The research described in this Thesis was carried out within the BIOCOSM project (Biocatalysis for oils and fats in cosmetics) funded by Cariplo Foundation and Innovhub in partnership with the University of Milano, Consorzio Italbiotec and Bict s.r.l. (Integrated research on industrial biotechnologies and bioeconomy - joint Call 2017). Lecithin is a mixture of phospholipids (PL) containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA) and minor amounts of lysophosphatidylcholine (LPC). Vegetable lecithin are derived from oleaginous seeds (i.e. soybean, sunflower, rapeseed etc.) after oil extraction/refining and are used as emulsifiers in a vast range of applications. Lecithin represent also a biological renewable feedstock for high-added value products such as PL and glycerophosphoric acid esters. Glycerophosphoric acid esters such as glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) are PL derivatives in which both the fatty acid chains esterified with glycerol are chemically or enzymatically removed. GPC is a cognitive enhancer, whereas GPI is a naturally occurring antinflammatory agent. Herein we reported on chemical and enzymatic approaches for the obtainment of GPC and GPI. Since lecithin is a complex mixture of PL, the analytical characterization of the starting material, as well as the development of an efficient analytical method for reaction and downstream monitoring was crucial. To this aim, TLC, LC-MS, HPLC and NMR spectroscopy (1H-NMR, 31P-NMR) techniques were used (in collaboration with the University of Milano). In particular, 31P NMR resulted to be the technique of choice for the analysis of PL and glicerophosphoric acid esters. The complexity of the substrate (sunflower lecithin) and the evidence that PL are structurally related make difficult PL separation from lecithin as well as the recovery of PL-derivatives from the reaction mixture; this constraint was partly circumvented by submitting lecithin to a multi-step fractionation protocol in order to obtain a PC-enriched lecithin and a PI-enriched lecithin as reaction substrates. The approach here described, which represents a novelty in the view of submitting lecithin to a (bio)transformation, allowed to reduce the interference of PL different from PC or PI, and to maximize the formation of the target product. A biocatalytic approach has been designed and performed using lipases/phospholipases both for the preparation of GPC and GPI. GPC was obtained by using Novozym 435, an immobilized lipase B from Candida antarctica, in tert-butanol; this solvent has never been reported before for this biotransformation. Besides being well tolerated by lipases, tert-butanol is acknowledged as a “preferred” solvent for its “green” status. GPC was obtained in 30% yield. On the other hand, the bioprocess for the synthesis of GPI was carried out by using enzymes selected upon an extensive screening (in collaboration with Bict S.r.l.). However, the natural feature of lecithin as emulsifier represented the main bottleneck in setting up the reaction conditions and the downstream step. Although we succeeded in obtaining pure GPI (yield: 25%) upon this enzymatic reaction in fully aqueous medium, a great effort is still necessary to tune all the reaction parameters and to optimize the purification step. It is noteworthy that the use of ion exchange chromatography resulted to be successful for the recovery of GPI. This result, although is still to be optimized, is promising in the view of a preparative biotransformation on a larger scale. In parallel, chemical routes to the synthesis of GPI and GPC were also pursued. Glycerophosphoric acid esters can be obtained after alkaline hydrolysis of lecithin/phospholipids: GPC was obtained starting from PC-enriched lecithin with good yield (88%), whereas hydrolysis of PI-enriched lecithin afforded GPI in 33% yield.
Questo progetto di Tesi è stato realizzato nell’ambito del progetto BIOCOSM (Biocatalysis for oils and fats in cosmetics) finanziato da Fondazione Cariplo e Innovhub-SSI (Integrated research on industrial biotechnologies and bioeconomy - joint Call 2017). Partner del progetto sono l’Università degli Studi di Milano, il Consorzio Italbiotec e l’azienda Bict S.r.l. Le lecitine sono miscele di fosfolipidi (PL) contenenti principalmente fosfatidilcolina (PC), fosfatidiletanolammina (PE), fosfatidilinositolo (PI), acido fosfatidico (PA) oltre a acidi grassi e gliceridi. Le lecitine vegetali sono sottoprodotti dell’estrazione dell’olio da semi oleaginosi come soia, girasole, colza etc.; sono ampiamente utilizzate come emulsionanti nell’industria alimentare e farmaceutica. Inoltre le lecitine possono essere considerate una fonte (feedstock) rinnovabile per l’ottenimento di prodotti ad alto valore aggiunto come fosfolipidi e esteri dell’acido glicerofosforico. Gli esteri dell’acido glicerofosforico come la glicerilfosforilcolina (GPC) e il glicerilfosforilinositolo (GPI) sono derivati dei fosfolipidi privi degli acidi grassi che esterificano il glicerolo. GPC è una molecola con attività stimolante delle funzioni cognitive (cognitive enhancer), mentre GPI è un antinfiammatorio naturale indicato per il trattamento di patologie infiammatorie cutanee. In questa Tesi sono descritte alcune vie sintetiche, chimiche ed enzimatiche, sviluppate per ottenere GPC e GPI a partire da lecitina di girasole. Prima di effettuare la (bio)trasformazione della lecitina è stato necessario caratterizzare la matrice vegetale e mettere a punto un metodo analitico per il monitoraggio delle reazioni e del processo di downstream. Grazie alla collaborazione con l’Università degli Studi di Milano, la lecitina è stata caratterizzata mediante LC-MS, spettroscopia NMR (1H NMR, 31P NMR), HPLC e TLC. In particolare, la spettroscopia 31P NMR è risultata la tecnica di elezione per l’analisi di fosfolipidi e degli esteri glicerofosforici. L’estrema complessità del substrato (lecitina) e il fatto che i PL comprendono gruppi di molecole strutturalmente correlate rendono molto difficile la separazione delle “famiglie” di PL dalla lecitina e anche la loro trasformazione selettiva. L’isolamento e la purificazione di questi prodotti e dei loro derivati dalla miscela di reazione è altrettanto complessa. Per questo motivo la lecitina è stata pre-trattata mediante un frazionamento multi-step per ottenere una frazione di lecitina arricchita in PC e una frazione arricchita in PI da utilizzare come substrati per ottenere, rispettivamente, GPC e GPI, consentendo di ridurre l’interferenza di PL diversi da PC e PI e di massimizzare la formazione dei prodotti. Le biotrasformazioni sono state catalizzate da lipasi e/o fosfolipasi. GPC è stata ottenuta utilizzando Novozym 435, una lipasi B da Candida antarctica , in tert-butanolo. Questo solvente non è mai stato utilizzato nell’idrolisi enzimatica di lecitine per l’ottenimento di GPC. Oltre a essere ben tollerato dalle lipasi, il tert-butanolo è considerato un “preferred solvent” per le sue caratteristiche di basso impatto ambientale. GPC è stato isolato con una resa del 30%. Il bioprocesso per la sintesi di GPI è stato effettuato utilizzando enzimi selezionati in seguito a uno screening (in collaborazione con Bict S.r.l.). Le proprietà emulsionanti della lecitina hanno rappresentato la principale difficoltà per la messa a punto delle condizioni di reazione e di downstream. La reazione è stata effettuata in un medium completamente acquoso e il prodotto è stato ottenuto con una resa pari a 25% dopo cromatografia a scambio ionico. Parallelamente, GPC e GPI sono stati ottenuti anche mediante sintesi chimica “convenzionale” mediante idrolisi alcalina della lecitina arricchita (resa: 88% per GPC e 33% per GPI).
Dalla lecitina agli esteri degli acidi glicerofosforici: approcci chimici ed enzimatici
MEMA, KLODIANA
2019/2020
Abstract
The research described in this Thesis was carried out within the BIOCOSM project (Biocatalysis for oils and fats in cosmetics) funded by Cariplo Foundation and Innovhub in partnership with the University of Milano, Consorzio Italbiotec and Bict s.r.l. (Integrated research on industrial biotechnologies and bioeconomy - joint Call 2017). Lecithin is a mixture of phospholipids (PL) containing phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA) and minor amounts of lysophosphatidylcholine (LPC). Vegetable lecithin are derived from oleaginous seeds (i.e. soybean, sunflower, rapeseed etc.) after oil extraction/refining and are used as emulsifiers in a vast range of applications. Lecithin represent also a biological renewable feedstock for high-added value products such as PL and glycerophosphoric acid esters. Glycerophosphoric acid esters such as glycerophosphocholine (GPC) and glycerophosphoinositol (GPI) are PL derivatives in which both the fatty acid chains esterified with glycerol are chemically or enzymatically removed. GPC is a cognitive enhancer, whereas GPI is a naturally occurring antinflammatory agent. Herein we reported on chemical and enzymatic approaches for the obtainment of GPC and GPI. Since lecithin is a complex mixture of PL, the analytical characterization of the starting material, as well as the development of an efficient analytical method for reaction and downstream monitoring was crucial. To this aim, TLC, LC-MS, HPLC and NMR spectroscopy (1H-NMR, 31P-NMR) techniques were used (in collaboration with the University of Milano). In particular, 31P NMR resulted to be the technique of choice for the analysis of PL and glicerophosphoric acid esters. The complexity of the substrate (sunflower lecithin) and the evidence that PL are structurally related make difficult PL separation from lecithin as well as the recovery of PL-derivatives from the reaction mixture; this constraint was partly circumvented by submitting lecithin to a multi-step fractionation protocol in order to obtain a PC-enriched lecithin and a PI-enriched lecithin as reaction substrates. The approach here described, which represents a novelty in the view of submitting lecithin to a (bio)transformation, allowed to reduce the interference of PL different from PC or PI, and to maximize the formation of the target product. A biocatalytic approach has been designed and performed using lipases/phospholipases both for the preparation of GPC and GPI. GPC was obtained by using Novozym 435, an immobilized lipase B from Candida antarctica, in tert-butanol; this solvent has never been reported before for this biotransformation. Besides being well tolerated by lipases, tert-butanol is acknowledged as a “preferred” solvent for its “green” status. GPC was obtained in 30% yield. On the other hand, the bioprocess for the synthesis of GPI was carried out by using enzymes selected upon an extensive screening (in collaboration with Bict S.r.l.). However, the natural feature of lecithin as emulsifier represented the main bottleneck in setting up the reaction conditions and the downstream step. Although we succeeded in obtaining pure GPI (yield: 25%) upon this enzymatic reaction in fully aqueous medium, a great effort is still necessary to tune all the reaction parameters and to optimize the purification step. It is noteworthy that the use of ion exchange chromatography resulted to be successful for the recovery of GPI. This result, although is still to be optimized, is promising in the view of a preparative biotransformation on a larger scale. In parallel, chemical routes to the synthesis of GPI and GPC were also pursued. Glycerophosphoric acid esters can be obtained after alkaline hydrolysis of lecithin/phospholipids: GPC was obtained starting from PC-enriched lecithin with good yield (88%), whereas hydrolysis of PI-enriched lecithin afforded GPI in 33% yield.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12561