Protein engineering from a bioindustrial point of view

Protein engineering from a bioindustrial point of view
Donn N Rubingh
Work with proteins, particularly enzymes, is a rapidly growing
segment of the biotechnology industry. Directed evolution
promises to become an increasingly important strategy in their
development as it allows one to sidestep some of the difficult
questions relating the structural and functional properties of
such proteins to their industrial utility. It is also clear, however,
that greater understanding of how to engineer certain basic
enzyme properties, such as stability, activity, and surface
properties, is beginning to emerge, and this understanding
will make rational design more efficient. To engineer a
commercially useful protein many properties need to be
changed, and frequently these changes are interdependent.
Recent protein engineering studies on protease, amylase,
lipase and cellulase illustrate some of the progress in this
area.
Addresses
Procter and Gamble Co, Miami Valley Laboratory, PO Box 398707,
Cincinnati, OH 45239-8707, USA; e-mail: rubingh.dn@pg.com
Current Opinion in Biotechnology 1997, 8:417-422
http://biomednet.com/elecref/0956166900800417
0 Current Biology Ltd ISSN 0958-l 669
Abbreviation
CBD cellulose binding domain
Introduction
The importance of protein engineering in industry contin-
ues to grow as the number of applications of proteins ex-
pands, and the technology to efficiently discover proteins
with useful properties is better able to address industrially
relevant problems. Recent advances in directed evolution
are being implemented in many established industrial
laboratories as well as in start-up companies, augmenting
the rational design approach. Additionally, organisms
from extreme environments are becoming an important
source of new backbones for engineering proteins with
significantly different properties. The I?e tzowo design of
new proteins is just beginning to make an impact on
industrial applications.
The successfully engineered protein generally requires
a proper combination of properties. For example, a
detergent protease would require, at minimum, stability
in the presence of detergent and activity against certain
protein stains. Nevertheless, the control of a few basic
properties is a recurring theme in many applications.
Properties such as sufficient stability, high activity (in the
case of enzymes), and the ability to interact correctly with
surfaces are necessary for a variety of industrially important
proteins. Some significant advances in the understanding
417
of how to tailor these basic properties have occurred over
the past few years.
This review will focus on protein engineering for non-
pharmaceutical applications. Enzymes such as proteases,
amylases, lipases, cellulases, and xylanases have received
significant attention because of their importance in
cleaning products, starch processing, and pulp and paper
manufacturing. Recent advances in understanding these
enzymes will be reviewed. Other applications of protein
engineering to food processing, chemical synthesis, and
bioremediation are also beginning to find their way into
industrial processes; however, these will not be covered
here.
Strategies for protein engineering in
industrial settings
Rational design
Rational design was the earliest approach co protein
engineering and is still the most widely used way to
introduce desired properties into a protein of interest.
The strategy hinges on relating structure to function, fre-
quently via molecular modeling techniques. Advances in
rational design depend on progress in structure determina-
tion, improved modeling procedures, and significant new
insight into structure-function relationships. A promising
new technique for obtaining structural information on
hard to crystalize proteins is the two-dimensional (ZD)
crystallization technique that utilizes metal ion coordina-
tion to surface histidines in order to crystallize proteins
at interfaces [lo]. The importance of the micelle-vesicle
phase transition in the mechanism of 2D crystallization
for membrane proteins is provided in a recent paper by
Dolder eta/. [Z]. Modeling advances continue to be made
in such areas as free energy perturbation methods [3]
and molecular dynamics calculations [4]. A recent review
discusses the state of the art in comparative modeling,
threading and Ab initio protein structure prediction [S].
Directed evolution
For many industrial enzymes, the connection between the
desired use and the relevant properties of the enzyme
is often difficult co make. Additionally, many questions
on the relationship of protein properties to structure are
still unanswered. In this context the directed evolution
approach [6] is attracting increasing interest from industry.
Directed evolution, also called molecular evolution, sexual
PCR, and in vitro evolution, is the technique of preparing
protein variants by recombining gene fragments in vitro,
using PCR [7], expressing the protein and then selecting
or screening for those with improved properties. The
DNA shuffling technique, introduced by Stemmer [8],
sets the directed evolution approach apart from earlier
random mutagenesis and screening efforts. Crameri et

418 Protein engineering
a/. [9] have successfully applied directed evolution to
improve the whole cell fluorescence by green fluorescent
protein, which is widely used as a reporter for gene
expression and regulation.
An evolutionary approach has been used by You and
Arnold [lo] to give a 471-fold increase in the activity
of subtilisin E in 60% aqueous dimethyl formamide.
Their paper was important in demonstrating the possibility
of engineering enzymes to perform in organic solvents,
very different conditions from their natural environments.
Building upon this work, this group applied the directed
evolution approach to develop an esterase capable of
carrying out a particular deprotection step of an antibiotic
synthesis in aqueous-organic solvents [ll”]. At the time
of writing, two quite different ways of implementing
directed evolution have been published. The Arnold
group [lO,l l**] screens through single and then higher
multiples of variants to cover sequence space systemati-
cally. Stemmer’s group [6-91 relies on examining large
numbers of variants with multiple mutations using high
definition screens or selection to find the best one. The
preferred approach may depend on the nature of the
problem and the type of selection or screens that can be
developed. In fact, the development of high-throughput
screening methods along with screens that accurately
reflect the property that is being engineered for final use,
is a critical area for success when using directed evolution.
Not many publications of screening methods are available,
but a paper describing a simple and effective screening
method for proteases has recently been published [12’].
Sources of material to be engineered
In addition to the substantial effort made by enzyme
suppliers and industrial users to improve existing enzymes,
extremeophiles are being aggressively pursued to provide
new enzymes [13] that are highly thermostable, salt
tolerant, cold active, and so on, depending on the
environment of the native organism. Very interesting
enzymes may be isolated from bacteria on a worm that
resides near thermal vents in the ocean, and are expected
to have a much broader temperature profile of activity
than normal mesophilic or thermophilic enzymes [14].
Such enzymes could function effectively across the variety
of temperature conditions encountered in global laundry
applications.
The de nom design of proteins, although very challenging,
offers the broadest possibilities for new structures. In one
of the first practical applications, a de nova four helix
bundle protein [15] was designed to serve as a source
of scarce amino acids (methionine, threonine, lysine and
leucine) when expressed in the rumen bacteria of dairy
cows [16]. A related concept is to create small protein-like
molecules, either by copying natural motifs [17] or via
templates [l&19], which can serve as ‘molecular scaffolds’
for groups introducing functional properties. For example,
a natural scaffold consisting of the 37 amino acid toxin
from scorpions has been used to design a metal-bindi
site that mimics the one found in carbonic anhydrase [Zr
The successful introduction of the metal-binding si
supports the idea that protein engineering in this mann
may yield functional materials in the future.
Tailoring fundamental properties
Stability
Since the conditions in which industrially importa
proteins are used often differ from their natural e
vironment, stability is a necessary consideration 1
most engineered proteins. The introduction of disulfi
bonds [Zl], chemical cross-links [Z?‘], and salt bridg
[23] has been widely used to increase stability, althou,
not all disulfide bonds increase stability. Recent wo
on barnase by X-ray crystallography [24] and hydrog
exchange [‘25] shows that those disulfide bonds that do n
improve stability disrupt the local structure. The definiti
of an effective salt bridge interaction has perhaps be
sharpened by the recent observation by Tanner et al. [Z!
By comparing a number of glyceraldehyde phosphz
dehydrogenase structures from organisms with differe
heat tolerance they found a strong correlation betwe
increased thermostability and the number of hydrog
bonds involving charged sidechains and neutral partne
On the other hand, for the Arc protein [27], a buried s
bridge between arginine and glutamic acid was found
provide less stability than hydrophobic residues. Althou
not necessarily contradictory to Tanner’s observation
since this is presumably a charge+harge salt bridge, it dc
suggest that the contribution of salt bridges to stabil
remains unresolved.
Activity
Although improving the activity of an industrial enzyr
is often a primary goal, it is also one of the mc
complex. This is partly because in many applicatia
of enzymes the substrate is chemically complex a
heterogeneous. It is reassuring in such a context to fi
the principle of transition state stabilization verified
a relatively simple system. In a study of the hydroly
of acetylcholine by acetycholinesterase [ZS], the cataly
acceleration corresponding to 18 kcal mol-1 of transiti
state stabilization could be accounted for in terms
specific molecular interactions, as identified in the crys
structure of a transition state analog inhibitor with TOQC
californica acetylcholinesterase. A quantitative method
analyze the similarity of substrates and inhibitors, w
respect to their enzyme stabilized transition states,
the purpose of designing more effective transition st;
inhibitors has also recently been published [29].
For more complex reactions, such as the hydrolysis
protein amide bonds by a protease, much less is knov
In a revealing study on the proteolysis of variants o
single chain Monellin (a sweet protein consisting of
amino acids), the extent of proteolysis at a fixed til
correlated with the free energy of protein unfoldi

suggesting that in this case substrate unfolding may be rate
limiting [30]. Many industrial enzymes, such cellulases,
amylases, lipases, and even proteases in certain contexts,
work on insoluble substrates. In this context the rate of
substrate turnover may be diffusion limited and controlled
by enzyme mobility at the surface or by on/off enzyme
desorption rates [31]. These in turn are frequently related
to the surface properties of the enzyme and conditions at
the interface between enzyme and substrate [32].
Surface properties
Many industrially important enzymes act at surfaces.
Some, such as cellulases and xyianases, possess binding
domains that aid in adsorption of the enzyme. Lipases
are generally not active until adsorbed at an oil-water
interface (a phenomenon known as interfacial activation).
Some recent studies have attempted to understand the
driving forces for adsorption of some important commercial
enzymes such as Savinase (a detergent protease [33]), and
Lipolase (a detergent lipase [34]). These studies support
the importance of charge interactions in adsorption. Not
surprisingly, a number of protein engineering studies have
focused on changing charges to affect surface properties
[31,35]. A modeling study of cutinase and various charged
variants found a linear relationship between the free
energy of adsorption and the charge on the protein.
Additionally, the electric moment appears to play a
significant role in orienting cutinase in the electric field
near the substrate surface [36”].
An important issue, from both an industrial and academic
view point, is the extent to which the stability, activity
and surface properties are linked. Is it possible to
create an enzyme that gives good rates of proteolysis
at low temperature and is still stable and active at
high temperature? Recent results of Zhang et a/. on T4
lysozyme [37] suggest there may be a trade-off between
activity and stability; however, the bacteria near thermal
vents appear to have found a solution. Engineering a lipase
for improved activity at elevated temperatures might be
expected to involve understanding something about the
interrelation of all three properties mentioned above.
Protein engineering improvements in
industrial enzymes
Protease
Laundry and cleaning products account for over 40% of
the approximately one billion dollar industrial enzyme
market. Proteases have a long history in such products
and they account for the largest single enzyme market.
The mechanism of protease action that leads to better
protein-based stain removal is still not completely under-
stood, although recently a model has been proposed [38].
Protein engineering has been used to improve the stability
of BPN’ from Bacillus amyfoliquefaciens in the chelating
environment of the detergent by deleting the strong
calcium-binding site (residues 75-83) and re-stabilizing
the enzyme through interactions not involving metal-ion
Protein engineering from a bioindustrial point of view Rubingh 419
binding. Stability increases of greater than lOOO-fold in
10mM EDTA have been reported for this protease [39].
The surface properties of BPN’ have also been engi-
neered. It was found that variants containing mutations
that produce negative charges in the active site region of
the molecule adsorbed less strongly [31] and gave better
laundry performance [40].
Amylase
Amylases are primarily used to convert starch to various
sugar syrups, although they are used in textile desizing
and cleaning products as well. Different enzymes are
employed to obtain different end products. Thus a-amy-
lase is generally applied early in the process to yield
maltodextrins. Subsequent processing by glucoamylase
produces glucose, and further processing with B-amylase
yields maltose. Finally, application of glucose isomerase
gives fructose. The starch conversion process is carried out
at high temperature, and a number of protein engineering
studies have reported mutations that improve the thermal
stability of these enzymes [41]. A recent paper reported
that changing alanine to valine at position 209 and
histidine to tyrosine at position 133 increased the half-life
of Bascihs iicheniformis a-amylase ninefold at 9o’C 1421.
Positions discovered through random mutagenesis that
result in increased thermostability of a B-amylase from
barley have also been reported [43]. Furthermore, the
nature of the starch-binding domain in glucoamylase
from Aspetgifhs awamon’ was investigated by preparing
deletion variants. It was concluded that the entire region
was required to hydrolyze native starch, although the
deletions did not affect the activity on soluble starch or
the enzyme’s thermostability [44]. A glucoamylase with
improved thermostability has been prepared by making
glycine to alanine mutations within the a-helical secondary
structures of the molecule. These mutations are thought
to function by reducing helix flexibility [45].
Lipase
Lipase catalyses the hydrolysis (or synthesis) of insol-
uble esters. The primary use of lipase is in cleaning
applications, although its use in the chiral synthesis of
high value chemicals is also important. A comparison of
the experimental results of several site-directed variants
with structural modeling has provided much insight into
the catalytic mechanism of a fungal lipase from RI&opus
oryzae at the molecular level [46’]. In order to understand
lipase activity fully one must also take into account its
ability to interact with a macroscopic substrate, such as
a triglyceride surface. Most lipases are activated at the
oil (substrate)-water interface by a conformational change
in which a ‘lid’ is shifted to expose the hydrophobic
binding pocket of the enzyme [47]. Changes at Glu87
and Trp89 in this lid region have been reported to
alter activity [48*] of the lipase from Humico/a lanugittosa
(Lipolase). Surfactant and calcium sequestering agents,
such as sodium tripolyphosphate, reduce the activity
of current lipases lOO-lOOO-fold in laundry detergents

420 Protein engineering
[32]. Some progress in designing variants that reduce
this inhibition by creating favorable surfactant-enzyme
interactions have been reported to give improved laundry
performance [49].
Callulase and xylanase
Cellulases have become increasingly important in recent
years because of their ability to provide the soft feel
of stone washed jeans in textile processing, and fabric
care benefits (such as color crispness) when used in a
laundry detergent [SO]. Most commercial cellulases are
endoglucanases (promoting internal bond hydrolysis) and
contain a catalytic functional region and a cellulose-
binding domain (CBD) connected by a linker region.
Since cellulases have very poor activity against insoluble
cellulose without the binding domain, significant effort
has gone into understanding the adsorption and activity
relationship [Sl]. It is noteworthy that in this work better
activity was correlated with stronger adsorption, in contrast
to the studies mentioned earlier on proteases [31]. A
recent paper reported the free energies of binding of two
different CBDs and a fusion protein of the two. The
double CBD was found to bind much more tightly than
the separate domains [52]. A study of the role of Tyr169 in
the Trihodmna reesei cellobiohydrolase II catalytic domain
suggests that it plays an important role in distorting the
glucose ring into a more reactive conformation [53’].
Xylanases are used in the pulp and paper industry to
reduce the quantity of chemicals required for bleaching,
and thereby provide an environmentally preferred route
to pulp processing [54]. A good review of the different
families of xylanases and their structure and activity is
presented in a recent article [W] in Current Opinion
in Biotechno/ogy. In an important mechanistic study of
a xylanase from B. circulans, Lawson et a/. [56*] varied
the distance between the two catalytically active carboxyl
groups at the active site. They found the native distance
was optimum, but the fall off in activity was more
rapidly when the distance was increased than when it was
shortened.
Conclusion
It is clear that protein engineering has played a central
role in improving commercially important enzymes and
in finding new applications of proteins quite different
from their natural function. From an industrial perspective,
being able to rapidly identify such an enzyme is also
very important. Recent successes in the directed evolution
approach suggest it may be more efficient than rational
design. The possibility of bypassing the laborious task
of understanding the relationship(s) between protein
function and the envisioned application, as well as
sidestepping the difficult questions of how structure
and function are related, strengthens this perception.
Perhaps experience will teach that devising representative
high-throughput screens of the final application will be just
as difficult as the rational design of functional materials.
Meanwhile, many industries are looking carefully at the
directed evolution approach. Structure determination and
structure-function studies will continue to be essential
because most industrial problems are solved by a combi-
nation of approaches. Additionally, such information is the
source of ideas for new opportunities.
An understanding of the relationship between basic
properties and structure is far from complete. Never-
theless, improving protein thermostability is becoming
relatively routine. This is because earlier investigations
have identified a number of structural features influencing
thermostability, so that mutations with a high probability
of success are easy to identify and to test. Improving
enzyme activity is more difficult, since the structural pa-
rameters affecting activity are not as clearly defined. This
is particularly true where substrates are macromolecular
and insoluble, as they are for many of the important
industrial enzymes.
In the last section I tried to focus on a few of
the most important classes of industrial enzymes and
on important applications of each type. I hope it is
clear that developing improved enzymes is challenging,
requiring an understanding of structure, a knowledge of
structure-function relationships, and an appreciation of the
mechanism of action responsible for good performance in
the ultimate use. More importantly, I hope that this review
illustrates that real progress is being made in improving
commercially important enzymes.
Acknowledgements
I would like fo thank Phil Brode, Carolyn Burns, Rowan Grayling, Phil
Green, Charlie Saunders and Sancai Xie, who offered helpful comments on
the manuscript.
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422 Protein engineering
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