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Since information is lacking regarding substrate availability in the various sediment layers of Chott El Guettar, we may only speculate that this saline environment offers biomass of a less variable nature, probably mostly of algal origin, which could explain the lack of catalytic diversification of GH43 enzymes in H. orenii. To date, the crystal structures of 28 unique GH43 members have been determined, mainly including bacterial ��-xylosidases, xylanases, ��-l-arabinofuranosidases and arabinanases (http://www.cazy.org). Based on sequence similarity to other GH43 arabinofuranosidases (EC 3.2.1.55), the gene Olaparib chemical structure encoding the H. orenii glycosidase (Gene ID 7314382; UniProt accession No. B8CZV1) was suggested to be a putative ��-l-arabinofuranosidase (Kori, 2012 ?). Here, we report the cloning, overexpression, high-resolution crystal structure determination and preliminary biochemical characterization of the H. orenii GH43 glycosidase. 2.?Experimental procedures ? 2.1. Cloning, expression and purification ? The gene encoding the H. orenii glycosidase was cloned into the pNIC28-Bsa4 vector with a cleavable His6 tag and a Tobacco etch virus (TEV) protease cleavage site at the N-terminus (Savitsky et al., 2010 ?) using ligation-independent cloning (Doyle, 2005 ?). The recombinant plasmid was transformed into Escherichia coli bepotastine strain BL21(DE3) and the protein was overexpressed for 16�C18?h at 18��C following induction with 0.2?mM isopropyl ��-d-1-thiogalactopyranoside. The cells were lysed in 20?mM HEPES pH 7.0, 150?mM NaCl (lysis buffer) using an Avestin Emulsiflex-C3 system. The gene product was purified from the lysate using Ni�CNTA agarose resin (Invitrogen) eluted with lysis buffer containing 350?mM imidazole. The His6 tag and imidazole were removed by dialysis of the sample overnight in a dialysis bag (12�C14?kDa molecular-weight cutoff) containing TEV protease and 20?mM HEPES pH 7.0, 150?mM NaCl. The sample was rerun through the Ni�CNTA resin and the flowthrough containing the target protein was collected and concentrated. To remove remaining Ni2+ ions, 20?mM EDTA and 20?mM EGTA were added and a final step of gel filtration was performed using a HiLoad 26/60 Superdex 200 prep-grade column (GE Healthcare Life Sciences) equilibrated with 20?mM HEPES pH 7.0, 150?mM NaCl. Eluted fractions containing the H. orenii glycosidase ROCK inhibitor were collected in a 96-well deep-well microplate, pooled and concentrated using a Vivaspin centrifugal concentrator (10?kDa molecular-weight cutoff). 2.2. Stability assay using Thermofluor screening ? The effects of various parameters on the thermal stability against unfolding of the GH43 glycosidase were investigated using Thermofluor screening (Ericsson et al., 2006 ?). Specifically, the influence of pH (5.5�C9.0), metal ions (2?mM Mn2+, Mg2+, Ca2+ and Na+) and salt (0, 0.1, 0.5, 1.0, 2.0, 4.0 and 5.
