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Evolutionary Methods in Biotechnology. Clever Tricks for Directed Evolution. By Susanne Brakmann and Andreas Schwienhorst

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Evolutionary Methods in
Clever Tricks for
Directed Evolution.
By Susanne Brakmann and Andreas
Wiley-VCH, Weinheim 2004.
214 pp., hardcover
E 129.00.—ISBN
There is an ever-increasing demand for
identifying or evolving new industrial
enzymes capable of catalyzing processes
under a range of different conditions.
Examples include enzymes that are
stable and active over long periods of
time, those that are active in nonaqueous solvents, or those that can accept
and catalyze efficient turnover of various unnatural substrates. The “rational”
re-design by site-directed mutagenesis
requires a knowledge of the proteins
three-dimensional structure, as well as
the ability to predict the effects of
amino acid substitutions that would
yield the desired features. However,
despite numerous published studies
and experimental observations concerning folding and kinetic properties of proteins, there are still no general rules for
successful de novo protein design or
rational change of protein substrate specificity. F. H. Arnold recently stated that
“rational design has failed miserably at
helping us make useful proteins”. For
that reason, methods other than
“rational” protein design became popular by the 1980s. These are based on the
principle that natural evolution produces a large number of variants by mutation and subsequently selects the “fitAngew. Chem. Int. Ed. 2005, 44, 3649 – 3651
test” among them. These Darwinian
evolutionary processes can be mimicked
by using routine molecular biological
methods of mutation/recombination
and screening/selection in the test-tube,
thus allowing for rapid and direct isolation of biomolecules based on their
functional properties. This collection of
methods has been termed “directed evolution”, and provides a powerful tool for
the development of biocatalysts with
novel properties, without requiring an
understanding of their complicated
structure–function relationships or a
knowledge of enzyme structures or catalytic mechanisms.
In this context, the success of the
previously published book Directed
Evolution of Proteins, edited by Kai
Johnsson and Susanne Brakmann
(Wiley-VCH, 2002; ISBN 3-527-304231)—which describes technologies for
laboratory evolution, including methods
for making and characterizing gene
libraries, high-throughput screening,
and hybrid computational-evolutionary
optimization strategies—was not surprising. Therefore, one can without a
doubt expect equal success for this “successor” volume, Evolutionary Methods
in Biotechnology: Clever Tricks for
Directed Evolution, edited by Susanne
Brakmann and Andreas Schwienhorst
(Wiley-VCH, 2004). The reasons for
such expectations are simple: firstly,
the work consists of an extraordinary
collection of all the key methods now
used for directed evolution research;
secondly, it provides step-by-step procedures, details, “tricks, and ”tips“ (including troubleshooting) to minimize frustration and provide shorter ways to
experimental results; and thirdly, the
information that it contains is equally
valuable for newcomers and more experienced investigators.
As the editors explain in their introduction (Chapter 1), the work aims to be
a “cookbook” that focuses on methods
described in sufficient detail to serve as
“recipes” for successful directed-evolution experiments. On the other hand,
since the directed evolution is applied
in a variety of research areas, it is certainly difficult to balance different interests in such a multi-author book of limited size. Consequently, some important
topics are not covered, for example:
a) the use of directed molecular
tion to develop enabling technologies
for plant biologists (for example, to achieve insect tolerance, disease control,
and herbicide tolerance, to improve
crop yield and nutritional qualities);
b) screening technologies to develop
improved vaccines and medicines with
the potential for short-term and longterm therapeutic effects; or c) directed
evolution methods in which protein
diversity libraries are screened for soluble variants, as an alternative route to
obtaining soluble proteins. There is a
large potential audience of readers
who are interested in these practical
applications, and one can expect that
these and similar topics will be covered
in future editions.
The book can be divided into four
different subject areas: the generation
of gene libraries, selection and screening
techniques, computational methods, and
the important issue of practical procedures for patenting in biotechnology.
The contribution by S. Brakmann and
B. F. Lindeman (Chapter 2) provides a
short general overview as well as
simple protocols for generating molecular diversity, either by random mutagenesis or by suitably designed mutator
strains. In Chapters 3 and 4, the DNA
shuffling method that generates diversity by DNA recombination from individual genes is described by H. Suenaga,
M. Goto, and K. Furukava, while the
popular StEP variant (staggered extension processes), with its simple protocols, is described by M. Ninkovic. Chapters 5 and 6 describe some of the latest
developments in selection and screening
strategies. The FACS screening of combinatorial peptide and protein libraries
displayed on a microbial cell surface,
which allows large molecular repertoires
to be generated (usually over 1010), is
described by T. M. Adams, H. U.
Schmoldt, and H. Kolmar in Chapter 5,
then P. Soumillion (Chapter 6) describes
phage-display selection methods based
on the use of suicide substrates. Methods and protocols for screening and
selection of aptamers (that is, targetbinding nucleic acids) are described by
H. Fickert, H. Betat, and U. Hahn in
Chapter 7. The processes and methods
for selecting RNA catalysts with particular functions from large pools of
random sequences are described by
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
B. J. Holley and B. E. Eaton (Chapter 8).
Natural enzymes that are products
of biological evolution usually catalyze
a given reaction with high specificity
and enantioselectivity. While they are
perfectly compatible to perform their
physiological role, their activity and stability are often ill-suited for industrial
purposes. M. T. Reetz describes highthroughput approaches to the design of
enzymes with tailored enantioselectivity
as valuable tools for the production of
(Chapter 9). In Chapter 10, D. Tomandl
and A. Schwienhorst describe a computer program that “reverse translates”
a desired target set of amino acids in
order to enhance the identification of
molecules with desired properties in a
huge background of nonfunctional molecules in a particular library. The
enzymes, proteins, and catalytically
active biomolecules generated by the
techniques of directed evolution are of
great interest for commercialization by
the biotechnological industry. Therefore, legal protection of methods, techniques, and materials is of great importance in this research field. In this context, Chapter 13 (written by M. Leimkhler and H. W. Meyers) provides a
researcher in the field with a basic
understanding of the major patenting
aspects and practical guidelines needed
to protect an invention.
This hard-back book (accompanied
by supplementary material on CDROM for Chapters 10, 11, and 12),
with a convenient subject index, is
truly a highly practical handbook, a
“must have” for any laboratory practicing directed evolution of proteins, and
a methodological “cookbook” of high
Nediljko Budisa
Max-Planck-Institut fr Biochemie
Junior Research Group “Molecular
Martinsried (Germany)
DOI: 10.1002/anie.200485223
Chemistry and Technology of
Flavors and Fragrances
Edited by David J.
Rowe. Blackwell
Publishing, Oxford
2005. 352 pp.,
£ 95.00.—ISBN
Following the award of the 2004 Nobel
Prize in physiology or medicine to R.
Axel and L. B. Buck for their discoveries of odorant receptors and the organization of the olfactory system, this is
perfect timing to publish a book on
Chemistry and Technology of Flavors
and Fragrances. That is all the more
valid since the book is intended to give
a general introduction to the chemistry
of aroma compounds to readers who
are less familiar with this area, for example, graduate students or scientists from
other sectors of industry. The monograph is divided into 13 chapters, covering different topics that are specific to
the flavor and fragrance industry. As it
is the work of different authors, the
quality of the content varies from one
chapter to another. Therefore, the
reader may want to consult individual
parts of the book, rather than reading
it through in one session.
The conception of the book is mainly
based on molecular structures, and some
basic knowledge of organic chemistry is
required to understand the different
aspects. The most general chapter
(Chapter 4) covers aroma chemicals
composed of carbon, hydrogen, and
oxygen, which represents most of the
compounds used in the flavor and fragrance industry. The various classes of
compounds are arranged according to
their functional groups, with brief statements of their origins and preparation,
some olfactory characteristics, their
uses, and some problems that may be
encountered with them. The chapter is
informative and reads fluently, but
unfortunately remains quite superficial.
Additional references for further reading could have completed this overview.
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Besides the classification of compounds according to their chemical
functionalities, flavors and fragrances
are often grouped into families with similar olfactory properties. Good examples
of such a group are the musk compounds, which are didactically well
described in Chapter 7. Structures are
presented in their historical context,
and the evolution of new areas is indicated. The citation of some perfume
brands in which typical musks are commercialized makes a link to the real
world of fine perfumery. The chapter
also discusses some attempts that have
been made concerning the rational
design of odorants based on structure–
activity relationships. Another author
(Chapter 11) describes the design of fragrances based on the comparison of
their vibrational spectra. This approach
is in contradiction to the latest knowledge of chemoreception, the importance
of which has been highlighted by the
Nobel Prize award mentioned above.
The monograph puts a lot of emphasis on flavor chemicals, as shown by a
series of chapters covering, among
others, sulfur compounds and pyrazines.
One of the chapters gives an overview
on the generation of flavors in food
during cooking or fermentation processes (Chapter 3). The large number of
references given makes this chapter a
good starting point for further reading.
Another author discusses the chemistry
of sulfur compounds by evaluating the
main advantages and problems of some
key reactions, illustrated by a number
of well-selected concrete examples
(Chapter 6). Yet another chapter (Chapter 8) describes the most important
processes that are the basis for natural
flavorings, and gives various examples
of their preparation. It also defines
basic terms such as “natural” and “soft
The book also contains a chapter on
the world of taste and sensations (such
as tingling, cooling, bitterness, or astringency) and the interaction of molecules
with receptors giving a biological signal
(Chapter 9). This mainly structurally
based chapter is written from an organic
chemists point of view, and as such it
offers a good first introduction to this
topic, although the concept of the
tongue map is by now outdated.
Angew. Chem. Int. Ed. 2005, 44, 3649 – 3651
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andreas, schwienhorst, tricks, biotechnology, evolutionary, evolution, method, brakmann, directed, susanna, clever
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