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

Samuel J. Butler, Simon Birgersson, Mathias Wiemann, Monica Arcos-Hernandez, Henrik Stålbrand

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)154-166
Number of pages13
JournalProcess Biochemistry
Volume112
DOIs
Publication statusPublished - 2022

Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

Free keywords

  • β-Mannanase
  • Transglycosylation
  • Enzymatic synthesis
  • Enzyme engineering
  • Synergy
  • Galactomannan

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