PN-III-P1-1.1-TE-2019-1573
PROJECT CODE: PN-III-P1-1.1-TE-2019-1573, contract number TE 101 ⁄ 2020
PROJECT TITLE:
Green chemistry route for the enzymatic cascade synthesis of biodegradable oligoesters
Sinteza enzimatica unor oligoesteri biodegradabili printr-un procedeu verde de tip cascada
ACRONYM: GREEN_POLYGEL
PROJECT COORDINATOR: Politehnica University of Timişoara
PROJECT DIRECTOR: Ș.l dr. ing. Anamaria Todea - 2020 / Ș.l. dr. ing. Iulia Maria Păușescu - 2021

BUDGET: 431.900 RON (Public budget)
START TIME: 15/09/2020
END TIME: 14/09/2022

PROJECT LAYOUT

Cascade reactions catalyzed by multienzymatic systems strongly increased the researchers interest due to their unique potential for the environmentally benign production of chemicals and materials. In recent years considerable progress has been made for one pot systems and the synthetic power of several enzymes was demonstrated. Vegetable oils represent important bio-based raw-materials for polymer synthesis due to their universal availability, low price and biodegradability. The main objective of the project is to develop an efficient green and cost-effective route for the biocatalytic synthesis in one-pot system starting from castor oil and bio-based furan monomers using a combination of two enzymes. The proposed reaction system involves an innovative three cascade reaction system catalyzed by two enzymes of different classes: (i) hydrolysis of triglycerides, (ii) glycerol oxidation and (iii) synthesis of oligoesters. All the purposed reactions will be mediated by tailor-made immobilized enzymes that are non-toxic, recyclable and eco-friendly biocatalysts, by using green solvents or solventless systems. The resulted biodegradable oligoesters will present new functionalities and properties. The enzyme stabilization will be performed by covalent binding and the selectivity will be evaluated in terms of maximal catalytic efficiency, to increase the productivity of the process. The reaction products will be characterized in detail by several analytical techniques for structure confirmation and assessment of the physico-chemical properties and their biodegradability rate will be evaluated by two methods. The synthesized monomers and oligoesters will be used as starting materials for novel organogels preparation.

PROJECT TEAM:
Project leader: Ș.l. dr.ing. Anamaria Todea-2020 / Ș.l. dr. ing. Iulia Maria Păușescu - 2021
Postdoctoral researchers:
Ș.l. dr. ing. Iulia Maria Păușescu
Conf. dr. ing. Ionuț Valentin Ledeți
Asist. Dr. chim. Diana Maria Dreavă (Aparaschivei)
Ș.l. dr. ing. Ana Maria Pană
PhD Students:
Drd. Ing. Ioan Bîtcan
Drd. Ing. Ioana Cristina Benea
Drd. Ing. Ionuț Mihai Tănase
Students:
Andreea Petrovici

 OBJECTIVE:
The main objective of the project is to obtain new oligoesters in one-pot system starting from castor oil and bio-based furan monomers by a complete green route, using a combination of two enzymes. All the purposed reactions will be mediated by tailor-made immobilized enzymes that are non-toxic, recyclable and eco-friendly biocatalysts, by using green solvents or solventless systems. The resulted biodegradable oligoesters will present new functionalities and properties.

STAGES
Stage 1 (2020, 4 months)- Enzymatic synthesis and optimization of glycerol conversion in glyceric and tartronic acids

Stage 2 (2021, 12 months)- Increasing the stability of laccases by immobilization for the synthesis of hydroxy acids from glycerol. Biotransformation of triricinolein from castor oil and furan derivatives in oligoesters in a "one-pot" system using immobilized laccases and lipases.

Stage 3 (2022, 8 months)- Enzymatic and microbial degradation a new oligoesters. Preparation and characterization of some organogels with two or three components.

DELIVERABLES:
Stage 1
Experimental methodology for enzymatic synthesis of glycerin acids using laccases.
Protocol for immobilization of laccases by covalent bonding.
Stage 1 report.
Stage 2
Grams amounts of immobilized laccase.
Experimental methodology for conversion of triricinolein to oligoesters using immobilized enzymes
Optimized experimental methodology for biotransformation of vegetables oils using immobilized lipases and laccases.
Experimental design methodology for synthesis of oligo-esters in the one-pot system.
Grams of synthesized oligo-esters.
Stage 2 report.
Abstracts for two presentations at scientific conferences.
A scientific article submitted for publication / accepted in the journal with impact factor indexed in the Web of Science database.
Stage 3
Laboratory methodology for enzymatic and microbial biodegradation of oligoesters.
Laboratory methodology for preparation and characterization of organogels.
A scientific article submitted for publication /accepted in the journal with impact factor,indexed in the Web of Science database.
Abstracts for two presentations at scientific conferences.
Stage 3 report
Final report.

RESULTS 2020-2022

Abstract Stage 1 (2020)

In the first stage of the project, a series of studies on the oxidation reaction of glycerin to glyceric and tartronic acids were carried out, in accordance with the planned activities. Studies were also carried out on the development of a new catalyst by using functionalized methacrylic resin supports and magnetic nanoparticles in different variants for the immobilization of laccase from Aspergillus oryzae. To monitor the oxidation reaction, it was necessary to develop a liquid chromatographic analysis method, and in this sense several chromatographic columns were tested using different mobile phases at different temperatures. Based on the results obtained, an acid analysis protocol was developed. The increase in the efficiency of the oxidation reaction was achieved by using an initiator, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl, TEMPO, which favored the oxidation reaction, and the reaction yield was improved, obtaining results promising. Tartronic acid was characterized by FT-IR and 1H-NMR spectroscopy. Next, laccase immobilization studies were carried out on 7 supports, followed by the investigation of the biocatalysts obtained in the oxidation reaction of 2,6-dimethoxyphenol. Methacrylic resins functionalized with epoxy and amino groups were used, as well as magnetic nanoparticles that were functionalized with amino groups using 2 precursor silanes, resulting in 13 enzyme preparations. Two of the 5 magnetic biocatalysts were characterized by DLS. For all 13 biocatalysts, the operational stability was studied in 6 successive reaction cycles, two of them showing very good results. Based on the obtained results, a protocol was developed for the immobilization of laccases by covalent binding. In conclusion, all the objectives of this stage have been met.

Scientific report project TE101 - stage 1, 2020

Abstract Stage 2 (2021)

In the second stage of the project, a series of studies were carried out on the oxidation reaction of glycerin to glyceric and tartronic acids using all 4 commercially available laccases. Studies were also carried out on the development of a new catalyst by using functionalized methacrylic resin supports and magnetic nanoparticles in different variants for the immobilization of laccase from Trames versicolor. Two of the magnetic biocatalysts were characterized by SEM and DLS. For all 12 biocatalysts, the operational stability was studied in 6 successive reaction cycles, two of them showing very good results. Next, the enzymatic synthesis of new oligomeric compounds was studied starting from ricinoleic acid (RCA) or castor oil and 5-hydroxymethyl-2-furancarboxylic acid. Several lipases have been studied as biocatalysts, the best performing being the one from Pseudomonas stutzeri, unused until now in this type of reactions. Among the remarkable results obtained during these researches is the demonstration of the possibility to control the copolymer/homopolymer ratio by the appropriate choice of the native biocatalyst and the highlighting of the important role of water in these reactions. A "one-pot" cascade reaction system was also studied, in which it started from castor oil and using a single lipase both the hydrolysis of this oil and the transformation of the obtained RCA into oligoesters were achieved, thus opening the perspectives obtaining a varied range of new biocompounds. Structural analysis, demonstration of copolymer formation and its composition were performed using sophisticated instrumental techniques, such as 2D-NMR and MALDI-TOF MS. Significant from the engineering point of view of the studied reactions were the study of the influence of the solvent and the demonstration of the operational stability of the biocatalyst in several reaction cycles. The biodegradability of oligoesters was then studied in the presence of porcine pancreas lipase and the results indicate a decrease in molecular masses after the first 72h.

Scientific report project TE101 - stage 2, 2021

Abstract Stage 3 (2022)

During the third stage of the project, a series of studies on the immobilization of lipase from Ps. stutzers by adsorption and covalent binding and its testing in the one-pot reaction system. Next, the enzymatic synthesis of new oligomeric compounds using castor oil and 5-hydroxymethyl-2-furancarboxylic acid was studied. Structural analysis, demonstration of copolymer formation and its composition were performed using sophisticated instrumental techniques, such as 2D-NMR and MALDI-TOF MS. Other activities were initiated in this stage, focused on biodegradation of the synthesized polyesters and organogels preparation. Biodegradation studies were performed in liquid culture media, both in the presence of a native lipase from pig pancreas or a consortium of microorganisms collected from Bega river and sea water. For the microbial degradation studies systems equipped with sensors that measure the biochemical oxygen demand (BOD) were used. Promising results were obtained with in both cases, showing high biodegradation degrees after 21 days of incubation with a consortium of microorganisms taken from a natural environment. The resulted organogels were morphologically characterized.

Publications:

1. D. Dreavă, I.-C. Benea, I. Bitcan, A. Todea, E. Sisu, M. Puiu, F. Peter , Biocatalytic Approach for Novel Functional Oligoesters of ε-Caprolactone and Malic Acid, Processes 2021, 9(2), 232.
2. A. R. Buzatu, A.t E Frissen, L. AM van den Broek, A. Todea, M. Motoc, C. G. Boeriu, Chemoenzymatic synthesis of new aromatic esters of mono-and oligosaccharides, Processes, 2020, 8, 12, 1638.
3. I. Bîtcan, A. Petrovici, A. Pellis, S. Klébert, Z. Károly, L. Bereczki, F. Péter, A. Todea, Enzymatic route for selective glycerol oxidation using covalently immobilized laccases, Enzyme and Microbial Technology, 2022, under evaluation
4. I Păușescu, DM Dreavă, I Bîtcan, R Argetoianu, D. Dăescu, M. Medeleanu, Bio-Based pH Indicator Films for Intelligent Food Packaging Applications, Polymers, 2022, 14(17), 3622.

Conferences:

1. I. Bîtcan, A. Petrovici, A.Todea, D. Aparaschivei, I. Păușescu, F. Peter, Green Route Synthesis of Oligoesters from 5-Hydroxymethyl-2-Furan Carboxylic Acid and Ricinoleic Acid, The 6^th International Conference on Biocatalysis in Non-Conventional Media (BNCM 2021), 6-8 May 2021, Milan, Italy.
2. I. Bîtcan, A. Petrovici, A. Ștefan, A. Todea, Iulia Păușescu, F. Peter, Selective glycerol oxidation mediated by covalently immobilized laccases, International Forum on Industrial Biotechnology and Bioeconomy- IFIB 2021, 30 September 2021 - 1 October 2021, Trento, Italy.
3. I. Bitcan, A. Petrovici, A. Ștefan, A. Todea, I. Păușescu, F. Peter, Laccasses stabilization by covalent immobilization onto functionalized magnetic and sepabeads suports, New trends and strategies in the Chemistry of advanced materiais with relevance in bioloqical systems, tehnique and environmental protection" 13^th Edition, online, 7-8 October 2021, Timisoara, Romania.
4. I. Bîtcan, A.Todea, D. Dreavă, I. Păușescu, F. Peter, L. Nagy, S. Kéki Green synthesis and characterization of novel furan-based oligoesters for organogel applications, 9^th IUPAC International Conference on Green Chemistry (ICGC 2022), 5 - 9 September 2022, Athens, Greece.
5. D. Dreavă, I. Bîtcan, A. Petrovici, I. Păușescu, F. Peter, A. Todea, Optimization of furan-based oligoester enzymatic synthesis by design of experiments, 9^th IUPAC International Conference on Green Chemistry (ICGC 2022), 5 - 9 September 2022, Athens, Greece.

Summary of the project results

The project " Green chemistry route for the enzymatic cascade synthesis of biodegradable oligoesters" targeted as main objective the demonstration of a new concept for the valorization of vegetable oils, mainly castor oil, through the synthesis of new oligoesters containing hydroxyl groups and aromatic rings, in a system of enzymatic cascade reactions. This type of reactions leads to reaction products that can be exploited in the medical, pharmaceutical and food fields.

The methods and the evolution development of techniques in biotechnology, molecular and cellular genetics, etc., have led to the increasing use of enzymes for in vitro applications, for example as a catalyst in polymerization and polyesterification reactions.

The experimental studies were carried out in the Biocatalysis Laboratory at the Politehnica University of Timişoara.

Within the project two types of enzymes, lacasses and lipases, were used and stabilized by immobilization. The enzyme stabilization was be performed by covalent binding and adsorption and theur perfomances were tested for glycerol oxidation (laccases) and hydrolysis and polyesterification (lipases). The reaction products were characterized in detail by several analytical techniques including HPLC, gel permeation chromatography, MALDI-TOF MS, NMR and the structures of the products were confirmed. The novel furan-based oligoesters presented a well-defined arhitecture and medium molecular weights in the range of 1200-1500 Da.

The resulted oligoesters were used as starting materials for novel organogels preparation. The organogel formation in different solvents was demonstrated by rheological studies.