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Project CEEX : BIOTECH – Nr. 129 / 2006



PRESENTATION OF THE PROJECT

       
 


General presentation
 

        The aim of the project is to obtain by enzymatic synthesis the following amino acids isotopically labelled with 15N: 15N L-aspartic acid, 15N L glutamic acid, 15N L-serine and 15N L-methionine.

        For that purpose the following enzymes are considered: Aspartase, Glutamate dehydrogenase (GluDH), Alanine dehydrogenase (AlaDH) and Phenylalanine dehydrogenase (PheDH), together with Leucine dehydrogenase (LeuDH) and Glucose dehydrogenase (GlucDH), the last two enzymes being already obtained during a former project:Biotech/ 2003.

        Also proteins (15.000-40.000 DA) uniform labelled  with 15N and proteins specifically labelled with L-leucine-15N will be obtained by the recombinant DNA technology. The main problem of the enzymatic biosynthesis of labelled 15N L-amino acids is their obtaining at a reduced production cost, the obtaining of the highest possible yield of the enzymatic reaction.The project also involves the use of analysis methods for the caracterization of 15N L-amino acids: MS, IR, RX diffraction and NMR spectroscopy on the 15N nucleus. Using 2D NMR experiments will elucidate the 15N labelled protein structure.The main objective consists in the workout of the bio-technologies for the obtaining of L-amino acids of minimum 99 % purity and 99 at.% 15N isotopic concentration of 15N.
 

The Main objective :


       The aim of the project is to work out molecular bio-technologies for the obtaining of some L-amino acids isotopically labelled with 15N of minimum 99 % purity and 99 at.% 15 N isotopic concentration at.15N.
 

 
The specific research objectives proposed under the project :
 

1. Preliminary experiments concerning enzymatic biosynthesis of L-aspartic acid labelled with 15N, and preparation of Aspartase;

2. The elaboration of the molecular biotechnology for the preparation of L-aspartic acid -15N at 99 at.% 15N isotopic conc., 99% purity and high isotopic yield;

3. The elaboration of the molecular biotechnology for the preparation of L-glutamic acid-15N at 99 at.% 15N isotopic conc., 99% purity and high isotopic yield ; The characterization of labelled compounds with 15N by MS, IR, RX diffraction and NMR analysis;

4. The elaboration of the molecular biotechnology for the preparation of L-serine-15N at 99 at.% 15N isotopic conc., 99% purity and high isotopic yield; The biosynthesis of protein uniform labelled with 15N; MS, IR, RX diffraction and NMR studies on L-serine-15N;The determination of proteins-15N structure by 2D NMR analysis;

5. The elaboration of molecular biotechnology of L-methionine-15N at 99 at.% 15N isotopic conc., 99% purity and high isotopic yield; The protein biosynthesis specifically labelled with L-leucine-15N; SM, IR, RX diffraction and NMR studies on L-methionine-15N; The determination of protein structure labelled specifically with L-leucine-15N by 2D NMR analysis;

 

 
Measurable objective :
 

- biochemical characterization of the enzymes ( Km and Vmax ) obtained by recombinant DNA technology;

- determination of the parameters of enzymatic reaction synthesis of the L-amino acids (pH, temperature, reactant concentrations, etc);

- control of 15NH3 consumption in the biosynthesis of L-amino acids, theoretical and practical aspects;

- determination of the conditions and the purification method of 15N-L-amino acids;

- determination of the chemical and isotopic yield of the enzymatic synthesis reaction;

- measurement of the chemical purity and isotopic concentration of 15N-L-amino acids by MS, IR, RX and NMR;

- determination of the conditions for biosynthesis of proteins (15.000-40.000 DA) labelled uniformly with 15N and labelled specifically with L-leucine-15N;

- determination of 15N labelled proteins structure by 2D NMR experiments.

 

 

Stages and activities :

 

Stage I. Preliminary experiments concerning enzymatic biosynthesis of L- aspartic acid labelled with 15N.

 

  • Activity I.1. The determination of experimental conditions on biosynthesis of L-aspartic acid labelled with 15N ;
  • Activity I.2. Preparation of Aspartase and its biochemical characterization (Km and Vmax) ;

 

Stage II.  The elaboration of molecular biotechnology method for the preparation of L-aspartic acid -15N.

 

  • Activity II.1. Preparation of L-aspartic acid labelled with 15N at 99 at.% 15N isotopic conc. and min. 99% purity ;
  • Activity II.2. Preparation of Glutamate dehydrogenase and its biochemical characterization (Km and Vmax) ;
  • Activity II.3. High resolution 15N NMR characterization of L –aminoacids labelled with 15N.

 

 

Stage III. The elaboration of molecular biotechnology method for the preparation of L-glutamic acid -15N.

 

  • Activity III.1. Preparation of L- glutamic acid labelled with 15N at 99 at.% 15N isotopic conc. and min. 99% purity ;
  • Activity III.2. Preparation of alanine and phenylalanine dehydrogenases and their biochemical characterization (Km and Vmax);
  • Activity III.3. High resolution 15N NMR characterization of L-glutamic-15N and L-aspartic-15N acids.

 

 

 Stage IV. The elaboration of molecular biotechnology method for the preparation of L-serine -15N.

 

  • Activity IV.1. Preparation of L-serine  labelled with 15N at 99 isotopic conc. at.% 15N and min. 99% purity ;
  • Activity IV.2. The synthesis of uniform labelled proteins with 15N ;
  • Activity IV.3. 15N NMR analyses on L-serine-15N.

 

 

Stage V. The elaboration of molecular biotechnology method for the preparation of L-methionine -15N.

 

  • Activity V.1. Preparation of L-methionine  labelled with 15N at 99 isotopic conc. at.% 15N and min. 99% purity ;
  • Activity V.2. The synthesis of proteins specifically labelled with L-leucine-15N;
  • Activity V.3. 15N NMR analyses on L-methionine-15N ; 2D NMR spectra recording and processing for L-leucine-15N labelled proteines.
 

 
Results :
 

 


 
Participants :
 
Project Coordonator : INCDTIM Cluj-Napoca
Dr. Maria Chiriac – Director of the project
 
Dr. Palibroda Nicolae
Dr. Axente Damian
Dr. Bratu Ioan
Dr. Borodi Gheorghe
Dr. Moldovan Zaharia
Dr. Farcas Sorin
Dr. Dronca Simion
Ing. Chim. Marcu Cristina
Ing. Chim. Popa Flavia
Fiz. Schmutze Gabriella
Ec. Vlas Emilia
Ing. Albert Stefan
Tehn. Vermesan Eugenia
Tehn. Pop Ligia
Tehn. Golescu Viorica
Tehn. Medgyesi Edita
 
Partner 1 – Faculty of  Biology and Geology, UBB Cluj-Napoca
Prof. Dr. Popescu Octavian
 
Prof. Dr. Coman Nicolae
Conf. Dr. Dordea Manuela
Drd. Bel Simona Maria
Drd. Lupan Iulia
Drd. Kelemen Beatrice Simona
Drd. Ferencz Beatrix-Katalin
Drd. Bacila Ioan
Drd. Chilici Anet-Monica
Drd. Jakab Endre
Drd. Suteu Dana Cristina
Drd. Basangiu Florentina Bianca
Drd. Bucs Szilard Lehel
Drd. Nagy Csaba Istvan
Drd. Demeter Jozsef Szabolcs
Student masterand Chira Sergiu
Student masterand Sipos Botond
Student Maier Maria Virginia
Student Constantinescu Alice Mihaela
Student Meszaros Noemi
Ec. Gal Floare
Tehn. Pârjol Aida
 
Partner 2 – Faculty of  Physics, UBB- Cluj-Napoca
Prof. Dr. Simion SIMON
 
Dr. Mihaela Aluas
Drd. Mihai Vasilescu
Drd. Romulus Valeriu Flaviu Turcu
Drd. Dina Mariana Petrisor
Drd. Dorin Alexandru Udvar
Drd. Oana Ponta
Drd. Camelia Albon
Drd. Bogdan Frentiu
Student masterand Nicolae Corojan
 

 
Partners :
 
  • National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca (CO);
  • Faculty of  Biology and Geology, UBB Cluj-Napoca (P1);
  • Faculty of  Physics, UBB Cluj-Napoca (P2).

 


 
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