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.

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.

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 flavin-dependent 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.

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

Natural reactionsD

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New avenues in biocatalysisAmong 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

DRS FRANK HOLLMANN & CAROLINE PAUL

26 INTERNATIONAL INNOVATION

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.

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 oxidoreductase-catalysed 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

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.

MIMICKING NATUREOBJECTIVES

• To replace nature’s original cofactors with biomimetic compounds, while improving the effi ciency and reducing the cost of the use of oxidoreductases in biocatalysis

• To open the door to bio-orthogonal reactions and applications in the biomedical fi eld

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

Enzymicals AG

FUNDING

EU Seventh Framework Programme (FP7) – grant no. 327647 • German Federal Environmental Foundation (DBU)

CONTACT

Dr Frank HollmannAssistant Professor

Delft University of TechnologyDepartment of BiotechnologyJulianalaan 1362628 BL DelftThe Netherlands

T +31 642 683 053E f.hollmann@tudelft.nl

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.

INTELLIGENCE

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increase in interest as catalysts for the enantioselective reduction of various conjugated carbon-carbon double bonds, providing access to a broad range of valuable fi ne 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

the possible applications of the mimics for bio-orthogonal reaction sequences and in the biomedical fi eld.”

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 effi cient catalysts, yielding safer products and allowing for highly simplifi ed synthesis routes. If biocatalysis continues in this stride, it may not be too long before white becomes green biotechnology.

Designed to be better than nature

Biocatalysis is an increasingly recognised technology for chemical synthesis. Though ‘simple’ hydrolytic enzymes dominate the fi eld, 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 signifi cantly cheaper but can also be designed to be more stable and reactive than their natural counterparts.

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 effi ciency and practicability of biocatalysis using oxidoreductases.

before white becomes green biotechnology.

Biocatalysis is an increasingly recognised technology for chemical synthesis. Though ‘simple’ hydrolytic enzymes dominate the fi eld, 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

synthesis and understanding of biomimetic compounds for oxidoreductases.