An open reading frame (ORF) encoding the enzyme β-glucosidase from the extremely thermophilic bacterium Fervidobacterium islandicum has been identified, cloned and sequenced. The bgl1A gene was cloned in a pET-Blue1 vector and transformed in Escherichia coli, resulting in high-level expression of β-glucosidase FiBgl1A that was purified to homogeneity in a two-step purification. FiBgl1A is composed of 459 amino acid residues and showed high homology to glycoside hydrolase family 1 proteins. It exhibited highest activity towards p-nitrophenyl-β-D: -glucopyranoside with an optimum activity at pH 6.0 and 7.0 and at 90 °C. The enzyme is resistant to glucose inhibition. Furthermore, it did not require divalent cations for activity, nor was it affected by the addition of p-chloromercuribenzoate (10 mM), EDTA (10 mM), urea (10 mM) or dithiothreitol (10 mM). Addition of surfactants (with the exception of SDS) and a number of solvents enhanced the activity of FiBgl1A. It also displayed remarkable activity across a broad temperature range (80-100 °C). The thermoactivity and thermostability of FiBgl1A and its resistance to denaturing and reducing agents make this enzyme a potential candidate for industrial applications.

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni(2+)-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni(2+), activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD(+)/NADH, whereas no activity has been observed with NADP(+)/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn(2+). The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni(2+)-dependent activity towards 1,3-propanediol.

NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

Glaciecola sp. strain 4H-3-7+YE-5 was isolated from subseafloor sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecola sp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases, offering a high potential for plant biomass degradation.

Two members of the family Flavobacteriaceae were isolated from subseafloor sediments using artificial seawater with cellulose, xylan, and chitin as the sole carbon and energy sources. Here, we present the complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, which both encode putatively novel enzymes involved in cellulose, hemicellulose, and chitin metabolism.Members of the family Flavobacteriaceae are well known for degrading complex polymeric substrates in marine habitats (4). Two marine representatives of this family, Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, were isolated from subseafloor sediments at Suruga Bay (Japan) at depths of 31.4 and 41 m using a mixture of cellulose, xylan, and chitin as the sole carbon sources in enrichment cultures.To gain insight into the gene repertoire of these organisms, the complete genomes of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4 were generated at the Department of Energy (DOE) Joint Genome Institute (JGI) using Illumina (1) and 454 (3) technologies. For Krokinobacter sp. strain 4H-3-7-5, we sequenced an Illumina GAii shotgun library, which generated 38,286,828 reads (2,909 Mbp); a 454 Titanium standard library, which generated 205,742 reads; and a paired-end 454 library (average insert size, 11.1 Ϯ 2.8 kbp), which generated 287,950 reads, totaling 136.6 Mbp of 454 data. For Lacinutrix sp. strain 5H-3-7-4, we sequenced an Illumina GAii shotgun library, which generated 54,815,066 reads (4,165.9 Mbp); a 454 Titanium standard library, which generated 268,763 reads; and one paired-end 454 library with an average insert size of 17 kb, which generated 220,650 reads, totaling 152.2 Mbp of 454 data. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. The 454 Titanium standard data and the 454 paired-end data were assembled using Newbler version 2.3, while the Illumina sequencing data were assembled with Velvet versions 0.7.63 and 1.0.13 (5).

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Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.

Members of the carnobacteria have been extensively studied as probiotic cultures in aquacultures and protective cultures in seafood, diary, and meat. We report on the finished genome sequence of Carnobacterium sp. 17-4, which has been isolated from permanently cold seawater. The genetic information reveals a new circular bacteriocin biosynthesis cluster.