Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites -2 and -1. The known mobility of Trp at the -1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K-m on glucose derived substrates, while the negative effect on k(cat) is compensated by the Q88A mutation, which also contributes to a further reduction of K-m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase. (C) 2011 Elsevier Ltd. All rights reserved.