Enzyme Engineering

It was recently discovered that these tiny efficient machines - enzymes - can be modified to become robust enzymes that can revolutionize industrial processes. The ROBOX project specifically focuses on oxidative enzymes that can replace the chemical processes that the industry uses now. This is a complex challenge, but one that the ROBOX project has taken up - determined to make it a success.

Enzymes: What are they? What do they do?

Enzymes are biological catalysts: every form of life depends on many hundreds or thousands of different enzymes. They are molecular machines that facilitate or enable chemical reactions to occur - this is called catalysis. They can do this in astonishing specific and effective manner. Specific: they will only catalyze one type of reaction, converting one specific compound into something else, while not touching other compounds. Effective: they can easily speed up reactions by a million fold, converting billions of a specific compound molecules while just one enzyme molecule is needed. This means that they make things happen. 

What can we do with enzymes?

Enzymes are used in many, many products: washing powder, toothpaste, bread preparation, brewing beer. They are needed and used for: making medicines, sensors, plastics, biofuels, vitamins, improving food, producing antibiotics, developing healthcare products, and many more. Yet, the use of enzymes is limited, for a large part because enzymes from nature are not always suited for use in the final application. They have evolved to be active in their natural environment, and therefore often do not tolerate other conditions (e.g. high temperature, or acid conditions). As a consequence, enzymes often have to be tuned for their envisaged application. This can be done by enzyme engineering. 

What is enzyme engineering?

In the last few decades, techniques have been developed to study enzymes at molecular level. Recently, various methods have been developed that allow to engineer and redesign enzymes. We now know, for a fair number of enzymes, how they look, how they are able to perform chemical reactions and how they can be produced. With this it is possible to exchange each individual amino acid in an enzyme. This means that we can alter enzymes at atomic level at will. This is called enzyme engineering. 

ROBOX's results

ROBOX established a collection of stable enzymes, along with industrial conversion protocols. The project succeeded in: developing an enzyme capable of oxidizing glycerol, applying P450 enzymes to produce drug metabolites on a large scale, applying ADH and BVMO enzymes to produce novel fragrance molecules, as well as precursors for specialty and performance polymers.

In short: ROBOX has demonstrated the techno-economic viability of enzymatic bio-oxidation processes as a greener alternative to traditional chemical processes.

Work Packages

The ROBOX project has an integrated work-flow structure of 7 work packages. Find out more about the work packages below:  

Work Packages

Work Package 1

Enzyme engineering and identification of oxidative enzymes

Work Package 2

Enzyme Production

Work Package 3

Process design and validation

Work Package 4

Demonstration of Robust Biocatalytic Oxidations and Biooxidation Catalysts

Work Package 5

Process benchmarking & evaluation

Work Package 6

Project Exploitation, Intellectual Property Management, Public Engagement

Work Package 7

Project Management


Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

Aalbers, Friso S., and Marco W. Fraaije. 
ChemBioChem 20, no. 1 (2019): 20-28.

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Side-Chain Pruning Has Limited Impact on Substrate Preference in a Promiscuous Enzyme

Fürst, Maximilian JLJ, Elvira Romero, J. Rúben Gómez Castellanos, Marco W. Fraaije, and Andrea Mattevi. 
ACS catalysis 8, no. 12 (2018): 11648-11656.
DOI: 10.1021/acscatal.8b03793

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Overriding Traditional Electronic Effects in Biocatalytic Baeyer–Villiger Reactions by Directed Evolution

Li, Guangyue, Marc Garcia-Borràs, Maximilian JLJ Fürst, Adriana Ilie, Marco W. Fraaije, K. N. Houk, and Manfred T. Reetz. 
Journal of the American Chemical Society 140, no. 33 (2018): 10464-10472.

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Co‐immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self‐sufficient oxidative biocatalyst

Solé, Jordi, Gloria Caminal, Martin Schürmann, Gregorio Álvaro, and Marina Guillén. 
Journal of Chemical Technology & Biotechnology 94, no. 1 (2019): 244-255.

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The crystal structure of P450-TT heme-domain provides the first structural insights into the versatile class VII P450s

Tavanti, Michele, Joanne L. Porter, Colin W. Levy, J. Rubén Gómez Castellanos, Sabine L. Flitsch, and Nicholas J. Turner.
Biochemical and biophysical research communications 501, no. 4 (2018): 846-850.

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A whopping 19 international partners (universities, companies and institutes) were involved in this groundbreaking research project. This just goes to show how huge this project’s scope was, with so many parties that benefited from the research. 


Marco Fraaije
Margit Winkler
Team Member
Rolf Breinbauer
Team Member
Geert Deroover
Team Member
Andreas Vogel
Team Member
Sebastian Bartsch
Team Member
Sven Panke
Team Member
Martin Held
Team Member
John M Woodley
Team Member
Christian Leggewie
Team member
Robert Floor
Team Member
Simon Ellwood
Team Member
Martin Schürmann
Team Member
Arjan van Kampen
Team Member
Ulrich Schwaneberg
Team Member
Anton Glieder
Team Member
Gregorio Álvaro
Team Member
Andrea Mattevi
Team Member
Stefaan De Wildeman
Team Member
John Whittall
Team Member
Nick Turner
Team Member


Project Coordinator

This project is funded by
Grant no. 635734

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