Development of a fast, robust and sensitive spectrophotometric assay for measuring hydroxycinnamic acid decarboxylation reactions

master's thesis

• Title (croatian): Razvoj brze, robusne i osjetljive spektrofotometrijske metode za mjerenje reakcija dekarboksilacija hidroksicimetnih kiselina
• Author: Mirna Larva
• Mentor: Anita Slavica (mentor)
• Committee member: Renata Teparic (predsjednik povjerenstva)
• Committee member: Blaženka Kos (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2023-05-11, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Biocatalytic decarboxylation of bio-based hydroxycinnamic acids yields p-hydroxystyrene derivates, which are important precursors of antioxidants, epoxy-coatings, adhesives and other polymeric materials. Bacillus subtilis phenolic acid decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyses the cleavage of carbon dioxide from hydroxycinnamic acids such as p-coumaric, caffeic, ferulic and sinapic acid with high catalytic efficiency. Current methods to kinetically characterise the decarboxylation reactions rely on HPLC, MS, GC or NMR, which require extensive sample work-up while measuring only endpoint concentrations. In this work, we use a fast, robust and sensitive spectrophotometric assay that allows following decarboxylation reactions in real-time while avoiding product extraction. Absorption spectra of the pure reaction products were recorded after verifying the completeness of the reactions by TLC and NMR analyses. An optimized assay procedure was used to measure the kinetic constants (KM and Vmax values) of BsPAD for three substrates, which indicates the broad applicability of the assay.

Abstract (croatian)

Biokatalitičkom dekarboksilacijom prirodnih hidroksicimetnih kiselina dobiju se derivati p-hidroksistirena, koji su važni prekursori u proizvodnji antioksidansa, epoksi premaza, adheziva i drugih polimernih materijala. Dekarboksilaza fenolnih kiselina iz bakterije Bacillus subtilis (BsPAD) je enzim neovisan o kofaktoru, koji katalizira izdvajanje ugljičnog dioksida iz p-hidroksicimetnih kiselina poput p kumarinske, kofeinske, ferulinske i sinapinske kiseline uz visoku katalitičku učinkovitost. Metode koje se koriste za kinetičku karakterizaciju reakcija dekarboksilacije se temelje na korištenju HPLC, MS, GC ili NMR, i zahtijevaju opsežnu pripremu uzoraka, a njima se određuje samo krajnja koncentracija produkata ovih reakcija. U ovom diplomskom radu razvijena je brza, robusna i osjetljiva spektrofotometrijska metoda kojom se reakcija dekarboksilacije prati u realnom vremenu i kod koje nije potrebna ekstrakcija dobivenih produkata. Apsorpcijski spektri produkata snimljeni su nakon potvrde o kompletnoj dekarboksilaciji supstrata i to dvjema metodama - TLC i NMR. Optimizirana metoda korištena je za određivanje kinetičkih konstanti (KM i Vmax) za tri supstrata BsPAD, što ukazuje na široku primjenu ove razvijene metode.

• Keywords: p-hydroxycinnamic acids; phenolic acid decarboxylase; UV-VIS spectrophotometric assay
• Keywords (croatian): p-hidroksicimetne kiseline; dekarboksilaza fenolne kiseline; UV-VIS
• Language: english
• URN:NBN: urn:nbn:hr:159:342678
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-07-09 10:07:32