title: Improved transferase/hydrolase ratio through rational design of a family 1 β-glucosidase from Thermotoga neapolitana.
abstract: 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 to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable β-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, 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 an aqueous environment, while none displayed this property in the 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 a 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%.
authors.affiliation: Lund University, Department of Chemistry, Biotechnology, Lund, Sweden. pontus.lundemo@biotek.lu.se
authors.forename: Pontus
authors.initials: P
authors.lastname: Lundemo
authors.forename: Patrick
authors.initials: P
authors.lastname: Adlercreutz
authors.forename: Eva Nordberg
authors.initials: EN
authors.lastname: Karlsson
mesh_terms: D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006027:Glycosides / Q000096:biosynthesis*; D006867:Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA; D013501:Surface-Active Agents; D046949:Thermotoga neapolitana / Q000201:enzymology*; D014166:Transferases / Q000378:metabolism; D014867:Water / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D006027:Glycosides; D013501:Surface-Active Agents; D014867:Water; D014166:Transferases; D006867:Hydrolases; D001617:beta-Glucosidase