A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and <i>Bacteroides</i> GH36 <i>Bo</i>Gal36A α-Galactosidases

Abstract

The transglycosylation behavior and capacity of two clan GH-D α-galactosidases, BoGal36A from the gut bacterium Bacteroides ovatus and Aga27A from the guar plant, was investigated and compared. The enzymes were screened for the ability to use para-nitrophenyl-α-galactoside (pNP-Gal), raffinose and locust bean gum (LBG) galactomannan as glycosyl donors with the glycosyl acceptors methanol, propanol, allyl alcohol, propargyl alcohol and glycerol using mass spectrometry. Aga27A was, in general, more stable in the presence of the acceptors. HPLC analysis was developed and used as a second screening method for reactions using raffinose or LBG as a donor substrate with methanol, propanol and glycerol as acceptors. Time-resolved reactions were set up with raffinose and methanol as the donor and acceptor, respectively, in order to develop an insight into the basic transglycosylation properties, including the ratio between the rate of transglycosylation (methyl galactoside synthesis) and rate of hydrolysis. BoGal36A had a somewhat higher ratio (0.99 compared to 0.71 for Aga27A) at early time points but was indicated to be more prone to secondary (product) hydrolysis in prolonged incubations. The methyl galactoside yield was higher when using raffinose (48% for BoGal36A and 38% for Aga27A) compared to LBG (27% for BoGal36A and 30% for Aga27A).

Keywords

• transglycosylation
• α-galactosidase
• enzymatic synthesis