ORIGINAL PAPER Enzymes and proteins from extremophiles as hyperstable probes in nanotechnology: the use of D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis for sugars monitoring Luca De Stefano Æ Annalisa Vitale Æ Ilaria Rea Æ Maria Staiano Æ Lucia Rotiroti Æ Tullio Labella Æ Ivo Rendina Æ Vincenzo Aurilia Æ Mose’ Rossi Æ Sabato D’Auria Received: 30 October 2006 / Accepted: 29 November 2006 / Published online: 13 January 2007 Ó Springer 2007 Abstract The D-trehalose/D-maltose-binding protein (TMBP), a monomeric protein of 48 kDa, is one component of the trehalose and maltose (Mal) uptake system. In the hyperthermophilic archaeon Thermo- coccus litoralis, this is mediated by a protein-dependent ATP-binding cassette system transporter. The gene coding for a thermostable TMBP from the archaeon T. litoralis has been cloned, and the recombinant protein has been expressed in E. coli. The recombinant TMBP has been purified to homogeneity and characterized. It exhibits the same functional and structural properties as the native one. In fact, it is highly thermostable and binds sugars, such as maltose, trehalose and glucose, with high affinity. In this work, we have immobilized TMBP on a porous silicon wafer. The immobilization of TMBP to the chip was monitored by reflectivity and Fourier Transformed Infrared spectroscopy. Further- more, we have tested the optical response of the pro- tein-Chip complex to glucose binding. The obtained data suggest the use of this protein for the design of advanced optical non-consuming analyte biosensors for glucose detection. Keywords Trehalose/maltose-binding protein Á Biosensors Á Porous silicon Á Glucose Á Diabetes Á Archaeon Introduction The general interest in biomolecules, isolated from thermophilic organisms was originally due to the bio- technological advantages offered by the utilization of these highly stable molecules in industrial processes (Brock 1985). In fact, enzymes and proteins isolated from thermophilic microorganisms exhibit a high sta- bility in conditions usually used to denature proteins: high temperature, ionic strength, extreme pH-values, elevated concentration of detergents and chaotropic agents (Jaenicke 1994). In addition to the potential industrial applications, it is important to highlight that proteins and enzymes that are stable and active over 100°C represent good models to shed light on the molecular adaptation of life at high temperature (D’Auria et al. 1998). The D-trehalose/D-maltose-binding protein (TMBP) is one component of the trehalose (Tre) and maltose (Mal) uptake system, which, in the hyper- thermophilic archaeon Thermococcus litoralis, is med- iated by a protein-dependent ATP-binding cassette (ABC) system transporter (Xavier 1996). TMBP from T. litoralis is a monomeric 48 kDa two-domain mac- romolecule containing 12 tryptophan residues (Diez et al. 2001). TMBP shares common structural motifs Communicated by D. A. Cowan. The authors wish to dedicate this work to Prof. Ignacy Gryczynski, University of North Texas, TX, USA, for his outstanding contribution to the development of new sensing methodologies. L. De Stefano Á I. Rea Á L. Rotiroti Á I. Rendina Istituto di Microelettrica e Microsistemi, CNR, Napoli, Italy A. Vitale Á M. Staiano Á T. Labella Á V. Aurilia Á M. Rossi Á S. D’Auria (&) Istituto di Biochimica delle Proteine, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy e-mail: s.dauria@ibp.cnr.it 123 Extremophiles (2008) 12:69–73 DOI 10.1007/s00792-006-0058-6