Eloy Ferreras

• Autonomous University of Madrid

Screening of an Antarctic soil functional fosmid metagenomic library identified a novel bacterial gene, homologous to known Water Hypersensitivity (WHy) domains. The WHy domain is a typical component of Late Embryogenesis Abundant (LEA) proteins which occurs widely in both prokaryotes and in plant eukaryotes and are expressed under various stress c...

Metagenomic library screening, by functional or sequence analysis, has become an established method for the identification of novel genes and gene products, including genetic elements implicated in microbial stress response and adaptation. We have identified, using a sequence based approach, a fosmid clone from an Antarctic desert soil metagenome l...

The Antarctic continent is largely covered by an expansive ice sheet, but it harbors diverse terrestrial and aquatic habitats in the coastal ice-free continental margins. Here we present the draft genome of Microbacterium sp. CH12i, which was isolated from hypersaline, alkaline, and nutrient-rich groundwater from Cape Hallett, northern Victoria Lan...

The ancient bacterial lineage Thermus spp has a primitive form of outer membrane attached to the cell wall through SlpA, a protein that shows intermediate properties between S-layer proteins and outer membrane (OM) porins. In E. coli and related Proteobacteria, porins are secreted through the BAM (β-barrel assembly machinery) pathway, whose main co...

Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the th...

Optimum pH of HIS6::TthPAC. The TthPAC enzymatic activity was assayed at 65°C in the presence of 2.5 mM PenK and in 20 mM Britton-Robinson buffer at pH 3, 4, 5, 6, 7, 8 or 9.

Comparison of the kinetic data of TthPAC with other penicillin acylases. Data were measured at 40°C unless otherwise stated.

Chaperone elimination after co-expression with HIS6:: Tth PAC. Tth PAC was co-expressed with GroEL/ES in E. coli BL21 cells. Total soluble protein fraction was incubated in the absence (lanes 1–4) or in the presence of 5 mM ATP/10 mM Cl2Mg (lanes 5–8) [41] for 2 h at 4°C. In order to separate the Tth PAC from the detached GroEL/ES a 0-, 30-, 45- or...

Heterologous expression of the chimeric protein SpEco-PACTthin E. coli cells.TthPAC α- and β-subunit were immunodetected in the cytoplasmic fraction (upper and middle panels) and in the periplasmic fraction (lower pannel) of E. coli cells from BL21 strain(1, A and G); Rosetta-gami2 strain (2, B and H); BL21 strain co-expressing GroEL/ES and trigger...

Sequence alignment of PKAs and characterized PGAs. TthPKA, Thermus thermophilus HB27 PKA [TTC1972]; AutaPKA, Actinoplanes utahensis PKA [P29958]; SlavPKA, Streptomyces lavendulae PKA [AY611030]; EcoPGA, Escherichia coli PGA [P06875]; AfaePGA, Alcaligenes faecalis [ADD11517]; PretPGA, Providencia rettgeri PGA [AAP86197]; KcitPGA, Kluyvera citrophila...

Thermal inactivation course of TthPAC. Solutions containing 0.08 mg/ml of purified TthPAC were incubated in 50 mM phosphate buffer pH 7.5 at 65 and 75°C. The residual hydrolytic activity was determined using 5 mM penicillin K as substrate.

Table S2. Primers used in this work.

Room temperature ionic liquids (ILs) can affect enzyme activity in some enzyme-catalyzed reactions, however the effects of these cosolvents on the enzymes are not clearly understood. Using β-galactosidase from Thermus thermophilus HB27 (TTP0042), we found an important change from the classical regioselectivity of the transglycosylation reaction wit...

Cell extracts from ten different strains of Thermus thermophilus have been screened for glycosidase activity. Among these, the sequenced strain HB27 hydrolyzed a wide variety of glycosides and increased six fold its β-glycosidase activity when grown with cellobiose in nutrient-limited media. We selected five genes encoding (putative) glycosidases (...

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes...