This research study is designed to synthesize γ-glutamyl methionine, immobilizing the bacterial γ-glutamyl transpeptidase from Bacillus subtilis (BsGGT), in glyoxyl-agarose (GLX-AG). Protein concentration, through Bradford colorimetric assay and activity of BsGGT by spectrophotometer assay were performed. In our investigation, several supports were analyzed to immobilize BsGGT, such as agarose-based and Sepabeads-based supports. Considering the results obtained from the various immobilization tests, the glyoxyl-agarose, thanks to its hydrophilic nature, gave a higher yield in term of immobilized protein than the other supports. Once the immobilization of BsGGT-GLX-AG was obtained, the analytical synthesis of γ-glutamyl methionine monitoring it is in HPLC (High Performance Liquid Chromatography) was performed. Following and the product obtained was recovered from the reaction and purified by ion exchange chromatography. The fractions collected from the column were monitored by TLC (Thin Layer Chromatography) analysis and detected using ninhydrin. Finally, the NMR was performed to confirm the purity of the synthesized γ-glutamyl methionine.
Questo studio di ricerca è progettato per sintetizzare la γ-glutamil metionina, immobilizzando la γ-glutamil transpeptidasi batterica da Bacillus subtilis (BsGGT), in gliossil-agarosio (GLX-AG). Sono state eseguite le concentrazioni di proteine, attraverso il saggio colorimetrico di Bradford e l'attività di BsGGT mediante saggio spettrofotometrico. Nella nostra indagine, sono stati analizzati diversi supporti per immobilizzare BsGGT, come i supporti a base di agarosio e quelli a base di Sepabeads. Considerando i risultati ottenuti dalle varie prove di immobilizzazione, il gliossil-agarosio, grazie alla sua natura idrofila, ha dato una resa in termini di proteina immobilizzata maggiore rispetto agli altri supporti. Una volta ottenuta l'immobilizzazione di BsGGT-GLX-AG, è stata eseguita la sintesi analitica del monitoraggio della γ-glutamil metionina in HPLC (High Performance Liquid Chromatography). Successivamente il prodotto ottenuto è stato recuperato dalla reazione e purificato mediante cromatografia a scambio ionico. Le frazioni raccolte dalla colonna sono state monitorate mediante analisi TLC (Thin Layer Chromatography) e rilevate utilizzando ninidrina. Infine, l'NMR è stato eseguito per confermare la purezza della γ-glutamil metionina sintetizzata.
Immobilizzazione di gamma-glutamil transpeptidasi da Bacillus subtilis per la sintesi di sostanze kokumi.
FIUMARA, MARILENA
2019/2020
Abstract
This research study is designed to synthesize γ-glutamyl methionine, immobilizing the bacterial γ-glutamyl transpeptidase from Bacillus subtilis (BsGGT), in glyoxyl-agarose (GLX-AG). Protein concentration, through Bradford colorimetric assay and activity of BsGGT by spectrophotometer assay were performed. In our investigation, several supports were analyzed to immobilize BsGGT, such as agarose-based and Sepabeads-based supports. Considering the results obtained from the various immobilization tests, the glyoxyl-agarose, thanks to its hydrophilic nature, gave a higher yield in term of immobilized protein than the other supports. Once the immobilization of BsGGT-GLX-AG was obtained, the analytical synthesis of γ-glutamyl methionine monitoring it is in HPLC (High Performance Liquid Chromatography) was performed. Following and the product obtained was recovered from the reaction and purified by ion exchange chromatography. The fractions collected from the column were monitored by TLC (Thin Layer Chromatography) analysis and detected using ninhydrin. Finally, the NMR was performed to confirm the purity of the synthesized γ-glutamyl methionine.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/20.500.14239/13570