Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Improved production of alkaline and solvent-stable proteases from a halotolerant Exiguobacterium isolate through heterologous expression.

Halophiles are excellent sources of detergent proteases that are attributed to stability in alkaline pH, salts, surfactants, and hydrophobic solvents. The lower enzymatic yields and tedious downstream processes necessitate the search for newer halophilic sources. We have previously reported a halotolerant Exiguobacterium sp. TBG-PICH-001, which secretes solvent-tolerant alkaline protease/s. The present study describes the heterologous expression of two protease genes, namely, rsep metalloprotease (WP_195864791, 1.23 Kb) and tpa serine protease (WP_195864453, 0.879 Kb) genes. These were cloned into the pET 22b + plasmid vector and expressed in Escherichia coli BL21(DE3). The recombinant proteases rsep and tpa showed respective yields of 6.3 and 6.7 IU/mg, 11 and 12-fold higher than the crude native protease/s from TBG-PICH-001. These showed soluble expression at 46 and 32 KDa, respectively. These were purified to homogeneity through Ni-NTA-affinity chromatography. The purified proteases were characterized for properties like pH & temperature optima and stability, substrate specificity, kinetic parameters, and detergent attributes. They showed affinity towards various substrates with a respective Km of 392 and 301 µM towards casein. The recombinant proteases exhibited stability in the alkaline pH (7-10), surfactants, metal ions, detergents, and hydrophobic solvents, rendering their suitability as detergent additives.

Proteases influence colony aggregation behavior in Vibrio cholerae.

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183 mg/mL and 4.904 U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4 kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Expression levels of serine proteases, their homologs, and prophenoloxidase in the Eriocheir sinensis with hepatopancreatic necrosis syndrome (HPNS) and their expression regulation by Runt.

The occurrence of hepatopancreatic necrosis syndrome (HPNS) has seriously affected the sustainable development of Chinese mitten crab (Eriocheir sinensis) farming industry. Limited studies have focused on the immune responses in crabs with HPNS. Serine proteases (SPs) and SP homologs (SPHs) play important roles in the innate immunity of crustaceans. This study investigated the effects of HPNS on the expression levels of genes related to prophenoloxidase (proPO) activation system, and the relationship between Runt transcription factor and the transcriptions of these genes. Eight SPs and five SPHs (SPH1-4, Mas) were identified from E. sinensis. SPs contain a catalytic triad of "HDS", while SPHs lack a catalytic residue. SPs and SPHs all contain a conservative Tryp_SPc domain. Evolutionary analysis showed that EsSPs, EsSPHs, EsPO, and EsRunt were clustered with SPs, SPHs, POs, and Runts of other arthropods, respectively. In crabs with HPNS, the expression levels of six SPs (1, 3, 4, 6, 7, and 8), five SPHs, and PO were significantly upregulated in the hepatopancreas. The knockdown of EsRunt could evidently decrease the expression levels of four SPs (3, 4, 5 and 8), five SPHs (SPH1-4, Mas), and PO. Therefore, the occurrence of HPNS activates the proPO system. Furthermore, the expression levels of partial genes related to proPO system were regulated by Runt. The activation of innate immune system may be a strategy for crabs with HPNS to improve immunity and fight diseases. Our study provides a new understanding of the relationship between HPNS and innate immunity.

Cynomolgus macaque model of neuronal ceroid lipofuscinosis type 2 disease.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal-recessive fatal neurodegenerative diseases that occur in children and young adults, with symptoms including ataxia, seizures and visual impairment. We report the discovery of cynomolgus macaques carrying the CLN2/TPP1 variant and our analysis of whether the macaques could be a new non-human primate model for NCL type 2 (CLN2) disease. Three cynomolgus macaques presented progressive neuronal clinical symptoms such as limb tremors and gait disturbance after about 2 years of age. Morphological analyses using brain MRI at the endpoint of approximately 3 years of age revealed marked cerebellar and cerebral atrophy of the gray matter, with sulcus dilation, gyrus thinning, and ventricular enlargement. Histopathological analyses of three affected macaques revealed severe neuronal loss and degeneration in the cerebellar and cerebral cortices, accompanied by glial activation and/or changes in axonal morphology. Neurons observed throughout the central nervous system contained autofluorescent cytoplasmic pigments, which were identified as ceroid-lipofuscin based on staining properties, and the cerebral cortex examined by transmission electron microscopy had curvilinear profiles, the typical ultrastructural pattern of CLN2. These findings are commonly observed in all forms of NCL. DNA sequencing analysis identified a homozygous single-base deletion (c.42delC) of the CLN2/TPP1 gene, resulting in a frameshifted premature stop codon. Immunohistochemical analysis showed that tissue from the affected macaques lacked a detectable signal against TPP1, the product of the CLN2/TPP1 gene. Analysis for transmission of the CLN2/TPP1 mutated gene revealed that 47 (49.5%) and 48 (50.5%) of the 95 individuals genotyped in the CLN2-affected macaque family were heterozygous carriers and homozygous wild-type individuals, respectively. Thus, we identified cynomolgus macaques as a non-human primate model of CLN2 disease. The CLN2 macaques reported here could become a useful resource for research and the development of drugs and methods for treating CLN2 disease, which involves severe symptoms in humans.

Heterologous expression and characterization of Bacillus velezensis SW5 serine protease involved in the hydrolysis of anchovy protein.

BACKGROUND: Bacillus velezensis SW5, with good enzyme production ability, was isolated and identified in our laboratory from fermented fish sauce. Its galactosidase has been expressed in Escherichia coli, which could hydrolyze lactose in milk. The present study aims to express a novel serine protease gene (SPr-SW5) of this strain by Bacillus subtilis WB800N, and applies the expressed enzyme in hydrolysis of anchovy to prepare antioxidant substances, aiming to alleviate the waste of low-value fish resources. RESULTS: SPr-SW5 with the open reading frame of 1353 bp encodes a serine protease (SPr-SW5) with 450 amino acids. The theoretical molecular weight and isoelectric point are 47.2 kDa and 5.22, respectively. The successful expression of SPr-SW5 in B. subtilis WB800N was confirmed by a skim milk plate test. Its optimal temperature and pH were 50 °C and 8.0, respectively. SPr-SW5 activity was increased by Ca2+ and Zn2+ , but inhibited by Fe3+ . Furthermore, SPr-SW5 was tolerant to 1% Tween-40 and Tween-80; however, its activity was strongly inhibited by 10 mm phenylmethylsulfonyl fluoride. Additionally, SPr-SW5 could be capable of hydrolyzing anchovy, the hydrolysate (AHP) at 10 g L-1 , with 2,2-diphenyl-1-picrylhydrazyl and hydroxyl (·OH) scavenging rates of 73.21% and 79.71%, displaying good antioxidant activity. CONCLUSION: The novel SPr-SW5 was successfully expressed in B. subtilis WB800N. It exhibited excellent temperature stability and good tolerance to several metal ions. In addition, the anchovy hydrolyzed by expressed SPr-SW5 has good antioxidant ability. Overall, this research lays a good foundation for SPr-SW5 with respect to exploration and application in the food industry as enzyme preparation. © 2023 Society of Chemical Industry.

Screening, characterization, and kinetic studies of a serine alkaline protease from kitchen wastewater bacteria P2S1An and evaluation of its application in nutraceutical production.

This present investigation aimed at characterizing the biochemical potential and kinetic study of the protease isolated from kitchen wastewater bacteria, P2S1An. The enzymatic activity was optimum when incubated for 96 h, at 30°C and pH 9.0. The enzymatic activity of the purified protease (PrA) was 10.47-folds that of crude protease (S1). PrA was about 35 kDa in molecular weight. The broad pH and thermal stability, chelators, surfactants and solvent tolerance, and favorable thermodynamics suggested the potentiality of the extracted protease PrA. Thermal activity and stability were enhanced in presence of 1-mM Ca2+ ion at high temperatures. The protease was a serine one as its activity was completely diminished in presence of 1-mM PMSF. The Vmax , Km , and Kcat /Km suggested stability and catalytic efficiency of the protease. PrA hydrolyzes fish protein with 26.61 ± 0.16% of peptide bond cleavage after 240 min, comparable to Alcalase 2.4L (27.13 ± 0.31%). PRACTITIONER POINTS: A serine alkaline protease PrA was extracted from kitchen wastewater bacteria Bacillus tropicus Y14. Protease PrA showed significant activity and stability in a wide temperature and pH range. Protease showed great stability towards additives like metal ions, solvents, surfactants, polyols, and inhibitors. Kinetic study showed that the protease PrA had a prominent affinity and catalytic efficiency for the substrates. PrA hydrolysed fish proteins into short bioactive peptides which signify its potential in the formation of functional food ingredients.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PURPOSE: Neutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study. METHODS: Two NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3. RESULTS AND DISCUSSION: The zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method. CONCLUSION: The NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

The Repeating, Modular Architecture of the HtrA Proteases.

A conserved, 26-residue sequence [AA(X2)[A/G][G/L](X2)GDV[I/L](X2)[V/L]NGE(X1)V(X6)] and corresponding structure repeating module were identified within the HtrA protease family using a non-redundant set (N = 20) of publicly available structures. While the repeats themselves were far from sequence perfect, they had notable conservation to a statistically significant level. Three or more repetitions were identified within each protein despite being statistically expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel ß-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these ß-barrels, but the use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 Å (mean and median). Our findings support Dayhoff's hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains.

Amino acid sequence of two new milk-clotting proteases from the macroalga Gracilaria edulis.

This study is aimed at identifying and characterising the proteases we previously extracted from the red seaweed Gracilaria edulis with the potential as milk-clotting enzymes. The protease extract was first analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Two protease bands with a molecular weight of 44 and 108 kDa were identified, and analysed using in-gel digestion and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS). Eight peptides from the LC-MS/MS analysis matched those in existing protein databases but they were not related to any protease of the genera Gracilaria and Hydropuntia. Further analysis revealed that more than 80% of the peptide sequence of the algal proteases matched with those from members of the bacteria kingdom, including Gallaecimonas and Alteromonas. Among these, twelve matching homolog proteases were identified as metalloprotease and serine protease. The results indicated that the algal proteases have a close relationship with both algae and bacteria, and suggest that the proteases might have resulted from past bacterial colonisation of the algae and subsequent horizontal gene transfer between bacteria and algae.

Hepatitis C Virus NS3/4A Inhibition and Host Immunomodulation by Tannins from Terminalia chebula: A Structural Perspective.

Terminalia chebula Retz. forms a key component of traditional folk medicine and is also reported to possess antihepatitis C virus (HCV) and immunomodulatory activities. However, information on the intermolecular interactions of phytochemicals from this plant with HCV and human proteins are yet to be established. Thus, by this current study, we investigated the HCV NS3/4A inhibitory and host immune-modulatory activity of phytocompounds from T. chebula through in silico strategies involving network pharmacology and structural bioinformatics techniques. To start with, the phytochemical dataset of T. chebula was curated from biological databases and the published literature. Further, the target ability of the phytocompounds was predicted using BindingDB for both HCV NS3/4A and other probable host targets involved in the immune system. Further, the identified targets were docked to the phytochemical dataset using AutoDock Vina executed through the POAP pipeline. The resultant docked complexes with significant binding energy were subjected to 50 ns molecular dynamics (MD) simulation in order to infer the stability of complex formation. During network pharmacology analysis, the gene set pathway enrichment of host targets was performed using the STRING and Reactome pathway databases. Further, the biological network among compounds, proteins, and pathways was constructed using Cytoscape 3.6.1. Furthermore, the druglikeness, side effects, and toxicity of the phytocompounds were also predicted using the MolSoft, ADVERpred, and PreADMET methods, respectively. Out of 41 selected compounds, 10 were predicted to target HCV NS3/4A and also to possess druglike and nontoxic properties. Among these 10 molecules, Chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose exhibited potent HCV NS3/4A inhibitory activity, as these scored a lowest binding energy (BE) of -8.6 kcal/mol and -7.7 kcal/mol with 11 and 20 intermolecular interactions with active site residues, respectively. These findings are highly comparable with Asunaprevir (known inhibitor of HCV NS3/4A), which scored a BE of -7.4 kcal/mol with 20 key intermolecular interactions. MD studies also strongly suggest that chebulagic acid and 1,2,3,4,6-Pentagalloyl glucose as promising leads, as these molecules showed stable binding during 50 ns of production run. Further, the gene set enrichment and network analysis of 18 protein targets prioritized 10 compounds and were predicted to potentially modulate the host immune system, hemostasis, cytokine levels, interleukins signaling pathways, and platelet aggregation. On overall analysis, this present study predicts that tannins from T. chebula have a potential HCV NS3/4A inhibitory and host immune-modulatory activity. However, further experimental studies are required to confirm the efficacies.

High-expression and characterization of a novel serine protease from Ornithinibacillus caprae L9T with eco-friendly applications.

In the current work, a novel thermophilic serine protease gene (P3862) from Ornithinibacillus caprae L9T was functionally expressed in Bacillus subtilis SCK6. The monomeric enzyme of about 29 kDa was purified to homogeneity with 43.91% of recovery and 2.81-folds of purification. Characterization of the purified protease revealed the optimum activity at pH 7 and 65 °C. The protease exhibited excellent activity and stability in the presence of Na+, Mg2+, Ca2+, ethanediol, n-hexane, Tween-20, Tween-80 and Triton X-100. P3862 displayed favorable caseinolytic activity, moderate keratinolytic activity but no collagenolytic activity. Besides, the homology model of P3862 possessed a globular configuration and characteristic of α/ß hydrolase fold, and displayed stable interactions with casein, glycoprotein and keratin rather than collagen. Moreover, the crude enzyme could completely dehair goatskin within 6 h, resulting in decrease in BOD5, COD and TSS loads by 72.86, 74.07, and 73.79%, respectively, as compared with Na2S treatment. Biocatalytic applications revealed that it could effectively remove egg-stains from fabrics at 37 °C for 30 min with low supplementation (300 U/mL), and was able to degrade the feathers of duck and chicken. Overall, these outstanding properties make P3862 valuable in the development of environmentally friendly biotechnologies .

The Q41R mutation in the HCV-protease enhances the reactivity towards MAVS by suppressing non-reactive pathways.

Recent experimental findings pointed out a new mutation in the HCV protease, Q41R, responsible for a significant enhancement of the enzyme's reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation is located rather far from the active site, and its involvement in the overall reaction mechanism is thus unclear. We used classical molecular dynamics and QM/MM to study the acylation reaction of HCV NS3/4A protease variants bound to MAVS and the NS4A/4B substrate and uncovered the indirect mechanism by which the Q41R mutation plays a critical role in the efficient cleavage of the substrate. Our simulations reveal that there are two major conformations of the MAVS H1'(p) residue for the wild type protease and only one conformation for the Q41R mutant. The conformational space of H1'(p) is restricted by the Q41R mutation due to a π-π stacking between H1'(p) and R41 as well as a strong hydrogen bond between the backbone of H57 and the side chain of R41. Further QM/MM calculations indicate that the complex with the conformation ruled out by the Q41R substitution is a non-reactive species due to its higher free energy barrier for the acylation reaction. Based on our calculations, we propose a kinetic mechanism that explains experimental data showing an increase of apparent rate constants for MAVS cleavage in Q41R mutants. Our model predicts that the non-reactive conformation of the enzyme-substrate complex modulates reaction kinetics like an uncompetitive inhibitor.

Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect.

The investigation of a plant glycosylated serine protease (EuRP-61) isolated from Euphorbia resinifera latex for potential antiplatelet and anticoagulation activities has been previously reported. In the present study, the protein sequence and native crystal structure of EuRP-61 were characterized. The structure was identified using single-wavelength anomalous diffraction with a refinement resolution of 1.7 Å (PDB ID: 7EOX). The main structural components of EuRP-61 were composed of three domains: catalytic, protease-associated (PA), and fibronectin type III (Fn3)-like domains. The crystal structure revealed that some loops in the PA and catalytic domains of EuRP-61 were different from the other subtilisin-like proteases (cucumisin and SBT3). These different loops might be involved in the general monomer formation of EuRP-61, substrate specificity, and maintenance of the catalytic domain. The Fn3-like domain may provide flexibility to the enzyme to bind with various substrates and cell receptors. Additionally, the active site of EuRP-61 consisted of the catalytic triad of Ser434, His106, and Asp32, similar to other serine proteases. The present study provides additional information and insight into the protease and antithrombotic activities of EuRP-61, which could contribute to further development of this enzyme for biomedical treatment.

Cloning and sequence analysis of a serine protease gene from Rhizoctonia solani Kühn AG5.

The present study aimed to identify the subtilisin-like proteases (SLPs) of Rhizoctonia solani Kühn potentially involved in the virulence of this phytopathogenic fungus, which has 14 anastomosis groups (AGs) responsible for many crop diseases. Through mycelial microscope observation and strain identification of pathogenic fungus MS-3, it was determined to be R. solani AG-5. Both 5' and 3' rapid amplification of cDNA ends were used to clone the serine protease gene RsSLP from R. solani AG-5. The full-length obtained for RsSLP was 1714 bp with an open reading frame of 1587 bp, encoding a protein of 528 amino acids with a molecular mass of 55.8 kDa. This protein contained a predicted signal peptide for secretion but lacked a transmembrane domain or membrane anchor site. Bioinformatics analysis identified this protein as a serine protease with the Peptidase_S8 and Inhibitor_I9 characteristic domains of SLPs. Phylogenetic analysis suggested that frequent gene duplications of the SLPs occurred in R. solani (RsSLP), and RsSLP shares characteristic sequence features with virulence factors of other phytopathogenic fungi. Because the secretory serine protease RsSLP from R. solani AG5 is similar to the virulence factors of other phytopathogenic fungi, its identification will be helpful in studies considering the roles of these proteases in pathogen virulence.

Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims' death or permanent disability. To better characterize the inflammatory process induced by this snake's venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1ß in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.