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The first hyperthermophilic D-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of D-arabitol, D-xylitol, D-ribulose, or D-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg(2+) or K(+). Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of D-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of D-arabitol to D: -ribulose and therefore belongs to the class of D-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product D-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, D-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors.

The phosphotriesterase-like lactonase (PLL) encoded by Vmut_2255 in the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia (VmutPLL), represents the only hyperthermophilic PLL homologue identified so far in addition to the previously characterized thermophilic PLLs from Sulfolobus spp. The Vmut_2255 gene was cloned, heterologously expressed in Escherichia coli ; the resultant protein purified and characterized as a 82 kDa homodimer (36 kDa subunits). The VmutPLL converted lactones and acyl-homoserine lactones (AHLs) with comparable activities. Towards organophosphates (OP) VmutPLL showed a promiscuous but significantly lower activity and only minor activity was observed with carboxylesters. The catalytic activity strictly depended on bivalent cations (Cd 2+ > Ni 2+ > Co 2+ > Mn 2+ > Zn 2+ ). Furthermore, VmutPLL showed a pH optimum around 8.0, a temperature optimum of 80 °C, and thermostability with a half-life of 26 min at 90 °C. In this work, the stereoselectivity of a PLL enzyme was investigated for the first time using enantiopure lactones. The VmutPLL showed a slight preference but not an exclusive specificity for the (R)-enantiomers of capro- and valerolactone. The high thermal stability as well as the broad substrate spectrum towards lactones, AHLs and OPs underlines the high biotechnological potential of VmutPLL.