Journal of Molecular Catalysis B: Enzymatic 47 (2007) 21–27 Transglycosylation reactions using glycosyl hydrolases from Thermotoga neapolitana, a marine hydrogen-producing bacterium Annabella Tramice, Edoardo Pagnotta, Ida Romano, Agata Gambacorta, Antonio Trincone ∗ Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Naples, Italy Received 27 December 2006; received in revised form 9 March 2007; accepted 19 March 2007 Available online 24 March 2007 Abstract The use of the extremophilic marine bacterium Thermotoga neapolitana (DSM 4359) for the bioproduction of hydrogen using complex carbo- hydrates as feedstock has been recently described in a patent. In this growth condition glycosyl hydrolases can be well expressed. An important issue to plan economical feasibility of biological hydrogen production comprises techniques for recovering useful materials such as physiologically active biomolecules from biomasses grown in large quantities. The present paper describes a series of enzymatic transglycosylation reactions performed using the crude homogenate of T. neapolitana. The study is focused on synthetic features of the transglycosylating enzymes. Xylosidase/xylanase activity seems to be the most abundant leading to convenient syntheses of interesting series of pure (-1,4)-xylooligosaccharides of different aglycones such as 1-hexanol (producing promising candidates for new surfactants), 9-fluorene methanol (obtaining anti-HSV agents), 1,4-butanediol (for the synthesis of new glycolipids), and geraniol (producing aroma compounds). Furthermore, the regioselectivity during galactose, fucose, glucose, and mannose enzymatic transfers is also investigated. The knowledge of synthetic characteristics of all these enzymes will be useful in the feasibility evaluation of large-scale processes of the biohydrogen production based on T. neapolitana. © 2007 Elsevier B.V. All rights reserved. Keywords: Glycosyl hydrolases; Xylosides; Thermotoga; Biocatalysis; Biohydrogen 1. Introduction Marine ecosystem is considered a quite unexplored source of biological material and can be also a surprising font of enzymes endowed with new and interesting catalytic activities for appli- cations in biocatalysis [1]. Marine sources are represented by marine microorganisms comprising bacterial extremophiles, and plants or animals. Thermotoga neapolitana (DSM 4359) is an extremophilic marine bacterium belonging to the Thermotogales order. The ability of this microorganism to produce hydrogen has been described in a patent specifically in an environmental- friendly process using complex carbohydrates as feedstock ∗ Corresponding author. Tel.: +39 081 8675095; fax: +39 081 8041770. E-mail address: antonio.trincone@icb.cnr.it (A. Trincone). [2]. Members of this order possess an array of impor- tant hyperthermophilic glycosyl hydrolases [3] that in the conditions adopted for hydrogen production can be well expressed. In the last few years, glycosyl hydrolases gained interest as applicative enzymes for the convenient synthesis of bio- logical relevant glycosidic linkages in the field of production of oligosaccharides [4,5]. They belong to inverting or retain- ing class on the basis of their mechanism of action. In the inverting mechanism, the hydrolysis occurs through a single dis- placement involving an oxocarbenium ion-like transition state. The retaining reaction mechanism involves a nucleophile group and an acid/base catalyst. This reaction proceeds by a double- displacement in which a covalent glycosyl-enzyme intermediate is firstly formed (glycosylation step) and then hydrolysed (deg- lycosylation step) in a general acid/base-catalysed process. Therefore, retaining glycosyl hydrolases can transglycosylate by 1381-1177/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2007.03.005