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

Hui-Yong Feng,Jullien Drone,Lionel Hoffmann,Vinh Tran,Charles Tellier,Claude Rabiller,Michel Dion

doi:10.1074/jbc.m502873200

Abstract

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.

Highlights

Glycosyltransferases and glycosidases constitute the two major classes of enzymes that can be used in such processes
Some limitations could still be noticed with this approach as follows. (i) aryl glycosides can be used as donors with formate or azide (21, 22), ␣-glycosyl fluorides are more often used because neither self-condensation nor hydrolysis of synthesis products can be observed
These donors are thermolabile, and when the reaction is catalyzed by thermophilic enzymes, high concentrations of enzyme must be used to compete efficiently with the spontaneous hydrolysis of ␣-glycosyl fluorides above 50 °C (18). (ii) The acceptor specificity seems often restricted to sugar such as aryl glycosides because almost no examples of transfer on natural disaccharides could be found with exoglycosynthases (17, 18)

Summary

EXPERIMENTAL PROCEDURES

Plasmids, and Media—Selection of ampicillin-resistant (100 ␮g/ml) Escherichia coli was made on LB agar plates. Expression of the tt␤gly gene was performed from the pBtac vector in the strain XL1 blue MRFЈ of E. coli. The expression plasmid containing the WT tt␤gly gene (GenBankTM accession number Y16753) (1.3 kb) under the control of the Ptac promoter and between the restriction sites EcoRI and PstI was termed pBBGly. Random and Directed Mutagenesis—Random mutations were introduced by mutagenic PCR (35). Primers D (5Ј-CAATTAATCATCGGCTCG) and F (5Ј-AATCTTCTCTCATCCGCC) flank the gene before the Ptac promoter and after the PstI restriction site. The reaction was thermocycled as follows: one cycle at 96 °C, 5 min, 30 cycles at 96 °C, 30 s; 55 °C, 30 s; 72 °C, 5 min. Mutagenized PCR products were digested by the EcoRI and PstI restriction enzymes and cloned back into the pBTac vector digested by the same enzymes. Saturation mutagenesis of codons 282, 339, 390, and 401 were carried

The abbreviations used are

RESULTS

DISCUSSION