Improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from Thermotoga neapolitana.

Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis one can obtain well-defined alkyl glycosides, something that is very difficult using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from Thermotoga neapolitana, TnBgl1A, from the well-studied glycoside hydrolase family 1 was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in aqueous environment while none displayed this property in presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17 % to 58 %.