Converting a β-Glycosidase into a β-Transglycosidase by Directed Evolution

Abstract

Directed evolution was applied to the ␤-glycosidase of Thermus thermophilus in order to increase its ability to synthesize oligosaccharide by transglycosylation. Wild-type enzyme was able to transfer the glycosyl residue with a yield of 50% by self-condensation and of about 8% by transglycosylation on disaccharides without nitrophenyl at their reducing end. By using a simple screening procedure, we could produce mutant enzymes possessing a high transferase activity. In one step of random mutagenesis and in vitro recombination, the hydrolysis of substrates and of transglycosylation products was considerably reduced. For certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative, whereas synthesis by transglycosylation on maltose and on cellobiose could reach 60 and 75%, respectively. Because the most efficient mutations, F401S and N282T, were located just in front of the subsite (؊1), molecular modeling techniques were used to explain their effects on the synthesis reaction; we can suggest that repositioning of the glycone in the (؊1) subsite together with a better fit of the acceptor in the (؉1) subsite might favor the attack of a glycosyl acceptor in the mutant at the expense of water. Thus these new transglycosidases constitute an interesting alternative for the synthesis of oligosaccharides by using stable and accessible donor substrates.

Degradation of Alkyl Methyl Ketones by Pseudomonas veronii MEK700

“…On the other hand, if the situation is similar to some glycosidases, there might be just one mutation necessary to convert the transferase activity into a hydrolysis activity. This was observed for a ␤-glycosidase of Thermus thermophilus, where several single mutations changed the main activity from hydrolysis action to transglycosylation and vice versa (18). Homoserine acetyltransferases play a role in de novo methionine biosynthesis of many bacteria.…”

Molecular basis of substrate specificity in family 1 glycoside hydrolases

“…Apart from that, residues indirectly involved in the interactions with the substrate may be important in the bglycosidase specificity (42,43). However, the identification of them is not simple due to b-glycosidases large number of residues.…”

“…The relevance of the residue that interacts with the basal platform of the subsite þ 1 was also demonstrated by the conversion of the Thermus thermophilus b-glycosidase (Ttbgly) into a transglycosidase using directed evolution (43). This conversion involved a better fit of the acceptor of the transglycosylation reaction in the subsite þ 1.…”

Correlation between thermostability and stability of glycosidases in ionic liquid

“…Overexpression and purification of the Tt gly enzyme were conducted as previously described (Feng et al 2005). The -galactosidase AgaB was overexpressed in XL1 blue cells and purified according to the procedure described in a previous paper (Dion et al 2001).…”

“…For this purpose, glycoside hydrolases have been particularly investigated but their use is limited by the competition between hydrolysis and transglycosylation reactions which are both catalyzed by these enzymes. To overcome this limitation, several protein engineering strategies, based on site directed mutagenesis (Mackensie et al 1998) or molecular directed evolution (Feng et al 2005), have been proposed to reduce and even eliminate the hydrolytic activities of glycosidases while keeping the transglycosylation activities. Other alternatives suggested to prevent the undesirable hydrolysis reaction were reducing the water activity by using organic solvents (van Rantwijk et al 1999) or using frozen buffer (ChiffoleauGiraud et al 1997).…”