Enzymatic glucosylation: sucrose glucosyltransferases and glucosidases in O- and S-glucoside synthesis

Glycosylation is considered as a useful method for improving chemical properties like solubility and volatility of compounds with interesting organoleptic or physiological properties. The aim of the research described in this thesis was to explore the enzymatic glycosylation of aglycones (nonsaccharide acceptor molecules). The initial focus is on the glucosylation of aromatic alcohols by non-Leloir glucosyltransferases like sucrose glucosyltransferases.
Several streptococcal sucrose glucosyltransferases (glucansucrases) were screened for transglucosylation activity using the flavonoid catechin as model aglycone and sucrose as an economically feasible glucosyl donor substrate (Chapter 2). Streptococcusmutans GS-5 glucosyltransferase-D (GTF-D) glucosylated catechin most efficiently (90% catechin yield). Three different catechin glucosides were isolated of which two catechin glucoside structures were spectroscopically elucidated; catechin-4'- O -α-D- and catechin-4',7- O -α-di-D-glucopyranoside. The structure of the third glucoside remained unsolved, although hydrolysis studies using Aspergillusniger amyloglucosidase suggested that catechin-7- O -α-D-glucopyranoside was formed. The acceptor specificity of GTF-D towards aromatic aglycones is restricted to compounds containing two adjacent aromatic hydroxyl groups, e.g. (substituted) catechol(s) (Chapter 3). This suggests that one hydroxyl group is involved in the interaction with GTF-D, while the other is available for formation of the glucosidic linkage. Compounds containing one hydroxyl group like phenol, irreversibly inhibit GTF-D transglucosylation activity.
To facilitate the transglucosylation of less water soluble aglycones, the addition of water miscible organic solvents (cosolvents) was studied (Chapter 3). Bis-2-methoxyethyl ether (MEE) was selected as the most appropriate cosolvent. MEE addition resulted in a 4-fold increase in catechin transglucosylation activity due to a 12-fold increase in catechin solubility. Addition of MEE (10-30% v/v) also enabled the glucosylation of catechol aglycones at otherwise inhibitory concentrations (200 mM). This was explained by assuming that the partitioning of the aglycone between solvent and enzyme was changed upon MEE addition.
The addition of bis-2-methoxyethyl ether also affected the formation rate and absolute amounts of glucan formed (Chapter 4). An increase of 20% in reducing sugars was observed using 20% (v/v) MEE. Besides an increase in sucrose hydrolysis there was also an increase the formation of high molecular weight glucan chains (10 ²-10 ³kDa). Linkage analysis showed that also the type of glucosidic linkage was affected upon MEE addition. Glucan formed in the presence of MEE contained an increased amount ofα(1,3) linkages. It was hypothesised that MEE affected glucan formation through modifying the GTF-D glucan binding domain.
The accumulation of fructose was shown to inhibit aglycon glucosylation and glucan formation. To overcome this inhibition, the fructose consuming yeasts Pichiapastoris and the mutant Saccharomycescerevisiae T2-3D were added (Chapter 2). Both yeasts are incapable of utilising sucrose. Due to the consumption of fructose during transglucosylation, an increase in glucoside yield and the maximum duration of catechin glucosylation was observed. Consequently, the consumption of sucrose by GTF-D increased. Eventually glucosylation yields by GTF-D could be engineered either by adding MEE or by fructose consuming yeasts (Chapter 2 and 3). Using MEE, the glucoside yield based on catechin decreased and the sucrose based yield increase. The addition of the yeasts resulted in an increased catechin based glucoside yield and a decreased sucrose based glucoside yield.
In the second part of this thesis the attention is focussed on glycosidases and the hydrolysis and formation of thioglucosides. The observed stability of thioglucosides towards enzymatic hydrolysis was used to screen for new thioglucoside active enzymes (Chapter 5). Using octylthioglucoside (OTG) as a carbon source for microbial growth, Sphingobacterium sp. strain OTG1 was isolated. In the cell free extract a novel thioglucoside hydrolase activity was observed, showing distinct characteristics compared to typicalβ- or thioglucosidases. Various thioglucosides were hydrolysed by the Sphingobacterium cell free extract, with almost the same activities.
In view of the aim of this thesis, the synthesis of glycosides, various glycosidases were screened for the glycosylation of 1-propanethiol (Chapter 6). Theβ-glucosidase from almond, Aspergillusniger and Caldocellumsaccharolyticum showed thioglucosylation activity using glucose and 1-propanetiol. The almond enzyme showed the highest activity (3μmol.min ^-1.mg ^-1). Only primary and secondary (10-fold slower reaction rate) aliphatic thiols were glucosylated. Of the different thiols examined, the glucosylation of furfuryl mercaptan is the most interesting because of the potential of the glucoside as a flavour precursor.
Because of the stability of thioglucosides towards most enzymatic hydrolases (glycosidases), an excess of aglycone otherwise frequently applied in reversed hydrolysis glycosylation is not necessary for thioglucosylation. Consequently, high yields up to 60% based on 1-propanethiol and 40% based on glucose were obtained for the synthesis of 1-propanethioglucoside.