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

Retaining beta-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 beta-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 degrees 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 beta-mannosides (with 1-3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar alpha-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.

Automated summary

The study demonstrated that the mutant R171K/E205D had a twofold higher propensity for transglycosylation with mannotetraose and allyl alcohol compared to wild-type TrMan5A. By employing enzyme synergy with GH27 guar alpha-galactosidase, there was a 2.1-fold increase in allyl mannosides and significant production of allyl galactopyranoside, leading to a 4.4-fold increase in overall yield of allyl glycosides.