Transglycosylation by β-mannanase TrMan5A variants and enzyme synergy for synthesis of allyl glycosides from galactomannan

• Published in: Process biochemistry (1991) Vol. 112; pp. 154 - 166
• Main Authors: Butler, Samuel J., Birgersson, Simon, Wiemann, Mathias, Arcos-Hernandez, Monica, Stålbrand, Henrik
• Format: Journal Article
• Language: English
• Published: Barking Elsevier Ltd 01-01-2022; Elsevier BV
• Subjects: Allyl alcohol; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Biologi; Biological Sciences; Biomaterials; Biomedical materials; Catalysis; Chemical synthesis; Enzymatic synthesis; Enzyme engineering; Enzymes; Galactomannan; Galactosidase; Glycopolymers; Glycosidases; Glycoside hydrolase; Glycosides; High-performance liquid chromatography; Liquid chromatography; Mannanases; Natural Sciences; Naturvetenskap; pH effects; Spectrometry; Substrates; Synergy; Transglycosylation; β-Mannanase; Transglycosylation; Enzymatic synthesis; Synergy; Galactomannan; Enzyme engineering; β-Mannanase
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2021.11.028

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•β-Mannanase TrMan5A variants catalysed synthesis of reactive allyl β-mannosides.•The substitutions R171 K/E205D increased the transglycosylation capacity two-fold.•TrMan5A can use galactomannan as donor for synthesis of allyl glycosides.•The yield increased four-fold using enzyme synergy by addition of α-galactosidase.
Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171K and R171K/E205D had pH optima between pH 4.0–5.3 and showed >80 % remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH2 column. R171K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1–3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.