Transglycosylation catalyzed by β-mannanases: Molecular aspects relevant for enzymatic synthesis

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title = "Transglycosylation catalyzed by β-mannanases: Molecular aspects relevant for enzymatic synthesis",
abstract = "β-Mannanases are a major type of enzyme involved in the hydrolysis and modification of β-mannans, a hemicellulose present in high amounts in e.g. softwoods such as spruce. The β-mannanases studied in this thesis belong to glycoside hydrolase (GH) families GH5 and GH26. In addition to hydrolysis, some β-mannanases can also catalyze transglycosylation whereby another molecule (acceptor) is transferred onto a saccharide (donor); this makes β-mannanases potentially useful in enzymatic synthesis of glyco-conjugates. To date, transglycosylation capacity with saccharides and alcohols as acceptors has been observed with several GH5 β-mannanases but not with any GH26 ones. The work included in this thesis aims to provide further insight into the molecular aspects of β-mannanase-catalyzed transglycosylation.GH5 and GH26 β-mannanases from different environments were studied. Comparative studies of three GH5 β-mannanases from the fungus Aspergillus nidulans (Paper I) revealed high transglycosylation capacity with saccharides as acceptors with the β-mannanase AnMan5B. However, AnMan5B could not use alcohols as acceptors (alcoholysis) in contrast to the other two studied β-mannanases AnMan5A and AnMan5C. A non-conserved tryptophan in the putative +2 subsite of AnMan5B may contribute to its comparably high transglyco-sylation capacity with saccharides as acceptors. In Paper IV alcoholysis was studied with AnMan5C as well as the Trichoderma reesei GH5 β-mannanase TrMan5A and its +2 subsite-engineered variant TrMan5A-R171K, where the R171K substitution has previously been shown to eliminate transglycosylation with saccharides as acceptors. TrMan5A was used to enzymatically synthesize alkyl mannosides with surfactant properties. Lower alcoholysis capacity was observed with TrMan5A-R171K compared to TrMan5A, indicating a potential role of Arg171 in subsite +2 in interactions with alcohols. Residues in the +2 subsite were also studied in Paper V where two tryptophans in the +2 subsite of the blue mussel GH5 β-mannanase MeMan5A were substituted with alanines. The +2 subsite substitutions were found to impair the hydrolytic activity of MeMan5A and reduced or eliminated transglycosylation with saccharides as acceptors, but did not appear to affect alcoholysis. Transglycosylation with saccharides as acceptors was also observed in Paper II with the GH5 β-mannanase BlMan5A from the human gut bacterium Bifidobacterium animalis subsp. lactis. However, no transglycosylation with saccharides as acceptors was observed with the GH26 β-mannanases BoMan26A or BoMan26B from the human gut bacterium Bacteroides ovatus in Paper III. Interestingly, BoMan26A was able to use methanol as acceptor, the first reported instance of alcoholysis capacity in GH26 β-mannanases to date.The results presented in this thesis give further insight into enzyme-acceptor interactions in aglycone (+) subsites in β-mannanase-catalyzed transglycosylation with saccharides and alcohols as acceptors. The thesis also discusses several factors potentially influencing transglycosylation capacity and acceptor specificity in GH5 and GH26 β-mannanases, which may increase the applicability of β-mannanases in enzymatic synthesis.",
keywords = "β-mannanase, glycoside hydrolase, enzymology, kinetics, MALDI-TOF MS, HPLC, transglycosylation, alcoholysis, enzyme engineering, protein structure, enzymatic synthesis",
author = "Johan Morrill",
note = "Defence details Date: 2017-10-20 Time: 13:15 Place: Lecture hall C, Center for chemistry and chemical engineering, Naturvetarv{\"a}gen 14, Lund External reviewer(s) Name: Biely, Peter Title: Dr. Affiliation: Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia --- ",
year = "2017",
language = "English",
isbn = "978-91-7422-544-0",
publisher = "Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology",
school = "Biochemistry and Structural Biology",