Converting a {beta}-glycosidase into a {beta}-transglycosidase by directed evolution.
Directed evolution was applied to the beta-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.
Feng
Hui-Yong
HY
Biotechnologie, Biocatalyse, Biorégulation (UMR CNRS 6204), Université de Nantes Faculté des Sciences et des Techniques, 2 Rue de la Houssinière, BP 92208, F-44322, Nantes Cedex 3, France.
Drone
Jullien
J
Hoffmann
Lionel
L
Tran
Vinh
V
Tellier
Charles
C
Rabiller
Claude
C
Dion
Michel
M
D002475:Cellobiose / Q000737:chemistry / Q000378:metabolism; D019020:Directed Molecular Evolution; D044644:Endo-1,3(4)-beta-Glucanase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D006027:Glycosides; D002475:Cellobiose; D008320:Maltose; D005964:Glucosyltransferases; D044644:Endo-1,3(4)-beta-Glucanase