Enzymology and Applied Microbiology Laboratory

Head: Nadia Bucurenci, PhD, senior researcher, nadiab@cantacuzino.ro

Staff

Adrian Onu, PhD, senior researcher, Adrian.Onu@cantacuzino.ro

Adriana Zoe Costache, PhD student, researcher, aradulescu@cantacuzino.ro

Ana-Maria Eftimie, MS, researcher, aeftimie@cantacuzino.ro

Florina Toma, MS, PhD student, researcher, tflorina@cantacuzino.ro

Short presentation

Basic activities: cloning, over expression, purification of bacterial proteins (enzymes).

Major scientific interests:

•  
  • bacterial nucleoside monophosphate kinase family as new therapeutic targets for antibacterial agents
  • new diagnostic methods using mono- or polyclonal antibodies or coupled enzymatic reactions (toxin sub-units, bacterial antigens etc.)
  • enzymatic regeneration of ATP and NADH in industrial enzymatic syntheses

Research report

Enzymatic regeneration of ATP in industrial enzymatic syntheses

Phosphorylation is often used in industrial enzymatic syntheses. ATP (phosphate donor) needs to be regenerated during the synthesis in order to have an economically convenient procedure.

Syntheses: phosphorylation of adenosine monophosphate to adenosine diphosphate, phosphorylation of guanosine to guanosine monophosphate,  phosphorylation of glucose to glucose-6-phosphate

ATP regeneration systems: creatine kinase / creatine phosphate; acetate kinase (AcK) / acetyl phosphate (AcP)

AcP: Na salt, Li salt, generated in situ

Generating AcP in situ: we cloned and used pyruvate oxidase, which catalyses the synthesis of AcP from pyruvate, phosphate and oxygen; to avoid damages caused by hydrogen peroxide (the other reaction product) we had to add several coupled reactions including a NADH regenerating system. The overall reaction involves AcK, pyruvate oxidase, catalase, alcohol dehydrogenase, glucose dehydrogenase and glucose mutarotase.

Results: The best yield (87%) obtained for the phosphorylation of glucose to glucose-6-phosphate, using acetyl phosphate generated in situ, proved that ATP was regenerated and the whole system was functional. 

Phosphorylation of Gua to GMP is still under study because the yield obtained was not satisfactory probably due to enzyme inhibition by reaction products or intermediary.

Development of a new reagent for tuberculosis (TB) skin testing using specific recombinant antigens

The traditional tuberculin skin test lacks specificity for TB. Cross-reactions are due to vaccination with BCG or to infection with other mycobacteria.

Development of new TB-specific skin test requires identification of M.tuberculosis (M.tb)-specific antigens that elicit DTH responses in TB patients but not in BCG vaccinated persons. Since culture filtrate antigens are usually potent in DTH-based immunoassays we chose proteins secreted by M.tb during its early and active growth phase.

Four such proteins (ESAT6, CFP10, MPT64, PPE68), already identified as being unique for M.tb, were found in M.tb H37Rv genome and the corresponding genes were cloned. The CFP10 protein was over expressed, purified and tested for DTH response in guinea pigs, with promising results.

Implementation and optimization of a technological process for obtaining therapeutic sera with F(ab’)₂ fragments against highly pathogenic bacterial and viral agents

The major objective of the project is the development of laboratory scale methods for obtaining therapeutic sera with divalent F(ab’)₂ fragments as active components.

The project has a distinct impact on the national health system because it offers the premises for autonomy at national level concerning the production of therapeutic sera used for the treatment of some severe acute diseases with bacterial or viral etiology .

Innovative technological solutions :

• harvesting of hyper-immune plasma by plasmapheresis
• fibrin/fibrinogen removing from plasma by cryoprecipitation
• controlled pepsin enzymatic digestion of plasma in acidic medium
• processing, purification and concentration methods with single-use systems
• flexible technological process adjustable for different antigen types

Antigens produced and characterized: tetanus anatoxin from Clostridium tetani TD21 (Lister Institute - London), anthrax antigen from Bacillus anthracis 34 F₂ (Sterne), viral antigens from H5N1 – NIBRG-14 strain (reassortant virus prepared by reverse-genetics from A/Vietnam/1194/04 and A/PR/8/34 – H1N1 strains).

Hyper-immune plasma (serum) obtained : horse serum and plasma against tetanus anatoxin, sheep serum against NIBRG – 14 H5N1 virus, sheep serum against PR/8/34 virus, horse serum and plasma against Bacillus anthracis 34 F₂.

Our research was supported by national (The Romanian Agency for Research) grants

Publications (2000 - 2008):

• Genetic and Biochemical Characterization of Salmonella enterica Serovar Typhi Deoxyribokinase - L.Tourneux, N.Bucurenci, C.Saveanu, P.A.Kaminski, M.Bouzon, E.Pistotnik, A.Namane, P.Marliere, O.Barzu, I.L.de la Sierra, J.Neuhard, A.M.Gilles, 2000, J.Bact., 182(4), 869 - 873
• Sugar specificity of bacterial CMP kinases revealed by crystal structures and mutagenesis of Escherichia coli enzyme - T.Bertrand, P.Briozzo, L.Assairi, A.Ofiteru, N.Bucurenci, H.Munier-Lehmann, B.Golinelli-Pimpaneau, O.Barzu, A-M.Gilles, 2002, J.Mol.Biol., 315, 1099-1110
• Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase - G.Labesse, N.Bucurenci, D.Douguet, H.Sakamoto, S.Landais, C.Gagyi, A-M.Gilles, O.Barzu, 2002, Biochem.Biophys.Res.Commun., 294, 173-179
• UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity - Cristina Gagyi, Nadia Bucurenci, Ovidiu Sirbu, Gilles Labesse, Mihaela Ionescu, Augustin Ofiteru,Liliane Assairi, Stephanie Landais, Antoine Danchin, Octavian Barzu and Anne-Marie Gilles, 2003, Eur.J.Biochem., 270, 3196–3204
• Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae 01 and non 01 in Romania - Anca Israil, Carmen Balotescu, Nadia Bucurenci, Nadia Nacescu, Claudia Cedru, Cornelia Popa, C.Ciufecu, 2003, Roum. Arch. Microbiol. Immunol., 62(3-4), 155-177
• Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains - Anca Israil, Carmen Balotescu, Maria Damian, Cristina Dinu and Nadia Bucurenci, 2004, Roum. Arch. Microbiol. Immunol., 63 (1-2), 63-77
• Characterization of Vibrio cholerae strains isolated in the Republic of Moldavia between 1995-1999 - Anca Israil, Carmen Balotescu, Ionela Alexandru, Radu Cojocaru, Nadia Bucurenci, Valeriu Chicu, Ludmila Mutoi, 2005, Roum. Biotechnol. Lett., 10 (6), 2441-2445
• Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase - Guillaume Hible, Louis Renault, Francis Schaeffer, Petya Christova, Adriana Zoe Radulescu, Cecile Evrin, Anne-Marie Gilles, Jacqueline Cherfils, 2005, J.Mol.Biol., 352, 1044-1059
• Structural and functional consequences of single amino acid substitutions in the pyrimidine base binding pocket of Escherichia coli CMP kinase - Augustin Ofiteru, Nadia Bucurenci, Emil Alexov, Thomas Bertrand, Pierre Briozzo, Hélène Munier-Lehmann, Anne-Marie Gilles, 2007, FEBS Journal, 274(13) , 3363–3373
• Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria - Evrin C, Straut M, Slavova-Azmanova N, Bucurenci N, Onu A, Assairi L,Ionescu M, Palibroda N, Barzu O, Gilles AM.,  2007, J.Biol. Chem, 282(10), 7242-7253
• Fluorescent Study of the Interaction between Staphylococcus aureus UMP kinase and UTP - C.Chilom, D. Gazdaru, N. Bucurenci, M. Straut, A. Popescu, 2007, J.of Optoelectronics and Advanced Materials, 9(8), 2585-2588
• Characterization of Guanylate Kinase from gram positive and gram negative microorganisms; preliminary results – Ana-Maria Ruxandra Eftimie, Florina Toma, Adriana-Zoe Costache and Nadia Bucurenci, 2007, Roum. Arch. Microbiol. Immunol.,  66(1-2), 22-25
• Stereoselective synthesis of L-[¹⁵N] amino acids with glucose dehydrogenase and galactose mutarotase as NADH regenerating system - Maria Chiriac, Iulia Lupan, N. Bucurenci, O. Popescu, N. Palibroda, Journal of Labelled Compounds and Radiopharmaceuticals , 2008, 51 (4) , 171-174