Genes encoding two different β-glucosidases of Thermoanaerobacter brockii are clustered in a common operon

Abstract

A 5.9-kb fragment of chromosomal DNA coding for β-glucosidase activity of the thermophilic anaerobe Thermoanaerobacter brockii was sequenced. Two genes, cglT and xglS, encoding a cellodextrin-cleaving β-glucosidase and a xylodextrin-degrading xylo-β-glucosidase, respectively, were located directly adjacent to each other. The 5' region contained two additional genes, cglF and cglG, whose products exhibited similarity to integral membrane proteins of metabolite transport systems. The two β-glucosidases, CglT and XglS, with deduced molecular masses of 52 and 81 kDa, belong to different families of glycosyl hydrolases. Both enzymes were overexpressed in Escherichia coli and could be detected after protein gel electrophoresis and activity staining. The enzyme CglT was purified by fast protein liquid chromatography and identified by N-terminal sequencing. The enzyme was thermostable at 60°C for at least 24 h, and the temperature optimum was 75°C. The k(i) for glucose inhibition was calculated to 200 mM. The enzyme released glucose from the nonreducing end of β-1,4-cello oligomers as well as from various disaccharides. CglT was active on glucosides, galactosides and on fucosides while XglS cleaved β-glucosides and β-xylosides as well. The cglT gene was also expressed in Bacillus subtilis, and the enzyme was mainly intracellular during exponential growth but was efficiently released into the supernatant after cultures entered the stationary phase.