L1.1

Enzymatic reactions and metabolites — synthetic advances and strategiesRoland Wohlgemuth

Sigma-Aldrich, Research Specialities, Industriestrasse 25, CH-9470 Buchs, Switzerlande-mail: Roland Wohlgemuth <roland.wohlgemuth@sial.com>

The universe of enzymes and metabolites in living bio-logical cells continues to play a central role in life sciences, technology and medicine. The fundamental importance of metabolites goes beyond their function as substrates for enzymes and includes regulation and sensing in metabolic pathways, regulating gene expression as indu-cers or in ri-boswitches and signalling within the cells, to other cells and to their envi-ronment.For the many metabolites present in biological systems, authentic material for unam-biguous assignment, charac-terization and functional investigation is essential. The syn-thetic strategies for the synthesis of densely and differen-tially functionalized small molecules require combinations of the best selective methods known. The interface of highly selective biocatalytic reaction platforms with organic synthesis is thereby im-portant [1, 2]. Interesting biochemi-cal blueprints for the design of synthetic routes are pro-vided by the known metabolic pathways, but the retrosyn-thetic analysis needs to take into account the practically realized biocatalytic reaction classes. The natural metabolic pathways have not only inspired both classical chemical and biocatalytic syntheses, but have also been a starting point for assembling a number of biocataly-tic steps by preparing the required enzymes and auxiliary reagents [3-7].The revitalization of this classical biochemistry area has been started with the focus on the central metabolites along major pathways and pathway intersections of heal-thy bi-ological systems. As the chirality of many important me-tabolites has biochemi-cal relevance, asymmetric synthesis is a major strategy [8]. It has been useful for a non-racemic metabolite to first synthesize the racemic form, then to de-velop the ana-lytical chiral separation and to synthesize the chiral metabolite in the third phase.Synthetic advances for metabolites glycolysis, pentose phosphate, shikimate, meva-lonate and non-mevalonate, isoprenoid, steroid and vitamin B6 pathways have been achieved by the development of new methods, ingredients and tools.References:1. Ward J, Wohlgemuth R (2010) Curr Organic Chem 14 (in press).2. Alphand A, Wohlgemuth R (2010) Curr Organic Chem 14 (in press).3. Wohlgemuth R (2009) Biotechnology J 9: 1253–1265.4. Richter N, Neumann M, Liese A, Wohlgemuth R, Eggert T, Hummel W (2009) ChemBioChem 10: 1888–1896.5. Richter N, Neumann M, Liese A, Wohlgemuth R, Weckbecker A, Eg-gert T. Hummel W (2010) Biotech Bioeng 106: 541–552.6. Wohlgemuth RJ (2009) Mol Catal B: Enzymatic 61: 23–29.7. Schell U, Wohlgemuth R, Ward JM (2009) J Mol Catal B: Enzymatic 59: 279–285.8. Wohlgemuth R (2010) Curr Opin Microbiol 13: 283–292.

Session 1: Technical Biochemistry

LecturesL1.2

Biotransformations of carbohydrates with hydrolasesAntonio O. Ballesteros

Departamento de Biocatalisis, Instituto de Catálisis CSIC, 28049 Madrid, Spainweb page Antonio O. Ballesteros www.icp.csic.es/abg

Carbohydrates are the most important class of renewable compounds. Sugar esters are non-ionic surfactants formed by a carbohydrate moiety as hydrophilic group and one or more fatty acids chains as lipophilic component. By con-trolling the esterification degree and the nature of fatty acid and sugar, it is possible to synthesize derivatives with different hydrophile-lipophile balance. Carbohydrate fatty acid esters have a vast number of applications in the food, polymers, cosmetic, oral care, detergent, and pharmaceu-tical industries; among them, sucrose esters are the most developed, investigated and produced in a higher scale.The enzyme chosen (within lipases or esterases) is of para-mount importance in the regioisomers obtained. Chemi-cal catalysis may interfere with the enzymatic process: The regioespecificity of both types of catalysts is strikingly dif-ferent, and may lead to mistakes. Methodologies for enzy-matic carbohydrate acylation (also applicable to other poly-hydroxilic compounds) need to find a medium in which a polar reagent (sugar) and a nonpolar acyl donor are solu-ble and may react in the presence of an appropriate (bio)catalyst; our method — a mixture of 2-methyl-2-butanol and DMSO (4:1) appears to be the best protocol report-ed. Concerning acyl donors, the best results were found by transesterification with an enol ester (i.e. a vinyl ester, which pushes forward the reaction by forming acetalde-hyde). A broad spectrum of interesting properties of su-crose esters have been investigated in our laboratory.References:Ferrer M, Cruces MA, Bernabe M, Ballesteros A, Plou FJ (1999) Biotechnol Bioeng 65: 10–16.Plou FJ, Cruces MA, Pastor E, Ferrer M, Bernabe M, Ballesteros A (1999) Biotechnol Lett 21: 635–639.Plou FJ, FerrerM, Ballesteros A (2003) In: Encyclopedia of Catalysis, Horvath I, ed, vol. 6, pp 483–506, John Wiley.Ferrer M, Soliveri J, Plou FJ, Lopez-Cortes N, Reyes-Duarte D, Chris-tensen M, Copa-Patiño JL, Ballesteros A (2005) Enzyme Micr Technol 36: 391–398.

45th Annual Meeting of the Polish Biochemical Society 3

L1.3

Biotransformations useful in the industryMagdalena Klimek-Ochab, Paweł Kafarski

Department of Bioorganic Chemistry, Faculty of Chemistry, Technical University of Wroclaw, Wrocław, Polande-mail: Paweł Kafarski <pawel.kafarski@pwr.wroc.pl>

Enzymes and whole cells of various organisms can catalyze virtually all chemical reactions thereby yielding products that meet industrial requirements. Biotransformations con-stitute an important segment of various branches of indus-try, in particular of pharmaceutical and chemical industries, which is confirmed by the recently observed tendency to the replacement of classical chemical synthesis processes with biotechnological methods or coupling chemical proc-esses with biotransformations to develop chemoenzymatic processes. Application of biocatalysis is fully competitive to using chemical catalysis and constitutes an important el-ement of “Green Chemistry” which is a common method of realization of the paradigm of “Sustainable Develop-ment”.Both microbial cells and partially purified enzyme prepa-rations are widely used in industrial biocatalytic processes. The principal biocatalysts are hydrolases (account for 44% all biocatalysts) and oxidoreductases (30%). It is to note that immobilized enzymes account for almost 50% com-mercial enzyme preparations. It is surprising that public databases like Biocatalysis of Accelrys or University of Min-nesota Biocatalysis Database contain more than 35.000 various chemical reactions catalyzed by enzymes. This information provides evidence that the number of biocatalysts that are potentially useful in organic synthesis is huge. However, only approximately 200 enzymes have been hitherto used in this field.The principal area of biotransformations is the resolution of racemic mixtures into pure enantiomers. It stems from the fact that over 50% potential drugs subjected to investi-gations in 2006 in three pharmaceutical companies such as Pfizer, GlaxoWellcome and AstraZeneca are chiral compounds. So it is obvious that the majority of products from enzyme-catalyzed synthesis reactions are chiral compounds (86%). Results of investigations in this area have either cognitive character (the development of new structural modifica-tions of molecules) or can be used in practice (methods of resolution of racemic mixtures into pure enantiomers, studies on the structure of chiral compounds, manipula-tion of biotransformation conditions to increase its stere-oselectivity and stereospecificity).The majority of industrial processes which employ bio-catalysts are conducted in the small-tonnage scale but the number of large-tonnage processes has been increasing which is illustrated by glucose isomerase-catalyzed con-version of glucose into fructose upon manufacturing of glucose-fructose syrups. This process yields annually more than 1 000 000 tons fructose.

L1.4

Metal ions in chemistry and biology of proteins involves in neurodegenerationHenryk Kozlowski, Lukasz Szyrwiel, Magdalena Taraszkiewicz, Ewa Gralka

Faculty of Chemistry, University of Wroclaw, Wroclaw, Polande-mail: Henryk Kozlowski <henrykoz@wchuwr.chem.uni.wroc.pl>

Neurodegenerative disorders are usually the fatal diseases. Understanding of these pathological events in the nerv-ous system is the leading challenge next 20–30 years. In many instances these diseases are associated with biologi-cally essential metals or the disorders in proteins that bind metal ions. There are numerous neurodegenerative diseases including prion related disorders which are very rare but have unusually vast interest in various studies. Prion dis-eases include spongiform encephalopathies in cattle and sheep, Kuru and Creutzfeld-Jacob syndrome in humans. Biological role of prion proteins (PrP) is still hardly un-derstood but it seems to be generally accepted that it may play a basic role in copper homeostasis and anti-oxidant enzymatic activity [1]. In the case of mammalian protein metal ions interact with prion protein by the N-terminal octa-repeat domain containing (PHGGGWGQ) octapep-tide repeats. The second metal binding domain is in the neurotoxic region containing two histidine residues [2, 3]. The unique biology of prion proteins and their relation to metal homeostasis has focused much of research on pro-teins and metallo-proteins derived from mammals down to fishes [4]. After relatively good understanding of mammali-an and avian protein interactions with Cu(II) ions the stud-ies were extended to prion proteins from Japanese puff-erfish [5] and zebrafish [6]. The His-rich regions of these proteins are basic for the metal binding and resulting anti-oxidant activity. A distinct increase in the binding of metal ions by fish proteins especially that of zebra-fish variant is observed. The His-rich regions of fragments of zebra fish PrP-rel-2 protein bind very effective not only Cu(II) but also Zn(II) ions in homo- and heteronuclear complexes [7]. The chemical studies strongly suggest the involvement of prion and prion-like proteins in metal ion homeostasis.References:1. Gaggelli E, Kozlowski H, Valensin D, Valensin G (2006) Chem Rev 106: 1955.2. Kozłowski H, Brown DR, Valensin G (2006) Metallochemistry of Neurode-generation, The Royal Society of Chemistry, Cambridge.3. Brown DR, Wong BS, Hafiz F, Clive C, Haswell SJ, Jones IM (1999) Biochem J 344: 1–5.4. Kozlowski H, Janicka-Klos A, Stańczak P, Valesnin D, Valensin G, Ku-lon K (2008) Chord Chem Rev 252: 1069–1078.5. Stańczak P, Valensin D, Porciatti E, Jankowska E, Grzonka Z, Molteni E, Gaggelli E, Valesnin G, Kozłowski H (2006) Biochemistry 45: 12227–12239.6. Gaggelli E, Jankowska E, Kozłowski H, Marcinkowska A, Migliorini C, Stańczak P, Valesnin D, Valesnin G (2008) J Phys Chem B 112: 15140–15150.7. Vaslensin D, Szyrwiel L, Camponeschi F, Rowinska-Zyrek M, Molteni E, Jankowska E, ASzymanska A, Gaggelli E, Valensin G, Kozlowski H (2009) Inorg Chem 48: 7330.

Abstracts4

L1.5

Bacterial diversity monitoring in membrane bioreactor’s activated sludge treating high concentration of petroleum organic compoundsAleksandra Ziembińska1, Sławomir Ciesielski2, Jarosław Wiszniowski1

1The Silesian University of Technology, Environmental Biotechnology Department, Akademicka 2, 44-100, Gliwice, Poland; 2University of Warmia and Mazury in Olsztyn, Department of Environmental Biotechnology, Słoneczna 45G, Olsztyn, Polande-mail: Aleksandra Ziembińska <aleksandra.ziembinska@polsl.pl>

Polycyclic aromatic hydrocarbons (PAHs) are known to be ubiquitous recalcitrants appearing in soil, water and air. It is widely known, that some of these chemicals can be mu-tagenic and cancerogenic, while all of them are harmful to aquatic life and cause risk to humans. These substanc-es directed to the water tracts and further to wastewater treatment plants can irreversibly inhibit sensitive biological processes and in effect cause the water treatment failure. Such a situation is a result of the harmful influence of PAHs to activated sludge — a mixture of microorganisms, responsible for sewage purification in biological wastewater treatment plants (WWTP). Observation of the bacterial diversity changes in activated sludge could explain how heavy is the polycyclic aromatic hydrocarbons’ influence on bacterial community. Due to the fact that activated sludge microorganisms are uncultur-able and it is impossible to study their biology with classical microbiological tools, direct molecular approach is needed. Denaturing gradient gel electrophoresis (DGGE) seems to be the most suitable technique for bacterial diversity analysis. In this method total bacterial DNA from activated sludge sample is used for PCR amplification, using specific primers. The PCR products are separated electrophoreti-cally in polyacrylamide gel containing increasing gradient of denaturants. The fingerprint obtained in DGGE shows the complexity of the bacterial community, due to the fact that PCR products are the same in length, but different in DNA sequence. They can be easily compared and the changeability of genotypes can be observed.The aim of this experiment was to monitor bacterial diver-sity and changeability in membrane bioreactor (MBR) deal-ing with wastewater contaminated with high concentration of polycyclic aromatic hydrocarbons. The research lasted for 6 months and it revealed that the organics removal was at the level of 99% during total length of experiment and the doses of PAHs between 0.05 ml/l wastewater to 0.5 ml/l seems to stimulate biodiversity growth. The concentration of PAHs above 1 ml/l waste-water leads to dramatic change of bacterial biodiversity and nitrification failure. Probably PAHs can serve as an addi-tional carbon source for bacteria till the level of 1 ml/ l of wastewater. Other explanation could be the possibility of storing these substances inside the flocs. When the concen-tration of PAHs reached the threshold of harmfulness to bacteria it occurred as biodiversity decrease.

L1.6

Utilization of biomass for the preparation of environmentally friendly polymer materials Andrzej Okruszek

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Polande-mail: Andrzej Okruszek <andrzej.okruszek@p.lodz.pl>

The Project is being prepared by the Consortium com-posed of several research groups under the supervision of prof. A. Okruszek (Technical University of Łódź). The ma-jor participants of the Consortium are research teams from the Technical University of Łódź, headed by professors: I. Krucińska, L. Ślusarski, M. Zaborski, T. Antczak and P. Walczak. In addition, to the Consortium belong research groups from the Institute of Biopolymers and Chemical Fibres in Lodz (dr. D. Ciechańska), Centre of Molecular and Macromolecular Studies PAS in Lodz (prof. P. Kubisa, prof. A. Duda, prof. A. Gałęski), University of Agriculture in Krakow (prof. A. Libik, prof. A. Lepiarczyk), and Central Mining Institute in Katowice (prof. K. Czaplicka-Kolarz, dr. H. Rydarowski). The Project is envisaged for the time frame 2009–2013. The leading idea of the Project involves the utilization of various kinds of biomass (eg. potatoes, sugar beets, grains of cereals and corn, straw, oily materi-als) and their transformation by biotechnological methods, including either enzymatical or microbiological processes, into environmentally friendly polymer materials. Thus, it is planned to prepare cellulose nanofibre materials from cel-lulose biomass via an enzymatic process. These nanofibre materials will be further transformed into innovative com-posite materials. Other enzymatic processes will be em-ployed for transformation of oil-plant biomass into oligo-diols/polyols, which will form the basis for the preparation of new biodegradable copolyesters. From these polyesters, new functional textiles and nonwovens will be prepared, with potential sanitary or technical application. Further utilization of biomass involves its stereoselective micro-bial transformation into L-lactic acid. This product will be chemically transformed into L,L-lactide, which will be later polymerized to form tactical polylactide, a substrate for various fibres and thermoforming. Some attempts will be made to use the new polymeric materials and the prepared textiles/nonwovens/composites for preparation of geo-textiles and agrotextiles. Also, controlled biodegradation of obtained polymeric/fibrous products will be attempted.Acknowledgements: Project POIG 01.01.02-10-123/09 partially financed by the European Union within the European Regional Development Fund.The Project is envisaged for the time frame 2009–2013.

45th Annual Meeting of the Polish Biochemical Society 5

L1.7

Enzymes as molecular targets for antifungal chemotherapyI. Gabriel1, J. Olchowy1, K. Kur1, P. Szweda1, W. Rypniewski2, J. Raczyńska2, S. Milewski1

1Gdansk University of Technology, Department of Pharmaceutical Technology and Biochemistry, Narutowicza 11/12, 80-233 Gdańsk, Poland; 2Polish Academy of Sciences, Institute of Bioorganic Chemistry, Noskowskiego 12/14, 61-704 Poznań, Polande-mail: Iwona Gabriel <iwona.gabriel@wp.pl>

Identification and characterization of enzymes which are essential for fungal survival and/or pathogenicity is crucial for the rational design of antifungal drugs. In the present communication we report results of our studies on Candida albicans enzymes catalyzing crucial steps in two metabolic pathways: (A) a four-step cytosolic biosynthesis of UDP-GlcNAc, providing building blocks for chitin formation and (B) the α-aminoadipate pathway of L-lysine biosynthe-sis. Molecular studies on GlcN6P synthase (Gfa1p), catalysing the first committed step in the (A) pathway were performed. Fragments 1-345 and 346-712 of Gfa1p, were identified as functional glutamine amidohydrolase (GAH) and isomer-ase (ISOM) domains, respectively. X-ray crystallography of ISOM revealed its homotetrameric structure; the same was found for the whole enzyme in solution by means of SAXS measurements. Spectrofluorimetric studies of the isolated domains and the whole enzyme revealed that the binding site for the allosteric inhibitor, UDP-GlcNAc, is located at ISOM and phosphorylation of the S208 residue by protein kinase A results in an enhanced rate of glutamine amide hydrolysis by GAH. Finally, comparison of catalytic ac-tivities of V711FGfa1p, Δ709-712Gfa1p, W97FGfa1p and W97GGfa1p to those of the native Gfa1p and the isolated domains provided evidence for the intramolecular channel connecting GAH and ISOM of Gfa1p, with W97 residue functioning as a molecular gate. A group of inhibitors, structural analogues of fructoseimine-6-P intermediate were designed, synthesized and found to inhibit GlcN6P synthase and growth of human pathogenic fungi. The separate studies concerned enzymes catalyzing ini-tial steps of (B): homocitrate synthase (HCS), homoaco-nitase (HA), homoisocitrate dehydrogenase (HICD) and α-aminoadipate aminotransferase (AadAT). Two izozymes of C. albicans HCS, Lys21p and Lys22p, were isolated as oligoHis fusions and characterized in details. Growth phe-notype in vitro and virulence in the murine model of the constructed lys21Δ/lys22Δ mutant of C. albicans were de-termined to verify a target potential of the enzyme. A gene encoding AadAT was identified and initial characterization of catalytic properties of the gene product was performed. Potential inhibitors of the AAP enzymes were designed and tested for their enzyme inhibitory properties and fun-gal growth inhibitory effect.

L1.8

Structures of complexes of the chagasin with proteolitic enzymes as potential targets for drug designGrzegorz Bujacz

Institute of Technical Biochemistry, Technical University of Łódź, Stefanowskigo 4/10, 90-924 Łódź, Polande-mail: Grzegorz Bujacz <gdbujacz@p.lodz.pl>

Chagasin is a cysteine protease inhibitor identified in Trypanosoma cruzi, the parasite that causes a Chagas’ disease. The parasite also expresses a protease - cruzipain. Chagasin is associated with cruzipain and takes part in regulation of its activity. At the site of infection, chagasin is secreted out-side of the parasite and interacts with host proteases. Chagasin belongs to a new structural family of inhibitors. Although it resembles some well-characterized cysteine protease inhibitors, such as cystatins, it has a unique amino acid sequence and a completely different structure. The in-hibitory epitope of chagasin is composed of 3 loops, which interact with the catalytic cleft of the enzyme, with only the central loop inserted directly into the catalytic center. We have determined high-quality structures of chagasin in complexes with the human proteases cathepsin L and B, which are potential target enzymes during T. cruzi infection as well with plant cysteine protease — papain, the struc-ture of which is almost identical to the catalytic domain of cruzipain. Cathepsin B shows both endo- and exopeptidase activity, the latter due to a unique occluding loop restricting access to the active site cleft. The crystal structure of cathepsin B in complex with the T. cruzi inhibitor chagasin clarifies the mode by which natural protein inhibitors manage to overcome this obstacle. Chagasin is a more potent inhibi-tor of cathepsin B than cystatins. Inhibition of the enzyme involves a conformational change of the occluding loop. The structures of chagasin in complexes with proteolytic enzymes provide a detailed view of how the parasite pro-tein inhibits host enzymes that may be of paramount im-portance as the first line of host defense. The high level of structural and functional similarity between cathepsins L, B and between papain and cruzipain offers interesting clues as to how the cysteine protease activity of the parasite could be targeted. Nowadays designing the drugs on the basis of the crystall structure of proteins involved in pathological processes is very common. The structure of chagasin with proteolitic enzymes will help parasitologists explain its role in the life cycle of the parasite and drug designers to disrupt the mo-lecular mechanism of Chagas’ disease. Additionally, cathe-psins L and B are overexpressed in a number of immune and cancer diseases and designing of inhibitors based on the chagasin complexes may create a possible way to con-trol their activity.

Abstracts6

L1.9

Mechanisms of biochemical reactions in the light of modeling and isotope effectsPiotr Paneth

Institute of Applied Radiation Chemistry, Faulty of Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Polande-mail: Piotr Paneth <piotr.paneth@gmail.com>

Understanding molecular details of enzymatic catalysis is fundamental to rational drug design, in particular when transition state analogs are considered. Studies of the mechanisms of enzyme-catalyzed reactions using isotope effects comprise two aspects. First, reaction rates differ for reactant isotopologues and these differences result in kinetic isotope effects. Their magnitude may be used to elucidate rate-determining transition state structure and/or relative rates of steps involved in the overall catalysis. Sec-ond, equilibrium between free and enzyme-bound reactant may also be perturbed by isotopic substitution. While this is a complication in interpretation of kinetic isotope effects these binding isotope effects can provide valuable infor-mation regarding interactions between the reactant and the protein active site. In this communication we illustrate both aspects of isotope effects studies of enzyme-catalyzed re-actions on the example of recent projects carried out in our laboratory. The attempts to understand the mechanism of the dehalogenation step catalyzed by selected dehaloge-nases by comparing theoretical values of chlorine, isotope effects with the available experimental data will be used to illustrate the use of kinetic isotope effects. The analysis of the pattern of binding isotope effects of pyruvate and oxa-mate with lactate dehydrogenase will illustrate the use of binding isotope effects.

L1.10

Creating novel enzymes for glycoside synthesis by protein engineering and directed evolutionManu De Groeve1,2, Tom Desmet1, Wim Soetaert1

1InBio.be - Centre of Expertise Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, 9000 Ghent, Belgium; 2Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Polande-mail: Manu R.M. De Groeve <manu.degroeve@p.lodz.pl>

Glycosylation can significantly influence the properties of a chemical compound. In the case of pharmaceuticals, gly-cosylation can improve the pharmacokinetic properties of a drug and induce drug targeting to specific organs, result-ing in less side effects and smaller required doses. This research was part of a larger project in which the goal is to develop a generic glycosylation technology based on glycoside phosphorylases (GPs). Here, the Cellulomonas uda cellobiose phosphorylase (CP) enzyme was used as a model. It is a member of CAZY family GH-94 and cat-alyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate (αGlc1P) and glucose. Since most natural phosphorylases have a relatively narrow substrate specificity, they should be improved by protein engineer-ing. Therefore, a suitable high-throughput screening (HTS) system was developed based on the measurement of re-leased inorganic phosphate. It was applied to an E649 satu-ration library to identify CP enzyme variants with acceptor specificity towards anomerically substituted glucosides. An enzyme variant containing an E649C mutation was found, displaying high activity towards several non-native accep-tors. Also the donor specificity of C. uda CP was modified from αGlc1P towards a-galactose 1-phosphate (αGal1P), resulting in so-called lactose phosphorylase (LP) enzymes that have not yet been found in nature. Further broadening of acceptor specificity towards β-glucosides could be achieved by mutating residues near the active site entrance and screening for acceptor specifi-city towards octyl β-glucoside. A mutant containing five amino acid mutations was found which displays broad acceptor specificity towards various β- and α-glucosides, including alkyl β-glucosides, aryl β-glucosides and me-thyl α-glucoside.Finally, αGal1P, methyl β-cellobioside, methyl β-lactoside and lactose were produced, purified and crystallized with the created enzyme variants. From these glycosides, methyl β-lactoside has most interesting properties since it was re-ported to have anti-cancer effects.

45th Annual Meeting of the Polish Biochemical Society 7

L1.11

Hfq bacterial gene expression regulator, crucial in short RNAs pathways, is involved in cellulose biosynthesis in Gluconacetobacter xylinusKatarzyna Kubiak1, Małgorzata Rogalska1, Julian Sikorski2, Alina Krystynowicz1, Stanisław Bielecki1

1Institute of Technical Biochemistry, Department of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland; 2Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerlande-mail: Katarzyna Kubiak <katarzyna.kubiak@p.lodz.pl>

Gluconacetobacter xylinus is well known producer of bacte-rial cellulose (BC) — nanocrystalline, highly elastic and mechanically durable polymer. BC is biotechnological ma-terial attractive for medicine as it is chemically pure, bio-compatible and easy to fabricate in any size and shape. Na-tive and modified cellulose is routinely produced in ITB and tested in external and internal medical applications [1]. Even though BC secretion by Ga. xylinus is intensive, BC production process efficancy is unsatisfactory. Further op-timization is impossible without complete understanding of molecular regulation of BC biosynthesis. Cellulose synthase (CS) operon (bcsABC/D) is sequenced in many strains but the only proven regulatory mechanism of ithis enzyme activity is allosteric interacion of CS regula-tory subunit (BcsB) with cyclic-di-GMP. The level of c-di-GMP in bacterial cell is regulated by the opposite actions of diguanylate cyclases (DGC) and phosphodiesterases A (PDEA) and their activity in Ga. xylinus is connected with redox conditions of the cell [3, 4]. Genetic modifications introduced in Ga. xylinus so far concerned metabolic path-ways leading to final polymerization of UDP-glucose and brought no significant changes in overall cellulose pro-ductivity. We hypothesized that level of BC secretion may be regulated in similar way as polysaccharide secretion in biofilm formation in other Enterobacter species and may be related to cellular stress response. Short RNAs are powerful gene expression regulators, ac-tive in bacterial environmental signal and stress response pathways. Sequences of such molecules are species-specific but they are often cooperating with highly evolutionary conserved Hfq chaperone protein. First identified target for Hfq (and its RNA partner DsrA) was RpoS gene, cod-ing sigma factor S. Here we show that Hfq is involved in cellulose biosynthesis in Ga. xylinus. We bring evidence for presence of hfq gene in Ga. xylinus genome (HM004551) and changes of its expression under conditions influenc-ing cellulose secretion. Finally we show that deletion of this gene altered cellulose biosynthesis. These observa-tions suggest that regulatory pathways engaging Hfq (and sRNAs) may play important role in bacterial cellulose se-cretion what opens new ways for genetic modifications of productive strains. References:1. Krystynowicz A et al. (2002) J Ind Microbiol Biotechnol 29: 189-95; 2. Römling U (2002), Res Microbiol 153: 205–212.3. Chang AL et al. (2001) Biochemistry 40: 3420–3426.4. Yaning Qi et al. (2009) Biochemistry 48: 10275–10285.

Posters

P1.1

A new method for measuring the expression of intracellular glucocorticoid receptors in living thymocytes by flow cytometryEwelina Kiernozek1, Ewa Kozłowska1, Magdalena Markowska2, Anna Kowalik1, Nadzieja Drela1

1Department of Immunology; 2Department of Animal Physiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Polande-mail: Ewelina Kiernozek<ekiernozek@biol.uw.edu.pl>

Endogenous glucocorticoids (GC) are involved in numer-ous physiological processes such as induction of apoptosis and maintaining the homeostasis of the immune system. GC regulate the activity of the immune system during inflammation and are involved in the development of T lymphocytes in the thymus. The main source of endog-enous GC is adrenal cortex, but they are produced also by cortical thymic epithelial cells (TECs). Recently, the syn-thesis of these hormones by the thymocyte itself has been demonstrated. Growing body of evidences demonstrate that the endo- and exogenous glucocorticoids act primarily through cytoplasmic receptors for glucocorticoids (cGCR). Glucocorticoids, depending on the strength and type of signals received by T cells, can act pro-or anti-apoptotically. Natural regulatory T cells (nTreg) are less sensitive to glu-cocorticoid-induced apoptosis than other populations of T lymphocytes. It seems that GC may regulate nTreg cell sup-pressive properties, but it is unclear whether there is a di-rect correlation between the level of glucocorticoid recep-tors and the suppressive activity of nTregs. Thus, the aim of our study was to develop a method, which allows the investigation of the expression of intracellular receptors for GC in living T cells. For the identification of glucocorti-coid receptors, we applied the technique of flow cytometry based on the use of a synthetic hormone, dexamethasone, conjugated with a fluorochrome. The fluorochrome-dex-amethasone complex enters the cell, and binds to intracel-lular GC receptors. We used this method for the evaluation of the expression of GC receptors in the main thymocyte subsets: DN CD4-CD8-, DP CD4+CD8+, SP CD4+CD8-, SP CD4+CD8- and CD4+CD25+ thymocytes containing thymus-deriving natural Tregs. For the study male and fe-male C57BL/6 mice have been used. This method allows the simultaneous determination of the level of expression of intracellular glucocorticoids receptors, and the pheno-type of the cells, as well as tracking of the changes de-pendent on culture conditions. We compared the results of our studies with those of other authors using Western blot technique and flow cytometry analysis of permeabilized T cells labeled by fluorochrome-conjugated monoclonal anti-GCR antibody.Acknowledgements:This study was supported by Grant No. N N303 395836 from the MNiSW (Poland).

Abstracts8

P1.2

Catalytic properties of the lipases from newly isolated strains Rhizomucor variabilis and Fusarium solani for biodiesel productionRenata Bancerz, Agata Sieczkowska, Jerzy Rogalski

Department of Biochemistry, Maria Curie-Skłodowska University, 20-031 Lublin, Polande-mail: Renata Bancerz <rwiater@tlen.pl>

Lipases (EC.3.1.1.3, triacylglycerol acylhydrolases) are a group of enzymes, which hydrolyze triacylglycerols to free fatty acids and glycerol in oil-water emulsion. In the pres-ence of organic solvents, the hydrolytic reaction can be replaced by interesterification and transesterification reac-tions [1]. These kind of the enzymes possesses very high regiospecificity and enantioselectivity towards to theirs substrates [2]. It open the possibility of lipases in appli-cations in food technology, in the detergent and chemical industries, and also in biomedical sciences [3]. Biochemical and molecular characterization of a number of lipases of different sources has brought to light great deal of het-erogeneity in them with regard to specificity, amino acid sequence and catalytic properties.The aim of the present work was to study the biocata-lytic properties and stability of lipases produced by newly strains isolated from the soil (Rhizomucor variabilis and Fusar-ium solani). These two lipases had essentially the same optimal temper-atures and pH, substrate specificities but different pH and thermal stabilities. Enzymes from R. variabilis and F. solani had broad substrate specificity towards both triglyceride substrates and oils since they efficiently hydrolyzed short-chain and long-chain fatty acids but greater preference was observed for the short-chain fatty acids. Tributyrin was found to be the best substrate for the both lipases, among those tested. Lipases produced by Rhizomucor variabilis and Fusarium solani showed the highest stability in the organic solvents with log P value more than 3.5.The ability of these lipases to catalyze the production of fatty acid ethyl esters was also investigated. The effects of enzymes dosage, solvent types, concentration and molar ratio of substrates, and reusability of the lipase prepara-tions in esterification were studied. Degree of esterification between oleic acid and ethanol under optimal conditions reached 87% and 94.2% for lipases from Rhizomucor vari-abilis and Fusarium solani, respectively.References:1. Joseph B et al. (2008) Biotechnol Adv 26: 457–470.2. Gupta R et al. (2003) Biotechnol Appl Biochem 37: 63–71.3. Jaeger KE et al. (2002) Curr Opin Biotechnol 13: 390–397.

P1.3

Structural investigation of β-fructofuranosidase from probiotic Bifidobacterium longumAnna Bujacz, Grzegorz Bujacz, Marzena Krzepkowska-Jędrzejczak, Izabela Redzynia, Stanisław Bielecki

Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Łódź, Polande-mail: Anna Bujacz <bujacza@p.lodz.pl>

We present the structural investigation of β-fructo furano-sidase from Bifidobacterium longum, a unique enzyme, which allows these probiotic bacteria to function in the human digestive system. Bifidobacteria are found in human and animal gastrointestinal tracts as well as in the oral cavity and the vagina. They are among the first bacteria colonizing the sterile digestive system of newborns and they become predominant microorganisms in breast-fed infants. β-fructofuranosidase enzyme belongs to the glycoside hy-drolase family GH32. All these enzymes share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The β-sheets creating the β-propeller are located radialy and pseudosymmetrically around the central axis. Each of them consists of 4 antiparallel β-strands. Overall shape of this domain is cylindrical with a “funnel” shaped channel in-side. The first strand in each blade is closest to the central axial channel, running from the deepest part of it to the opposite side of the molecule. Each fourth strand located on the external surface of this module is connected with the first strand of the next blade. The enzyme investigated by us has an additional N-terminus 19 amino acid α-helix, which distinguishes it from other already known members of the GH32 family. Structural comparison can highlight the modifications of the enzyme that allow the bacteria to be active in the digestive system of a human host. The GH32 family shows a wide range of substrate specificity. The active site is located in the center of the β-propeller domain, in the bottom of the “funnel”. This binding site, responsible for tight fructose binding, is very conserved. The enzyme formed different crystal forms depending on the crystallization conditions. The crystal structures of the β-fructofuranosidase apo form and in complex with a product of hydrolysis — fructose, produced by soaking apo crystals in raffinose, were determined at the resolution of ~1.8 Å.The apo structure was solved by an atypical molecu-lar replacement method using hybrid model created from two different enzymes to which the sequence identity was ~25%. The catalytic domain came from Thermotoga maritima (1W2T), while Aspergillus awamori (1Y4W) provided the β sandwich domain.References:1. Hopkins MJ, MacFarlane GT (2002) J Medic Microbiol 51: 448–454.2. Alberto F, Jordi E, Henrissat B, Czjzek M (2006) Biochem J 395: 457–462.

45th Annual Meeting of the Polish Biochemical Society 9

P1.4

A novel mRNA interferase encoded in toxin-antitoxin system of Staphylococcus aureus targeting tetranucleotide sequenceMichal Bukowski1, Benedykt Wladyka1, Robert Lyzen2, Agnieszka Szalewska-Palasz2, Anna Rojowska1, Grzegorz Dubin3, Adam Dubin1

1Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; 2Department of Molecular Biology, University of Gdansk, Kładki 24, 80-822 Gdansk, Poland; 3Department of Microbiology Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Polande-mail: Michał Bukowski <wladykab@interia.pl>

Small bacterial proteins exhibiting endoribonuclease activ-ity, called also mRNA interferases, are widespread among Prokaryota and currently comprise an object of insightful research. Many of these enzymes are capable to specifically recognise a short sequence of mRNA and catalyse hydroly-sis of sugar-phosphate backbone within it, which leads to transcript degradation and translation inhibition. These factors often posses corresponding protein inhibitors and then consist toxin-antitoxin systems. Activation of such systems triggered by stress conditions switches bacterial metabolism over to a dormant state and can influence bio-film formation and integration through adhesion modula-tion. These phenomena allow the persistence and recovery of microbial colonies, which is important not only during natural progression of an infection but also in the case of antibiotic treatment. Moreover, the demonstration of the relation between toxin-antitoxin systems occurring among virulent strains and the pathogenesis development seems to be a fact of great importance. Taking under considera-tion the key role played by mRNA interferases in regulation of metabolism of bacterial cell, this research gives an op-portunity for better understanding of the physiology of the organisms focusing on the functional analysis of an mRNA interferase called PemKSa and encoded within pemIKSa operon, which is located in pCH91 plasmid of Staphylococcus aureus CH91. The operon is a classic example of toxin-an-titoxin operon encoding toxic for host metabolism protein PemKSa and its inhibitor PemISa. The described enzyme is characterised by a high specificity toward the recognised cleavage tetranucleotide sequence of single stranded RNA. Bioinformatic analysis of the sequence occurrence in cod-ing sequences of the genome of S. aureus ED98, which also codes for the described toxin-antitoxin system, elucidated its potential relation with the strain virulence. These find-ings suggest existence of correlation between activation of PemKSa and induction of bacteriostasis followed by the loss of biofilm integration and an increase in adhesion to host proteins and have pointed possible future directions for further research.References:This work was supported partially by grants N N302 130734 (to AD) and N N301 032834 (to GD) from the Polish Ministry of Science and Higher Education.

P1.5

New strategies in theconstruction of DNA, RNA and protein microarraysBarbara Cegielska1, Marcin K. Chmielewski2, Wojciech T. Markiewcz2, Marek Figlerowicz1

1Center of Excellence for Nucleic Acid-Based Technologies CENAT, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Poland; 2Laboratory of Bioconjugate Chemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, Polande-mail: Barbara Cegielska <barceg@ibch.poznan.pl>

In the advancement of high throughput methods to study biological systems, microarrays have demonstrated dramat-ic progress over the past decade. Microarrays provide the ability to measure the expression of thousands of genes in parallel, even if only a small amount of biological mate-rial is given. As a result it is the main reason, why DNA microarrays have become a powerful tool for studying gene expression and various aspects of genomics. However, traditional DNA microarray technology is not without its challenges and limitations and that is why this technique is constantly improved. For microarrays produced using spotting technologies, much attention has to be given to the development of slide surfaces, attachment chemistry and spotting solutions. The main target are arrays with high sensitivity and low variability. Application of the optimal and reliable methods ensuring effective binding of nucleic acid probes with slide surface is one of the key factors war-ranting high quality results. Developments in the field of microarrays occur at a rapid pace and some novel solutions may offer suggestions of new strategies. The new approach is the idea of synthesis of microarrays using (TPG). This is very attractive concept of chemical synthesis, which is based on removal TPG by temperature in aqueous environ-ment with neutral pH.

Abstracts10

P1.6

Determination of cell permeabilization exposed in vitro tonanosecond-duration high electric field pulsesAnna Choromańska1, Aude Silve2,Nina Skołucka1, Lluis M. Mir2

1Wroclaw Medical University, Department of Medical Biochemistry, Wroclaw, Poland; 2Laboratory of Vectorology and Antitumor Therapies, UMR 8203 CNRS, University Paris-Sud, Institut Gustave-Roussy, 94805 Villejuif, France e-mail: Anna Choromańska <awawrzuta@gmail.com>

The barrier function of plasma membrane of DC-3F cells exposed to nsPEF(nanosecond pulsed electric fields) was examined using two differentmethods, cloning efficacy test after electropermeabilization in thepresence of various concentrations of bleomycin, or the release ofcalcein from cells preloaded with this fluorescent molecule. Weinvesti-gated the effect of 3, 30 and 3000 nanopulses (np) with anelectric generator delivering 7.4 kV on cuvettes with a 1mm gapbetween the electrodes. Three types of pulse repetition frequencywere compared: 10, 100 and 1000 Hz. Cloning efficacy test afterelectropermeabilization with 30 nM of bleomycin showed that cellswere killed with all types of repetition frequency afterelectropermeabilization with 3 and 30 np. The same exposureconditions were not sufficient to observe permeabilization by calceinliberation. Indeed, to observe a decrease in the fluorescence of the-cells, a higher number of pulses (3000 np) was required. In thatcause an effect of frequency was observed.

P1.7

Analytical applications of nucleic acids electroactivityM. Filipiak, M. Tichoniuk, D. Gwiazdowska, M. Ligaj

Chair of Biochemistry and Microbiology, Poznan University of Economics, Poznan, Polande-mail: Marian Filipiak <marian.filipiak@ue.poznan.pl>

Electrochemical properties of nucleic acids have been pre-sented in some review papers [1]. In this work few exam-ples of analytical application of this feature are presented and discussed. DNA oxidation on carbon electrode emerges in irrevers-ible oxidation of guanine and adenine and typical DNA voltammogram shows two distinctive peaks of these two bases. Guanine is the easiest oxidized base and its signal is always present in voltammograms of DNA fragments. The presence of thymine or cytosine in oligonucleotides is recorded in voltammogram when their contribution is significantly high, exceeding 50% of all bases [2]. Some low molecular compounds interact with DNA what leads often to nucleic acid damage. Electrochemical DNA biosensor was developed for detecting specific toxic sub-stance o-dianisidine [3]. Detection was accomplished by monitoring guanine signal of double-stranded deoxyribo-nucleic acid immobilized on carbon paste electrode. In the presence of o-dianisidine guanine response was reduced to the extent proportional to the analyte concentration. Hy-bridization biosensors are usually destined for the detection of microorganisms. In this work an electrochemical bio-sensor for the detection of Aeromonas hydrophila, foodborne pathogen of emerging importance is presented. The detec-tion layer of this biosensor was a gold electrode modified by a self assembled monolayer of thiolated single-stranded DNA probe and alkanethiol as diluent. DNA hybridization biosensors are supposed to be also very useful in detec-tion of genetically modified food. In this work the biosen-sor for the detection of bar gene coding fosphinothricin herbicide resistance is presented. Single-stranded 19-mer DNA probe specific to bar gene was covalently attached by 5'-phosphate end to the surface of modified carbon paste electrode [4]. References: 1. Boussicault F et al (2008) Chem Rev 108: 2622–2645.2. Stempkowska I et al (2008) Bioelectrochem 70: 488–494.3. Jasnowska J et al (2004) Bioelectrochem 64: 85–90.4. Ligaj M et al (2008) Bioelectrochem 74: 32–37.

45th Annual Meeting of the Polish Biochemical Society 11

P1.8

Geomyces sp. P7 lipase as useful enzyme in chemical synthesisTomasz Florczak1, Maurycy Daroch2, Marianna Turkiewicz1, Lesley A Iwanejko2

1Technical Biochemistry Institute, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Łódź, Poland; 2School of Biological Sciences, University of Liverpool, L69 7ZB Crown Street, Liverpool, UKe-mail: Tomasz Florczak <tofman@op.pl>

The lipase synthesized by the Antarctic filamentous fungus Geomtces sp. P7 from the pure culture collection at the In-stitute of Technical Biochemistry of Technical University of Lodz was found to be efficient catalyst of enantiose-lective transesterification of secondary alcohols. Gas chro-matography of products of transesterification reaction of 1-phenylethanol and vinyl acetate, catalyzed by the lipase revealed more than 90% enantioselectivity and almost 50% yield of kinetic resolution (KR) of the racemate.Characterization of the purified enzyme proved its kinetic adaptation to low temperature. At the same time, the en-zyme displayed the surprising capability of renaturating. This result is contradictory to the common opinion that molecules of psychrophilic enzymes generally demon-strate the high flexibility. With an exception of the catalytic center, the remaining part of the molecule of the cold-adapted enzyme can contain more hydrophobic amino acid residues. The similar conclusion was drawn by the Japanese scientists, who studied thermostable enzymes from psy-chrophiles [1]. The isolated lipase in all certainty is an unique biocatalyst, but cultivation of the strain producing this enzyme is dif-ficult due to the very slow growth, low biomass yield, and relatively low optimum growth temperature of around 10oC. For these reason the gene of this lipase was ex-pressed with fusion domain (because of the easy peptide purification) in the Sacharomyces cerevisiae system. To isolate the lipase gene sequence the Lambda Zap cDNA Synthesis Kit (Staratagene) and SMART cDNA (Clontech) synthesis kit were used. In the next step the library was sequenced by pirosequencing and obtained contigs were analyzed by BLAST program. The selected contigs were isolated again from the cDNA library and completed by PCRs reactions. The complete genes were ligated with PYES2.1/V5-HIS-TOPO vector and transformed into BJ5465 Sacharomyce cerevisiae strain. The resulting proteins were compared with native lipase.References:1. Oikawa T et al (2003) J Mol Catal B: Enzymatic 23: 65–70.

P1.9

Screening of the oxidizing activity in non-enzymatic fraction of wood rotting fungiMarcin Grąz, Tomasz Sosnówka

Department of Biochemistry, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Polande-mail: Marcin Grąz <graz@umcs.lublin.pl>

Basidiomycetes fungi are very efficient in wood degradation. Biodegradation of lignocellulose has received attention because of the potential utility of wood sources, and also the need for wood decay protection. Wood rotting fungi secrete an array of oxidative and hydrolytic enzymes in-volved in lignocellulose degradation. But enzymes are too large to penetrate intact wood and initiate lignocellulose degradation. Thus, in the initial step of wood decay the low molecular weight diffusible active agents have to be involved. As low molecular weight agents, organic acids, peptides, reactive oxygen species (Fenton system, Fe2+/H2O2), siderophores and phenolate compounds are con-sidered [1]. All functions of these low molecular weight compounds are not fully explained but their ability to syn-ergistic action with ligninolytic enzymes is confirmed [2]. In our report the low molecular weight fractions (LMFr) (MW < 10 kDa) from 20 species of brown and white rot fungi were screening for their ability to oxidizing phenolic substrates. We have found non-enzymatic oxidative agents in LMFr of tested wood rotting fungi, which could belong to the group of the low molecular weight oxidative peptide factors found recently in fungi [3].References:1. Arantes et al. (2009) Int Biodeter Biodegr 63: 478–483.2. Arantes V, Milagres AMF (2007) Enzyme Microbial Technol 42: 17–22.3. Wang W, Gao PJ (2003) J Biotechnol 101: 119–130.

Abstracts12

P1.10

Selection and induction of Saccharomyces cerevisae for the increase in the ethanol tolerance and productionJan Fiedurek, Anna Gromada, Marcin Skowronek, Olimpia Klikocka

Maria Curie-Skłodowska University in Lublin, Department of Industrial Microbiology, Lublin, Polande-mail: Anna Gromada <robertgromada@vp.pl>

Bio-ethanol production by yeast is a growing industry due to energy and environmental demands. The successful per-formance of alcoholic fermentations depends on the abil-ity of the used yeast strains to cope with a number of stress factors occurring during the process. The objectives of the present study were to select the best ethanol-producing yeast strains from our collection and to improve yeast fermentation performance by adaptation.Microorganisms can adapt to perturbations of the sur-rounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the cul-ture medium. A laboratory strain of S. cerevisiae ER was cultivated in the medium containing a low ethanol con-centration, followed by repetitive cultivations in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repeti-tive cultivations, the yeast cells adapted to the high ethanol concentration showed high biomass accumulation in the medium containing the same ethanol concentration, in comparison to that of the non-adapted strain. The most active strain Saccharomyces cerevisiae MR-A resistant to etha-nol stress was characterized by a high resistance to acid (pH 2.0), oxidative (1% and 2% of H2O2) and high temperature (45oC and 50oC) stress. During cultivation under all stress conditions considerably increased viability was observed, the level of which ranged widely from about 1.04 to 3.94 in comparison with the parent strain Saccharomyces cerevisiae MR.Some of the cultural parameters affecting ethanol fermen-tations on sucrose were standardized. At an initial sucrose concentration of 150 g/l in the mineral medium containing yeast extract, (NH4)2SO4 at 30°C and within 72 h, the most active S. cerevisiae ER-A strain reached an ethanol concen-tration of 80 g/l, an ethanol productivity of 1.1 g/l/h, an ethanol yield (% of theoretical) 99.26. I was significantly higher in comparison with the parent strain (ethanol yield 71.96% and productivity of 0.08 g/l/h).The studies presented above seem to confirm a high ef-ficiency of described method of yeast adaptation for the increase in the ethanol production.

P1.11

Collaboration between two ClpB proteins in resolubilization of inclusion bodies in Escherichia coli cellsIzabela Guenther, Sabina Kędzierska-Mieszkowska

University of Gdańsk, Department of Biochemistry, Gdańsk, Polande-mail: Izabela Guenther <guenther@biotech.ug.gda.pl>

Bacterial AAA+ ATPase ClpB is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chap-erone system. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not con-tain the substrate-interacting N-terminal domain. E. coli produces two isoforms of ClpB to optimize the activity of this chaperone. In our studies we examined the ability of the two differ-ent-sized ClpB homologues to solubilize inclusion bod-ies in E. coli cells. We used a recombinant protein VP1-β-galactosidase (VP1LAC) that is enzymatically active and its production results into cytoplasmic inclusion bodies. We found that the most efficient solubilization of inclu-sion bodies occurs in the presence of both ClpB proteins. After 3 h incubation at 30°C about 75% of VP1LAC was present in the soluble cell fraction, whereas in the clpB mu-tant producing ClpB95 or ClpB80 alone it was 50% and 30%, respectively. An enzymatic activity of the recom-binant protein was also the highest in the ΔclpB mutant cells expressing ClpB80/ClpB95. These results confirm our previous observations that two isoforms of ClpB synergistically cooperate in vivo and pro-duce a highly efficient aggregate-reactivating chaperone system.

45th Annual Meeting of the Polish Biochemical Society 13

P1.12

Structural proteins of bacteriophage A5WUrszula Jankiewicz1, Magdalena Witkowska2, Małgorzata Łobocka1,2, Wiesław Bielawski1

1Warsaw University of Life Sciences - SGGW, Warsaw, Poland; 2Institute of Biochemistry and Biophysics PAS, Warsaw, Polande-mail: Urszula Jankiewicz <urszula_jankiewicz@sggw.pl>

In view of the increasing prevalence of antibiotic resist-ance among many pathogenic bacteria, the acquisition of new bacteriophages and their characterization with practi-cal application in mind is becoming of increasing impor-tance. A5W, a polyvalent Staphylococcus aureus phage, belongs to the family Myoviridae. The objective of this study was to identify the structural proteins of the phage.Phage lysates were purified using ion exchange on ani-onite bed (HPLC). Fractions containing active phages were analyzed by one and two-dimensional electrophoresis in polyacrylamide gels. Protein bands obtained after staining the gels with silver compounds or Coomassie blue were analyzed by mass spectometry (MS Laboratory, IBB). Alto-gether, 14 phage A5W structural proteins were identified, ten of which showed a high degree of amino acid sequence overlap (about 60%). In terms of quantity, the dominat-ing protein was that of the capsid, followed by the main protein forming the tail fibers. A detailed analysis of the data allows to conclude that at least two of the identified phage A5W proteins are subject to post-translational modi-fications.

P1.13

A new approach to heterocyclic aromatic amines determination by the use of electrochemical DNA biosensorJoanna Jasnowska-Małecka, Marian Filipiak

Poznań University of Economics, Department of Biochemistry and Microbiology, Poznań, Poland e-mail: Joanna Jasnowska Małecka <j.jasnowska@ue.poznan.pl>

Heterocyclic aromatic amines (HAA) are carcinogenic and mutagenic compounds, isolated and identified in protein-rich food such as meat and fish prepared with typical household cooking practices. HAAs are formed as a result of chemical reactions of creatine, sugars and amino acids at temperatures over 150oC. One of HAA which is among the most potent mutagens ever tested in Amens bacterial reversion assay and present on the list of IRCA as probably carcinogenic to humans is 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Moreover, IQ presents properties for dou-ble stranded DNA (dsDNA) intercalation. The aim of the work was developing of easy-to-operate, cheap dsDNA electrochemical biosensor for rapid deter-mination of IQ.The experiments were conducted with the use of square-wave voltammetry and tree-electrode system. The analyti-cal procedure consisted of four steps: 1) Pretreatment of carbon paste electrode (CPE) by po-larization. 2) Preparation of biodetection layer responsible for recog-nition of planar 2-amino-3-methylimidazo[4,5-f]quinoline in the sample by dsDNA immobilization on the surface of CPE. 3) Immersion of biosensor for 1 min interaction in the sample or buffer and washing.4) Recording of electrochemical signal of IQ oxidation.Results achieved with dsDNA biosensor were satisfactory. Recorded electrochemical signal was of good reproducibil-ity (RSD 3–13 %). The IQ peak potential at +0.78 V indi-cated DNA intercalation as it shifted slightly into positive potentials after 1 min interaction with DNA. Moreover, ac-cumulation of IQ in dsDNA layer enabled determination of IQ signal after transfer of washed biosensor into elec-trochemical cell. IQ peak was dependent on IQ concentra-tion in the range from 1 to 500 μM. The linear relation between IQ peak and its concentration was between 1 and 10 μM. The limit of detection was 0.1 μM. Direct electrochemistry of IQ with non-modified CPE gave the oxidation peak at +0.75 V with low reproduc-ibility (RSD 30 %). The limit of detection was 1 μM. The analyte did not undergo accumulation at the surface of CPE. There was no signal of IQ observed after transfer of washed electrode to electrochemical cell. Acknowledgements:The work was supported by MNISW grant no. 2PO6T 008 30, Poland.

Abstracts14

P1.14

Enzymatic and molecular properties of a bifunctional enzyme involved in thiamine (vitamin B1) biosynthesis in Saccharomyces cerevisiae-thiamine phosphate synthase/5-(2-hydroxyethyl)-4-methylthiazole kinaseEwa Kowalska1, Mariusz Olczak2, Andrzej Kozik1

1Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Analytical Biochemistry, Kraków, Poland; 2University of Wrocław, Faculty of Biotechnology, Department of Biochemistry, Wrocław, Polande-mail: Ewa Kowalska <ewa.b.kowalska@uj.edu.pl>

Thiamine occurs in all living organisms. It serves as a pre-cursor of thiamine diphosphate (TDP) which functions as a cofactor of many enzymes involved in the main meta-bolic pathways such as glycolysis and tricarboxylic acid cy-cle. Plants and microorganisms can synthesize thiamine de novo while for animals and humans it is an essential nutrient (vitamin B1) that must be acquired with food. Thiamine biosynthesis involves the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyri-midine diphosphate (HMP-PP) and 5-(2-hydroxyethyl)-4-methylthiazole phosphate (HET-P) which are then condensed into thiamine monophosphate (TMP). These pathways are well recognized in bacteria but in a model eu-karyotic organism - the baker’s yeast Saccharomyces cerevisiae - many steps remain poorly characterized. In S. cerevisiae, TMP synthase, the enzyme which catalyzes the condensation of pyrimidine and thiazole moieties, is encoded by the thi6 gene and possesses an additional activ-ity of HET kinase. In the current work, this dual-function protein was obtained by an overexpression in Escherichia coli using the IMPACTTM (Intein Mediated Purification with an Affinity Chitin-binding Tag) expression system and puri-fied by affinity chromatography and high performance gel filtration. Two enzymatic activities of THI6 protein were character-ized in terms of basic kinetic constants, with an interest-ing finding that TMP synthase activity is uncompetitively inhibited by excess of one of the substrates (HMP-PP), ATP and the reaction product, inorganic pyrophosphate. It is also strongly inhibited by UTP. In contrast, the HET kinase activity followed the classical Michaelis-Menten ki-netics and no inhibitors could be identified to date. A bioinformatic analysis of the THI6 sequence suggested that these two activities were located to separate domains, the N-terminal TMP synthase domain and C-terminal HET kinase domain. The models of the overall folds of THI6 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modeling server, on the basis of known three-dimensional structures of two Bacillus subtilis proteins, ThiE (TMP synthase) and ThiK (HET kinase). The essential roles of several amino acid residues for both enzymatic activities of THI6 protein were additionally confirmed by site-directed mutagenesis.

P1.15

Glucosamine-6-phosphate synthase with an oligoHis insertKarolina Kwiatkowska, Justyna Czarnecka, Marek Wojciechowski, Sławomir Milewski

Department of Pharmaceutical Technology & Biochemistry, Gdansk University of Technology, 11/12 Narutowicza, 80-233 Gdansk, Polande-mail: Karolina Kwiatkowska <karolina.kwiatkowska85@gmail.com>

Glucosamine-6-phosphate (GlcN-6-P) synthase known also as L-Glutamine: D-fructose-6-phosphate amidotrans-ferase, catalyzes the first committed step in the pathway leading to the ultimate formation of UDP-GlcNAc. The final product (GlcN-6-P) of this pathway is an activated precursor of numerous macromolecules containing amino sugars, including chitin and mannproteins in fungi, pepti-doglycan and lipopolysaccharides in bacteria, and glycopro-teins in mammals. The important differences between prokaryotic and eu-karyotic forms of the enzyme include a substantially larger size of the latter and sensitivity of the latter but not the former to the feedback inhibition by UDP-GlcNAc. The enzyme has been proposed as a promising target in antifun-gal chemotherapy as well as for the treatment of insulin-independent diabetes mellitus [1].Our studies have been focused on GlcN-6-P synthase from human pathogenic fungi Candida albicans. Determination of the molecular structure of this protein is of crucial im-portance for the possibility of rational design of enzyme inhibitors. Structure of the isomerase domain (ISOM) of C. albicans enzyme was already determined [2] but that of the entire protein is not known, due to the problems with its purification and crystallization. In our approach to fa-cilitate and optimize purification of the enzyme we have designed and constructed several recombinant versions of C. albicans GlcN-6-P synthase containing appropriately lo-cated internal oligoHis fragments. Insertion of oligoHis at the interdomain undecaapeptide linker resulted in an inac-tive enzyme. On the other hand, replacing residues 655-670 of ISOM of the wild-type protein with a hexahistydyl fragment afforded active GlcN-6-P synthase. The result-ing construct overproduced in E. coli could be effectively purified to near homogeneity in a rapid procedure involv-ing metal chelate chromatography. Molecular and catalytic properties of the isolated protein were characterized. References:1. Milewski S (2002) Biochim Biophys Acta 1597: 173–192.2. Raczyńska J et al. (2007) J Mol Biol 372: 672–688.

45th Annual Meeting of the Polish Biochemical Society 15

P1.16

Multiplex polymerase chain reaction for detection of Aeromonas hydrophilaMarta Ligaj, Daniela Gwiazdowska, Mariusz Tichoniuk, Sebastian Sacharowski, Marian Filipiak

Chair of Biochemistry and Microbiology, Poznan University of Economics, Al. Niepodleglosci 10, 61-875 Poznan, Polande-mail: Marta Ligaj <m.ligaj@ue.poznan.pl>

Aeromonas hydrophila has been included in the Contaminant Candidate List of the Environmental Protection Agency (EPA) of potential waterborne pathogens. This bacterium was frequently isolated from aquatic environment, includ-ing ground, surface and drinking water and from differ-ent fresh and raw foods as vegetables, meat, milk and dairy products. A. hydrophila may cause several types of infection, especially is dangerous for children and persons with im-munosuppression (Naharro et al., 2010). During the last years the number of recognized species of the genus Aeromonas has rapidly expanded. Conventional methods used for identification of the Aeromonas species may lead to obtain inappropriate results. To overcome these difficulties we developed a detection system including a multiplex PCR with rpoD, 16S rRNA and aerA genes. The rpoD gene encoding s70 factor (which is one of the sigma factors that confer promoter-specific transcription initia-tion on RNA polymerase) and 16S rRNA gene (encoding a small ribosomal subunit) could be suitable genetic mark-ers for this bacteria identification. Aerolysin gene (aerA) is used for determining potential pathogenicity of A. hy-drophila (Yogananth et al., 2009). The amplified fragments sizes were 584 base-pair (bp) for rpoD gene, 356 bp for 16S rRNA gene and 156 bp for aerA gene. Multiplex PCR system enabled accurate detection of pathogenic strains of A. hydrophila and may be used to distinguish this bacterium from other related species. References:Naharro G et al. (2010) Molecular detection of Foodborne Pathogens (Liu D, ed) 273–288, Taylor & Francis Group, CRC Press USA.Yogananth N et al (2009) Global J Biotechn Biochem 4: 51–53. Acknowledgements:This work was supported by The Polish Ministry of Science and Higher Education (Grant No. N N312 206736).

P1.17

Determination of human fibrinogen N-homocysteinylation sites using mass spectrometric methodsŁukasz Marczak1, Marta Sikora1, Joanna Suszyńska-Zajczyk1, Maciej Stobiecki1, Hieronim Jakubowski1,2

1Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, Poznań, Poland; 2Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USAe-mail: Łukasz Marczak <lukasmar@ibch.poznan.pl>

Homocysteine (Hcy), a sulfur-containing non protein ami-no acid, has become a focus of intense studies in the con-text of human pathophysiology. An abnormally elevated concentration of total Hcy (> 15 uM) is termed hyperho-mocysteinemia and has been recognized as a risk factor for a number of human diseases, including cardiovascular dis-ease. A cyclic thioester of Hcy, homocysteine-thiolactone (HTL), spontaneously forms N-Hcy-protein adducts, in which Hcy is N-linked to the e-amino group of protein lysine residues. Protein N-homocysteinylation affects pro-tein structure and function, causes cellular toxicity, elicits autoimmune response leading to the creation of antibodies against N-Hcy-proteins, and causes aggregation of N-Hcy-fibrinogen in thrombosis, etc.To monitor the protein N-homocysteinylation process we decided to use mass spectrometric methods. The constant development of mass spectrometers in the field of bio-logical experiments made them a perfect tool for protein analysis. ESI or Maldi ionization methods together with quadrupole and ToF analysers became tools of choice for protein identification and posttranslational modifications detection. Also, development of new methodology for quantitative analysis, including application of stable isotope coding of proteins, gives mass spectrometry a chance to become a leading tool also in this field.In presented research we utilized earlier developed in our group approach to localize N-homocysteinylation sites of proteins. To evaluate the homocyteinylation sites of human fibrinogen standard protein was modified with homocysteine thiolactone in vitro and control samples were prepared as well. All analyzes of peptides derived from trypsin digestion were performed on mass spec-trometers, both Maldi-ToF and LC-MS/MS systems were used for qualitative analysis and only LC-MS was applied for quantitation of modified peptides. Confirmation of N-homocysteinylation of predicted peptides was possi-ble due to analysis of fragmentation obtained in tandem mass spectrometer (Q-ToF hybrid analyzer). For purposes of further detailed analysis fibrinogen was separated from human plasma samples and digested with trypsin. To fish out only N-homocysteinylated peptides new method us-ing affinity chromatography on aldehyde resin was applied. Persons with high homocysteine level in blood were taken as donors and the correlation between level of fibrinogen homocysteinylation and concentration of homocysteine in blood was measured.

Abstracts16

P1.18

MSSCP technique in searching MAL gene polymorphisms in industrial strains of Saccharomyces cerevisiae yeastAnna Misiewicz1, Sylwia Wróblewska-Kabba2, Anna Goncerzewicz1

1Institute of Agricultural and Food Biotechnology, Department of Microbiology, Culture Collections of Industrial Microorganisms, Rakowiecka 6, 02-532 Warsaw, Poland; 2Laboratorium Genetyki Medycznej, Centrum Badań DNA Sp. z o.o., Mickiewicza 31, 60-835 Poznań, Polande-mail: Anna Misiewicz <misiewicz@ibprs.pl>

Several important industrial applications of the yeast Sac-charomyces cerevisiae, such a brewing and producing of dis-tilled beverages, rely on efficient fermentation of starch hydrolysates rich in glucose and in the alfa-glucosides mal-tose and maltotriose. In brewer’s wort the most abundant fermentable sugar is maltose (50 to 60%), followed by mal-totriose (15 to 20%) and glucose (10 to 15%). The rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. Maltose fermenta-tion in Saccharomyces cerevisiae requires the presence of one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4 i MAL6. Each locus consists of up to three functionally distinct genes encoding maltose permease, a maltase and a transcriptional activator. Using of cheap, useful, and sensitive screening technique MSSCP (Multitemperature Single Strand Conformation Polymorphism) to DNA poly-morphism or change of sequence identification allow to choose strains do further sequence analysis. After MSSCP of 241-600 nucleotide region of MAL31 U2/L2 gene from 4 strains (KKP 169, 171, 172 and 181) were analyzed by sequence analysis. Our results were compared with data from Blast database to verify. Two substitution in KKP 169 strain was found, substitution of 361 nucleotide in GCT codon changes it to TCT codon, than changes serine to va-line. In KKP 171 strain four substitution were found, one of them change codon CTC/TCT and aminoacid alanine to serine. Seven substitition was found in KKP 172 strain, two from them may have influence on secondary structure of protein. In KKP 181 strain one substitution of 364 nu-cleotide changes serine in alanine. MSSCP analysis is use-ful metod in screening of MAL gene polymorphisms in industrial strain yeasts.

P1.19

Identification and properties of the psychrophilic bacterium Desulfolatea psychrophila single-stranded DNA binding proteinsMarta Nowak, Marcin Olszewski, Józef Kur

Gdańsk University of Technology, Department of Microbiology, Narutowicza 11/12, 80-233 Gdańsk, Polande-mail: Marta Nowak <molszewski@pg.gda.pl>

To study the biochemical properties of SSB from Desul-folatea psychrophila (DpsSSB), we have cloned the ssb genes obtained by PCR and have developed Escherichia coli over-expression systems. The gene consists of an open read-ing frame of 426 nucleotides encoding SSB protein of 142 amino acids with a calculated molecular mass of 15.6 kDa and it is the smallest known bacterial SSB protein indenti-fied to date. The amino-acid sequence of DpsSSB exhib-its 41% identity and 56% similarity to Escherichia coli SSB. In analysis by gel filtration chromatography we show that DpsSSB is functional as homotetramer, with each monomer encoding one single-stranded DNA binding domains (OB-fold). In fluorescence titrations with poly(dT), it binds sin-gle-stranded DNA with a binding site size of about 19–25 nt depending on the salt concentration, and fluorescence is quenched by about 90%. Thermostability with half-lives of about 15 min at 90°C and 5 min at 100°C makes SSB from Desulfolatea psychrophila the most thermostable SSB protein among psychrophilic and mesophilic bacteria indentified to date. DpsSSB is more thermostable even than SSB from T. aquaticus and T. thermophilus offering an very attractive alternative for theirs in numerous applications in diverse molecular biology and analytical methods.

45th Annual Meeting of the Polish Biochemical Society 17

P1.20

The presence of UDPGP in membrane fraction of Gluconacetobacter xylinus E25Marzena Nowakowska, Stanisław Bielecki

Institute of Technical Biochemistry, Technical University of Lodz, Łódź, Polande-mail: Marzena Nowakowska <nowakowska.marzena@wp.pl>

Synthesis of extracellular polysaccharides is one of the phenomena connected with bacterial cells. Among the mi-croorganisms producing cellulose (Rhizobium, Agrobacterium and Sarcina), Gluconacetobacter xylinus E25 is the best producer of this polymer. The unique properties of cellulose are con-firmed by many potential applications (chemical industry, food, pulp, cosmetic) and medicine. G. xylinus E25 cells lose their ability to produce cellulose in agitated culture condi-tion. However, it was also observed that Cel- cells could reverse to cellulose producing forms in static culture condi-tions. It was shown that noncellulose-producing Cel- forms lacked intracellular glucose-1-phosphate uridylyltransferase (UDPGP) enzyme which was responsible for synthesis of UDP-glucose, neccesary for the production of cellulose. In this study we carried out the separation of membrane proteins fraction of three G. xylinus forms (Cel+, Cel- and revertant) by two-dimensional (2-D) gel electrophoresis. On the basis of the comparison of 2-D proteins profiles of revertant cells (which were cultured with and without the addition of stress factor — ethanol) spots comprising two up-regulated protein were selected from the ethanol-treated probe. The analysis of chosen spots has revealed the presence of UDPGP in the membrane fraction of revertants cells. The similar protein spots layout was also observed both in 2-D separation of membrane proteins of cellulose-synthesizing forms (Cel+) and non-producing cellulose (Cel–) cells. Thus the results suggest that these two selected protein spots identified in revertants cells are present in membrane fraction of two others types of G. xylinus E25 cells (Cel+ and Cel–). Nevertheless, because of the fact that so far no information about UDPGP in Cel- mutant has been noted, we chose one of the two spots (corresponding to analyzed spot of revertant form) and analyzed by mass spectrometry. The obtained results have confirmed our supposition. Thus we can suppose that not only intracellular UDPGP (playing an important role in cel-lulose biosynthesis) but probably also isoforms of UDPGP associated with membrane fraction, which functions in eve-ry type of cells independently of their ability to produce cellulose, exists in G. xylinus E25 cells. We can also suppose that identified UDPGPs may play other role (in osmotol-erance, thermotolerance, e.g.) than those present only in cellulose-producing Cel+ cells.

P1.21

Aromatic amino acid aminotransferase from the Antarctic strain Psychrobacter sp. B6-gene isolation and heterologous expressionKarolina M. Nowakowska-Sapota, Marianna Turkiewicz

Institute of Technical Biochemistry, Technical University of Lodz, Lodz, Polande-mail: Karolina Nowakowska Sapota <karolina.nowakowska@gmail.com>

Cold-adapted enzymes produced by psychrophilic micro-organisms display the high catalytic efficiency at low tem-perature and usually they are thermolabile. These prop-erties cause that the application of psychrozymes offers considerable potential to the biotechnology, i.a. because of the possibility to reduce energy input and to increase the stereoselectivity of chemical reactions.Within the scope of this research we carried out the bioin-formatic analysis of the known and deposited in the NCBI database genomes of psychrophilic bacteria from the ge-nus Psychrobacter to find genes of enzymes with potential industrial importance. As an objective of further detailed studies we selected the aromatic amino acid aminotrans-ferase (AroAT).AroAT is an enzyme with the broad substrate specificity and plays a key role in tyrosine and phenylalanine biosyn-thesis. This enzyme is applied mainly in the pharmaceuti-cal industry in production of natural amino acids and their analogues, for example L-homophenylalanine, and also chi-ral intermediates to be used in production of medicines, such as L-2-aminobutyric acid. In the food industry AroAT is used along with other enzymes in cheese-ripening proc-esses.Until now we isolated the aromatic amino acid aminotrans-ferase gene (aroAT), using PCR techniques, from the Antarctic bacterial strain, Psychrobacter sp. B6 belonging to the pure cultures collection at the Institute of Technical Biochemistry of Technical University of Lodz. Sequence of this gene was determined (GenBank, Accession No. GQ253123) and it showed 87% identity with the sequence of AroAT gene from P. cryohalolentis and P. arcticus. Analysis of the amino acid sequence of AroAT protein, deduced from the aroAT ORF, showed that it displayed character-istics typical of psychrozymes. The gene of cold-adapted AroAT was cloned into an expression vector from the pET series and the recombinant protein with carboxy-terminal His-tag was successfully expressed in E. coli host. Cur-rently, we have been working on developing a procedure for AroAT protein purification by affinity column chroma-tography and investigating the activity of the recombinant enzyme.

Abstracts18

P1.22

Novel single-stranded DNA-binding proteins from extreme psychrophilic bacterium Psychromonas ingrahamii 37Marcin Olszewski, Marta Nowak, Natalia Maciejewska, Józef Kur

Gdańsk University of Technology, Department of Microbiology, Narutowicza 11/12, 80-233 Gdańsk, Polande-mail: Marcin Olszewski <molsza@wp.pl>

We report the identification and characterization of the single-stranded DNA binding protein (SSB) from extreme psychrophilic bacterium Psychromonas ingrahamii 37 (Pin-SSB) that grows expotentially at –12°C and may well grow at even lower temperatures. PinSSB is one of the largest known bacterial SSB protein consisting 222 amino acid residues with a calculated molecular mass of 25.1 kDa. The analysis by gel filtration chromatography revealed single peak with a molecular mass of about 77.5 kDa that indi-cates PinSSB is functional as homotetramer containing one single-stranded DNA binding domain (OB-fold) in each monomer. Psychromonas SSB possesses a high sequence homology to Escherichia coli SSB (45% identity and 58% similarity). Fluorescence spectroscopy assays indicated that PinSSB binds single-stranded DNA with a binding site size of 8–10 nucleotides only, depending on the salt concen-tration. Bacterial SSBs proteins indentified to date have at least twice as big ssDNA-binding site. It indicates that very probably PinSSB as huge homotetramer can binds ssDNA in special manner. The half-lives of PinSSB was 5 min at 60°C and 1 min at 70°C. These results showed that PinSSB as the first characterized SSB from strictly psychrophilic microorganizm is partly thermostable SSB protein with unique very short ssDNA-binding site, offering an attrac-tive alternative for other SSB proteins in their applications for molecular biology techniques.

P1.23

Mutagenic and crystallographic characterization of rHint-1, the phosphoramidase with the P-S cleavage activity Magdalena Ozga, Rafał Dolot, Agnieszka Krakowiak, Renata Kaczmarek, Wojciech J. Stec

Centre of Molecular and Macromolecular Studies of the Polish Academy of Science, Department of Bioorganic Chemistry, Lodz, Polande-mail: Magdalena Ozga <mozga@cbmm.lodz.pl>

The histidine triad nucleotide binding protein 1 (Hint-1) is an enzyme of the first branch of the HIT super family that catalyses the hydrolysis of P-N bond in AMP-lysine, AMP-alanine, AMP-morpholidate substrates with the rate of ca.~ 1 nmol/min/mg and adenosine- 5'- monophospho-ramidate (AMP-NH2) with the rate of 1.71 nmol/min/mg [1]. Recently, we reported that in the cleavage reaction of the P-diastereoisomers of 5'-O-[N-(tryptophanylamide)phos-phoramidothioate catalyzed by the wt rHint-1, the hydroly-sis of P-N bond was accompanied by a parallel loss of the sulfur from the resulting AMPS [2]. In an independent experiment we have demonstrated that the wt Hint-1 hydrolyzes the AMPS with the rate of ca. 0.2 nmol/min/mg resulting in formation of AMP and H2S as final products. Previous crystallographic studies on rHint-1 gave a proof that the motif HfHfHff containing three histidines (H) separated by hydrophobic amino acids (f) is involved in nu-cleotide substrate binding and hydrolysis [1, 3]. The activ-ity of Hint-1 depends on the presence of the conserved middle histidine (His-112 for rHint-1) and the mechanism of P-N bond cleavage was revealed by Krakowiak et al [3]. In our extended studies on desulfuration process and the P-S cleavage ability of Hint-1 enzyme, we have used mu-tagenic approach to generate Hint-1 His114 mutants. In order to determine the role of His114 in catalysis of the P-N and P-S bonds cleavage, we have measured the activ-ity of wt rHint1 and its His114 mutants towards AMP-Lys and AMPS. On the other hand we are also interested in the mechanism of rHint-1 action in P-S bond cleavage of AMPS. The crys-tallographic studies on the structure of rHint-1 enzyme in complex with AMPS are in progress. References:1. Bieganowski P et al. (2002) J Biol Chem 277: 10852–10860. 2. Krakowiak A et al. (2007) Chem Comm 2163–2165. 3. Krakowiak A et al. (2004) J Biol Chem 279: 18711–18716.

45th Annual Meeting of the Polish Biochemical Society 19

P1.24

Partial purification and characterization of Sinorhizobium laccaseAnna Pawlik1, Katarzyna Baran1, Monika Marek-Kozaczuk2, Jerzy Wielbo2, Jerzy Rogalski1

1Departament of Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland; 2Departament of Genetics and Microbiology, Maria Curie-Skłodowska University, Lublin, Polande-mail: Anna Pawlik <anpawlik@gmail.com>

Laccases ( benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are the most numerous members of the mul-ticopper protein family, that oxidaze variety of aromatic compounds. Because of their broad substrate specifity and interesting chemical properties (e.g.: high thermostability, alkaline pH-stable activity), laccases harbor great potential to biotechnological processes. These enzymes are used in paper and pulp, food, cosmetic, textile and petrochemical industry, nanobiotechnology, medical diagnostics and for bioremediation and clinical purposes [1].Laccases have mostly been isolated and characterized from plants and fungi. The presence of laccase and laccase-like multicopper oxidases (LMCO) has been recently reported in insects as well as in bacteria. Experimental data and whole genome analysis suggest that laccases are widespread in prokaryotes. Among bacteria enzymes with laccase-like activity have been described participating in cell pigmen-tation, sporulation, phenolic substances and UV radiation resistance, electron transport and also metal oxidation [2]. Despite the knowledge about the occurrence of prokaryo-tic laccases, until now, only few bacterial laccases have been characterized so far.The main goal of this work was to contribute to a better knowledge of these enzymes with respect to potential bio-technological application of laccases.Our earlier studies showed laccase activity in bacteria from the genus Rhizobium collected from Lublin region soils. Si-norhizobium L3,8 cultures were grown in TY medium sup-plemented with copper to the stationary phase and peri-plasmic extract was prepared as described elsewhere [3]. Proteins were precipitated with (NH4)2SO4 and initially purified through gel filtration chromatography steps. The effect of pH and temperature on enzyme activity was ex-amined. Substrate specificity was measured using classical laccase substrates. The Km and Vmax values were calcu-lated from double reciprocal plots. In this study the basic physico-chemical properties of bacterial LMCO were de-termined. L3,8 laccase is a thermo- and pH- stable protein with promising biotechnological application.References:1. Couto SR, Herrera JLT (2006) Biotechnol Adv 24: 500–513.2. Mayer AM, Staples RC (2002) Phytochem 60: 551–565.3. Rosconi F, Fraguas LF, Martinez-Drets G, Castro-Sowinski S (2005) Enzyme Microbial Technol 36: 800–807.

P1.25

Screening for production of bioflocculants in some strains of NocardiaMarzanna Paździoch-Czochra, Agata Rębisz

Department of Biochemistry, Maria Curie-Skłodowska University, Lublin, Polande-mail: Marzanna Paździoch-Czochra <marzanna.pazdzioch-czochra@poczta.umcs.lublin>

Bioflocculants are biodegrable macromolecular polymers produced by algae, bacteria, actinomycetes and fungi dur-ing their growth. A large number of them were reported to belong polysaccharides, functional proteins, glycoproteins and glycolipids. They are known for their ability to clarify turbid water and due to bioflocculants may be practically applied in drinking water and wastewater treatment, the food and fermentation processes. Compared with inor-ganic flocculants, bioflocculants have special advantages such as biodegrability, non-toxicity, harmlessness and lack of secondary pollution. In the present study ten strains of Nocardia was screened out for bioflocculating activity. The cells were cultivated, as reported by Kurane et al. (1986) in media with various sugar and nitrogen components. The investigation revealed that all tested strains produced bioflocculant. The highest bioflocculating activity achieved for kaolin suspension was found in Nocardia autotrophica (89) after 10 days of cultiva-tion. The most preferred carbon and nitrogen source for this strain were glucose and NH4Cl, respectively. More than 90% of bioflocculating activity was released into superna-tant, which indicated that bioflocculant was an extracellu-lar product. The effects of cation concentration were also evaluated. The bioflocculating activity was stimulated in the presence bivalent cations but was inhibited by univalent cations. Preliminary structural studies showed that biofloc-culant produced by Nocardia autotrophica was composed of lipids. The obtained results suggested that bioflocculant might find possible application in wastewater treatment.

Abstracts20

P1.26

Isolation of high quality RNA from nanocellulose producing Gluconacetobacter xylinus cellsMarta Peplińska, Stanisław Bielecki

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences Technical University of Lodz, Lodz, Polande-mail: Marta Peplińska <m.peplinska@gmail.com>

Advanced methods in molecular biology allow for studying gene expression in bacterial cells, leading to a better under-standing of microbial metabolism and physiology. We have chosen SSH (selective subtractive hybridization) technique [1] to study differential gene expression of two Ga. xylinus strains. The Ga. xylinus E25 strain is deposited at pure culture collection of Technical University of Lodz and it is used for cellulose wound dressings production un-der static conditions. Nevertheless, agitated cultures cause appearance of cellulose non-producing forms (Cel-) [2]. Contrary to E25, Ga. xylinus strain NQ5 (ATCC53582) is able to synthesize the cellulose, even with agitation. In our opinion, the comparative transcriptomics can pro-vide new insights into the molecular determinants of bac-terial nanocellulose biosynthesis and regulation of this process in bacteria Ga. xylinus. SSH technique requires mRNA as a starting material. Therefore, in our initial studies we optimized extraction of high quality total RNA and mRNA from cellulose produc-ing Ga. xylinus cells. Methods for isolation of intact RNA with high purity from microorganisms are problematic. Bacterial mRNA, represent only 2% of total RNAs, is dif-ficult to extract because of its short half life, rapid degrada-tion by RNases and general lack of polyA [3]. We tested several modifications of RNA extraction pro-tocols and chose the best procedure for isolation of total RNA and mRNA from Ga. xylinus.This work will be valuable in further studies concerning comparative analysis of gene expression profiles.References:1. De Long SK et al. (2008) Appl Environ Microbiol 74: 225–232.2. Krystynowicz A et al. (2005) Acta Biochim Pol 52: 691–698.3. Schoenberg DR (2007) Nat Chem Biol 3: 535–536.

P1.27

Isolation, purification and preliminary crystallographic studies of two major proteins from mulberry silkworm (Bombyx mori L.) hemolymphAgnieszka J. Pietrzyk1, Anna Bujacz2, Małgorzata Łochyńska3, Mariusz Jaskólski1, Grzegorz Bujacz1,2

1Center of Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; 2Technical University of Lodz, Faculty of Biotechnology and Food Sciences, Insitute of Technical Biochemistry, Lodz, Poland; 3Institute of Natural Fibres and Medicinal Plants, Poznan, Polande-mail: Agnieszka Pietrzyk <pietrzyk.aga@gmail.com>

Bombyx mori L. is an insect of high industrial importance as the source of natural silk fiber. Thus, studies of its devel-opment are both of scientific and practical interest. Major hemolymph proteins are specific to the final instar larvae stage of mulberry silkworm B. mori, when they accumu-late at high titre. The molecular mass of most of them is around 30 kDa. These proteins are often essential for the development of the insect. An example is provided by ju-venile hormone binding protein (JHBP), which is involved in the protection and transport of juvenile hormone (JH) from the site of its synthesis (corpora allata) to target tissues. The presence of the hormone in the hemolymph maintains the larval stage of the insects, whereas decreased titre of JH in the hemolymph leads to metamorphosis. JHBP from B. mori is a homologue of JHBPs from pests such as Galle-ria mellonella, Manduca sexta and Heliothis virescens. These pests present a serious problem for agriculture, thus the structure of the JHBP protein is an important target in the design of novel, specific insecticides.Two 30 kDa proteins from the fifth instar larvae hemol-ymph of B. mori were isolated. The purification protocol involved two chromatography steps. First, gel filtration on Superdex 200 pg resin was carried out and the fractions containing 30 kDa proteins were collected. Next, the pro-teins were separated according to their pI by ion exchange chromatography on Q Sepharose resin. During this proc-ess, two protein peaks were obtained. One of them cor-responds to JHBP, as confirmed by JH-binding activity assays. The second peak corresponds to a protein labeled BmX, which belongs to the low-molecular-mass lipopro-tein family. The crystallization conditions for these proteins were established using the vapor diffusion method and a large set of crystallization buffers. To improve the mor-phology of the JHBP crystals, various additives were also tried. Preliminary X-ray diffraction experiments for these crystals were carried out at cryogenic temperatures using synchrotron radiation. Integration and scaling of the in-tensity data allowed to determine the unit cell parameters and space group symmetry. The crystals of JHBP are or-thorhombic, C2221, and diffract X-rays to 3.0 Å. The crys-tals of BmX are triclinic and diffract X-rays to 1.9 Å. Fur-ther analysis are in progress.Acknowledgements:Project co-funded by the European Union within the European Regional Development Fund.

45th Annual Meeting of the Polish Biochemical Society 21

P1.28

Signal peptidase activates SplB protease during secretionKatarzyna Pustelny1, Grzegorz Dubin3, Justyna Stec-Niemczyk1, Grzegorz M. Popowicz2, Jan Potempa3, Adam Dubin1

1Department of Analytical Biochemistry and 3Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2 Max-Planck Institute of Biochemistry, Martinsried, Germanye-mail: Katarzyna Pustelny <kpustelny@interia.pl>

Staphylococcus auresu is a common and dangerous human pathogen. Among other virulence factors, S. aureus se-cretes up to twelve proteolytic enzymes. Six of those had been thoroughly characterized and their role in virulence is well documented. To the contrary, very little is known about the remaining six proteases encompassed in a single spl operon. High sequence and structure homology to other virulence factors make them interesting target for investi-gation. One of the interesting staphylococcal secreted pro-teins is SplB, a highly specific serine protease. The sequence of mature SplB protease is proceeded by a 36 aa long signal peptide which directs the enzyme into extracellular com-partment. Here we demonstrate that the SplB signal pep-tide plays role not only in protein trafficking but also in inhibition of enzyme activity. Analysis of different length recombinant proteins: 1) full length — signal peptide con-taining protease, 2) SplB artificially extended at N-terminus with two amino acids, and 3) mature SplB, demonstrates markedly decreased activity of first two proteins. The crys-tal structures of second and third proteins show that the removal of N-terminal extension permits formation of a salt bridge and creation of a unique hydrogen-bond net-work. Mutating residues involved in the hydrogen-bond network mimics the decreased activity of the signal pep-tide containing protease directly demonstrating its role in enzyme activity. Such changes affect both the kinetics of hydrolysis and substrate recognition but the direct molecu-lar mechanism is different than the changes observed upon activation of chymotrypsinogen. Since all other proteases secreted by S. aureus contain either specific intracellular inhibitors or significant size profragments, which help to protect the bacterial cytoplasm against deleterious prote-olysis by misdirected enzymes while no inhibitors of Spl proteases were demonstrated, we hypothesize that signal peptide inhibition is a physiological mechanism of cyto-plasm protection against those enzymes.

P1.29

Comparison of papain inhibition by chagasin and cystatin BIzabela Redzynia1, Anna Ljunggren2, Anna Bujacz1, Magnus Abrahamson2, Mariusz Jaskolski3,4, Grzegorz Bujacz1,4

1Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland; 2Department of Laboratory Medicine, Division of Clinical Chemistry and Pharmacology, Lund University, Sweden; 3Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland; 4Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Polande-mail: Izabela Redzynia <izared@p.lodz.pl>

Chagasin is a cysteine protease inhibitor identified in Trypanosoma cruzi, the parasite that causes the irreversible medical condition known as Chagas disease. The parasite expresses a papain-like cysteine protease called cruzipain. Chagasin is associated with cruzipain and takes part in reg-ulation of its activity. At the site of infection, chagasin is secreted outside of the parasite and interacts with host pro-teases. Chagasin belongs to a new, recently defined struc-tural family of cysteine protease inhibitors. Although in size and function it resembles some well-characterized cysteine protease inhibitors, such as cystatins, it has a unique amino acid sequence and a completely different structure.The high resolution crystal structure of the chagasin-pa-pain complex demonstrates an inhibitory wedge composed of three loops which form a number of contacts responsi-ble for the high affinity binding. The structural information of the investigated structures can provide a detailed view of how the parasite protein inhibits host enzymes and will allow comparison of the molecular mechanism of chagasin with other evolutionary distant cysteine protease inhibitors. Comparison of the chagasin structure in apo form and in complexes with proteases enables the analysis of the con-formational changes of its molecule after binding to the enzyme.Comparison with the structure of papain in complex with human cystatin B reveals that, despite an entirely different fold, the two inhibitors utilize very similar atomic interac-tions, leading to essentially identical affinities for the en-zyme. Comparisons of the chagasin-papain complex with high-resolution structures of chagasin in complexes with cathepsins and falcipain allowed the creation of a consen-sus map of the structural features that are important for ef-ficient inhibition of papain-like enzymes. The comparisons also revealed a number of unique interactions that can be used to design enzyme-specific inhibitors. Since papain ex-hibits high structural similarity of the studied C1 proteases to the catalytic domain of the T. cruzi enzyme cruzipain, the chagasin-papain complex provides a reliable model of chagasin-cruzipain interactions. Such information, coupled with our identification of specificity-conferring interac-tions, should be important for the development of drugs for the treatment of the devastating Chagas disease caused by this parasite.References:Redzynia I et al. (2009) FEBS J 276: 793–806.

Abstracts22

P1.30

The combination of bleomycin and electropermeabilization by means of nanosecond pulsed electric fieldsNina Skołucka1, Aude Silve2, Anna Choromańska1, Lluis M. Mir 2

1Wroclaw Medical University, Department of Medical Biochemistry, Wroclaw, Poland; 2 Laboratoire Vectorologie et Transfert de Gènes, UMR 8121 CNRS, Institut Gustave-Roussy, 94805 Villejuif Cédex, Francee-mail: Nina Skołucka <nina.skolucka@gmail.com>

Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity. In this study we in-vestigated the effect of nanosecond pulses on DC-3F cell line. Determination of cell permeabilization was examined using cloning efficacy test after electropermeabilization in the presence of 30 μM of bleomycin. We examined the effect of 3, 30, 300, 3000 and 10 000 nanopulses (np) with 1000 Hz repetition frequency and an electric generator de-livering 7.4 kV on cuvettes with a 1mm gap between the electrodes. Cloning efficacy test after electropermeabiliza-tion with 30 μM of bleomycin showed that cells were killed after each number of pulses, while in the same exposure conditions but without bleomycin cells survived.

P1.31

Utilization of biomass for preparation of environmentally friendly polymer materialsŁukasz Stańczyk, Katarzyna Struszczyk, Mirosława Szczęsna-Antczak, Małgorzata Rzyska, Agnieszka Włodarczyk, Barbara Sikora, Małgorzata Piotrowicz-Wasiak, Dariusz Hiler, Tadeusz Antczak

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Polande-mail: Łukasz Stańczyk <lukasz.stanczyk@p.lodz.pl>

The objective of task 2.2 of POIG.01.01.02-10-123/09 project is the development of production procedure of microbial lipase preparations, e.g. from lipolytic Mucor circinelloides and Mucor racemosus strains from pure culture collection at ITB TUL, in a large laboratory scale. These lipase preparations will be used in production of polyols from plant oils, e.g. by means of selective acidolysis of oil by saturated fatty acids and directed hydrolysis of lipid oli-gomers. It is planned to develop appropriate lipase preparations in-cluding the following ones:Mycelium of Mucor filamentous fungi immobilized in a po-rous carrier in the form of uniform thin foams with open porosity and the large internal surface. These foams bear-ing the immobilized, intracellular lipase will be used in plant oil acidolysis and as a partition in a specially designed reac-tor to be used in the hydrolysis of oligolipids in a bi-phasic system water- organic solvent. Dehydrated, ground (particles with a diameter close to 3 μm) and additionally stabilized Mucor mycelium. The Mu-cor mycelium is known to catalyze hydrolysis of ester bonds in lipds at the interface of bi-phasic systems.Water-soluble Mucor lipases obtained by extraction of pro-teins from mycelium with aqueous detergent solutions. Commercial (nonspecific and sn-1,3 specific) lipases immo-bilized within the scope of this project on porous carriers. These preparations will be tested along with Mucor lipases in processes of fatty acid exchange in triacylglycerols and in hydrolysis of lipid oligomers.Processes of production of immobilized preparations of Mucor lipases will be optimized in terms of: culture medium composition, method of agitation in a fermentor, type of porous carrier (pore size, shape and dimensions of the po-rous carrier), conditions of de-fatting and de-hydration of mycelium (including pH of lipase microenvironment and water activity (aw) of preparation). It is also planned to de-velop a quick method enabling analysis of the degree of colonization of the porous material by filamentous fungi based on digital techniques of image analysis. One of ob-jectives of this task is also the development of a method of long-term storage and activation of immobilized prepara-tions of Mucor lipases. Task 2.2. Enzymes for production of polyols from biomass derived from oleaginous plants.Acknowledgements:Project POIG 01.01.02-10-123/09 partially financed by the European Union within the European Regional Development Fund. The project is envisaged for the time frame 2009-2013.

45th Annual Meeting of the Polish Biochemical Society 23

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Biotransformation of chitosanKatarzyna Struszczyk, Łukasz Stańczyk, Radosław Bonikowski, Dariusz Hiler, Mirosława Szczęsna-Antczak, Tadeusz Antczak

Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Lodz, Polande-mail: katarzyna struszczyk <katarzyna.struszczyk@p.lodz.pl>

Chitosan is a N-deacetylated derivative of chitin. Basic properties of this biopolymer, resulting in its diverse indus-trial applications, are biocompatibility, bioactivity and bio-degradability. Chemical, physical or enzymatic degradation of chitosan produces oligoaminosaccharides – that have many interesting properties. These chitosan derivatives dis-play numerous biological activities: antibacterial, antifun-gal, antiviral, antitumor and antioxidant, exert fat lowering effect and stimulate immune system. Fractions with low polymerization degree are very well soluble at neutral pH. Enzymatic method of chitosan hydrolysis (unlike chemical and physical methods) yields products with an intended de-gree of polymerization and N-deacetylation — parameters which determine their biological activity. Our studies showed that enzymatic preparations of Mucor circinelloides and Mucor racemosus were applicable to produc-tion of chitosan oligomers with different polymerization degrees: low-molecular weight chitosan, chitooligosaccha-rides and D-glucosamine. These compounds produced in continuous bioconversion processes are components of many cosmetics and pharmaceutical agents sought after by companies.Acknowledgements:This work is part-financed by the European Union within the European Regional Development Fund, project ”Biotransformations for pharma-ceutical and cosmetics industry” No. POIG.01.03.01-00-158/09-01.

P1.33

Characterization of Phlebia lindtneri cellobiose dehydrogenaseJustyna Sulej, Joanna Sarna, Karolina Żuber, Jerzy Rogalski

Department of Biochemistry, Maria Curie-Skłodowska University, Lublin, Polande-mail: Justyna Sulej <justynasulej@o2.pl>

Cellobiose dehydrogenase (CDH; EC 1.1.99.18; cellobiose: (acceptor) 1-oxidoreductase) is an extracellular haemofla-voenzyme produced by a number of lignocellulolytic fungi growing on cellulose. It is a monomeric glycoprotein con-sisting of two domains, C-terminal flavin domain with a non-covalently bound FAD and N-terminal heme domain containing a cytochrome b type heme.These domains are joined by protease sensitive peptide linker rich in hydroxy amino acids. CDH oxidises the reducing end of cellobi-ose, lactose and cello-oligosaccharides to their correspond-ing 1,5-lactones, which are subsequently hydrolysed to the carboxylic acids. Because of its attractive and unique properties, CDH has several technical applications, e.g. in biosensors for the sensitive and selective detection of toxic diphenols or lactose, in bioremediation for the degradation of recalcitrant pollutants including munitions and polyacr-ylate polymers, or in biocatalysis for the preparation of or-ganic acids [1].The present study has been carried out to explore the pos-sible biotechnological applications of the fungus as a CDH producer. Although CDH has been reported from many fungi, most of these organisms are low producers. Phlebia lindtneri from Fungi Collection of the Biochemistry De-partment, UMCS, a new source of cellobiose dehydroge-nase is an efficient producer of the enzyme. The Optimi-zation of the cultural conditions was studied in 24-wells plates. Maximum CDH production in Phlebia lindtneri cultures was observed after 8 day of incubation (35°C) using basic culture medium with 0.2% Avicel and 1% (NH4)2HPO4. The enzyme has been purified from culture fluid and its physicochemical properties have been studied.References:1. Ludwig R et al. (2004) Appl Microbiol Biotechnol 64: 213–222.

Abstracts24

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Application of DNA electrochemical biosensor for the detection of foodborne pathogen Aeromonas hydrophilaMariusz Tichoniuk, Marta Ligaj, Daniela Gwiazdowska, Marian Filipiak

Chair of Biochemistry and Microbiology, Poznan University of Economics, Poznan, Polande-mail: Mariusz Tichoniuk <mariusz.tichoniuk@ue.poznan.pl>

The fast and reliable detection of foodborne pathogens is one of the most desirable goals in contemporary food qual-ity assessment. Aeromonas hydrophila is specially hazardous because of its psychrophilic nature and ability to produce a broad range of virulence factors (e.g. toxins like aero-lysin). The conventional approaches of its detection suf-fer from numerous disadvantages amongst other time- and labour-consuming. Therefore, there is permanent demand for rapid and reliable alternative methods of this microor-ganism detection. In our work we make an attempt to develop electrochemi-cal biosensor for A. hydrophila identification. The recogni-tion interface consisted of single-stranded DNA probe (ssDNA probe), specific for the target aerolysine gene fragments, and diluent molecules, 6-mercapto-1-hexanol (MCH). The general characterization of recognition inter-face was performed by electrochemical capacitance meas-urement. The better organized monolayer, the lower capac-itance values were measured. For example, electric capacity for gold surface modified with ssDNA probe equaled 10 μF ∙ cm–2. Introduction of MCH into detection layer led to the capacitance decrease to the value of 4 μF ∙ cm–2. Detection of target nucleic acid fragments based on DNA duplex formation on the electrode surface which was mon-itored using electroactive indicators, methylene blue (MB) and Hoechst 33258. The first one revealed higher affinity to ssDNA probe than DNA duplex. Voltammetric response of accumulated MB enabled efficient discrimination be-tween the DNA samples isolated from A. hydrophila and other reference microorganisms. Hoechst 33258 indicator showed higher accumulation rate for DNA duplexes than ssDNA probes. The voltammetric signal after successful hybridization reaction was almost two times higher than in the presence of ssDNA probes. The presented biosensor enabled detection of nucleic acid fragments specific for A. hydrophila aerolysin gene. Estab-lished approach provided promising features towards the development an DNA hybridization biosensor for the de-tection of foodborne pathogens. Acknowledgements:This work was supported by the Polish Ministry of Science and Higher Education (Grant No. N N312 206736).

P1.35

Potential application of staphylococcal pCH91 plasmid in biotechnologyBenedykt Wladyka1, Weronika M. Ilczyszyn1,2, Joanna Pogwizd2, Anna Rojowska1, Natalia Malachowa2, Emilia Bonar1, Klaudia Polakowska2, Grzegorz Dubin2, Adam Dubin1

Department of 1Analytical Biochemistry and 2Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Polande-mail: Benedykt Wladyka <wladykab@interia.pl>

Among the branches of modern biotechnology, produc-tion of recombinant proteins is undoubtedly one with the fastest growth. A success in the field may be achieved by minimizing the costs and maximizing the income. The production process, therefore, should be based on inex-pensive media and results in high amount of recombinant proteins in relatively short time. To achieve this goal, the most widespread method is the application of bacteria cells transformed with a plasmid coding for protein of interest under the control of a strong promoter. However in the biotechnological industry, contrary to a laboratory scale, the application of antibiotic pressure for stable plasmid maintenance and promoters requiring expensive inducers are not welcomed. The strain Staphylococcus aureus CH91 overproduces an ex-tracellular cysteine protease which is encoded in the sta-bly maintained pCH91 plasmid. Bioinformatical analysis revealed an operon coding for possible plasmid addiction system. Further biochemical studies confirmed that the module consists of a stable toxin and its proteinaceous la-bile inhibitor. Here we provide data for an application of the promoter of cysteine protease gene in construction of various vectors enabling inducer-free homo- (S. aureus) and heterologous (Bacillus subtilis) overexpression of recombinant proteins as exemplified by cysteine and serine protease like B proteases as well as green fluorescent protein. Moreover, a possible application of toxin-antitoxin system in stable plasmid maintenance without antibiotic pressure was tested. Us-ing replica plate method we showed that when the toxin coding gene was introduced to Escherichia coli genome the plasmid coding for the antitoxin was stably propagated to the daughter cells without a need for external pressure. Ad-ditionally, the plasmid coding for plasmid addiction system and green fluorescent protein under the control of cysteine protease promoter introduced to S. aureus RN4220 strain was stably maintained in antibiotic-free bacterial culture over a week. In summary, two components of staphylococ-cal pCH91 plasmid were successfully applied in the con-struction of vectors useful for biotechnological production of recombinant proteins.Acknowledgements:This work was supported partially by grants N N302 130734 (to AD) and N N301 032834 (to GD) from the Polish Ministry of Science and Higher Education.

45th Annual Meeting of the Polish Biochemical Society 25

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Influence of the Congo Red’s binding to polymers conditions on the colour of fluorescenceGrzegorz Zemanek, Anna Jagusiak, Barbara Piekarska, Pawel Spólnik, Barbara Stopa

Chair of Medical Biochemistry, Medical College, Jagiellonian University, Kraków, Polande-mail: Grzegorz Zemanek <mbzemane@cyf-kr.edu.pl>

Congo Red is a bis-azo dye, which molecules associate with one another in a determined and ordered way while found in water solutions. Hydrophobic core of the sin-gular molecule, consisting of aromatic rings, bonds with aromatic rings of other dye molecules, and originates su-pramolecular liquid crystal. Arisen micelle takes a form of a ribbon, in which, side amino- and sulfo- groups endowed with electric charge are exposed towards hydrophilic sol-vent and they are able to create hydrogen bonds with it. Shape and plasticity of ribbon-like micelle, resulting from no covalence bonds between monomer molecules enables it to adjust to exposed and ordered hydrophobic structures inside the sphere protein molecules. Due to the large size of a molecule, supramolecular dyes, as opposed to non ag-gregating substances of similar chemical structure, have limited capacity to penetrate inside most native proteins. In water solutions, Congo Red does not demonstrate any fluorescence, but it`s behavior changes after its association with polymers. In case of Congo Red association with cel-lulose, amyloidal proteins and denatured proteins - red flu-orescence appears. It is characterized by high intensity and persistence. Identification of particular conditions enabling Congo Red molecules to emit visible light, may allow us to understand better the mechanism responsible for that dye’s association with both: protein and non-protein ligands. The discovery of association mechanism may explain not only a spatial configuration of supramolekular dye mol-ecules, but also a structure with which this dye associates. We examined changes in fluorescence intensity in relation to particular substances associating with Congo Red and conditions of these associations. As a Congo Red’s base we used proteins, polysaccharides and cyanoacrylate. To be able to evaluate the intensity of bonds, during tests, we de-vised a method of quantitative measurement of changes in the color of fluorescence on microscopic images, after dig-ital acquisition of image from fluorescent microscope. We submitted the results to statistic analysis after mathematical compensation of background interference for individual images.