Engineering of xylose reductase based on ancestral sequence reconstruction

master's thesis

• Title (croatian): Inženjerstvo ksiloza reduktaze temeljeno na rekonstrukciji ancestralnih sekvenci enzima
• Author: Ema Ćurin
• Mentor: Anita Slavica (mentor)
• Committee member: Jasna Novak (predsjednik povjerenstva)
• Committee member: Renata Teparić (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: 2025-03-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Phylogenetic tree was constructed by using 350 extant xylose reductases and ancestral sequence reconstruction, and for 15 of them kinetic parameters for D-xylose as substrate and NADH as cofactor were selected. Based on these criterion, five ancestors, N0, N5, N15, N17 and N22, were chosen for xylose reductase engineering. All five His-tagged proteins were expressed in E. coli BL21(DE3) and purified. N15 and N22 showed the desired substrate selectivity with NADH as a cofactor. D-xylose was converted faster by N17 when NADH rather than NADPH was used as the cofactor. Among the five ancestors, N15 showed the highest thermostability (Tm = 74.5°C). When compared to specific extant enzymes, N15, N17 and N22 exhibit lower specific activity and catalytic efficiency for D-xylose and NADH. Although N17 holds high promises because of its NADH over NADPH preference, these three ancestors at this stage are to be improved for industrial xylitol production.

Abstract (croatian)

Rekonstrukcijom 350 sekvenci ksiloza reduktaze formirano je filogenetsko stablo i za 15 enzima odabrani su kinetički parametri za supstrat (D-ksiloza) i kofaktor (NADH). Temeljem ovih kriterija odabrano je pet ancestora, N0, N5, N15, N17 i N22, za inženjerstvo ksiloza reduktaze. Nakon što je pet proteina s His-tagom eksprimirano u E. coli BL21(DE3), slijedilo je njihovo pročišćavanje. Enzimi N15 i N22 su pokazali selektivnost prema D-ksilozi z NADH kao kofaktor. N17 brže konvertira D-ksilozu uz NADH nego uz NADPH kao kofaktor. Između pet ancestora, N15 ima najveću termostabilnost (Tm = 74.5°C). Ukoliko se usporede s postojećim enzimima, N15, N17 i N22 iskazuju nižu specifičnu aktivnost i katalitičku učinkovitost za D-ksilozu i NADH. Iako kataliza pomoću N17 ksiloza reduktaze ima potencijal primjene zbog afiniteta prema NADH, potrebna su daljnja istraživanja tri ancestora s ciljem optimiranja prije njihove eventualne primjene u industrijskoj proizvodnji ksilitola.

• Keywords: xylose reductase; ancestral sequence reconstruction; NADH; D-xylose
• Keywords (croatian): ksiloza reduktaza; rekonstrukcija ancestralnih sekvenci; NADH; D-ksiloza
• Language: english
• URN:NBN: urn:nbn:hr:159:239011
• 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: 2025-03-07 09:37:46