The Oxygreen project

Oxygreen - Effective redesign of oxidative enzymes for green chemistry

Introduction

Life is based on the ability of living systems to perform an enormous array of chemical reactions. This ability is made possible by the existence of enzymes, which are proteins that are capable of accelerating (i.e. catalyzing) chemical reactions achieving rates that are simply beyond the limits of “classical” chemical methods. Such a terrific catalytic power is coupled to an exquisite degree of specificity: enzymes are extremely effective in dealing only with certain molecules and in avoiding the generation of unwanted byproducts. These features make enzymes obviously attractive for industrial applications. Among the most widely used and relevant chemical reactions are processes that uses oxygen. We are used to think of oxygen as the molecule that enables respiration. However, one must keep in mind that oxygen is one of the most widely used natural “oxidants” (i.e. molecules that “extract electrons” from another molecule) at the heart of a plethora of most diverse chemical reactions. This ability is made possible by the existence of enzymes, which are proteins that are capable of accelerating (i.e. catalyzing) chemical reactions achieving rates that are simply beyond the limits of “classical” chemical methods. Such a terrific catalytic power is coupled to an exquisite degree of specificity: enzymes are extremely effective in dealing only with certain molecules and in avoiding the generation of unwanted byproducts. These features make enzymes obviously attractive for industrial applications. Among the most widely used and relevant chemical reactions are processes that uses oxygen. We are used to think of oxygen as the molecule that enables respiration. However, one must keep in mind that oxygen is one of the most widely used natural “oxidants” (i.e. molecules that “extract electrons” from another molecule) at the heart of a plethora of most diverse chemical reactions.

Aim

The goal of OXYGREEN is to gather the knowledge on the functions of selected enzymes that use oxygen to perform industrially relevant reactions. This knowledge is intended to be applied for the development of enzyme tools of interest for industrial and biotechnological applications. The project is based on three main cornerstones:

1. Biochemistry to understand enzyme function;
2. genetics and microbiology to modify (engineer in technical jargon) the enzymes of interest as to perform the desired reactivities; and 
3. biotechnology and chemistry to develop the technical tools for the application and usage of the modified enzymes and make them attractive for end-user industrial applications.

Main results

So far, the research efforts in the OXYGREEN project have resulted in: 

• New knowledge on how enzymes work – We have performed an in-depth analysis of the oxidative enzymes under investigation using a variety of tools that are available to modern biochemists. This research has led us to have a detailed description of how the enzyme functions at the atomistic level - we can visualize atom by atom how enzymes are capable to use oxygen to modify the molecules they act upon. Among the results, we have produced a movie intended for educational and teaching purposes that illustrates the progresses and advancements generated by our studies. The movie on how a monooxygenase operates at molecular level is publically available at www.unipv.it/biocry/movies.php.

• New computational tools to assist in enzyme engineering – We have developed new tools to allow researchers to effectively and quickly grasp the information generated by our studies. This is a key issue because, as common in modern biology, the amount of the data is so huge that they have to be stored and organized in a proper way as to make them accessible and useful. More specifically, in our project we have constructed a web-based database (the MuteinDB), and improved a tool to assist in engineering enzymes (Mutagenesis Assistant Program, MAP3D). These tools allow any user to address questions such as: I am interested in a certain oxidative reaction, are there enzymes or modified enzymes useful for this purpose? I am using a certain enzyme, what are the “mutants” (i.e. modifications) that have been already generated and could they be useful for my goals? Which method should I use to prepare an enzyme mutant library? These tools are free to use and can be accessed via the internet at muteindb.genome.tugraz.at and map.jacobs-university.de/map3d.html.

• New methods for screening enzyme libraries – In the logic of the project, the development of tools for modifying in a target-oriented way the OXYGREEN oxidative enzymes is paralleled to the development of chemical tools to effectively evaluate the properties of the enzymes and their modified variants (mutants). Most important, a key aim of this section of the project is to design these tools to make them useful for highly automated systems. This is clearly a requirement for industrial applications that have to employ these enzymes for many different processes and transformation of many different molecules.

Expected final results

Enzyme engineering is a key technology for obtaining cleaner, cheaper and more robust (safer) industrial processes. By the OXYGREEN project, new efficient methods are provided that allow efficient engineering of enzymes. The project itself also generates new oxidative biocatalysts that are suited for novel and 'green' synthesis routes.

Potential impact and use

The results of the OXYGREEN-project will broaden the application area of oxidative biocatalysis. If stable and highly active enzymes, well integrated in the structure and physiology of whole cells, can be developed, oxidative biocatalytic systems can become easily applicable. With these biocatalytic tools the organic chemist and process engineer may be as eager to integrate a selective oxygenase in their process for selective oxidations as they are now with commonly used hydrolytic enzymes.