Natural nucleotides such as inosine-5’-monophosphate (5’-IMP) and guanosine-5’-monophosphate (5’-GMP) are used as food additives due to their activity as flavor enhancers.They elicit an umami taste in humans and synergistically increase the intensity of the umami taste of monosodium glutamate. Nucleoside and nucleotide analogues are synthetic, chemically modified molecules used as drugs in antiviral and anticancer chemotherapy; nucleotide analogues are frequently used as prodrugs of their parent nucleosides because the ionic feature of the phosphate group significantly enhances the solubility of poorly water-soluble nucleosides.The synthesis of nucleosides and nucleotides is wearisome and time consuming. Specifically, the selective formation of 5′-phosphate esters of nucleosides mostly rely on the chemical phosphorylation of protected nucleosides by POCl3 in the presence of trialkylphosphates (such as (Et)3PO4 or (Me)3PO4) as solvents. Environmental issues associated to the use of these reagents and solvents, moderate to poor regioselectivity of the reaction, and the need of tedious protection/deprotection steps have prompted the search of novel approaches to streamline the synthesis of nucleotides. In cells, the phosphate group is selectively introduced by phosphotransferases (kinases).In addition, some enzymes that hydrolyze phosphorylated compounds are also able to perform the opposite reaction (transphosphorylation reaction).For this reason, they can be used in a synthetic mode (i.e. as biocatalysts for nucleotide synthesis).Furthermore, enzymes work under mild conditions and, generally speaking, produce less waste.Thus, enzymatic reactions usually fall within the “green chemistry” approach. Bacterial non specific acid phosphatases (NSAPs) are a group of enzymes that catalyze the hydrolysis of phosphoester bonds. Some of them exhibit a phosphotransferase activity; thus, they are able to transfer a phosphate group from a donor molecule (e.g. phosphomonoesters or pyrophosphate) to an acceptor, such as nucleosides, alcohols, and carbohydrates.In this work, the non specific acid phosphatase from Morganella morganii (PhoC; EC 3.1.3.2) was recombinantly expressed in E. coli and purified by immobilized metal affinity chromatography.The biocatalytic characterization of this enzyme was carried out by assessing its activity in the hydrolysis of p-Nitrophenylphosphate (p-NPP) and investigating its substrate scope.A set of purine and pyrimidine deoxy- and ribonucleosides was screened as potential substrates of PhoC-Mm in the phosphotransferase reaction using pyrophosphate (PPi) as the phosphate donor.Surprisingly, PhoC-Mm shown a very narrow substrate specificity. On this ground, the study of the reaction parameters for setting-up a PhoC-Mm-catalyzed phosphorylation was carried out by using inosine as the “model” substrate.Enzyme concentration, temperature, pH, PPi concentration, were investigated as variables of the synthesis of 5’-IMP. Due to the reversibility of the reaction, the challenge was suppressing (or, at least, slowing down) the phosphatase activity in favor of the phosphotransferase activity. To this aim, the use of vanadate as a competitive inhibitor of phosphate was also assayed.A slight improvement of the bioconversion yield was achieved by using a large excess of PPi. With the aim to carry on the study of PhoC-Mm, which started with this Master thesis, and the application of this enzyme in biocatalysis, along with other phosphatases, preliminary immobilization tests of PhoC-Mm have been also performed. These experiments have revealed that the use of an epoxy carrier (Relizyme-112) or an aldehyde carrier (glyoxyl-agarose) resulted in a poor activity recovery.A systematic study on the enzyme immobilization will be thus necessary to find the optimal carrier that can preserve the catalytic activity of PhoC-Mm and improve the stability of the biocatalyst under the experimental conditions.
I nucleotidi 5’-monofosfato, tra cui inosina-5’-monofosfato (5’-IMP) e guanosina-5’-monofosfato (5’-GMP), sono utilizzati come additivi alimentari e intermedi per la sintesi di API (Active Pharmaceutical Ingredients). La fosforilazione dei nucleosidi per l’ottenimento dei corrispondenti nucleotidi mediante metodi chimici convenzionali è complessa: richiede passaggi di protezione e deprotezione, l’uso di reagenti e solventi tossici e condizioni sperimentali drastiche. La fosforilazione mediata da enzimi avviene, al contrario, in condizioni di reazione blande (es. pH, temperatura), in ambiente prevalentemente acquoso ed è selettiva. Le fosfatasi acide batteriche non specifiche (Non Specific Acid Phosphatases) sono enzimi che catalizzano, in natura, l’idrolisi di legami fosfoesterei ma, in determinate condizioni, mostrano attività fosfotransferasica, cioè sono in grado di trasferire un gruppo fosfato da una molecola donatrice (ad esempio da un fosfomonoestere o dal pirofosfato) a un accettore come nucleosidi, esosi, pentosi e alcoli. In questa tesi, la fosfatasi acida non specifica da Morganella morganii (PhoC; E.C. 3.1.3.2) è stata espressa in forma ricombinante e purificata. L’attività enzimatica è stata determinata mediante la reazione di idrolisi del p-Nitrofenilfosfato (pNPP), utilizzato come substrato standard. L’attività fosfotransferasica è stata determinata verso un set di desossi- e ribonucleosidi purinici e pirimidinici utilizzando pirofosfato (PPi) come donatore di fosfato. Questo studio ha evidenziato che PhoC-Mm ha una specificità di substrato molto ristretta dal momento che fosforila solo l’inosina. Lo studio della biotrasformazione è stato quindi effettuato utilizzando l’inosina come substrato modello. I parametri studiati comprendono concentrazione dell’enzima, pH, temperatura e concentrazione di PPi. Esperimenti preliminari di immobilizzazione dell’enzima su supporti epossidici e aldeidici hanno fornito una scarsa activity recovery suggerendo la necessità di intraprendere uno studio sistematico di immobilizzazione comprendente vari tipi di carrier e diversa binding chemistry.
Applicazione della fosfatasi acida da Morganella morganii (PhoC-Mm) in biocatalisi: preparazione, caratterizzazione e studio della specificità di substrato.
DE PASCALI, ARIANNA
2018/2019
Abstract
Natural nucleotides such as inosine-5’-monophosphate (5’-IMP) and guanosine-5’-monophosphate (5’-GMP) are used as food additives due to their activity as flavor enhancers.They elicit an umami taste in humans and synergistically increase the intensity of the umami taste of monosodium glutamate. Nucleoside and nucleotide analogues are synthetic, chemically modified molecules used as drugs in antiviral and anticancer chemotherapy; nucleotide analogues are frequently used as prodrugs of their parent nucleosides because the ionic feature of the phosphate group significantly enhances the solubility of poorly water-soluble nucleosides.The synthesis of nucleosides and nucleotides is wearisome and time consuming. Specifically, the selective formation of 5′-phosphate esters of nucleosides mostly rely on the chemical phosphorylation of protected nucleosides by POCl3 in the presence of trialkylphosphates (such as (Et)3PO4 or (Me)3PO4) as solvents. Environmental issues associated to the use of these reagents and solvents, moderate to poor regioselectivity of the reaction, and the need of tedious protection/deprotection steps have prompted the search of novel approaches to streamline the synthesis of nucleotides. In cells, the phosphate group is selectively introduced by phosphotransferases (kinases).In addition, some enzymes that hydrolyze phosphorylated compounds are also able to perform the opposite reaction (transphosphorylation reaction).For this reason, they can be used in a synthetic mode (i.e. as biocatalysts for nucleotide synthesis).Furthermore, enzymes work under mild conditions and, generally speaking, produce less waste.Thus, enzymatic reactions usually fall within the “green chemistry” approach. Bacterial non specific acid phosphatases (NSAPs) are a group of enzymes that catalyze the hydrolysis of phosphoester bonds. Some of them exhibit a phosphotransferase activity; thus, they are able to transfer a phosphate group from a donor molecule (e.g. phosphomonoesters or pyrophosphate) to an acceptor, such as nucleosides, alcohols, and carbohydrates.In this work, the non specific acid phosphatase from Morganella morganii (PhoC; EC 3.1.3.2) was recombinantly expressed in E. coli and purified by immobilized metal affinity chromatography.The biocatalytic characterization of this enzyme was carried out by assessing its activity in the hydrolysis of p-Nitrophenylphosphate (p-NPP) and investigating its substrate scope.A set of purine and pyrimidine deoxy- and ribonucleosides was screened as potential substrates of PhoC-Mm in the phosphotransferase reaction using pyrophosphate (PPi) as the phosphate donor.Surprisingly, PhoC-Mm shown a very narrow substrate specificity. On this ground, the study of the reaction parameters for setting-up a PhoC-Mm-catalyzed phosphorylation was carried out by using inosine as the “model” substrate.Enzyme concentration, temperature, pH, PPi concentration, were investigated as variables of the synthesis of 5’-IMP. Due to the reversibility of the reaction, the challenge was suppressing (or, at least, slowing down) the phosphatase activity in favor of the phosphotransferase activity. To this aim, the use of vanadate as a competitive inhibitor of phosphate was also assayed.A slight improvement of the bioconversion yield was achieved by using a large excess of PPi. With the aim to carry on the study of PhoC-Mm, which started with this Master thesis, and the application of this enzyme in biocatalysis, along with other phosphatases, preliminary immobilization tests of PhoC-Mm have been also performed. These experiments have revealed that the use of an epoxy carrier (Relizyme-112) or an aldehyde carrier (glyoxyl-agarose) resulted in a poor activity recovery.A systematic study on the enzyme immobilization will be thus necessary to find the optimal carrier that can preserve the catalytic activity of PhoC-Mm and improve the stability of the biocatalyst under the experimental conditions.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/20.500.14239/24810