Natural reactions
Two years ago, you started a project
examining the use of synthetic mimics
in enzyme-catalysed reactions. Can you
discuss these investigations in relation to
the nicotinamide cofactor?
Many interesting biocatalysts require the
nicotinamide cofactor; however, it is inherently
expensive to use, which motivated our group
to find a cheaper, functional alternative.
Therefore, we resumed work on the synthetic
nicotinamides that had been reported in
the 1930s and had been largely put aside
in biocatalysis.
What attracted you to biocatalysis?
How has your academic background as
organic chemists prepare you for your
current research?
As organic chemists, we have always been
fascinated by enzymes because of their inherent
high selectivity. Enzymes enable organic
chemists to perform challenging reactions that
are otherwise chemically difficult; for example,
selective oxyfunctionalisation reactions on
non-activated hydrocarbons, chemoselective
reduction and oxidations and, of course, highly
enantioselective transformations. Compared
to R&D in an industrial setting, we enjoy the
freedom to pursue ‘funky ideas’.
In our mind, biocatalysis is an integral part of
organic chemistry. Today, there are already
numerous examples of biocatalytic processes
that have replaced chemocatalysed reactions
and have led to purer products, requiring fewer
production steps while achieving a higher
product quality. We expect that this trend will
continue and that more industrial processes
will be based on biocatalytic reactions.
Eventually, chemocatalysis and biocatalysis will
go hand in hand.
Can you outline the main aims of the
Biocatalysis group at the Delft University of
Technology in The Netherlands?
‘Green chemistry’ is one of our leitmotifs. Our
research on the fundamental understanding
of enzymes and their application in organic
chemistry forms the basis for the use of
enzymes in industrial processes. Our research
projects range from fundamental insight
into enzymatic principles to engineering
enzymes and integrating them into novel
synthetic procedures.
For those who may not know, could you
explain ‘white biotechnology’ and reflect on
the potential of this technology in reducing
resource consumption?
Our definition of white biotechnology is simply
the use of biocatalysts to produce chemical
products. We believe that white biotechnology
has enormous potential to make the chemical
industry more economically and ecologically
sustainable. However, it is not inherently
greener than conventional chemical reactions
and the environmental benefit has to be
evaluated on a case-by-case basis.
DRS FRANK HOLLMANN & CAROLINE PAUL
Organic chemists Drs Frank Hollmann and Caroline Paul discuss the renewal of academic and
industrial interest in biocatalysis. They describe their investigations into novel enzymatic reactions
and elaborate on white biotechnology’s potential to change the chemical industry
After some initial disappointment while
reproducing published protocols, we found
success in using these mimics with other
enzyme classes, particularly the old yellow
enzyme family, which is a group of flavindependent redox biocatalysts that offer
many industrial applications. In this area, we
actually found that the mimics were not only
cheaper substitutes, but they also performed
just as well if not better than the natural
cofactor. Currently, we are working on the
next generation of mimics that would clearly
outperform the costly and unstable natural
cofactors in terms of price, stability and activity.
In addition to old yellow enzymes, we have
successfully applied the mimics to a range
of other enzyme classes such as peroxidases,
monooxygenases and dehydrogenases.
What value does collaboration add to your
research? Are there any key collaborators
that you would specifically like to mention?
Collaboration is incredibly important to our
research. We would not be able to do our work
without our connections in academia and
industry. Currently, we actively collaborate
with universities and centres all across Europe,
from the UK to Italy. In particular, we are very
excited about the commercialisation of some of
our mimics by the company Enzymicals AG in
Greifswald, Germany.
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DRS FRANK HOLLMANN & CAROLINE PAUL
New avenues in biocatalysis
Among the resurgence in biocatalytic studies, research into non-conventional concepts for biocatalysis
at Delft University of Technology is increasing the scope for employing enzyme-induced catalysis
CHEMICAL CATALYSIS IS at the heart of
entire industries, setting off reactions that
lead to usable, sellable products. However, the
traditional production methods that chemical
and pharmaceutical industries employ present
many problems; for example, they consume
enormous amounts of resources and generate
waste on a vast scale. In recent decades, ‘white
biotechnology’ has become the great hope
in transforming the methods industry uses
into cleaner, greener techniques through the
introduction of biocatalysis. Unlike classical
chemical catalysis, natural catalysts like
enzymes are capable of carrying out reactions
on organic compounds that limit the occurrence
of unwanted side-reactions – the cause of so
much waste. Currently enjoying a great surge
of interest from industrial ventures hoping
to achieve sustainability, biocatalysis has
consequently become a highly dynamic and
rapidly evolving field, permeating into almost
every facet of organic chemistry. One field in
particular where biocatalysis appears to be
having a large impact is in redox chemistry.
SCRUPULOUS SELECTION
Drs Frank Hollmann and Caroline Paul are
currently working on a novel approach for
enzyme-mediated oxidation and reduction
reactions, a field that has expanded significantly
over the last two decades. As Assistant Professor
within the Biocatalysis group at Delft University
of Technology, Hollmann is working with Paul
– a postdoctoral Fellow at the University’s
Department of Biotechnology – to probe the
wide potential of enzymes in many industrial
processes and unearth the exciting possibilities
that introducing enzymes into synthetic
procedures may afford.
26INTERNATIONAL INNOVATION
A major problem with chemical catalysts is
that they tend to discriminate less, causing
undesirable side-reactions. This means that
industry spends a great deal of time and energy
cleaning up product impurities. Selectivity is not
as big of an issue for enzymes; in fact, as Hollmann
states: “Selectivity is the main reason to use
biocatalysis in organic chemistry”. Compared
to chemical catalysts, enzymes are inherently
more chemoselective and regioselective under
relatively mild reaction conditions. Their greater
chemoselectivity means they can affect a single
functional group and leave the other potentially
reactive groups unchanged, while enzymes’
regioselectivity makes them capable of reacting
to differences between the functional groups
situated in the various regions of the molecule
on which it is acting.
PHARMACEUTICAL INDUSTRY INTEREST
The most interesting characteristic that
enzymes present by far, especially for the
pharmaceutical industry, is their degree of
enantioselectivity. Enantiomers act as a pair of
isomeric molecules known as stereoisomers;
the enantiomers are reflections of each other,
but one cannot be superimposed over the other.
These special molecules are of great interest to
organic chemists because often the biological
activity of one enantiomer will be different to
that of its counterpart; therefore, while one
enantiomer may produce the effects desired
to create a drug, the other may have no effect
at all, or even have adverse effects. Because of
this, from a pharmaceutical point of view, it is
hugely desirable to create products composed
of a single enantiomer – enantiopures. The
natural selectivity of enzymes means they can
preferentially produce one enantiomer to a
greater degree than their chemical cousins. In
addition to enabling access to products of higher
quality, biocatalysis also bears the promise of
significantly reducing the traditionally very high
waste streams of the pharmaceutical industry,
while replacing toxic reagents and solvents with
environmentally less demanding ones.
EVEN BETTER THAN THE REAL THING
Across history, there have been many examples
of the benefits of enzymatic catalysis. In fact,
Hollmann and Paul have recently revisited the
work on synthetic nicotinamides started by
Paul Karrer in 1937. Oxidoreductases – enzymes
that catalyse the transfer of electrons from
on molecule to another – need the cofactor
β-nicotinamide adenine dinucleotide to provide
or accept electrons; however, there are still major
challenges concerning cofactor regeneration,
and thus they are expensive to use. Initially,
scientists synthesised nicotinamide cofactor
mimics (mNADHs) to simulate oxidoreductasecatalysed reactions so they could investigate
the mechanisms of the reaction. Then in the
1970s, mNADHs were intentionally used in
enzyme-catalysed reactions for the first time,
albeit at very limited success – mostly because
the ‘wrong enzymes’ had been studied.
Picking up from the last flourish of work into
mNADHs in the 1990s, Hollmann and Paul have
demonstrated that these mimics, when applied
to the ‘right enzymes’, are not only crucial as
models for studying enzymatic reactions –
they are also cheaper, simpler and more viable
alternatives to well established regeneration
systems. The first family of enzymes the
researchers studied were old yellow enzymes.
Recently, they have been enjoying a dramatic
INTELLIGENCE
MIMICKING NATURE
OBJECTIVES
increase in interest as catalysts for the
enantioselective reduction of various conjugated
carbon-carbon double bonds, providing access
to a broad range of valuable fine chemicals
used in the agrochemical, cosmetic and
pharmaceutical industries. “We were astonished
to see that mNADHs not only are cheaper than
the natural cofactors but also enable faster and
more selective reactions,” Hollmann enthuses.
Overall, inexpensive synthetic mimics are
looking highly desirable for a broad range of uses
from metal-free redox reactions to therapeutics
and biomedical applications.
Subsequently, Hollmann and Paul have begun
applying mimics to other enzymes in an
effort to exploit their synthetic potential for
biocatalysis, in particular the in situ generation
of hydrogen peroxide (H2O2) to promote
peroxidase reactions. Using two cytochrome
P450 peroxygenases from Bacillus subtilis and
Clostridium acetobutylicum, the pair found an
interesting pattern of behaviour in the P450
enzymes’ reactivity that bypasses the need for
both a nicotinamide cofactor and regeneration
system, since the enzymes use H2O2 as an
oxidant to form the catalytically active ferric
hydroperoxy species from the resting state of the
enzyme. Through this, Hollmann and Paul have
provided a proof-of-concept that it is possible
to apply synthetic mimics to peroxygenases to
form the H2O2 necessary for the reaction in situ.
A BRIGHT FUTURE
Currently, Hollman and Paul’s lab are developing
more exciting applications of mNADHs and
extending them to further enzyme classes. “We
have only scratched the surface of what may be
possible with our concept,” Paul states excitedly.
“Currently, we are demonstrating the practical
feasibility of the mNADHs up to kilogram-scale
synthesis. In this respect, we are collaborating
with Enzymicals AG, an emerging company in
Greifswald, Germany, to commercialise the
mimics. Additionally, we are very excited about
• To replace nature’s original cofactors with
biomimetic compounds, while improving the
efficiency and reducing the cost of the use of
oxidoreductases in biocatalysis
• To open the door to bio-orthogonal reactions
and applications in the biomedical field
KEY COLLABORATORS
Professor Nigel Scrutton, Centre of Excellence
for Biocatalysis, UK • Dr Dirk J Opperman,
University of the Free State, South Africa •
Professor Dr Willem van Berkel, Wageningen
University, The Netherlands • Professor Dr
Thomas R Ward, Basel University, Switzerland
• Professor Dr Bernhard Hauer, University of
Stuttgart, Germany • Professor Dr Vlada B
Urlacher, University of Dusseldorf, Germany •
Dr Dirk Tischler, Freiberg University of Mining
and Technology, Germany
PARTNERS
The cofactor mimic mNAD (top) and natural cofactor
NAD (bottom). The synthetic mimic compared to the
natural cofactor retains the nicotinamide moiety that is
crucial for hydride transfer.
Enzymicals AG
FUNDING
EU Seventh Framework Programme (FP7) – grant
no. 327647 • German Federal Environmental
Foundation (DBU)
the possible applications of the mimics for
bio-orthogonal reaction sequences and in the
biomedical field.”
CONTACT
Despite the potential of white biotechnology
to revolutionise the wasteful production
techniques traditionally used in the chemical
and pharmaceutical industries, the ‘greenness’
of biocatalytic reactions cannot be taken for
granted. In some cases, the use of enzymes is
no more benign than the use of chemicals. As
a renewable feedstock, however, their adoption
instantly provides a more sustainable path
to take. Even more importantly, sometimes
enzymes are simply more efficient catalysts,
yielding safer products and allowing for highly
simplified synthesis routes. If biocatalysis
continues in this stride, it may not be too long
before white becomes green biotechnology.
Delft University of Technology
Department of Biotechnology
Julianalaan 136
2628 BL Delft
The Netherlands
Designed to be better than nature
Biocatalysis is an increasingly recognised technology for chemical
synthesis. Though ‘simple’ hydrolytic enzymes dominate the field,
oxidoreductases are catching up in popularity. These enzymes are
somewhat more complicated, as they require stoichiometric amounts of
costly and instable nicotinamide cofactors. To date, breaking this ‘cofactor
habit’ has not been pursued widely. However, synthetic nicotinamide
mimics (mNADHs) are not only significantly cheaper but can also be
designed to be more stable and reactive than their natural counterparts.
Dr Frank Hollmann
Assistant Professor
T +31 642 683 053
E [email protected]
FRANK HOLLMANN studied chemistry at the
University of Bonn, Germany. After his PhD
at the Swiss Federal Institute of Technology
(ETH Zurich) and completing a postdoctoral
research position at Max Planck Institute for
Coal Research, Germany, he worked as group
leader at Evonik Industries in Essen, Germany.
Since 2008, he has been Assistant Professor
at the Delft University of Technology. His
research interests are centered on the use of
oxidoreductases in organic synthesis.
CAROLINE PAUL studied biological chemistry
at the University of Toronto, Canada. Her PhD in
Bioorganic Chemistry at University of Oviedo,
Spain, led her to a postdoctoral research position
with Frank Hollmann at Delft University of
Technology as a Marie Curie Fellow. Her current
research interests revolve around the design,
synthesis and understanding of biomimetic
compounds for oxidoreductases.
mNADHs may open up a new development wave in biocatalysis, adding
cofactor engineering to the well-established protein engineering and
reactor-reaction engineering. Like these, cofactor engineering may have
dramatic effects on the efficiency and practicability of biocatalysis
using oxidoreductases.
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