Identification and characterization of cellodextrin phosphorylases for their application in synthesis of reducing-end thiol-modified cello-oligosaccharides

master's thesis

• Title (croatian): Identifikacija i karakterizacija celodekstrin fosforilaza za njihovu primjenu u sintezi tiol-modificiranih celo-oligosaharida
• Author: Mislav Sušac
• 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: 2023-03-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Four promising cellodextrin phosphorylases (CdPs) were identified as potential candidates for synthesis of reducing-end thiol-modified cello-oligosaccharides (retmco). The enzymes catalyzed two reactions - synthesis of the retmco and hydrolysis of glucosyl donor. CdP from Spirochetae thermophila (StCdP) proved to be the best candidate (the highest synthesis and the lowest hydrolase activity). Further improvement was achieved by the dual-enzyme cascade reaction, performed by cellobiose phosphorylase from Clostridium udae (CuCbP) and StCdP. The purity of synthesized 1-thio-cello-oligosaccharides >95% was confirmed with 1H-NMR, and average degree of polymerization was estimated to be 9.3. CdPs with low hydrolase activity have tyrosine in catalytic loop adjacent to the active site, while CdPs with high hydrolase activity have cysteine in this specific position, as confirmed by three single-site mutants. Additional improvement in the synthesis of the retmco was attained by the next dual-enzyme cascade reaction, performed by CuCbP and CdP from Clostridium cellulose mutant C626T.

Abstract (croatian)

Identificirane su četiri celodekstrin fosforilaze (CdPs) kao potencijalni kandidati za sintezu tiol-modificiranih celo-oligosaharida (retmco). Ovi enzimi kataliziraju reakciju u dva smjera - sintezu retmco i hidrolizu molekule donora glukozilne podjedinice. Ponajbolji kandidat (koji je imao najvišu aktivnost sintetaze i najnižu hidrolaznu aktivnost) je bila CdP iz Spirochetae thermophila (StCdP). Daljnje poboljšanje sinteze retmco postignuto je primjenom kaskadne reakcije s dva enzima - celobioza fosforilazom iz Clostridium udae (CuCbP) i StCdP. Čistoća sintetiziranog 1-tio-celo-oligosaharida iznad 95 % je potvrđena 1H-NMR analizom i prosječni stupanj polimerizacije dobivenog 1-tio-celooligosaharida procijenjen je na 9,3. CdPs sa niskom hidrolaznom aktivnosti imaju tirozin u katalitičkoj omči blizu aktivnog mjesta enzima, dok CdPs sa visokom hidrolaznom aktivnosti imaju cistein na ovoj specifičnoj poziciji, kao što je potvrđeno s tri točkaste mutacije. Daljnje poboljšanje sinteze retmco realizirano je primjenom kaskadne reakcije s dva enzima - CuCbP i mutanta C626T CdP iz Clostridium cellulose.

• Keywords: cellodextrin phosphorylases; reducing-end thiol-modified cello-oligosaccharides; cellulose; dual-enzyme cascade reaction
• Keywords (croatian): celodekstrin fosforilaze; tiol-modificirani celo-oligosaharidi; celuloza; kaskadna reakcija s dva enzima
• Language: english
• URN:NBN: urn:nbn:hr:159:241474
• 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: Closed access
• Created on: 2024-06-27 10:16:58