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Enzymatic degradation of collagen is of great industrial and environmental significance; however, little is known about thermophile-derived collagenolytic proteases. Here, we report a novel collagenolytic protease (TSS) from thermophilic Brevibacillus sp. WF146. The TSS precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, a β-jelly roll (βJR) domain, and a prepeptidase C-terminal (PPC) domain. The maturation of TSS involves a stepwise autoprocessing of the N-terminal propeptide and the PPC domain, and the βJR rather than the PPC domain is necessary for correct folding of the enzyme. Purified mature TSS displayed optimal activity at 70°C and pH 9.0, a half-life of 1.5 h at 75°C, and an increased thermostability as the NaCl concentration increased up to 4 M. TSS possesses an increased number of surface acidic residues and ion pairs, as well as four Ca(2+)-binding sites, which contribute to its high thermostability and halotolerance. At high temperatures, TSS exhibited high activity toward insoluble type I collagen and azocoll but showed a low gelatinolytic activity, with a strong preference for Arg and Gly at the P1 and P1’ positions, respectively. Both the βJR and PPC domains could bind but not swell collagen, and thus facilitate TSS-mediated collagenolysis via improving the accessibility of the enzyme to the substrate. Additionally, TSS has the ability to efficiently degrade fish scale collagen at high temperatures. IMPORTANCE Proteolytic degradation of collagen at high temperatures has the advantages of increasing degradation efficiency and minimizing the risk of microbial contamination. Reports on thermostable collagenolytic proteases are limited, and their maturation and catalytic mechanisms remain to be elucidated. Our results demonstrate that the thermophile-derived TSS matures in an autocatalytic manner and represents one of the most thermostable collagenolytic proteases reported so far. At elevated temperatures, TSS prefers hydrolyzing insoluble heat-denatured collagen rather than gelatin, providing new insight into the mechanism of collagen degradation by thermostable collagenolytic proteases. Moreover, TSS has the potential to be used in recycling collagen-rich wastes such as fish scales.

Background: Cucumber target leaf spot (TLS), caused by Corynespora cassiicola (C. Cassiicola), is a serious disease in cucumber (Cucumis sativus) production worldwide. Therefore, cultivating new varieties of TLS resistance of C. sativus is an important goal of cucumber breeding. Previous studies have shown that subtilisin-like protease (SUBP) plays an important role in response to C. Cassiicola infection in resistant plants. Objective: In this study, the full-length cDNA of the CsSUBP gene was cloned, and the prokaryotic expression vector was successfully constructed in order to study the effects of subtilisin. Futhermore, vital clues regarding CsSUBP gene involved in TLS resistance of C. sativus are gained from the bioinformatics assay. Method: The CsSUBP gene was identified by sequencing with the intermediate vector pMD18 by designing specific primers and PCR amplification techniques. The prokaryotic expression vector pET30a-CsSUBP was further constructed and identified by colony PCR and EcoR V and SalⅠ double digestion. Result: The primary structure of CsSUBP was predicted and analyzed by bioinformatics analysis. The results showed that CsSUBP was weakly acidic protein, N-terminal signal peptide region, including a Inhibitor_I9 domain domain. Conclusion: The pET30a-CsSUBP prokaryotic expression vector was constructed successfully. This study is convenient for the study of prokaryotic expression and its kinase activity.

Background Rolling of leaves (RL) is a phenomenon commonly found in grasses. Morphology of the leaf is an important agronomic trait in field crops especially in rice; therefore, majority of the rice breeders are interested in RL. There are only few studies with respect to RL of wheat and barley; however, the information regarding the genetic base of RL with respect to the shape of leaf in rye is lacking. To the best of our knowledge, this is the first study on the localization of loci controlling RL on high density consensus genetic map of rye. Results Genotypic analysis led to the identification of 43 quantitative trait loci (QTLs) for RL, grouped into 28 intervals, which confirms the multigenic base of the trait stated for wheat and rice. Four stable QTLs were located on chromosomes 3R, 5R, and 7R. Co-localization of QTL for RL and for different morphological, biochemical and physiological traits may suggests pleiotropic effects of some QTLs. QTLs for RL were associated with QTLs for such morphological traits as: grain number and weight, spike number per plant, compactness of spike, and plant height. Two QTLs for RL were found to coincide with QTLs for drought tolerance (4R, 7R), two with QTLs for heading earliness (2R, 7R), one with α-amylase activity QTL (7R) and three for pre-harvest sprouting QTL (1R, 4R, 7R). The set of molecular markers strongly linked to RL was selected, and the putative candidate genes controlling the process of RL were identified. Twelve QTLs are considered as linked to candidate genes on the base of DArT sequences alignment, which is a new information for rye. Conclusions Our results expand the knowledge about the network of QTLs for different morphological, biochemical and physiological traits and can be a starting point to studies on particular genes controlling RL and other important agronomic traits (yield, earliness, pre-harvest sprouting, reaction to water deficit) and to appoint markers useful in marker assisted selection (MAS). A better knowledge of the rye genome and genes could both facilitate rye improvement itself and increase the efficiency of utilizing rye genes in wheat breeding. Electronic supplementary material The online version of this article (10.1186/s12863-018-0665-0) contains supplementary material, which is available to authorized users.

AprBp is a 1146 bp gene of Bacillus pumilus strain 7Р that encodes an extracellular serine proteinase, characterized by significant similarity with subtilisins. The start sites of transcription (T) and translation (GTG) of the gene have been identified. A regulatory network for controlling proteinase gene expression has been established. Proteinase AprBp was isolated from the culture fluid at different growth phases, and the primary structure of the protein was identified at the variuos phases of growth. In the stationary phase, the enzyme forms dimers and is very stable. A feed additive based on B. pumilus proteinase is being developed for poultry farming

Phytaspases belong to the family of plant subtilisin-like proteases and are distinct from other family members, as they have strict and rarely occurring aspartate cleavage specificity and unusual localization dynamics. After being secreted into the apoplast of healthy plant tissues, phytaspases are able to return back into cells that have been committed to cell death due to a variety of biotic and abiotic stresses. It was recently discovered that retrograde transport of phytaspases involves clathrin-mediated endocytosis. Here, consequences of phytaspase internalization were studied. Proteolytic activity of phytaspases in the apoplast and intracellular protein fractions obtained from Nicotiana benthamiana leaves containing either endogenous phytaspase only or transiently producing Nicotiana tabacum phytaspase-EGFP protein (NtPhyt-EGFP) was determined. We demonstrated that triggering phytaspase internalization by antimycin A-induced oxidative stress is accompanied by re-distribution of phytaspase activity from the apoplast to the cell interior. Inhibition of clathrin-mediated endocytosis by co-production of the Hub protein prevented phytaspase internalization and phytaspase activity re-localization. Specificity of endocytic uptake of phytaspases was demonstrated by the co-production of an apoplast-targeted mRFP protein marker, which retained its apoplastic localization when phytaspase internalization was essentially complete. Overproduction of NtPhyt-EGFP, but not of the proteolytically inactive phytaspase mutant, per se caused moderate damage in young Nicotiana benthamiana seedlings, whereas antimycin A treatment induced a pronounced loss of cell viability independent of the NtPhyt-EGFP overproduction. Interestingly, inhibition of clathrin-mediated endocytosis abrogated cell death symptoms in both cases. In contrast to stress-induced internalization of tobacco phytaspase, Arabidopsis thaliana phytaspase-EGFP protein (AtPhyt-EGFP) was spontaneously internalized when transiently produced in N. benthamiana leaves. The AtPhyt-EGFP uptake was dependent on clathrin-mediated endocytosis as well, the internalized protein being initially visualized within the membranous vesicles. At later time points, the EGFP tag was cleaved off from AtPhyt, though the elevated level of intracellular AtPhyt proteolytic activity persisted. Our data, therefore, point to clathrin-mediated endocytosis as a means to deliver proteolytically active phytaspases into plant cells. It would be interesting to learn whether or not phytaspases are unique among the large family of plant subtilisin-like proteases in their ability to utilize retrograde trafficking.

Highlights • StSBTc-3 activity is optimized using response surface methodology. • StSBTc-3 has fibrinogenolytic activity at physiological conditions. • StSBTc-3 has potential use in biomedical devices. • rsm package with R is a suitable tool to perform plant protease activity optimizations., The aim of this study was to optimize in vitro conditions to enhance fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3). The effects of StSTBc-3 concentration (0.2–5 μM), pH value (6–10) and temperature (35–50 °C) on fibrinogenolytic activity were studied through response surface methodology (RSM). We obtained a model that predicts the response accurately. The relationship between enzyme concentration and fibrinogenolytic activity was linear, while the main effect from pH and temperature on the response was quadratic. From the RSM generated model the optimum pH was 8 and the optimum temperature was 43 °C, while higher concentrations of enzyme produce higher activities. Under optimum conditions there were no statistically significant differences between the experimental responses and the ones predicted from the model. This model also predicts the activity under physiological conditions. These results confirm that StSTBc-3 is a good candidate to be considered for therapeutic uses. The generated model will be useful for biotechnological purposes.

Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

Graphical abstract, Highlights • Homology models of four SUB1 orthologues from P. falciparum species were produced. • We analyzed the binding mode of our previous difluorostatone inhibitors to six SUB1. • In vitro activity of our difluorostatone-based inhibitors was correctly predicted. • We derived a structure-based pan-SUB1 pharmacophore, and validated it in silico. • We confirmed that development of pan-SUB1 inhibitors is a feasible task., Plasmodium falciparum subtilisin-like protease 1 (SUB1) is a novel target for the development of innovative antimalarials. We recently described the first potent difluorostatone-based inhibitors of the enzyme ((4S)-(N-((N-acetyl-l-lysyl)-l-isoleucyl-l-threonyl-l-alanyl)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine (1) and (4S)-(N-((N-acetyl-l-isoleucyl)-l-threonyl-l-alanylamino)-2,2-difluoro-3-oxo-4-aminopentanoyl)glycine (2)). As a continuation of our efforts towards the definition of the molecular determinants of enzyme-inhibitor interaction, we herein propose the first comprehensive computational investigation of the SUB1 catalytic core from six different Plasmodium species, using homology modeling and molecular docking approaches. Investigation of the differences in the binding sites as well as the interactions of our inhibitors 1,2 with all SUB1 orthologues, allowed us to highlight the structurally relevant regions of the enzyme that could be targeted for developing pan-SUB1 inhibitors. According to our in silico predictions, compounds 1,2 have been demonstrated to be potent inhibitors of SUB1 from all three major clinically relevant Plasmodium species (P. falciparum, P. vivax, and P. knowlesi). We next derived multiple structure-based pharmacophore models that were combined in an inclusive pan-SUB1 pharmacophore (SUB1-PHA). This latter was validated by applying in silico methods, showing that it may be useful for the future development of potent antimalarial agents.

© 2016 Pleiades Publishing, Ltd. Using the expression vector pGP382 containing a constitutive promoter (P degQ36 ) and an affinity tag (Strep-tag), we have obtained highly purified recombinant Bacillus pumilus 3-19 proteinases with different substrate specificities: glutamylendopeptidase (GseBp), subtilisin-like protease (AprBp), and metalloendopeptidase (MprBp). The products of the hydrolysis of the ß-amyloid peptide by the bacterial proteases from B. pumilus have been studied. The findings on the potential of the practical application of these bacterial enzymes as the agents preventing the development of the Alzheimer's disease are presented.

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.O isolado brasileiro de Beauveria bassiana (CG425) que apresenta alta virulência contra a broca do café (CBB) foi analisado quanto à produção de proteases degradadoras de cutícula, tipo-subtilisina (Pr1) e tipo-tripsina (Pr2). O crescimento fúngico foi realizado em meio contendo nitrato e em meio contendo cutícula da broca em condições de pH tamponado e não tamponado. Em meio não tamponado, suplementado com cutícula, o pH da cultura caiu e as atividades de Pr1 e Pr2 foram detectadas somente em valores de pH igual ou superior a 5,5. Em culturas tamponadas, as atividades Pr1 e Pr2 foram superiores em meio suplementado com cutícula, comparativamente as atividades em meio contendo nitrato. As atividades Pr1 e Pr2 ocorreram predominantemente no sobrenadante de cultivo. Os dados obtidos sugerem que Pr1 e Pr2 produzidas pelo isolado CG425 são induzidas por componentes da cutícula da broca do café (CBB), e que o pH da cultura influencia a expressão destas proteases, indicando a ocorrência de um mecanismo eficiente de secreção por este fungo. Os resultados obtidos neste estudo aumentam o conhecimento a respeito da produção de proteases por B. bassiana CG425, abrindo novos caminhos para o estudo do papel de proteases na virulência contra a broca do café durante o processo de infecção.

In this study effects of genotype, explants size and the composition of the culture medium on regeneration capacity of apexes of etiolated potato tubers sprouts were estimated. We observed positive correlation among survival, growth and development of plant tissues in vitro and the size of plant explants. When the cultivation medium was supplemented by RNase A (1-10 μg/ml) and ribonucleases from Bacillus pumilus (RNAse Bp, 1 μg/ml) 35% increase of the number of the regenerants was observed. Enzymes with ribonuclease activity at low concentrations (1-10 μg/ml) stimulated regenerative and morphogenic processes. In contrast neither plant regeneration dynamic nor plant morphogenesis were changed when cultivation medium was supplemented by other Bacillus pumilus proteolytic enzymes (subtilisin-like protease (AprBp), glutamyl endopeptidase (GseBp) and metalloendopeptidase (MprBp) in concentration of 1 μg/ml.

Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canis λEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2 , were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1 , SUB2 , and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1 , SUB2 , and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.

© 2014 Pleiades Publishing, Ltd. The features of the expression of the subtilisin-like protease from Bacillus pumilus have been studied. Potential binding sites for the transcription factor DegU∼P have been identified in the regulatory region of the gene of the enzyme, which were optimized for the interaction with this regulatory protein. The expression of the modified gene of the extracellular subtilisin-like protease has been investigated. It has been shown that the modification of one of the sites increased the expression of the protease twofold. It has been concluded that the optimization of the promoter leads to an increase in the expression of the subtilisin-like protease.

"nBackground: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study."nMethods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR."nResults: The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes."nConclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes. &nbsp

A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process. O isolado brasileiro de Beauveria bassiana (CG425) que apresenta alta virulência contra a broca do café (CBB) foi analisado quanto à produção de proteases degradadoras de cutícula, tipo-subtilisina (Pr1) e tipo-tripsina (Pr2). O crescimento fúngico foi realizado em meio contendo nitrato e em meio contendo cutícula da broca em condições de pH tamponado e não tamponado. Em meio não tamponado, suplementado com cutícula, o pH da cultura caiu e as atividades de Pr1 e Pr2 foram detectadas somente em valores de pH igual ou superior a 5,5. Em culturas tamponadas, as atividades Pr1 e Pr2 foram superiores em meio suplementado com cutícula, comparativamente as atividades em meio contendo nitrato. As atividades Pr1 e Pr2 ocorreram predominantemente no sobrenadante de cultivo. Os dados obtidos sugerem que Pr1 e Pr2 produzidas pelo isolado CG425 são induzidas por componentes da cutícula da broca do café (CBB), e que o pH da cultura influencia a expressão destas proteases, indicando a ocorrência de um mecanismo eficiente de secreção por este fungo. Os resultados obtidos neste estudo aumentam o conhecimento a respeito da produção de proteases por B. bassiana CG425, abrindo novos caminhos para o estudo do papel de proteases na virulência contra a broca do café durante o processo de infecção.

Phylogenetic analyses suggest that, among the members of the Eurosid I clade, nitrogen-fixing root nodule symbioses developed multiple times independently, four times with rhizobia and four times with the genus Frankia. In order to understand the degree of similarity between symbiotic systems of different phylogenetic subgroups, gene expression patterns were analyzed in root nodules of Datisca glomerata and compared with those in nodules of another actinorhizal plant, Alnus glutinosa, and with the expression patterns of homologous genes in legumes. In parallel, the phylogeny of actinorhizal plants was examined more closely. The results suggest that, although relationships between major groups are difficult to resolve using molecular phylogenetic analysis, the comparison of gene expression patterns can be used to inform evolutionary relationships. In this case, stronger similarities were found between legumes and intracellularly infected actinorhizal plants (Alnus) than between actinorhizal plants of two different phylogenetic subgroups (Alnus/Datisca).

Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus . The gene was functionally expressed in Escherichia coli , and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.