Browsing by Subject "structural analysis"

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  • Kontro, Jussi; Lyra, Christina; Koponen, Milla Jasmin; Kuuskeri, Jaana; Kähkönen, Mika; Wallenius, Janne; Wan, Xing; Sipilä, Jussi; Mäkelä, Miia; Nousiainen, Paula Annukka; Hilden, Kristiina (2021)
    Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70 degrees C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.
  • Juvonen, Minna (Helsingfors universitet, 2011)
    The aim of the present study was to implement a tandem mass spectrometry (MS/MS) method for determining glycosidic bond linkage positions of neutral isomeric glucooligosaccharides. The methods for structural analysis and the fragmentation mechanisms of oligosaccharides in MS/MS-analysis was reviewed. The MS/MS-spectra of oligosaccharide contains glycosidic bond and cross ring cleavage fragment ions. The linkage position can be determined by cross ring cleavage fragment ions of cationized or anionized oligosaccharides. The study started by analyzing the typical MS/MS-spectra of model isomeric disaccharides with different linkage positions. The data was then used to assess the MS/MS-spectra of model tri- and tetrasaccharides. The similarity of MS/MS fragmentation patterns of the model disaccharides and the tri- and tetrasaccharides would enable the linkage position analysis by the applied MS/MS method. The MS/MS-analyses were carried out by using electron spray ionization with ion trap mass spectrometer in both positive and negative mode. In positive mode the oligosaccharides were analyzed as sodium and lithium adduct ions while chloride adduct ions were used in negative mode analysis. The different linkage positions of disaccharides were characterized by the different MS/MS fragmentation profiles in positive and negative ionization methods. The reducing end linkage positions of tri- and tetrasaccharides were easily identified by comparing their MS/MS fragmentation patterns with the MS/MS-spectra of model disaccharides. The other linkages in the tri- and tetrasaccharide were identified by using negative mode. In positive mode the identification of other linkages was not possible because the cross ring cleavage fragment ions were formed mostly from the reducing end. The linkages of tri- and tetrasaccharides could not be identified by MS3-analysis due to formation of isomeric glycosidic bond fragment ions as the charge can be retained in the reducing or non-reducing end. The results indicated that the applied MS/MS method was suitable for determing the glycosidic bond linkages of oligosaccharides in negative mode. In positive mode only the reducing end linkage can be determined.