This project aims to develop a highly efficient enzyme for the conversion of xylose from cellulosic wastes into high-value biosynthetic intermediates. By optimising the catalytic efficiency of phosphoketolase and designing an enzymatic reactor, the project aims to provide a sustainable method of utilising lignocellulosic residues.
The project focuses on the innovative use of lignocellulosic wastes, which are abundant but difficult to process due to their complex composition of lignin, hemicellulose and various monosaccharides such as xylose. Traditional methods often resort to incineration of these residues, leading to environmental problems. The main objective of this research is to develop a highly efficient enzyme, phosphoketolase, to convert xylose into high-value biosynthetic intermediates. This enzyme will be obtained from Leuconostoc mesenteroides, Synechocystis and Pseudomonas aeruginosa and its genes will be cloned, expressed, purified and characterised. In addition, an enzymatic reactor will be designed to optimise this conversion process.
The reactor will combine a centrifugal heterogeneous unit with classical immobilised enzyme units, optimised by a factorial design of experiments. The aim is to achieve significant improvements in the conversion yield of xylose to valuable intermediates such as glyceraldehyde-3-phosphate and acetyl phosphate. This project builds on previous research in the Department of Enzymology which demonstrated the potential of using phosphoketolase in conjunction with xylose isomerase and xylulose kinase.
The successful implementation of this project could lead to the reactivation or establishment of new lignocellulosic waste processing facilities in Romania, benefiting industries such as cellulose production and potentially attracting small and medium enterprises interested in sustainable bioprocessing. Ultimately, this research aims to provide an environmentally friendly and economically viable solution to the over-valuation of lignocellulosic residues.
Head of Department
Prof. Dr. Stefan Szedlacsek is the Head of the Enzymology Department at the Institute of Biochemistry of the Romanian Academy. He holds a PhD degree in Biotechnology from Polytechnical University of Bucharest as well as a MSc in Organic Synthesis (Polytechnical University, Bucharest) and MSc in Mathematics (University of Bucharest). As a visiting scientist, he performed research in the field of cholesterol metabolism at the University of Illinois at Urbana-Champaign (USA), where he succeeded to evidence a new pathway in the metabolism of oxysterols. He is an “Alexander von Humboldt” fellow and worked in Germany, in the Institute of Biochem More...
Project coordinator: Prof. Dr. Stefan Szedlacsek
Partners:
1. University Babeş-Bolyai Cluj-Napoca; Project manager P1: Prof. Dr. Octavian Popescu
2. University „Politehnica” Bucharest ; Project manager P2: Prof. Dr. Ovidiu Muntean
3. University Sapientia, Miercurea Ciuc; Project manager P3: Prof. Dr. Lányi Szabolcs
Objective 1: Obtention and purification of recombinant phosphoketolase from Leuconostoc mesenteroides, Synechocystis, Pseudomonas aeruginosa and Lactococcus lactis
Objective 2: General enzymatic characterization and determination of the kinetic parameters for phosphoketolase from Leuconostoc mesenteroides, Synechocystis, Pseudomonas aeruginosa si Lactococcus lactis
Objective 3: Determination of the conditions for optimal enzymatic activity of phosphoketolases from Leuconostoc mesenteroides, Synechocystis, Pseudomonas aeruginosa si Lactococcus lactis.
Objective 4: Obtention of an optimized, artificial phosphoketolase
Objective 5: Obtention and kinetic characterization of xylose izomerase, xylulose kinase and phosphoketolase immobilised on inert support.
Objective 6: Construction of a laboratory scale enzymatic reactor for xylose conversion into compounds important for industrial biosyntheses
In this project, a laboratory-scale device for the enzymatic conversion of xylose into biotechnologically important compounds: acetyl-phosphate and glyceraldehyde-3-phosphate, was developed using an enzyme system consisting of three immobilised enzymes: xylose isomerase, xylulose kinase and phosphoketolase.
Xylose isomerase was purchased commercially and xylulose kinase and phosphoketolase were produced in the laboratory. Phosphoketolase genes were cloned from Leuconostoc mesenteroides, Synechocystis sp. and Pseudomonas aeruginosa.
The corresponding enzymes were expressed, purified and kinetically characterised. A comparative study of enzyme stability data, optimal reaction conditions and kinetic parameters was performed to determine the optimal enzyme for our project.
Several reaction conditions were tested for all phosphoketolases. The effects of pH, TPP concentration and temperature were investigated and the Km and kcat constants were determined. All phosphoketolases have an optimal pH in the range 6.5-7, optimal TPP concentration 1 mM - 5 mM and optimal temperature - 37°C. Comparison of the kinetic parameters of the phosphoketolases led to the conclusion that the phosphoketolase from L. mesenteroides was the most stable.
All three enzymes, xylo-isomerase, xylulokinase and phosphoketolase, were immobilised on an inert support in three unsegregated columns. Preliminary experiments carried out on the three linked columns in series showed the conversion of xylose to acetyl-phosphate and glyceraldehyde-3-phosphate.
In conclusion, this project provides a promising biotechnological process for the valorisation of cellulosic waste.
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TECHNOLOGY |
The stage of realisation |
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Optimised reactor for the enzymatic conversion of xylose into products of biotechnological use |
Laboratory scale reactor |
International Congresses:
(1)13th European Congress on Biotechnology; 16-19 September 2007; Barcelona, Spain; Poster presentation: "Enzyme-coupled procedure for xylose conversion into biotechnologically important intermediates"; Authors: G.Petrareanu, M.C. Balasu, S.E. Szedlacsek.
(2)Congress of the International Union of Crystallography, August, 23-31, 2008; Osaka, Japan; Poster presentation: "Expression, purification and crystallization of phosphoketolase from L. lactis"; Authors: G. Petrareanu, M. Balasu, R. Badea, S. Szedlacsek.
Seminars, Conferences:
(1)The Annual International Conference of the Romanian Society of Biochemistry and Molecular Biology; 1-3 June 2006; Constanta, Romania; Work presented: "Enzymatic characterization of phosphoketolases from Leuconostoc mesenteroides and Lactoccocus lactis"; Authors: M.C. Balasu, G.Petrareanu, S.E. Szedlacsek.
(2) Seminars of the Institute of Biochemistry; Work presented: "Study of some phosphoketolases – Substrate synthesis and kinetic characterization"; Author: M.C. Balasu.
(3) The Annual International Conference of the Romanian Society of Biochemistry and Molecular Biology; May, 2008 - Bucharest; Romania; Work presented: "Kinetic characterization of immobilized enzymes involved in a procedure of xylose conversion"; Authors: Petrareanu G., Balasu M., Szedlacsek S.
Workshops:
(A) The completion of the first phase of the OMEXIL project and the details of the research in phase II.
(1) "Molecular optimization of a central enzyme in the metabolic pathway of xylose conversion. Optimized reactor for the enzymatic transformation of xylose resulting from cellulosic waste". Author: Szedlacsek S.
(2) "Models applicable to the design of enzymatic reactors"; Author: N. Iosif
(3) "Cloned phosphoketolases from P. aeruginosa and Synecocystys sp."; Author: O. Popescu
(4) "Expression and preliminary activity of the enzymes studied: xylose isomerase, xylulose kinase and phosphoketolase. Preparations for the construction of an experimental enzyme reactor, model"; Authors: L. Szabolcs, Szép Alexandru.
(B) Tools of Molecular Biology for Applied Enzymology.
(1) "Preparation, purification and enzymatic characterization of phosphoketolase – an enzyme with an increased biotechnological potential; Authors: Szedlacsek S, Petrareanu G, Balasu M,.
(2) "Design of enzymatic reactors"; Authors: Nagy I, Muntean O,.
(3) "Optimization of heterologous expression, purification and characterisation of xylulose kinase"; Authors: Lanyi S, Abraham B, Mikossy I, Petreareanu G, Balasu M, Szedlacsek S,.
Articles publication:
(1) "Cloning and expression of the gene encoding phosphoketolase in Pseudomonas aeruginosa 15442"; Authors: Cs. I. Nagy, Iulia Lupan, Beatrix-Katalin Ferencz, O. Popescu, Journal: Annals of the West University of Timisoara, Series of Chemistry, 16(3), 73-80, 2007
(2) "Cloning of the gene for phosphoketolase (xfp) from Synechocystis sp. PCC6803"; Authors: Cs. I. Nagy, Iulia Lupan, Beatrix Ferencz, O. Popescu,; Journal: Annals NSCB, XI, 348-352, 2006
Romanian Academy