properties of enzymes have already
promoted their introduction into several industrial products and
processes. Recent developments in biotechnology, particularly in
areas such as protein engineering and directed evolution, have
provided important tools for the efficient development of new
enzymes. This has resulted in the development of enzymes with
improved properties for established technical applications and in
the production of new enzymes tailor-made for entirely new areas
of application where enzymes have not previously been used.
Research and Development, Novozymes A/S, Krogshoejvej 36,
2880 Bagsvaerd, Denmark
Current Opinion in Biotechnology 2002, 13:345–351
0958-1669/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
century, aimed specifically at the production of enzymes by
use of selected production strains, made it possible to manufacture enzymes as purified, well-characterized preparations
even on a large scale. This development allowed the
introduction of enzymes into true industrial products and
processes, for example, within the detergent, textile and starch
industries. The use of recombinant gene technology has
further improved manufacturing processes and enabled the
commercialization of enzymes that could previously not be
produced. Furthermore, the latest developments within
modern biotechnology, introducing protein engineering and
directed evolution, have further revolutionized the development of industrial enzymes (Figure 1). These advances have
made it possible to provide tailor-made enzymes displaying
new activities and adapted to new process conditions, enabling
a further expansion of their industrial use. As illustrated in
Table 1, the result is a highly diversified industry that is still
growing both in terms of size and complexity.
DOI 10.1016/S0958-1669(02)00328-2
The enzyme industry as we know it today is the result of a
rapid development seen primarily over the past four decades
thanks to the evolution of modern biotechnology. Enzymes
found in nature have been used since ancient times in the
production of food products, such as cheese, sourdough, beer,
wine and vinegar, and in the manufacture of commodities such
as leather, indigo and linen. All of these processes relied on
either enzymes produced by spontaneously growing microorganisms or enzymes present in added preparations such as
calves’ rumen or papaya fruit. The enzymes were, accordingly,
not used in any pure or well-characterized form. The development of fermentation processes during the later part of the last
The majority of currently used industrial enzymes are
hydrolytic in action, being used for the degradation of
various natural substances. Proteases remain the dominant
enzyme type, because of their extensive use in the detergent and dairy industries. Various carbohydrases, primarily
amylases and cellulases, used in industries such as the
starch, textile, detergent and baking industries, represent
the second largest group [1]. As illustrated in Figure 2, the
technical industries, dominated by the detergent, starch,
textile and fuel alcohol industries, account for the major
consumption of industrial enzymes. Overall, the estimated
value of the worldwide use of industrial enzymes has
grown from $1 billion [1] in 1995 to $1.5 billion in 2000 [2].
This growth, however, has stagnated in some of the major
Figure 1
The steps involved in classical versus state-ofthe-art development of enzymes.
Classical enzyme development
Present enzyme development
Creating biological
Nature’s diversity
Molecular evolution
Primary screening
Secondary screening
Classical mutagenesis
Creating expression
Up-scaling process
Up-scaling process
Current Opinion in Biotechnology
Protein technologies and commercial enzymes
Table 1
Enzymes used in various industrial segments and their applications.
Enzyme class
Detergent (laundry and dish wash)
Glucose isomerase
Pectin methyl esterase
Glucose oxidase
Acetolactate decarboxylase
Pectate lyase
Glucose oxidase
Protein stain removal
Starch stain removal
Lipid stain removal
Cleaning, color clarification, anti-redeposition (cotton)
Mannanan stain removal (reappearing stains)
Starch liquefaction and saccharification
Glucose to fructose conversion
Cyclodextrin production
Viscosity reduction (fuel and starch)
Protease (yeast nutrition – fuel)
Milk clotting, infant formulas (low allergenic), flavor
Cheese flavor
Lactose removal (milk)
Firming fruit-based products
Fruit-based products
Modify visco-elastic properties
Bread softness and volume, flour adjustment
Dough conditioning
Dough stability and conditioning (in situ emulsifier)
Dough stability and conditioning (in situ emulsifier)
Dough strengthening
Dough strengthening, bread whitening
Biscuits, cookies
Laminated dough strengths
Phytate digestibility – phosphorus release
De-pectinization, mashing
Juice treatment, low calorie beer
Maturation (beer)
Clarification (juice), flavor (beer), cork stopper treatment
Denim finishing, cotton softening
Bleach termination
Excess dye removal
Pitch control, contaminant control
Biofilm removal
Starch-coating, de-inking, drainage improvement
Bleach boosting
De-inking, drainage improvement, fiber modification
De-gumming, lyso-lecithin production
Resolution of chiral alcohols and amides
Synthesis of semisynthetic penicillin
Synthesis of enantiopure carboxylic acids
Unhearing, bating
Antimicrobial (combined with glucose oxidase)
Bleaching, antimicrobial
Starch and fuel
Food (including dairy)
Animal feed
Pulp and paper
Fats and oils
Organic synthesis
Personal care
technical industries, first of all the detergent industry [2].
The fastest growth over the past decade has been seen in
the baking and animal feed industries, but growth is also
being generated from applications established in a wealth
of other industries spanning from organic synthesis to
paper and pulp and personal care. This review will, segment
by segment, discuss the most important recent developments
in the technical use of enzymes and will consider the
most recent technological advances that have facilitated
these developments.
New technologies for enzyme discovery
Natural microorganisms have over the years been a great
source of enzyme diversity. The developments in bioinformatics and the availability of sequence data have increased
immensely the efficiency of isolating an interesting gene
Industrial enzyme applications Kirk, Borchert and Fuglsang
from nature. Rational protein engineering and the possibility
of introducing small changes to proteins, on the basis of their
structure and the related biochemical and biophysical
properties, introduced a new valuable tool to enzyme
optimization in the 1980s. Directed evolution is the latest
addition to the toolbox (for a recent review see [3]). The
more or less random introduction of mutations generates
variant libraries that are subsequently exposed to a screening
or selection procedure. The isolated, improved variants from
one round of screening are then used as starting material
in the following rounds of recombination and/or new
diversity generation (Figure 1). Recently, various attempts
at understanding the important parameters in directed evolution have emerged and successful examples of combining
rational engineering with directed evolution have been
reported [4,5••]. Usually, the new, exciting technology is
predicted to out-compete the existing technologies, but we
expect that time will demonstrate how the combined use of
rational design, directed evolution and nature’s diversity will
be far superior to any lone-standing technology.
Figure 2
Animal feed
Current Opinion in Biotechnology
Segmentation of the industrial enzyme market. In the year 2000, the
enzyme market totalled $1.5 billion. The technical industries segment
comprises the detergent, starch, textile, fuel alcohol, leather, and pulp
and paper industries.
The detergent industry
Their use as detergent additives still represents the
largest application of industrial enzymes, both in terms of
volume and value. The major component is proteases, but
other and very different hydrolases are introduced to
provide various benefits, such as the efficient removal
of specific stains (Table 1). Constantly, new and improved
engineered versions of the ‘traditional’ detergent enzymes,
proteases and amylases, are developed. These new
second- and third-generation enzymes are optimized to
meet the requirements for performance in detergents, the
composition of which is also constantly developed. In
particular, the compatibility of enzymes with detergent
components (i.e. typically stability properties) is addressed,
but their ability to function at lower temperatures has also
been amongst the recently reported improvements. To
save energy, the temperature used in household laundering
and automated dishwashers has been reduced in recent
years. This often results in problems with efficient
cleaning and stain removal that enzyme technology can
help overcome.
Recent examples of second-generation detergent enzymes
include the development of novel amylases that have
enhanced activity at lower temperatures and alkaline pH,
while maintaining the necessary stability under detergent
conditions. These enzymes were developed by the
combined use of microbial screening and rational protein
engineering [6]. Proteases displaying activity at low
temperatures have been isolated from nature, but have
also been evolved in the laboratory by a directed evolution
approach [7]. Furthermore, from a starting material of
26 subtilisin proteases Ness and coworkers [8••] utilized
one round of DNA shuffling to isolate new proteases with
various improved properties. The improvements included
characteristics very relevant for detergent proteases
(i.e. improved activity and stability at alkaline pH).
The most recent introduction of a new enzyme class into a
detergent has been the addition of a mannanase — the
result of a joint development between Procter and Gamble
and Novozymes [9•]. This enzyme helps remove various
food stains containing guar gum, a commonly used
stabilizer and thickening agent in food products.
Enzymes for starch conversion
The enzymatic conversion of starch to high fructose corn
syrup is a well-established process and provides a beautiful
example of a bioprocess in which the consecutive use of
several enzymes is necessary. The enzymes utilized in the
starch industry are also subjected to constant improvements.
The first step in the process is the conversion of starch to
oligomaltodextrins by the action of α-amylase. The concomitant injection of steam puts extreme demands on the
thermostability of the enzyme. Using traditional α-amylases,
the pH has to be adjusted to an undesirable high level
and calcium must be added to stabilize the enzyme. New
α-amylases with optimized properties, such as enhanced
thermal stability, acid tolerance, and ability to function
without the addition of calcium, have recently been developed [6,10,11•] offering obvious benefits to the industry.
Engineering efforts have also been undertaken to develop
improved versions of the enzymes used later in the process
(i.e. glucoamylase and glucose isomerase [12,13]).
Fuel alcohol production
In the alcohol industry, the use of enzymes for the production
of fermentable sugars from starch is also well established.
Over the past decade, there has been an increasing interest
in fuel alcohol as a result of increased environmental
concern, higher crude oil prices and, more acutely, by the
ban in certain regions of the gasoline additive methyl
Protein technologies and commercial enzymes
Figure 3
Catalase (bleach clean-up)
Raw fabric
Peroxidase (removal
of excess dye)
Neutral cellulase
Acid cellulase
Enzymes used in various unit operations in
textile wet processing and the manufacturing
of Denim.
Finished fabric
Finished blue jeans
Current Opinion in Biotechnology
tert-butyl ether (MTBE), which can be interchanged
directly with ethanol [14–16]. Therefore, intense efforts
are currently being undertaken to develop improved
enzymes that can enable the utilization of cheaper and
partially utilized substrates such as lignocellulose, to make
bio-ethanol more competitive with fossil fuels [17,18].
The cost of enzymes needed to turn lignocellulose into
a suitable fermentation feed-stock is a major issue, and
current work focuses both on the development of enzymes
with increased activity and stability as well as on their
efficient production. Huge governmental programs have
been launched in the United States by the Department of
Energy to support these developments, spurred by the
general emphasis on reducing pollution and the need to
work towards fulfilling the Kyoto protocol.
Textile applications
In the textile industry a completely new enzymatic
activity has recently been introduced. This industry is
under considerable environmental pressure owing to its
large energy and water consumption and subsequent
environmental pollution. One of the most energy- and
water-consuming steps in the processing of cotton is the
scouring step, the removal of various remaining cell-wall
components on the cellulose fibers performed at high
temperature and under strong alkaline conditions. An
alternative, enzyme-based process performed at much
lower temperatures and using less water has now been
developed based on a pectate lyase [19•]. The positive
environmental impact of the new process was recognized
by a grant of the United States Presidential Green
Chemistry Challenge Award in 2001. Following the
introduction of this step, enzymes have now been
introduced into most of the major steps in the manufacturing of cotton textiles (see Figure 3). The use of these
enzymes has benefited both the textile industry and
the environment.
Enzymes for the feed industry
The use of enzymes as feed additives is also well established. For example, xylanases and β-glucanases have been
used throughout the past decade in cereal-based feed
for monogastric animals which, contrary to ruminants, are
unable to fully degrade and utilize plant-based feeds
containing high amounts of cellulose and hemicellulose.
During recent years focus has been on the utilization of
natural phosphorus bound in phytic acid in cereal-based
feed for monogastrics. Better utilization of total plant phosphorus, of which 85–90% is bound in phytic acid, is only
obtained by adding the enzyme phytase to the feed. The
attention on this enzyme has increased dramatically during
recent years owing to the bovine spongiform encephalopathy
(BSE)-related bone-meal ban in many countries, which has
effectively removed the main traditional source for inorganic
phosphorus. Also, several western countries with intensive
animal production have adopted standards for the release
of phosphorus into the environment. As the addition of
phytase to feeds results in a significant reduction of the
phosphorus outlet from monogastrics, the phytases have
grown to become the largest enzyme segment in the feed
industry. Besides the direct effects on phosphorus uptake
and secretion, indirect effects on the uptake of other
nutrients are also being recognized [20•,21].
The most recent advances in feed enzymes have been
aimed at improvements in the applicability and performance of phytases [22]. New fungal phytases have been
identified with 4–50-fold higher specific activities than
previously reported [23•]. Alternative approaches for the
development of more effective enzymes have been to
increase the catalytic activity of fungal phytases by
site-directed mutagenesis; for example, on the basis of
three-dimensional structural studies, the specific activity
of the Aspergillus fumigatus phytase was increased fourfold
[24]. In order for the enzymes to be applicable for use in
Industrial enzyme applications Kirk, Borchert and Fuglsang
pelleted feed products, they have to survive high temperatures
(above 80°C) during pelleting for short periods of time. A
novel approach used to achieve thermal stabilization has
been the construction of a ‘consensus phytase’ based on
homologies among various phytases. This enzyme exhibits
an increase in thermal stability to around 80°C [25•].
Still, phosphorus utilization is not the only issue of concern
to the animal feed industry; continuous effort is put into
obtaining increased nutritional value from various feed
sources, for example, by increasing the digestibility of the
protein in soybean meal. It is likely that in the future we
will see new and different hydrolytic enzymes applied in
the feed industry to increase the value of feed stock, thus
lowering the energy consumption and pollution per live
stock to the benefit of the environment.
Enzymes for the food industry
As indicated in Table 1, applications of enzymes in the
food industry are many and diverse, ranging from texturizing
to flavoring. Common to more or less all food applications,
the enzymes are applied to processed food products as
processing agents upstream from the final product. Several
advances have been made in the optimization of enzymes
for existing applications and in the use of recombinant
protein production to provide efficient mono-component enzymes that do not have potential detrimental
Recently, much work has been carried out on the application
of transglutaminase as a texturing agent in the processing
of, for example, sausages, noodles and yoghurt, where
cross-linking of proteins provides improved viscoelastic
properties of the products [26•]. An obstacle, which
may prevent even wider usage, is the currently limited
availability of the enzyme in industrial scale. At present
only the transglutaminase from Streptoverticillium sp. is
commercially available at a reasonable scale, and work is
ongoing to increase the availability of the enzyme by
recombinant production in Escherichia coli [27].
Within the baking industry there is an increasing focus
on lipolytic enzymes [28,29]. Recent findings suggest that
(phospho)lipases can be used to substitute or supplement
traditional emulsifiers, as the enzymes degrade polar
wheat lipids to produce emulsifying lipids in situ. Also,
efforts are currently devoted towards the further understanding of bread staling and the mechanisms behind the
enzymatic prevention of staling when using α-amylases
and xylanases [30]. Studies have confirmed previous findings showing that water-binding capacity and retention in
the starch and hemicellulose fractions of the bread, being
the substrates of α-amylases and xylanases, respectively, to
be critical for maintaining softness and elasticity. The
recently determined three-dimensional structure of the
widely applied amylase for antistaling (Novamyl) provided further insight into the mechanism of enzyme action
[31••]. This amylase is probably capable of degrading
amylopectin to a degree that prevents re-crystallization
after gelatinization, without completely degrading the amylopectin network which provides the bread with elasticity.
Besides the above-mentioned advances, a few entirely
new applications within the food industry should be
mentioned, although little, if any, literature is publicly
available. The use of laccase for clarification of juice (laccases
catalyze the cross-linking of polyphenols, resulting in an
easy removal of polyphenols by filtration) and for flavor
enhancement in beer are recently established applications
within the beverage industry. It is likely that the functional
understanding of different enzyme classes will provide
new applications within the food industry in the future.
Processing of fats and oils
In the fat and oil industries, several new enzyme-based
processes have recently been introduced. Even though the
use of immobilized lipases in the interesterification of
triglycerides was first described in the 1980s, the process
has not been sufficiently cost-effective to be introduced in
true large-scale applications, for example, in the production
of margarine. Although enzyme production has become
much more efficient, the cost of immobilization has
remained an obstacle. Recent developments have, however,
changed this picture. A new process for immobilizing lipases
based on the granulation of silica has dramatically lowered
process costs, and procedures based on this new material
are now being implemented for the production of commodity fats and oils with no content of trans-fatty acids
[32]. Another recently introduced process is the removal of
phospholipids in vegetable oils (‘de-gumming’), using a
highly selective microbial phospholipase [33•]. This is yet
another example where the introduction of an enzymebased step has enabled both energy and water savings for
the benefit of both the industry and the environment.
Enzymes for organic synthesis
Chemical synthesis is an area where the use of enzyme catalysis has long been seen as having great promise. Even so, the
chemical industry has been slow to implement enzyme-based
processes and the use of enzymes in the chemical industry is
still low compared with other industries. At present, however,
we are seeing very significant growth in this area and enzymebased processes are now, finally, being widely introduced for
the production of a diversity of different chemicals; one key
example is in the production of single-enantiomer intermediates used in the manufacture of drugs and agrochemicals [34].
This market is characterized by a very high degree of fragmentation, as very few enzymes have applicability in a broad
range of different processes. Recently introduced enzymebased processes include the use of lipases for the production
of enantiopure alcohols and amides, nitrilases for the
production of enantiopure carboxylic acids, and acylases for
the production of new semisynthetic penicillins [34]. As many
companies are currently at an early stage in the exploitation of
enzyme-based catalysis, many new developments are expected
in this area over the next few years.
Protein technologies and commercial enzymes
Conclusions and perspectives
As outlined above, enzymes are currently used in several
different industrial products and processes and new
areas of application are constantly being added. Thanks to
advances in modern biotechnology, enzymes can be
developed today for processes where no one would have
expected an enzyme to be applicable just a decade ago.
Common to most applications, the introduction of
enzymes as effective catalysts working under mild conditions results in significant savings in resources such as
energy and water for the benefit of both the industry in
question and the environment. In a world with a rapidly
increasing population and approaching exhaustion of many
natural resources, enzyme technology offers a great potential
for many industries to help meet the challenges they will
face in years to come.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
14. Jolly L: The commercial viability of fuel ethanol from sugar cane.
Int Sugar J 2001, 103:117-143.
15. Taylor F, Mcaloon AJ, Craig JC, Yang P, Wahjudi J, Eckhoff SR:
Fermentation and costs of fuel ethanol from corn with quick-germ
process. Appl Biochem Biotechnol 2001, 94:41-49.
16. Taylor F, Kurantz MJ, Goldberg N, Mcaloon AJ, Craig JC: Dry-grind
process for fuel ethanol by continuous fermentation and
stripping. Biotechnol Prog 2000, 16:541-547.
Zaldivar J, Nielsen J, Olsson L: Fuel ethanol production from
lignocellulose: a challenge for metabolic engineering and process
integration. Appl Microbiol Biotechnol 2001, 56:17-34.
18. Wheals AE, Basso LC, Alves DMG, Amorim AV: Fuel ethanol after
25 years. Trends Biotechnol 1999, 17:482-487.
19. Tzanov T, Calafell M, Guebitz GM, Cavaco-Paulo A: Biolpreparation

of cotton fabrics. Enzyme Microb Technol 2001, 29:357-362.
This work describes the successful substitution of traditional chemical processes by the introduction of pectinases for biopreparation of cotton fabrics.
20. Lei XG, Stahl CH: Nutritional benefits of phytase and

dietary determinants of its efficacy. J Appl Anim Res 2000,
The paper discusses the beneficial gains of utilizing phytase for animal feed,
in a fair and critical manner, and provides a nice overview on this particular
usage of phytase.
21. Kies AK, van Hemert KHF, Sauer WC: Effect of phytase on protein
and amino acid digestibility and energy utilization. Worlds Poult
Sci J 2001, 57:109-126.
Godfrey T, West SI: Introduction to industrial enzymology. In
Industrial Enzymology, edn 2. Edited by Godfrey T, West S. London:
Macmillan Press; 1996:1-8.
22. Lei XG, Stahl CH: Biotechnological development of effective
phytases for mineral nutrition and environmental protection. Appl
Microbiol Biotechnol 2001, 57:474-481.
McCoy M: Novozymes emerges. Chem Eng News 2000, 19:23-25.
Tobin MB, Gustafsson C, Huisman GW: Evolution: the ‘rational’
basis for ‘irrational’ design. Curr Opin Struct Biol 2000, 10:421-427.
Voigt CA, Kauffman S, Wang ZG: Rational evolutionary design: the
theory of in vitro protein evolution. Adv Protein Chem 2000,
23. Lassen SF, Breinholt J, Østergaard PR, Brugger R, Bischoff A,

Wyss M, Fuglsang CC: Expression, gene cloning and
characterization of five novel phytases from four Basidiomycete
fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp. and
Trametes pubescens. Appl Environ Microbiol 2001, 67:4701-4707.
Describes an entirely new group of fungal phytases and their properties.
One of these phytases has recently been commercialized for application in
animal feed.
Altamirano MM, Blackburn JM, Aguayo C, Fersht AR: Directed
evolution of a new catalytic activity using the α/β-barrel scaffold.
Nature 2000, 403:617-622.
The elegant combination of rational engineering and directed molecular evolution are used for the introduction of a new catalytic activity in an enzyme.
Bisgaard-Frantzen H, Svendsen A, Norman B, Pedersen S, Kjærulff S,
Outtrup H, Borchert TV: Development of industrially important
α-amylases. J Appl Glycosci 1999, 46:199-206.
Wintrode PL, Miyazaki K, Arnold FH: Cold adaptation of a
mesophilic subtilisin-like protease by laboratory evolution. J Biol
Chem 2000, 275:31635-31640.

Ness JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV,
Stemmer WPC, Minshull J: DNA shuffling of subgenomic
sequences of subtilisin. Nat Biotechnol 1999, 17:893-896.
This work describes the shuffling of a large family of homologous genes
and analysis of the resulting functional diversity. Screening of a rather small
library resulted in improvements for five different properties.
9. McCoy M: Soaps & detergents. Chem Eng News 2001,

An update on the latest developments within the detergent industry also
introducing the latest new detergent enzyme, a mannanase.
10. Shaw A, Bott R, Day AG: Protein engineering of α-amylases for
low pH performance. Curr Opin Biotechnol 1999, 10:349-352.
11. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C: Probing

structural determinants specifying high thermostability in Bacillus
licheniformis α-amylase. J Mol Biol 2000, 301:1041-1057.
The elegant use of suppressors aided the construction and analysis of thermostability of 175 amylase variants. Several stabilizing mutations were identified.
24. Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L,
van Loon APGM, Pasamontes L: Optimization of the catalytic
properties of Aspergillus fumigatus phytase based on the
three-dimensional structure. Protein Sci 2000, 9:1304-1311.
25. Lehmann M, Kostrewa D, Wyss M, Brugger R, D’Arcy A,

Pasamontes L, van Loon APGM: From DNA sequence to improved
functionality: using protein sequence comparisons to rapidly
design a thermostable consensus phytase. Protein Eng 2000,
An interesting new approach for designing enzymes with improved properties.
A significant thermal stabilization is obtained compared with the parent
phytase backbones.
26. Kuraishi C, Yamazaki K, Susa Y: Transglutaminase: its utilization in

the food industry. Foods Rev Int 2001, 17:221-246.
Industrial application of transglutaminase is still in its infancy. This paper
provides a nice overview of some of the first applications of this enzyme in
the food industry.
Yokoyama K, Nakamura N, Seguro K, Kubota K: Overproduction of
microbial transglutaminase in Escherichia coli, in vitro refolding,
and characterization of the refolded form. Biosci Biotechnol
Biochem 2000, 64:1263-1270.
28. Collar C, Martinez JC, Andreu P, Armero E: Effect of enzyme
associations on bread dough performance. A response surface
study. Food Sci Technol Int 2000, 6:217-226.
29. Monfort A, Blasco A, Sanz P, Prieto JA: Expression of LIP1 and LIP2
genes from Geotricum species in baker’s yeast strains and their
application to the bread-making process. J Agric Food Chem
1999, 47:803-808.
12. Sauer J, Sigurdskjold BW, Christensen U, Frandsen TP,
Mirgorodskaya E, Harrison M, Roepstorff P, Svensson B:
Glucoamylase: structure/function relationships and protein
engineering. Biochem Biophys Acta 2000, 1543:275-293.
30. Andreu P, Collar C, Martínez-Anaya MA: Thermal properties of
doughs formulated with enzymes and starters. Eur Food Res
Technol 1999, 209:286-293.
13. Hartley BS, Hanlon N, Jackson RJ, Rangrajan M: Glucose isomerase:
insight into protein engineering for increased thermostability.
Biochem Biophys Acta 2000, 1543:294-335.
31. Dauter Z, Dauter M, Brzozowski AM, Christensen S, Borchert TV,
•• Beier L, Wilson KS, Davies GJ: X-ray structure of Novamyl, the
five-domain ‘maltogenic’ α-amylase from Bacillus
Industrial enzyme applications Kirk, Borchert and Fuglsang
stearothermophilus: maltose and acarbose complexes at 1.7 Å
resolution. Biochemistry 1999, 38:8385-8392.
Describes the determination of the three-dimensional structure of a maltogenic
α-amylase widely applied for providing antistaling effects in white bread. Structural
insight into the unique specificity and performance of this enzyme is also provided.
33. Clausen K: Enzymatic oil-degumming by a novel

microbial phospholipase. Eur J Lipid Sci Technol 2001,
The use of phospholipases for oil-degumming is described with focus
on the introduction of the first enzyme of microbial origin for this application.
32. Christensen MW, Andersen L, Kirk O, Holm HC: Enzymatic
interesterification of commodity oils and fats: approaching the
tonnes scale. Lipid Technol News 2001,7:33-37.
34. Schmidt A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B:
Industrial biocatalysis today and tomorrow. Nature 2001,
Bioc 384 – Fall 2019
Dr. Miesfeld
Readings in Biochemical Sciences 1 – Extra Credit Opportunity (EC1)
Due in D2L drop box as .doc, docx, or .pdf file by 11:00pm on Tuesday, October 22, 2019.
Late submissions will not be accepted by email – all submissions must be through D2L website.
Note that all DropBox files are automatically evaluated by for plagiarism against all
web documents using a D2L function ( You must only include your answers,
do not repeat the questions. Moreover, DO NOT a) copy (or quote) from the journal article, b)
copy from another student, or c) copy from any internet source that you find or subscribe to;
all of these activities are clearly plagiarism. You need to use your own words for all answers.
PLAGIARISM; taking someone else’s work or ideas and passing them off as one’s own.
Your name and last four digits of your student ID number MUST be included at the top of
the first page to be eligible for extra credit points.
Read the two attached articles: How Enzymes Work by Dagmar Ringe and Gregory Petsko,
(2008) Science 320:1428-1429, Industrial Enzyme Applications by Ole Kirk, Torben Borchert,
and Clause Fuglsang, (2002) Current Opinion Biotech 13:345-351.
Your combined answers to all of these questions should be in the range of ~300-600 words.
1. What five lessons were learned from studying the structure and function of lysozyme?
2. What types of biochemical experiments were used to provide evidence for these five lessons?
3. Industrial grade enzymes are a multibillion-dollar business.
3A. Describe the primary difference between classical enzyme development, and present
enzyme development, with regard to isolating desirable enzymes with improved
biochemical properties.
3B. What three steps are required in both development processes to scale up from
mutant isolation to a commercialized product ready for the market? Briefly describe each
of the three steps.
4. Briefly describe the role of enzymes in each of the three biochemical process listed below and
include the name of at least one enzyme used in that process.
4A. The detergent industry
4B. Starch conversion
4C. Textile applications

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