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.
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.
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.
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This innovation is the result of the project
Title: Effective Redesign Of Oxidative Enzymes For Green Chemistry
Organisations and people involved in this eco-innovation.
Please click on an entry to view all contact details.
Role in project: Project Coordination
Contact person: Dr. POUTSMA Jan
Contact person: Dr. BOROWCYK Jadwiga
BIOLOG LIFE SCIENCE INSTITUTE, FORSCHUNGSLABOR UND BIOCHEMICA- VERTRIEB GMBH
Contact person: Dr. GENIESER Hans-Gottfried
DECHEMA GESELLSCHAFT FUER CHEMISCHE TECHNIK UND BIOTECHNOLOGIE E.V.
Contact person: Dr. SCHRADER Jens
DSM INNOVATIVE SYNTHESIS BV
Contact person: Mrs. WEIJER VAN DE Ine
DSM RESEARCH B.V.
Contact person: Dr. JANSEN Johan H.m.
Contact person: Dr. DUETZ Wouter
Contact person: Dr. FROSSARD Dominique
JACOBS UNIVERSITY BREMEN GMBH
Contact person: Mr. KIESCHNICK Ronald
RHEINISCH-WESTFAELISCHE TECHNISCHE HOCHSCHULE AACHEN
Contact person: Prof. SCHMACHTENBERG Ernst
TECHNISCHE UNIVERSITAET DORTMUND
Contact person: Mr. NIEHAGE Detlef
TECHNISCHE UNIVERSITAET GRAZ
Contact person: Prof. GLIEDER Anton
TECHNISCHE UNIVERSITAET WIEN
Contact person: Prof. MIHOVILOVIC Marko
UNIVERSITA DEGLI STUDI DI PAVIA
Contact person: Prof. MATTEVI Andrea