Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escheria coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°

A bacteriophage detection tool for viability assessment of Salmonella cells

Available online 7 September 2013Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods

Molecular studies on protein- and carbohydrate-converting ezymes from thermophilic bacteria

Microorganisms that are able to grow at hightemperatures are calledthermophiles(>55°C) orhyperthermophiles(>80°C). Growth at high temperatures involvesthermostableenzymes, and the question how this stabilization takes place at a structural level has played a central role in research of these organisms. In addition, the interest in applying them inindustry as biocatalysts has largely increased.The bacterial domain holds a few (hyper)thermophilicmembers, and the organisms that were used in this study, Fervidobacterium pennivorans , F. gondwanense and Thermotogamaritima , belong to theThermotogalesorder. They grow optimally from 65 to 80°C and are able to use a large variety of carbohydrates and peptides, for which they possess a range of enzymes. This research describes the detailed molecular and biochemical characterization of enzymes that enable growth of these organisms on, occasionally, remarkable substrates like pectin or chicken feathers. The study on these so-called thermozymes is an example of an integrated approach in which bioinformatics, molecular genetics, structural analysis, DNA microarray analysis, biochemistry and fermentation has been combined

Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostabilit

Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin

Structure-forming fibrous proteins like keratins, gelatins and collagens are degraded only by a few proteases as their tight packing limits access to the potential cleavage sites. To understand the keratin degradation in detail, we describe the first crystal structure of a keratin-degrading enzyme (keratinase), fervidolysin, from Fervidobacterium pennivorans as an immature form with propeptide (PD)-bound. The 1.7 Angstrom resolution crystal structure shows that the protease is composed of four domains: a catalytic domain (CD), two P-sandwich domains (SDs), and the PD domain. A structural alignment shows a distant relationship between the PD-CD substructure of fervidolysin and pro-subtilisin E. Tight binding of PD to the remaining part of the protease is mediated by hydrogen bonds along the domain surfaces and around the active cleft, and by the clamps to SD1 and SD2. The crystal structure of this multi-domain protein fervidolysin provides insights into proenzyme activation and the role of non-catalytic domains, suggesting a functional relationship to the fibronectin (FN)-like domains of the human promatrix metalloprotease-2 (proMMP-2) that degrades the fibrous polymeric substrate gelatin. (C) 2003 Elsevier Ltd. All rights reserved

The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer

The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonase

Characterisation and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritime

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 °C and a temperature optimum of 80 °C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U·mg1). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed

Cloning and expression of islandisin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escheria coli

A gene encoding a subtilisin-like protease, designated islandisin, from the extremely thermophilic bacterium Fervidobacterium islandicum (DSMZ 5733) was cloned and actively expressed in Escherichia coli. The gene was identified by PCR using degenerated primers based on conserved regions around two of the three catalytic residues (Asp, His, and Ser) of subtilisin-like serine protease-encoding genes. Using inverse PCR regions flanking the catalytic residues, the gene could be cloned. Sequencing revealed an open reading frame of 2,106 bp. The deduced amino acid sequence indicated that the enzyme is synthesized as a proenzyme with a putative signal sequence of 33 amino acids (aa) in length. The mature protein contains the three catalytic residues (Asp177, His215, and Ser391) and has a length of 668 aa. Amino acid sequence comparison and phylogenetic analysis indicated that this enzyme could be classified as a subtilisin-like serine protease in the subgroup of thermitase. The whole gene was amplified by PCR, ligated into pET-15b, and successfully expressed in E. coli BL21(DE3)pLysS. The recombinant islandisin was purified by heat denaturation, followed by hydroxyapatite chromatography. The enzyme is active at a broad range of temperatures (60 to 80°C) and pHs (pH 6 to 8.5) and shows optimal proteolytic activity at 80°C and pH 8.0. Islandisin is resistant to a number of detergents and solvents and shows high thermostability over a long period of time (up to 32 h) at 80°C with a half-life of 4 h at 90°C and 1.5 h at 100°