Enzymatic thioxyloside synthesis: characterization of thioglycoligase variants identified from a site-saturation mutagenesis library of Bacillus circulans xylanase.
Thioglycoligases are engineered enzymes for the synthesis of thioglycosides that are derived from retaining glycosidases by replacing the acid/base catalyst. The optimal choice of substitution for the acid/base mutant is currently unknown, so to investigate this question a complete acid/base library of the model glycosidase Bacillus circulans xylanase (Bcx) was generated by using site-saturation mutagenesis. A novel screening approach combining active site titration with semiquantitative product analysis by thin layer chromatography was established and used to evaluate specific activities of each mutant enzyme within crude cell lysates. The six most active Bcx variants were analyzed in more detail, a pH optimum of 8.5 was established and the identity of reaction products was confirmed. Optimal choices for substitution were small, preferably polar amino acids such as threonine, cysteine, and serine. We discuss the resultant data in the context of previously published studies on thioglycoligases.
Armstrong
Zachary
Z
Department of Chemistry, University of British Columbia, Vancouver BC, Canada.
Reitinger
Stephan
S
Kantner
Terrence
T
Withers
Stephen G
SG
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009154:Mutation; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D006027:Glycosides; D013438:Sulfhydryl Compounds; C010807:xylosides; D043364:Endo-1,4-beta Xylanases