Protein engineering from a bioindustrial point of view

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.
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
1. Frey W, Schief WR Jr, Pack DW, Chao-Tsen C, Chilkoti A, Stayton
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metal-ion coodination by naturally occurring surface histidines.
Proc Nat/ Aced Sci USA 1996, 9X4937-4941.
The use of histidines to nucleate two-dimensional (2D) crystals extends the
range of soluble proteins which may be crystallized in this manner. In addition
to providing materials for structure determination by electron diffraction, 2D
crystals can serve as seeds for exitaxial growth of three-dimensional crystals.
Such crystals could also form the basic structures of biosensors.
Dolder M, Engel A, Sulauf M: The micelle to vesicle transition of
lipids and detergents in the presence of a membrane protein:
towards a rationale for 2D crystallization. FEBS Leti 1996,
362:203-206.
Sun Y-C, Veenstra DL, Kollman PA: Free energy calculations
of the mutation of Ile + Ala in barnase: contributions to the
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Elamrani S, Berry MB, Phillips GN Jr, McCammon JA: Study of
global motions in proteins by weighted masses molecular
dynamics: adenylate kinase as a test case. Proteins 1996,
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Moult J: The current state of the art in protein structure
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