Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-beta-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 A degrees C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a beta-glucosidase resulted in the unexpected generation of a thermostable, highly specific beta-fucosidase, concomitant with large increases in beta-glucosidase, beta-cellobiosidase, alpha-arabinofuranosidase, beta-mannosidase, beta-glucuronidase, beta-xylopyranosidase, and beta-fucosidase activities and a dramatic decline in 6-phospho-beta-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-beta-glycosidase and to generate a thermostable beta-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.