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 enzyme systems that catalyze stereospecific synthesis of alkylaromatic alcohols. Currently the main research effort is concentrated on three enzymes, namely: Ethylbenzene Dehydrogenase (EBDH), Phenylethanol Dehydrogenase (PEDH), Steroid C25 Dehydrogenase (S25DH), Tungsten Aldehyde Oxidoreductase (AOR) and Benzlsuccinate Synthase (BSS). Recently JLBEC expanded its research into the field of bacterial synthetized bioplastics and its functionalization for biomedical applications. The projects conducted in JLBEC are funded by the National Center of Science (OPUS, SONATA BIS, SONATA, PRELUDOUM, BETHOVEEN LIFE), the National Center of Research and Development (LIDER, TECHMATSTRATEG) as well as funds from industrial partners.
The Laboratory provides also additional platform for integration of scientific society, joining together equipment potential of Institute of Catalysis and Surface Chemistry Polish Academy of Science and Institute of Plant Physiology Polish Academy of Sciences.
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).
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.
(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.
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 main aim of the project is development and experimental verification of mechanistic hypothesis describing catalytic activity of cholest-4-en-3-one Δ1-dehydrogenase. The enzyme belongs to the class of 3-ketosteroid Δ1-dehydrogenases (Δ1-KSDH), that initiates central pathway of cholesterol mineralization (opening of the steroid ring A) in denitrifying β-proteobactera, Sterolibacterium denitrificans. Enzymes of the Δ1-KSDH class catalyze oxidative dehydrogenation in the ring A of the steroids. In order to test the mechanistic hypothesis we conduct kinetic test with native and mutated enzymes, conduct crystallization of the enzyme and solving its structure as well as theoretical modeling of the reaction using QM, MD, QM:MM and QM:MD techniques. The kinetic tests are conducted in close cooperation with a group of professor Grażyna Stochel from Faculty of Chemistry JU and professor Marco Fraaije from University in Groningen. The studies of enzyme tertiary structure are conducted in cooperation of Colloids group of professor Zbigniew Adamczyk and with Institute of Plant Physiology PAS. We also conduct applied research toward development of industrial methods for steroid dehydrogenation in cooperation with Life Science University in Wroclaw.
The NCN OPUS-2016/21/B/ST4/03798 project introduced following equipement to the JLBEC:
- FPLC Biorad NGC Quest 10 Plus System
- Shimadzu UV-2700 spectrometer
Tungsten aldehyde oxidoreductase was isolated from bacteria Aromatoleum aromaticum. The aim of the conducted research is elucidation of the reaction mechanism catalyzed by the enzyme, i.e. oxidation of aldehydes to carboxylic acids. The AOR exhibits unusual to that class of enzyme resistance to O2 inactivation as well as much more complex subunit structure compared to already known AOR from archaea from Pyrrococus or Thermococus. The research project is conducted within frame of Interdisciplinary Environmental Doctoral Studies FCB and NCN Preludium project 2017/27/N/ST4/02676. It concentrates on biochemical and structural characterization of the enzyme and its tungsten cofactor. Project is conducted in a close cooperation with a group of professor Johann Heider from Marburg University.
Polyhydroxyalkanoates (PHAs) are biopolymers that represent one of the leading bioproduct sectors. PHAs represent a class of optically active biodegradable polyesters accumulated by numerous bacteria as isolated intracellular granules or as extracellular net-like structures. PHAs serve primarily as a reserve of carbon and energy. The chemical structure of PHA can be described as a linear formation of (R) -3-hydroxy acids, where the carboxyl group of one monomer forms an ester bond with the hydroxyl group of the adjacent monomer. PHA can be divided into several categories depending on the criterion used. Considering the size of the PHA monomer, it is divided into short (C3-C5; scl-PHA), medium (C6-C14; mcl-PHA) or long (> C14; lcl-PHA) chain polymers.
Chemical structure of PHA, where R1 and R2 are different side chains
Our research focuses on PHA polymers as well as their monomers. Ongoing work involves biocatalysis for the functionalization of PHA polymers and its monomers. We use enzymes from the group α / β-hydrolases - lipases, thanks to which we create new materials based on PHA. Bioplastic is subjected to enzymatic functionalization with selected drugs, on the other hand, monomers by biocatalysis are attached to sugar molecules. The research aims to create new materials for tissue regeneration, dressing materials, new anti-cancer drugs and surfactants.
Four research projects are carried out in a given topic:
- NCN Sonata project "Biosynthesis of new lactose esters via lipases. Characteristics of their physicochemical and anti-cancer properties" No. 2015/17 / D / ST4 / 0051
- Project Know "Biocatalytic synthesis of sugar esters"
- NCBiR Lider project "New functionalized biopolymers for medical applications" No. 0090 / L-7/2015
- TECHMATSTRATEG project "Technology for biorefining vegetable oils for the production of advanced composite materials" No. 407507/1 / NCBR / 2019
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.
- 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.
- 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.
- Prof. Johann Heider - Laboratory of Microbial Biochemistry, Fachbereich Biologie, Philipps-Universität Marburg, Germany
- Prof. dr. hab. Andrzej M. Skoczowski - Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland
- Prof. Grażyna Stochel - Physicochemistry of Coordination Compounds Group, Chemistry Department of Jagiellonian University, Kraków, Poland.
- Prof. Matthias Boll - Mikrobilogie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Germany
- POIG project "Biotransformations for pharmaceutical and cosmetic industry"