Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain.

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2's biochemical properties, providing valuable insights into its serine protease domain.

Bypass gastroplasty impacts oral health, salivary inflammatory biomarkers, and microbiota: a controlled study.

OBJECTIVES: Knowledge about the impact of gastroplasty on oral health and salivary biomarkers is limited. The aim was to prospectively evaluate oral health status, salivary inflammatory markers, and microbiota in patients undergoing gastroplasty compared with a control group undergoing a dietary program. MATERIALS AND METHODS: Forty participants with obesity class II/III were included (20 individuals in each sex-matched group; 23-44 years). Dental status, salivary flow, buffering capacity, inflammatory cytokines, and uric acid were assessed. Salivary microbiological analysis (16S-rRNA sequencing) assessed the abundance of genus, species, and alpha diversity. Cluster analysis and mixed-model ANOVA were applied. RESULTS: Oral health status, waist-to-hip ratio, and salivary alpha diversity were associated at baseline. A subtle improvement in food consumption markers was observed, although caries activity increased in both groups, and the gastroplasty group showed worse periodontal status after three months. IFNγ and IL10 levels decreased in the gastroplasty group at 3 months, while a decrease was observed in the control group at 6 months; IL6 decreased in both groups (p < 0.001). Salivary flow and buffering capacity did not change. Significant changes in Prevotella nigrescens and Porphyromonas endodontalis abundance were observed in both groups, while alpha diversity (Sobs, Chao1, Ace, Shannon, and Simpson) increased in the gastroplasty group. CONCLUSIONS: Both interventions changed in different degrees the salivary inflammatory biomarkers and microbiota, but did not improve the periodontal status after 6 months. CLINICAL RELEVANCE: Although the observed discrete improvement in dietary habits, caries activity increased with no clinical improvement in the periodontal status, emphasizing the need of oral health monitoring during obesity treatment.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Cationic Geminoid Peptide Amphiphiles Inhibit DENV2 Protease, Furin, and Viral Replication.

Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Analysis of peptidase activities of a cathepsin B-like (TcoCBc1) from Trypanosoma congolense.

The substrate specificity of TcoCBc1 was evaluated using two internally quenched fluorescent peptide libraries with randomized sequences designed to detect carboxydipeptidase (Abz-GXXZXK(Dnp)-OH) and endopeptidase (Abz-GXXZXXQ-EDDnp) activities at acidic and neutral pHs, respectively. All the data obtained with TcoCBc1 were compared with those of human cathepsin B, including the pH profiles of the hydrolytic reactions. The most relevant observation is the preference of TcoCBc1 for substrates with a pair of acidic amino acids at positions P(2) and P(1) for its carboxydipeptidase activity and the well acceptance for E and D at P(1) position for endopeptidase activity. These peculiar preferences for negatively charged groups of TcoCBc1 and its requirements for carboxydipeptidase activity were also observed on Abz labeled analogues of bradykinin (Abz-RPPG(↓)FSAFR-OH, Abz-RPPG(↓)FS(↓)AF-OH, Abz-RPPG(↓)DE(↓)AF-OH) and angiotensin I (Abz-DR(↓)VYIHAFHL-OH), where (↓) indicates the cleavage site. TcoCBc1 was modeled based on the atomic coordinates of the cathepsin B from Trypanosoma brucei and the positively charged environment in TcoCBc1 catalytic site contrasts with the negatively charged environment in human cathepsin B. The preferences of S1 and S2 subsites of TcoCBc1 for acidic amino acids have to be taken into consideration for future studies of physiological roles of TcoCBc1 as for instance in apoptotic processes of Trypanosoma congolense.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision.

OBJECTIVES: Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the detection of carbapenemase activity directly from Gram-negative colonies. Based on this principle, we evaluated the performance of MALDI-TOF MS for rapid detection of carbapenemase activity directly from positive blood culture vials. METHODS: A total of 100 blood culture vials were randomly selected. MALDI-TOF MS carbapenemase assay results were confirmed by the detection of carbapenemase-encoding genes. RESULTS: A total of 110 bacterial isolates were recovered. The MALDI-TOF MS carbapenemase assay identified 21 of 29 (72.4%) of the carbapenemase-producing isolates directly from the blood culture vials, especially those encoding KPC-2 (100%) and SPM-1 (100%), after a 4 h incubation period. Although the majority of OXA-23-producing Acinetobacter baumannii isolates were not identified on day 1, all isolates were identified as carbapenemase producers directly from the colony on the next day. CONCLUSIONS: The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.

Atlantic Forest's and Caatinga's semiarid soils and their potential as a source for halothermotolerant actinomycetes and proteolytic enzymes.

Actinomycetes are versatile about their metabolism, displaying high capacity to produce bioactive metabolites. Enzymes from actinomycetes represent new opportunities for industrial applications. However, proteases from actinomycetes are poorly described by literature. Thereby, to verify proteolytic potential of actinomycetes, the present study aimed the investigation of bacterial isolates from Caatinga and Atlantic Forest rhizosphere. Fluorescence resonance energy transfer (FRET) peptide libraries were adopted for the evaluations, since they are faster and more qualitative methods, if compared with others described by most reports. A total of 52 microorganisms were inoculated in different culture media (PMB, potato dextrose agar, brain heart infusion agar, Starch Casein Agar and Reasoner's 2A agar), temperatures (12, 20, 30, 37, 45 and 60°C), and saline conditions (0-4 M NaCl), during 7 days. The actinomycetes named as AC 01, 02 and 52 were selected and showed enzymatic abilities under the peptide probes Abz-KLRSSKQ-EDDnp and Abz-KLYSSKQ-EDDnp, achieving enhanced performance at 30 °C. Biochemical parameters were established, showing a predominance of alkaline proteases with activity under saline conditions. Secreted proteases hydrolysed preferentially polar uncharged residues (Y and N) and positively charged groups (R). Phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid inhibited the proteins, a characteristic of serine (AC 01 e 02) and metalloproteases (AC 52). All selected strains belonged to Streptomyces genera. In summary, actinomycete strains with halophilic proteolytic abilities were selected, which improve possibilities for their use in detergent formulations, food processing, waste management and industrial bioconversion. It is important to highlight that this is the first report using FRET libraries for proteolytic screening from Caatinga and Atlantic Forest actinobacteria.

Internally quenched fluorescent peptide libraries with randomized sequences designed to detect endopeptidases.

Identification of synthetic peptide substrates for novel peptidases is an essential step for their study. With this purpose we synthesized fluorescence resonance energy transfer (FRET) peptide libraries Abz (or MCA)-GXXXXXQ-EDDnp and Abz (or MCA)-GXXZXXQ-EDDnp, where X consists of an equimolar mixture of all amino acids, the Z position is fixed with one of the proteinogenic amino acids (cysteine was excluded), Abz (ortho-aminobenzoic acid) or MCA ([7-amino-4-methyl]coumarin) is the fluorescence donor and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) is the fluorescence acceptor. The peptide libraries MCA-GXXX↓XXQ-EDDnp and MCA-GXXZ↓XXQ-EDDnp were cleaved as indicated (↓) by trypsin, chymotrypsin, cathepsin L, pepsin A, and Eqolisin as confirmed by Edman degradation of the products derived from the digestion of these libraries. The best hydrolyzed Abz-GXXZXXQ-EDDnp sublibraries by these proteases, including Dengue 2 virus NS2B-NS3 protease, contained amino acids at the Z position that are reported to be well accepted by their S(1) subsite. The pH profiles of the hydrolytic activities of these canonical proteases on the libraries were similar to those reported for typical substrates. The FRET peptide libraries provide an efficient and simple approach for detecting nanomolar concentrations of endopeptidases and are useful for initial specificity characterization as performed for two proteases secreted by a Bacillus subtilis.

Enzymatic diversity of filamentous fungi isolated from forest soil incremented by sugar cane solid waste.

Fungi are natural degraders of organic matter which can produce enzymes for many industrial and biotechnological applications. In this context, crude enzymatic extracts of fungal isolates were evaluated regarding their hydrolytic and ligninolytic abilities. The fungal strains were isolated from soil samples from Atlantic Rain Forest Park incremented with sugar cane biomass (filter cake), which allowed the selection of efficient lignocellulolytic enzymes. A total of 190 fungi were isolated and evaluated by endocellulase screenings. Thirteen fungi were selected about their hydrolytic and ligninolytic abilities. Among them, three isolates showed xylanolytic activity. Eleven of the isolates were selected by their cellulolytic abilities. Proteolytic enzymes were also detected for three fungi, allowing the classification as metalloprotease and serine protease. The isolates SPZPF3_47 (Mucor sp.), SPZPF1_129 (Byssochlamys nivea) and SPZPF1_141 (Paecilomyces saturatus) were selected for further investigation on their lignin peroxidase abilities. KM, Vmax and kcat apparent for lignin peroxidases were also determined. The strain of Mucor sp. (SPZPF3_47) was highlighted since this fungal genus was not well described about its isolation in the adopted conditions in our study, and showing ligninolytic abilities.

Evaluation of the histamine H3 receptor antagonists from LINS01 series as cholinesterases inhibitors: Enzymatic and modeling studies.

Histamine is involved in several central nervous system processes including cognition. In the last years, H3 receptor (H3 R) antagonists have been widely explored for their potential on dementias and other cognitive dysfunctions, and the cooperative role between histamine and acetylcholine neurotransmissions on cognitive processes is widely known in literature. This motivated us to assess the potential of 1-[(2,3-dihydrobenzofuran-1-yl)methyl]piperazines (LINS01 compounds) as inhibitors of cholinesterases, and thus this work presents the inhibitory effect of such compounds against acetyl (AChE) and butyrylcholinesterase. A set of 16 selected compounds were evaluated, being compounds 2d and 2e the most potent inhibitors of both cholinesterases (IC50 13.2-33.9 µM) by competitive mechanism, as indicated by the kinetic assays. Molecular docking simulations suggested that the allylpiperazine and dihydrobenzofuran motifs present in these compounds are important to perform π-interactions with key tryptophan residues from the enzymes, increasing their affinity for both H3 R and cholinesterases. Metric analysis support that compound 2d (LINS01022) should be highlighted due to its balanced lipophilicity (ClogP 2.35) and efficiency (LE 0.32) as AChE inhibitor. The results add important information to future design of dual H3 R-cholinesterases ligands.

Studies on the catalytic mechanism of a glutamic peptidase.

Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln(53) and Glu(136) that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P(1)' position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S(1) subsite of SGP. These results and the pH profiles of SGP activity showed that its S(1) subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp(57) and/or Asp(43) as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu(136)) activated water, and establish a fundamental role of the S(1) subsite interactions in promoting catalysis.

Yellow fever virus NS2B/NS3 protease: hydrolytic properties and substrate specificity.

Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P'1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k(cat)/K(m) of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity - a unique feature among worldwide prominent flavivirus - was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k(cat)/K(m). The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates.

Kinetic analysis of salting activation of a subtilisin-like halophilic protease.

The secreted extracellular subtilase SR5-3 from Halobacillus sp. bacterium, isolated from the high-salt environment of Thai fish sauce, was utilized as a model halophilic serine protease. The dependence of salt activation on the size and structure of substrates was evaluated assaying the enzyme with Suc-AAPF-MCA and with the Fluorescence Resonance Energy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp. Solvent isotope effects (SIE) and the thermodynamic parameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease in the presence of salts were also performed. All the obtained results support the notion that the salting out effect is responsible for the halophilic character of SR5-3, and the magnitude of its hydrolytic activity is mainly derived from the improvement of catalytic and/or interaction steps depending on the nature and size of the substrates, principally if they occupy the substrate prime subsites.

Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition.

The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na2SO4, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.

Hexadecane biodegradation of high efficiency by bacterial isolates from Santos Basin sediments.

The aim of the study was the investigation of bacterial diversity from sediments collected at Santos Estuarine System, regarding to their abilities for hexadecane biotransformation. Hexadecane is a medium-chain linear alkane, considered as a model molecule for hydrocarbon biodegradation studies. It is a component from aliphatic fraction of crude petroleum, commonly related to environmental contamination by diesel oil. Santos Basin is an area with historical petroleum contamination. In the present work, sediment samples from this area were inoculated in artificial seawater (ASW), containing hexadecane as carbon source. Six bacterial isolates were selected as resistant to hexadecane. Chromatographic results showed biodegradation indexes above 97%. After 48 h of culture, five of them could degrade >80% of the initial hexadecane added. These isolates were characterized by 16S rDNA gene sequencing analysis. The following species were found: Bacillus amyloliquefaciens, Staphylococcus epidermidis, Micrococcus luteus, Nitratireductor aquimarinus, and Bacillus pumilus.

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.