Enzymes. Chemicals. Solutions. N EW P ROT Kick-off meeting: Partner profile Enzymicals AG Dr. Rainer Wardenga

Start-up history Spin off from the Institute of Biochemistry at the University of Greifswald Founded in August 2009 as corporation Management Dr. Ulf Menyes (CEO) Dr. Rainer Wardenga (CSO) Supervisory Board Prof. Uwe Bornscheuer (Chairman) Dr. Dagmar Braun Dr. Jürgen Eck Ownership structure Shareholders and strategic investor 1 Northern Germany, Biotechnikum Greifswald Company Info

1 Company Info Northern Germany, Sinetific Campus Greifswald Start-up history Spin off from the Institute of Biochemistry at the University of Greifswald Founded in August 2009 as corporation Management Dr. Ulf Menyes (CEO) Dr. Rainer Wardenga (CSO) Supervisory Board Prof. Uwe Bornscheuer (Chairman) Dr. Dagmar Braun Dr. Jürgen Eck Ownership structure Shareholders and strategic investor

Management and staff Dr. U. Menyes (Chemist), CEO Dr. R. Wardenga (Biologist and Biotechnologist), CSO Dipl. S. Weber (Economist), finance and controlling Biotechnologists (main focus molecular biology) Biotechnologist (main focus biocatalysis) Chemical technical assistant Biological technical assistant 2 Company Info Scientific staff Company management

Business Segments 3 Company Info

Institute of Biochemistry at the University of Greifswald (Prof. Bornscheuer) B.R.A.I.N. AG HERBRAND PharmaChemicals GmbH Institute of Chemistry at the University of Rostock (Prof. Kragl) Institute of Marine Biotechnology e.V. Greifswald 4 Partners

Research & Development Along the Value Chain 5 Core competence in protein science and development of biocatalytic process

6 Research & Development Along the Value Chain Identification of suitable catalysts Method development and routine application of HPLC, CE, GC and GC-MS Design and application of spectrophotometric assays Synthesis of screening substrates Screening of enzymes or strain collections Search in literature or pathway databases Search of known or novel motives in sequence databases Amplification of novel genes from genomic DNA De-novo genome sequencing and analysis

7 Cloning, expression and purification Fully equipped molecular laboratories State of the art expertise in molecular cloning Huge repertoire of expression vectors Well-founded knowledge of a range of recombinant expression systems Approved solutions for proteins which are difficult to express Screening of refolding conditions for aggregated proteins Production capacity up to 30 L-scale Catalysts are available in different grades of purity Purification via affinity-chromatography or fusion-tags Research & Development Along the Value Chain

8 Cloning, expression and purification in collaboration with B.R.A.I.N AG Peptide and Protein Production in  Escherichia coli / Bacillus sp.  Pseudomonas stutzeri / Pichia pastoris  Streptomyces sp.  Aspergillus sp. Production Systems: Optimized Production of Peptides or Proteins in Industrial Scale Research & Development Along the Value Chain

9 Biochemical, physiochemical and structural characterization Determination of catalytic properties and kinetic parameters Characteristics in temperature- and pH-profiles Investigation of storage and process stability Properties via gel-electrophoresis and blotting Secondary structure via CD-spectrophotometry Screening for crystallization conditions via commercial kits Access to MALDI-TOF, 2D-gel-electrophoresis General immobilization studies Research & Development Along the Value Chain

10 Protein engineering Selection of best engineering approaches for designated targets Approaches often inspired by structural alignments or molecular modeling Homology-model construction and hot-spot identification Rational design of variants by site-directed mutagenesis Directed evolution with high quality variant libraries Selection assays and library screening with high-throughput systems HT-screening of mutant-libraries Access to 3-laser flow cytometer with cell sorter Research & Development Along the Value Chain

11 Application of best catalysts paired with optimized chemistry Innovative synthetic routes using organic synthesis and biotransformation Custom manufacturing of fine and special chemicals up to the multi-kg scale Catalysis in multi-phase systems or ionic liquids Membrane processes for catalyst recovery and downstream processing Research & Development Along the Value Chain

12 Application of best catalysts paired with optimized chemistry Innovative synthetic routes using organic synthesis and biotransformation Custom manufacturing of fine and special chemicals up to the multi-kg scale Catalysis in multi-phase systems or ionic liquids Membrane processes for catalyst recovery and downstream processing Scope of our biocatalytic tollbox Research & Development Along the Value Chain

13 N EW P ROT : Objectives (R)-selective amine-transaminases Catalyze the transfer an amino group from a primary amine to a prochiral ketone or aldehyde Usable for the resolution of racemic amines or the synthesis of chiral amines Pyridoxal-5‘-phosphate dependent Typical amines : alanine, ethylamine, 1- and 2-propylamine, 1- and 2-butylamine ….

14 (R)-selective amine-transaminases Catalytic tool box for the evaluation of synthesis routes Conversion of aliphatic, arylaliphatic and aromatic ketones with strict (R)-selectivity pH-optima range between 7.0 and 8.5 Alanine serves as amine donor Products obtained by asymmetric synthesis with ECS-ATA N EW P ROT : Objectives

15 Baeyer-Villiger monooxygenases Flavoenzymes from the class of oxidoreductases Oxidation of linear, cyclic and aromatic ketones to esters or lactones Usable with cofactor regeneration or as whole cell system Known applications: Biooxidation of functionalized cycloketones Biooxidation of bicyclic ketones or chiral  -substituted cycloketones Kinetic resolution of racemic  -substituted cycloketones Regiodivergent biooxidation of fused bicyclobutanones N EW P ROT : Objectives

16 Baeyer-Villiger monooxygenases Catalytic tool box for the evaluation of synthesis routes Baeyer-Villiger oxidation of aromatic and aliphatic ketones, able to perform sulfur oxidations High regio- and enantioselectivity, NAD(P)H-dependent Scope of the ECS biooxidation-toolbox. Examples of products obtained from the corresponding ketones with ECS-M N EW P ROT : Objectives

Thank you for your attention Enzymicals AG, Walther-Rathenau-Straße 49a Greifswald Fon: +49(0) Fax: +49(0)