Joint Laboratory of Biotechnology and Enzymatic Catalysis

The Joint Laboratory of Biotechnology and Enzyme Catalysis combines the research potentials of Institute of Catalysis and Surface Chemistry and Institute of Plant Physiology, Polish Academy of Sciences. The research are focused on enzym systems that catalyse stereospecific synthesis of alkylaromatic alcohols. Currently the main research effort is concentrated on three enzymes, namely: Ethylbenzene Dehydrogenase (EBDH), Phenylethanol Dehydrogenase (PEDH) and Steroid C25 Dehydrogenase (S25DH). The reaserch conducted in JLBEC are a part of a greater POIG project "Biotransformations for pharmaceutical and cosmetic industry".

The Laboratory provides also additional platform for integration of scientific society, joining together equipment potential of ICSC, PAS and IPP, PAS.

EBDH

Ethylbenzene dehydrogenase (EBDH) is a  key enzyme in the mineralization of ethylbenzene by Aromatoleum aromaticum, a denitrifying bacterium related to the genera Azoarcus and Thauera. It is a molybdoenzyme belonging to the DMSO reductase family. Molybdenum enzymes are distinguished by the presence of a unique active site containing molybdenum atom, one or two pteridine cofactors and additional ligands (i.e. amino acid residues of Ser, Cys, SeCys or Asp and very often oxygen Mo=O ligand).

pterindin cofactor coordinating molybdenum atom

EBDH structure

Moco cofactor

EBDH structure

 

The main research topic is a study of ethylbenzene dehydrogenase (EBDH), the complex bacterial metalloenzyme (αβγ 164 kDa) containing monomolibdenum center (MoCo), iron-sulfur clusters and heme b559 prosthetic group. EBDH catalyses stereospecific hydroxylation of ethylbenzene to 1-(S)-phenylethanol. Recently, we have shown that EBDH oxidizes a range of aromatic or heterocyclic compounds with ethyl or propyl substituents to secondary alcohols.

PEDH

(S)-1-Phenylethanol dehydrogenase (PED) is a homotetrameric (108 kDa) NAD+/NADH dependent short-chain dehydrogenase/aldehyde reductase coming from a denitrifying bacterium Aromatoleum aromaticum (EbN1). It is the second biocatalysts in the pathway of EbN1 ethylbenzene mineralization and it catalyzes NAD+-dependent stereospecific oxidation of (S)-1-phenylethanol to acetophenone. 

 

 

The studies of PEDH focus on application of the enzyme in the biosynthesis of pure chiral alkylaromatic alcohols. Such application is possible due to the joint-patent hold together by Professor Johan Heider from Marburg University and BASF AG. The research conducted in JLBEC is aimed at optimization of reaction conditions for future application of EBDH in the industry.

S25DH

Steroid C25 dehydrogenase is an EBDH-like enzyme (αβγ heterotrimer containing molybdenum cofactor) isolated from faculatitive anaerobic ß-proteobacteria, Sterolibacterium denitrificans. S. denitrificans Chol-1ST (DSMZ 13999T) is  closely related to Thauera and Azoarcu sp., which utilize nitrates (or oxygen in aerobic conditions) as electron acceptors. It can use cholesterol as the sole carbon source. S25DH catalyzes a regioselective hydroxylation of tertiary C-25 atom carbon in cholesterol derivatives:

S25DH catalized hydroxylation of cholest-4-en-3-on to 25-hydroxycholet-4-en-3-on.

The research project is conducted in cooperation with prof. Matthias Boll from Freibug University in Germany.

MAIN RESEARCH TOPICS

  • Investigation of EBDH and PEDH substrates spectrum and analysis of their reaction products.
  • The study of kinetics and thermodynamics of enzyme's reduction and oxidation half-cycles.
  • Inhibition and deactivation study.
  • Enzyme immoblization
  • The modeling of reaction pathways with quantum chemical methods (QM and QM:MM).
  • Modeling of enzyme's reactivity with Quantitative Structure Activity Relationship (QSAR) approach.

METHODS

  • Investigation of enzyme activities towards different substrates basing on spectroscopic activity assay. Rapid mixing stopped flow kinetic measurements of half-cycle redox reaction in cooperation with Physicochemistry of Coordination Compounds Group of Prof. Grażyna Stochel from Chemistry department of Jagiellonian University.
  • FPLC and electrophoresis techniques for the enzyme purification.
  • LC-MS and GC-MS with RP and chiral columns for separation of reaction's products and identification of enzyme stereospecifity.
  • Liquid-liquid and solid phase exctractions (SPE) of the reaction mixtures for purification of the pure products.
  • Isothermal titration calorimetry for investigation of thermodynamics of enzyme reaction.
  • Quantum chemical modeling of the molybdenum active site's structure and verification of hypothetical reaction mechanism with various substrates.
  • Prediction of biological activity with Artificial Neural Networks and QSAR equations based on quantum chemical parameters.

MAIN ACHIEVMENTS

  • Aerobic procedure of EBDH purification.
  • Description of EBDH substrate spectrum, identification of reaction products with LC-MS and GC-MS methods.
  • Modeling of enzymes reactivity for substrate and inhibitors with Neural Networks sytems.
  • Formulation and veryfication of EBDH mechanism hypothesis based on kinetic results and quantum chemical modeling.

Raction mechanism - quantum chemical modeling

 

SCIENTIFIC COOPERATION