Innovation: Effective Redesign Of Oxidative Enzymes For Green Chemistry

Last update: 30.06.2013
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Keywords: 
chemical engineering, biochemistry, industrial biotechnology, flow cytometry, life sciences, oxyfunctionalization, biotechnology, industrial manufacture, enzyme redesign, high-throughput screening, oxygenase, biological resources, biocatalysis
Enzymes are extremely powerful natural catalysts able to perform almost any type of chemical reaction while being mild by nature and highly specific. In fact, the delicate functioning of enzymes forms the basis of every living creature. The catalytic potential of enzymes is more and more appreciated by the industry as many industrial processes rely on these sophisticated catalysts. However, the number of reactions catalyzed by enzymes is restricted as enzymes only have evolved to catalyze reactions that are physiologically relevant.

Furthermore, enzymes have adapted to the direct (cellular) environment in which they have to function (e.g. operative at ambient temperature, resilient towards proteolysis, catalytic turnover rate should fit with metabolic enzyme partners). This excludes the existence of enzymes that do not fit within boundaries set by nature. It is a great challenge to go beyond these natural boundaries and develop methodologies to design unnatural tailor-made enzymes. Ideally it should become possible to (re)design enzymes to convert pre-defined substrates. Such designer enzymes could theoretically exhibit unsurpassed catalytic properties and, obviously, will be of significant interest for industrial biotechnology.
So far, the research efforts in the OXYGREEN project have resulted in:

1. new knowledge on how enzymes work ? By the elucidation of new enzyme structures and bioinformatics analyses, new insights have been obtained on how enzymes function at atomic level. The generated knowledge is used as input for enzyme engineering.
2. new computational tools to assist in enzyme engineering. Several new bioinformatics tools have been developed to extend the toolbox that can be used for enzyme engineering. Important contributions are a web based database that contain information on mutant enzymes (MuteinDB) and an improved a tool to assist in preparing enzyme mutant libraries (MAP3D).
3. new methods for screening enzyme libraries ? For identifying optimised enzymes in an enzyme engineering project, efficient technologies that allow rapid and reliable detection are essential. Several new screening protocols have been developed in the OXYGREEN project that can be applied to the oxidative enzymes developed within OXYGREEN.

Main results from the current reporting period:

Work in work package one (WP1) has resulted in the elucidation of several new monooxygenase structures (steroid monooxygenase and phenylacetone monooxygenase and several complexed structures of these two biocatalysts). This has provided new and more detailed insight into the way how these enzymes bind their substrates and how catalysis is performed. Also substrate profiling of new monooxygenases has been performed showing differences among enzymes and providing more data on which residues determine the substrate selectivity of monooxygenases. This is used as input for the design of mutant enzymes. Furthermore, the MuteinDB has been completed and is now in the public domain. The database has integrated the data and tools that had been developed in the previous periods.

WP2: As other component for effective enzyme engineering, work has been done on improving the sequence saturation mutagenesis (SeSaM) methodology; a novel method to prepare mutant enzyme libraries. In this period emphasis was put at optimising, integrating and evaluating the newest SeSaM approach. By incorporating the use of a more suitable deoxyribonucleic acid (DNA) polymerase and the use of a new synthetic nucleotide (ribavirin-base), the SeSaM method has improved with respect to the quality of the generated library. A preliminary attempt was made to benchmark the SeSaM performance with known mutagenesis methods but firm conclusions await further study. Except for advancing SeSaM, also other mutagenesis approaches have been used to generate mutant libraries. For Baeyer-Villiger monooxygenases (BVMO) engineering, the newly developed OmniChange method was found to be effective to make a focused library in which multiple targeted residues could be mutated simultaneously. The first
screening of this library shows that such multi-side targeted libraries are relatively rich in mutant enzymes with different selectivities. A two-site targeted mutant library of a P450 monooxygenase revealed more active enzyme variants, confirming that with structure-inspired mutagenesis, high quality mutant libraries can be made.

WP3 and WP4: After preparative research in the first years of the project, the current reporting period has applied the developed methods for screening enzymes with improved properties. For P450 monooxygenases, screening for electrochemically assisted activity has successfully applied and a method for determining the exact identity of steroid hydroxylation products has been established. For BVMOs a newly developed screening methods was used to discover new enzyme activities, and for finding improved ketoglutarate dependent dioxygenases (KGDO) mutants the developed chromogenic assay has been applied with success. By this, the developed screening methods have been shown to be of help in the process of enzyme engineering: identifying interesting enzymes. Also the newly developed whole cell based screening methods (WP4) have been employed in the last period, resulting in the identification of improved P450s, BVMOs and KGDOs. Studies have been performed on stability and storage of
monooxygenases, strain development and biocatalytic performance (WP5). This provides the first leads for applying some of the generated enzymes and the first enzymes have been transferred to the involved industry for application tests.
The ultimate goal of the OXYGREEN project is twofold:

1. to advance current understanding of the functions of the enzymes that use oxygen to perform chemical reactions, and
2. to translate this knowledge into tools and protocols that can eventually lead to improved technologies for obtaining cleaner, cheaper and more robust (safer) industrial processes. The involvement in our project of a few well known chemical industries highlights our commitment to combine basic research with the development of improved technologies.

The generated enzyme derived tools will enable the organic chemist and process engineer to integrate a selected oxidative enzyme(s) in their process to make it more cost effective and environmentally friendly. In essence, it is the transfer of the capabilities that living organisms have developed in the course of evolution into processes used by chemical and biotechnology industries.

List of websites:

http://www.oxygreen.org

Collaboration sought: N/A

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This innovation is the result of the project

Title: Effective Redesign Of Oxidative Enzymes For Green Chemistry

Acronym: 
OXYGREEN

Runtime: 
01.05.2008 to 30.04.2013

Status: 
completed project

Organisations and people involved in this eco-innovation.

Please click on an entry to view all contact details.

RIJKSUNIVERSITEIT GRONINGEN

(Netherlands)

Role in project: Project Coordination

Contact person: Dr. POUTSMA Jan

Website: http://www.rug.nl

Phone: +31-503634142

Contact

BIOINFOBANK INSTITUTE

(Poland)

Contact person: Dr. BOROWCYK Jadwiga

Website: http://www.bioinfo.pl

Phone: +48-618653520

Contact

BIOLOG LIFE SCIENCE INSTITUTE, FORSCHUNGSLABOR UND BIOCHEMICA- VERTRIEB GMBH

(Germany)

Contact person: Dr. GENIESER Hans-Gottfried

Website: http://www.biolog.de

Phone: +49-421591355

Contact

DECHEMA GESELLSCHAFT FUER CHEMISCHE TECHNIK UND BIOTECHNOLOGIE E.V.

(Germany)

Contact person: Dr. SCHRADER Jens

Website: http://www.dechema.de

Phone: +49-9697564422

Contact

DSM INNOVATIVE SYNTHESIS BV

(Netherlands)

Contact person: Mrs. WEIJER VAN DE Ine

Phone: +31-630798316

Contact

DSM RESEARCH B.V.

(Netherlands)

Contact person: Dr. JANSEN Johan H.m.

Website: http://www.dsm.com

Phone: +31-464763286

Contact

ENZYSCREEN

(Netherlands)

Contact person: Dr. DUETZ Wouter

Phone: +31-715276348

Contact

EVONIK REXIM

(France)

Contact person: Dr. FROSSARD Dominique

Phone: +33-323814470

Contact

JACOBS UNIVERSITY BREMEN GMBH

(Germany)

Contact person: Mr. KIESCHNICK Ronald

Website: http://www.jacobs-university.de/

Phone: +49-4212004515

Contact

RHEINISCH-WESTFAELISCHE TECHNISCHE HOCHSCHULE AACHEN

(Germany)

Contact person: Prof. SCHMACHTENBERG Ernst

Website: http://www.rwth-aachen.de

Phone: +49-2418090490

Contact

TECHNISCHE UNIVERSITAET DORTMUND

(Germany)

Contact person: Mr. NIEHAGE Detlef

Phone: +49-2317552448

Contact

TECHNISCHE UNIVERSITAET GRAZ

(Austria)

Contact person: Prof. GLIEDER Anton

Website: http://www.tugraz.at

Phone: +43-3168734077

Contact

TECHNISCHE UNIVERSITAET WIEN

(Austria)

Contact person: Prof. MIHOVILOVIC Marko

Website: http://www.tuwien.ac.at

Phone: +43-15880115420

Contact

UNIVERSITA DEGLI STUDI DI PAVIA

(Italy)

Contact person: Prof. MATTEVI Andrea

Website: http://www.unipv.it

Phone: +39-0382985534

Contact