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Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative [alpha]-glucosidase gene (Trc0107), characterized the recombinant enzyme, and found that the preferred substrate was [alpha],[alpha]-l,l-trehalose, a new feature among [alpha]-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose ([alpha]-l,2 linkage), nigerose and turanose ([alpha]-1,3), leucrose ([alpha]-1,5), isomaitose and palatinose ([alpha]-1,6), and maltose ([alpha]-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous [alpha]-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the a-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehaiose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the [alpha]-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the [alpha]-glucosidase), indicating that the ct-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOFS-IMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.