Synthesis, characterization and kinetic study of silver and gold nanoparticles produced by the archaeon Haloferax volcanii.

AIMS: To evaluate the ability of the haloarchaeon Haloferax volcanii to produce Ag and Au nanoparticles (NPs) and to characterize the obtained material in order to find relevant properties for future potential applications. METHODS AND RESULTS: Nanoparticles were produced by incubating H. volcanii cells with the corresponding metal salt. In the presence of precursor salts, cultures evidenced a colour change associated to the formation of metallic nanostructures with plasmonic bands located in the visible range of the spectrum. X-ray fluorescence analysis confirmed the presence of Ag and Au in the NPs which were spherical, with average sizes of 25 nmol l-1 (Ag) and 10 nmol l-1 (Au), as determined by electronic microscopy. Fourier transformed infrared spectroscopy indicated that both types of NPs showed a stable protein capping. Ag NPs evidenced antibacterial activity and Au NPs improved the specificity of polymerase chain reaction reactions. Au and Ag NPs were able to reduce 4-nitrophenol when incubated with NaBH4 . CONCLUSIONS: Haloferax volcanii is able to synthesize metallic NPs with interesting properties for technological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate the ability of H. volcanii to synthesize metal NPs and constitutes a solid starting point to deepen the study and explore novel applications.

An alternative easy method for antibody purification and analysis of protein-protein interaction using GST fusion proteins immobilized onto glutathione-agarose.

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep).

AIMS: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease-catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent-tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. METHODS AND RESULTS: The tripeptide Ac-Phe-Gly-Phe-NH(2) was synthesized using Ac-Phe-OEt and Gly-Phe-NH(2) substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1.5 or 0.5 mol l(-1) NaCl. Purification and identification of the peptide product was achieved by RP-HPLC and ESI-MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. CONCLUSION: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. SIGNIFICANCE AND IMPACT OF STUDY: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.

Growth phase-dependent biosynthesis of Nep, a halolysin-like protease secreted by the alkaliphilic haloarchaeon Natrialba magadii.

AIMS: The alkaliphilic haloarchaeon Natrialba magadii secretes a halolysin-like protease (Nep) that is active and stable in high salt and in organic solvents, which represents a potential resource for biocatalysis in low water activity conditions. In this study, the effect of the growth stage on Nep biosynthesis was examined. METHODS AND RESULTS: Nep mRNA and extracellular protease activity were measured by RT-PCR and azocaseinolytic activity determination, respectively. Increased abundance in Nep mRNA was observed in Nab. magadii cells with culture age, which correlated with accumulation of extracellular protease activity. Moreover, a 'stationary phase behavior' on synthesis of Nep was evidenced in low-density cultures incubated with stationary phase medium. CONCLUSIONS: nep gene expression is up-regulated during the transition to the stationary phase in response to 'factors' (metabolite and/or regulatory molecule) occurring in high-density cultures of Nab. magadii. Although the identity of these molecules remains to be determined, preliminary evidence suggests that they are hydrophobic and stable in high salt and high pH values (3.5 mol l(-1) NaCl, pH 10). SIGNIFICANCE AND IMPACT OF STUDY: This study contributes to gain insight into the regulation of haloarchaeal protease biosynthesis, facilitating the large-scale production of this extremozyme for basic studies or potential applications.

Effect of nutritional conditions on extracellular protease production by the haloalkaliphilic archaeon Natrialba magadii.

AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.

Autoproteolytic activation of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

The haloalkaliphilic archaeon Natronococcus occultus produces an extracellular serine protease in the stationary growth phase and upon starvation. Two proteins immunologically related to the extracellular protease were detected into the cells: P200 and P190. P200 was detected at early stages of growth and its relative amount decreased as the culture reached the stationary growth phase, concomitantly with the appearance of P190 and proteolytic activity, suggesting that P200 may be the precursor of the secreted protease and P190 the mature enzyme. Both proteins were also detected in the culture medium. Conversion of inactive P200 into active P190 was attained in cell-free culture medium from stationary phase but not from exponential phase. This process was prevented in the presence of PMSF and could be attained by addition of purified mature extracellular protease to P200. Altogether these results indicate that activation of Natronococcus occultus extracellular protease may be autoproteolytic and that factor/s present in stationary phase culture medium may be required for this process.

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease.

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests that Natronococcus occultus extracellular protease may be a novel enzyme.

Extracellular protease of Natrialba magadii: purification and biochemical characterization.

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.

Detection and preliminary characterization of extracellular proteolytic activities of the haloalkaliphilic archaeon Natronococcus occultus.

Extracellular proteolytic activity was detected in the haloalkaliphilic archaeon Natronococcus occultus as the culture reached the stationary growth phase. Proteolytic activity was precipitated with ethanol and subjected to a preliminary characterization. Optimal conditions for activity were attained at 60 degrees C and 1-2 M NaCl or KCl. Gelatin zymography in the presence of 4 M betaine revealed a complex pattern of active species with apparent molecular masses ranging from 50 to 120 kDa. Experiments performed with inhibitors of the various groups of proteases indicated that the extracellular proteolytic enzymes of N. occultus are of the serine type. Individual protein species showed some differences in salt and thermal stability.