Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep∆CTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, Nep∆CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep∆CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase.

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3-1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.

The Lon protease from the haloalkaliphilic archaeon Natrialba magadii is transcriptionally linked to a cluster of putative membrane proteases and displays DNA-binding activity.

The ATP-dependent Lon protease is universally distributed in bacteria, eukaryotic organelles and archaea. In comparison with bacterial and eukaryal Lon proteases, the biology of the archaeal Lon has been studied to a limited extent. In this study, the gene encoding the Lon protease of the alkaliphilic haloarchaeon Natrialba magadii (Nmlon) was cloned and sequenced, and the genetic organization of Nmlon was examined at the transcriptional level. Nmlon encodes a 84 kDa polypeptide with a pI of 4.42 which contains the ATPase, protease and membrane targeting domains of the archaeal-type LonB proteases. Nmlon is part of an operon that encodes membrane proteases and it is transcribed as a polycistronic mRNA in N. magadii cells at different growth stages. Accordingly, NmLon was detected in cell membranes of N. magadii throughout growth by Western blot analysis using specific anti-NmLon antibodies. Interestingly, in electrophoretic mobility shift assays, purified NmLon bound double stranded as well as single stranded DNA in the presence of elevated salt concentrations. This finding shows that DNA-binding is conserved in the LonA and LonB subfamilies and suggests that Lon-DNA interaction may be relevant for its function in haloarchaea.

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.

Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep).

The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.

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.

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii.

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.

Haloarchaeal proteases and proteolytic systems.

Proteases play key roles in many biological processes and have numerous applications in biotechnology and industry. Recent advances in the genetics, genomics and biochemistry of the halophilic Archaea provide a tremendous opportunity for understanding proteases and their function in the context of an archaeal cell. This review summarizes our current knowledge of haloarchaeal proteases and provides a reference for future research.

Detection of an intracellular protease inhibitor in archaea.

Proteolytic activity and a subtilisin inhibitor (NSI) were detected in Natrialba magadii cells. The proteolytic activity was due to two different proteases: a approximately 90-kDa metallo protease (NMP) produced during exponential growth and a 246-kDa serine protease (NSP) detected in the stationary phase. Both proteases were detected in the cytosolic fraction. NSI activity was maximal during early stages of growth and decreased in the stationary phase. NSI is a 35-kDa thermosensitive protein; it inhibits NSP activity but has no effect on NMP, and it was detected as a soluble or membrane-bound protein depending on the growth phase. Our results suggest that NSI may regulate NSP activity in vivo and that this protease may have a role in stationary phase cells. To our knowledge, this is the first report on the occurrence of protease inhibitors in Archaea.

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.