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Biocatalytic flow reactors using extremophilic enzymes for a greener generation of aroma-compounds

Periodic Reporting for period 1 - AROMAs-FLOW (Biocatalytic flow reactors using extremophilic enzymes for a greener generation of aroma-compounds)

Reporting period: 2018-04-01 to 2020-03-31

Despite the innovative combination of flow reactor technology and biocatalysis can improve the performance of synthetic chemistry, little attention is paid to flow-biocatalysis applied to food chemistry, significantly reducing the important role of a green production of natural food compounds. The use of biocatalysts for the preparation of aroma-compounds, starting from natural substrates, classifies the products as natural, too. Based on this idea, AROMAs-FLOW created alternative, green and technologically-advanced synthetic procedures for the production of a number of natural aromas starting from cinnamaldehyde (in itself a natural compound) by combining the strong versatility of different enzymes with flow chemistry facilities. Multi-enzymatic cascade transformations, up to now reported in continuous only on analytical scale were scaled-up to multi-gram production making these methodologies extremely efficient. Taking into consideration the increased consumers’ awareness of health and nutrition conscious lifestyle, AROMAs-FLOW project represented an efficient alternative route for the preparation of natural aromas and fragrances. Among the advantages in comparison with traditional plant extraction, it allowed a complete independence from agriculture and possible shortages caused by local conditions such as climate, pesticides, wars, etc.. and strongly promoted the responsible care of natural resources. The reduction of working time as well as process costs for the obtainment of natural aroma-compounds, makes this strategy economically appealing for food industries.
During the 2-year project 3 enzymes were successfully cloned in E. coli, fully characterized and immobilized. While an alcohol dehydrogenase (HeADH-II), was identified in the genome of the halo-adapted bacterium Halomonas elongata, an enoate-reductase and an acyl-transferase were obtained from mesophilic microorganisms. The enzymes were characterized and the optimal reaction conditions investigated. An in-depth study towards covalent immobilization techniques was carried out in order to overcome the limits often encountered with free enzymes as well as to incorporate the biocatalysts into flow reactors.
During my first secondment at the University of Milan (Prof. Molinari, DeFENS) acetic acid bacteria were employed as green and clean oxidants for the preparation of cinnammic acid from cinnammaldehyde. Gluconobacter oxidans and Acetobacter aceti were selected as the best candidates and immobilized by entrapment (DALGEE). The whole-cell biocatalysts were finally tested in batch and subsequently employed under flow conditions.
Thanks to the longstanding experience of Molinari’s group in whole-cell biocatalyst mediated reactions, I was also trained in the application of moulds such as Aspergillus oryzae. Its mycelium-bound lipase(s) were employed for the preparation of a new set of aroma-compounds through direct acid-alcohol condensation.
The flow system set-up for the different reactions was designed together with Prof. Tamborini during my last secondment (University of Milan, DISFARM). The single flow-mode biotransformations were optimized in terms of temperature, pressure and catalyst reusability. In-line extractions and product purifications were added downstream the process to obtain advanced and reproducible flow strategies. During my last period at the University of Nottingham, the flow reactions studied so far as single steps were connected to develop a biocatalytic platform for the preparation of natural aromas starting from cinnamaldehyde.
Continuous biocatalyst-mediated reactions as well as in-line wok-up and purification steps were finally connected to obtain fully automated processes.
The combination of my previous experiences with the advanced training I gained throughout the project was an essential component for the achievement of my goal to become an independent researcher. Thanks to the results achieved with the Marie Curie fellowship, I am now able to apply for the Italian National Academic Qualification as Associate Professor (July 2020). The creative thinking and new ideas arisen during the 2-year fellowship gave me the background necessary to apply for an ERC starting grant during the next call.
During the 2-year fellowship, 10 papers linked to the AROMAs-FLOW project were published; 3 of them are editorials while 2 are invited papers for the dissemination of the advantages of flow biocatalysis to a generic audience. 4 additional manuscripts are still in preparation about the results obtained during the last period.
Data arisen from AROMAs-FLOW project were presented through oral and poster presentations at 4 international conferences (one of them as invited speaker) and 2 workshops. One of my poster presentations won the “Best Poster Award” and “Reaction & Engineering Poster Prize” during a meeting specialized on continuous flow reactions.
I was invited to give a lecture about enzyme immobilization and flow process intensification for students of PSF Master (Progettazione e Sviluppo dei Farmaci), session of Biocatalysis in Drug Discovery and Development at the University of Pavia.
PhD and undergraduate students have been trained during their experimental thesis on the applications, operational requirements, and synthetic advantages of flow biocatalysis.
The website of Prof. Paradisi as well as her social media were updated with the results of the project.
I was personally invited to attend a demonstration organized by the Syrris company to present the ASIA flow system, a different machine for continuous reactions from the Vapourtec systems used for the whole AROMAs-FLOW project.
The flow biocatalyzed reactions optimized within the project showed relevant advantages with respect to traditional chemical procedures. In particular, they fulfill all requirements to diminish environmental burden, reduce costs, and increase safety that are necessary to increment the sustainability of manufacturing processes. Non-hazardous materials were employed under safe reaction conditions with reduced risk and without toxic waste production. Flow chemistry, which has been recently introduced to advance the sustainability of organic synthesis, dramatically sped up the designed syntheses with a significant economic impact in terms of overall process cost, working hours, energy consumption. The presented strategy, applied in this case to the production of food compounds can be expanded to a wide range of different substrates, completely changing the approach to synthetic procedures. Enzymatic cascade transformations which, up to now, have been reported in continuous only on analytical scale were intensified to a multi-gram scale. The innovative AROMAs-FLOW project with its efficient and eco-friendly production of natural aroma-compounds encompasses both the need for alternative “natural” routes for flavor production, and a sustainable way of working and living. In agreement to Europe 2020 strategy which aims at “smart, competitive and inclusive growth” for the development of European economy, my AROMAs-FLOW promoted green chemistry, lowering production costs, chemical waste, and improving working efficiency. The combination of flow chemistry reactors and biocatalysis has demonstrated to be a powerful economic and sustainable alternative to conventional batch synthetic reactions, with a strong impact both on environment and on society.