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Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties., Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter., Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca 2+ , Mg 2+ , SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n -hexane and n -heptane. Enzymatic analysis revealed a K m of 2.89 ± 0.09 μM and a V max of 14.20 ± 0.12 nM•min -1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP., Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research.

The high content and quality of protein in Andean legumes make them valuable for producing protein hydrolysates using proteases from bacteria isolated from extreme environments. This study aimed to carry out a single-step purification of a haloprotease from Micrococcus sp. PC7 isolated from Peru salterns. In addition, characterize and apply the enzyme for the production of bioactive protein hydrolysates from underutilized Andean legumes. The PC7 protease was fully purified using only tangential flow filtration (TFF) and exhibited maximum activity at pH 7.5 and 40 °C. It was characterized as a serine protease with an estimated molecular weight of 130 kDa. PC7 activity was enhanced by Cu 2+ (1.7-fold) and remained active in the presence of most surfactants and acetonitrile. Furthermore, it stayed completely active up to 6% NaCl and kept ̴ 60% of its activity up to 8%. The protease maintained over 50% of its activity at 25 °C and 40 °C and over 70% at pH from 6 to 10 for up to 24 h. The determined K m and V max were 0.1098 mg mL -1 and 273.7 U mL -1 , respectively. PC7 protease hydrolyzed 43%, 22% and 11% of the Lupinus mutabilis, Phaseolus lunatus and Erythrina edulis protein concentrates, respectively. Likewise, the hydrolysates from Lupinus mutabilis and Erythrina edulis presented the maximum antioxidant and antihypertensive activities, respectively. Our results demonstrated the feasibility of a simple purification step for the PC7 protease and its potential to be applied in industrial and biotechnological processes. Bioactive protein hydrolysates produced from Andean legumes may lead to the development of nutraceuticals and functional foods contributing to address some United Nations Sustainable Development Goals (SDGs)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microbial alkaline proteases are dominating the global enzyme market with a share of over 65% due to their multifarious catalytic potentials. Hence, production of proteases with novel properties of commercial significance is highly desirable to meet the global enzyme demand. Here, we report the purification, characterization, and pilot-scale application of a serine protease from the desert soil bacterium Bacillus subtilis ZMS-2 with novel properties as dehairing agent in leather processing. The enzyme was purified 16.5-fold with a specific activity of 1543.5 U mg -1 and recovery percentage of 33.6% using ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. The purified enzyme was characterized as a metal ion-, surfactant-, and denaturant-compatible alkaline serine protease having a molecular weight of 36.1 kDa with an optimum activity at pH 8.5 and 60 °C. The catalytic activity of the enzyme was enhanced by Zn +2 (204%), Ag + (110%), H 2 O 2 (123%), Triton X-100 (110%), iso-octane (109%), chloroform (110%), ethanol (105%), ethyl acetate (110%), and acetonitrile (128%). During pilot-scale applications, the optimum condition was found to be a combination of enzyme (1.5%, 460 U mL -1 ), sodium sulfide (2%), and calcium hydroxide (lime) (3%). Under this condition, the time required for complete dehairing was 90 min. Chemoenzymatically processed skins exhibited better physical properties than chemically processed skin, including tensile strength (16.35 ± 6.68 N/mm), ball burst (452.88 ± 6.06 N/mm), percent elongation (38.85 ± 1.06 N), tear strength (50.16 ± 4.42 N/mm), and softness (6.5 mm). Electron microscopy analysis of the treated skin showed complete removal of hairs with roots, confirming the keratin specificity of the enzyme. Moreover, the enzyme-assisted dehairing process reduced chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) by 68, 77, 34, and 39%, respectively. Thus, the alkaline serine protease from B. subtilis ZMS-2 is a potential dehairing agent for the eco-friendly processing of animal skins on industrial scales., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia., Competing Interests: Declaration of competing interest Informed consent statement not applicable., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD 600  = 0.56,1303 U/mL), pH 8 (OD 600  = 0.83, 403.29 U/mL), 40 °C (OD 600  = 1.75, 1849.11 U/mL), fructose (OD 600  = 1.22, 1502 U/mL), and gelatin (OD 600  = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 μM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and β-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 μg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Ethnopharmacological Relevance: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported., Aim of the Study: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases., Materials and Methods: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically., Results: Seed proteins extract of S. saponaria was evaluated until 100 μg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects., Conclusion: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus , and 432 proteins by Lentinula edodes . Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca 2+ , Co 2+ , Mn 2+ , and DTT. It is inhibited by Fe 2+ , Cd 2+ , Cu 2+ , EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (K d ) and half lives (t 1/2 ) at 50-80 °C were in the range of 2.50 × 10 -3 to 5.50 × 10 -3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased β-sheets content., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu 2+ , Na + , Fe 3+ , Zn 2+ , Ba 2+ , K + and Mn 2+ , but inhibited by Fe 2+ , Mg 2+ and Ca 2+ . PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, β and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Proteases can be used in several biotechnological processes including detergent, food and leather industries. In the leather industry, dehairing is carried out by chemicals, which pollute the environment. Therefore, to make the hair removal process environmentally friendly, a protease produced by Aspergillus terreus has been purified, biochemically characterized and had an efficient ability to remove hair from bovine leather. The protease was produced using 1% wheat bran and was purified 2.3-fold using two chromatographic steps showing a molecular weight of 90 kDa. Optimal temperature and pH were 50 °C and 6.5, respectively. Thermal stability was up to 1 h at 50 °C. Protease was stable to detergents like Tween 80 and to organic solvents. The activity was activated by Ca 2+ and inhibited by Hg 2+ and Cu 2+ . The enzyme was classified as serine protease, by the inhibition by PMSF and was stable to reducing agents. It hydrolyzed casein, azocasein, BSA, egg albumin and BTpNA. The Km and Vmax values were 0.65 ± 0.03 mg/mL and 3.66 ± 0.18 μmol/min, respectively. Remarkable properties about temperature, pH, stability to detergents and reducing agents ensure that the protease from A. terreus can be an excellent candidate for industrial applications, particularly in the leather industry.

Background: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase., Objective: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme., Methods: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme., Results: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca 2+ and Mg 2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn 2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn 2+ ; DMSO <2%, v/v). The enzyme exhibited high resistance to Al 3+ , which might be explained by the soil properties of its host's residence. Circular dichroism confirmed the contribution of ions to the structure and activity., Conclusion: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated., Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated., Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study., Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with K m BApNA = 0.629 ± 0.02 mM and k cat /K m BApNA = 7.08 s-1/mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data., Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill's body was suggested., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The molecular mass of the enzyme, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, Fe 3+ , and Fe 2+ . The highest affinity exhibited by the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying its highest degrading activity towards the Aα-chains followed by Bβ chains and Cγ chains. The enzyme was also activated by plasminogen, indicating its ability to degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion into plasmin, or (b) by direct hydrolysis. It degraded thrombin, suggesting that it may act as an anticoagulant to prevent thrombosis. Taken together, our results indicate the potential of this enzyme in controlling cardiovascular disease.

Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/β-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/β-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9., (© 2019 John Wiley & Sons, Ltd.)

We found an elastolytic activity in the culture supernatant of Streptomyces sp. P-3, and the corresponding enzyme (streptomycetes elastase, SEL) was purified to apparent homogeneity from the culture supernatant. The molecular mass of purified SEL was approximately 18 kDa as judged by SDS-PAGE analysis and gel-filtration chromatography. Utilizing information from N-terminal amino acid sequencing of SEL and mass spectrometry of SEL tryptic fragments, we succeeded in cloning the gene-encoding SEL. The cloned SEL gene contains a 726 bp ORF, which encodes a 241 amino acid polypeptide containing a putative signal peptide for secretion (28 amino acid) and pro-sequence (14 amino acid). Although the deduced primary structure of SEL has sequence similarity to proteins in the S1 protease family, the amino acid sequence shares low identity (< 31.5 %) with any known elastase. SEL efficiently hydrolyses synthetic peptides having Ala or Val in the P1 position such as N -succinyl-Ala-Ala-(Pro or Val)-Ala-p-nitroanilide (pNA), whereas reported proteases by streptomycetes having elastolytic activity prefer large residues, such as Phe and Leu. Compared of kcat/Km ratios for Suc-Ala-Ala-Val-Ala-pNA and Suc-Ala-Ala-Pro-Ala-pNA with subtilisin YaB, which has high elastolytic activity, Streptomyces sp. P-3 SEL exhibits 12- and 121-fold higher, respectively. Phylogenetic analyses indicate that the predicted SEL protein, together with predicted proteins in streptomycetes, constitutes a novel group within the S1 serine protease family. These characteristics suggest that SEL-like proteins are new members of the S1 serine protease family, which display elastolytic activity.

Bacillus licheniformis W10 is a strain of biocontrol bacteria that was obtained from plant rhizosphere screening. In this study, we purified, identified, and carried out bioinformatics analysis of the W10 antifungal protein from Bacillus licheniformis. Mass spectrometry analysis was carried out by passing the antifungal protein through a high-resolution time-of-flight mass spectrometer. Mascot searches of the tandem mass spectrometry data identified this antifungal protein as a serine protease, and the 1347 bp gene encoding this protein was cloned. Bioinformatics analysis of this protein indicated that it contains 448 amino acid residues, has a molecular weight of 48,794.16 Da and an isoelectric point of 6.04, and is a hydrophilic protein. In the secondary and tertiary structure of this protein, the proportion of α-helices and β-folds is similar, and the protein possesses a Peptidase&#95;S8 conserved domain. Using BApNA as a substrate, it was found that the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) can inhibit the W10 antifungal protein. PMSF concurrently reduced the inhibitory effects of the antifungal protein on Botrytis cinerea, showing that the W10 antifungal protein possesses serine protease activity. The W10 antifungal protein has good thermal stability. The study implies potential of this enzyme for biocontrol of fungal plant pathogens., (Copyright © 2018. Published by Elsevier B.V.)

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2 4 , was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M PEG ), concentrations of PEG (C PEG ) and citrate (C CIT ), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG° m (-10.89 kJ mol -1 ), ΔH m (-5.0 kJ mol -1 ) and partition ΔS m (19.74 J mol -1 K -1 ) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.

Human coagulation factor XII, the initiating factor in the intrinsic coagulation pathway, is critical for pathological thrombosis but not for hemostasis. Pharmacologic inhibition of factor XII is an attractive alternative in providing protection from pathologic thrombus formation while minimizing hemorrhagic risk. Large quantity of recombinant active factor XII is required for screening inhibitors and further research. In the present study, we designed and expressed the recombinant serine protease domain of factor XII in Pichia pastoris strain X-33, which is a eukaryotic expression model organism with low cost. The purification protocol was simplified and the protein yield was high (~20 mg/L medium). The purified serine protease domain of factor XII behaved homogeneously as a monomer, exhibited comparable activity with the human βFXIIa, and accelerated clot formation in human plasma. This study provides the groundwork for factor XII inhibitors screening and further research.

A putative serine protease of T. spiralis (TsSP) was expressed in Escherichia coli and its potential as a diagnostic antigen was primarily assessed in this study. Anti-Trichinella IgG in serum samples from T. spiralis different animal hosts (mice, rats, pigs and rabbits) were detected on Western blot analysis with rTsSP. Anti-Trichinella antibodies were detected in 100% (30/30) of experimentally infected mice by rTsSP-ELISA. Cross-reactions of rTsSPELISA were not found with sera from mice infected with other parasites (S. erinaceieuropaei, S. japonicum, C. sinensis, A. cantonensis and T. gondii) and sera from normal mice. There was no statistical difference in antibody detection rate among mice infected with the encapsulated Trichinella species (T. spiralis, T. nativa, T. britovi, and T. nelsoni) (P>0.05). The results of rTsSP-ELISA showed that serum specific antibody IgG in mice infected with 100 or 500 T. spiralis muscle larvae (ML) were detectable early at 7-8 dpi, but not detected by ML ES antigen-ELISA prior to 10-12 dpi. Specific anti-Trichinella IgG was detected in 100% (18/18) of infected pigs by rTsSP-ELISA and ES-ELISA, but no specific antibodies was not detected in 20 conventionally raised normal pigs by two antigens. The results showed the rTsSP had the potential for early serodiagnosis of animal Trichinella infection, however it requires to be assayed with early infection sera of swine infected with Trichinella and other parasites.

Proteases, one of the largest groups of industrial enzymes occupy a major share in detergent industry. To meet the existing demands, proteases with efficient catalytic properties are being explored from bacteria residing in extreme habitats. Alkaline proteases are also considered as promising candidates for industrial sectors due to the activity and stability under alkaline and harsh environment. Therefore, a systematic review on experimental studies of bacterial proteases was conducted with emphasis on purification, characterization, cloning and expression and their suitability as detergent additive. Relevant searches using a combination of filters/keywords were performed in the online databases; PubMed, Science Direct, Scopus and Web of Science. Over thousands of research papers, 71 articles in Scopus, 48 articles in Science Direct, 18 articles in PubMed and 8 articles in Web of Science were selected with regard to bacterial extracellular proteases till date. Selected articles revealed majority of the studies conducted between the years 2015 and 17 and were focused on purification of proteases from bacteria. Among microbes, a total of 41 bacterial genera have been explored with limited studies from extreme habitats. Majority of the studies have reported the involvement of subtilisin-like serine proteases with effective properties for detergent industries. The studies revealed shifting of trend from purification to cloning to genetic engineering to meet the industrial demands. The present systematic review describes the proteases from extremophilic bacteria and use of biotechnological techniques such as site-directed mutagenesis and codon optimization to engineer enzymes with better hot spots in the active sites to meet industrial challenges.

Serine protease (SPs) is one of the immune enzyme's molecules that play a main role in the variation of a physiological process by controlling protease actions in vertebrates. For example, signaling cells, protector and improvement, which are included in melanization, are utilized to cascade with the meddling pathogens and defense the harmed tissue in insects. In this study, we explore the biochemical process of (SP-22) from Bombyx mori. Reverse-transcription polymerase chain reaction (RT-PCR) discloses that BmSP-22 is expressed in all tissues including the fat body. The formative expression profile of BmSP-22 reveal that BmSP-22 messenger RNA is expressed constitutively in larvae. Injection of recombinant BmSP-22 into B. mori larvae reduces significantly the transcript levels of antimicrobial peptides in the fat body. Our results suggest that BmSP-22 plays an important role in the innate immunity of B. mori and possibly in other insects., (© 2019 Wiley Periodicals, Inc.)

Background: Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration., Results: A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH 2 -terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 °C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500 L, respectively., Conclusions: This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.

A sarcoplasmic serine proteinase (SSP) was purified from threadfin bream (Nemipterus virgatus) belly muscle by ammonium sulfate precipitation and a series of chromatographies including Q-Sepharose, Phenyl Sepharose and Superdex 200. The SSP was purified 1967 folds with a yield of 4.8%. The molecular weight of the SSP was estimated to be 43.5 kDa and 22.5 kDa on SDS-PAGE under non-reducing and reducing conditions, respectively. The N-terminal amino acid sequence of the two protein bands were determined as IVGGYEXQPYSQAHQVSLNSGY and corresponded. It is suggested that the SSP exists as a homodimer. Optimum pH and temperature were 9.5 and 50 °C, using Boc-Val-Pro-Arg-MCA as a substrate. Substrate specificity and effects of inhibitors indicated that the SSP was a trypsin-like serine proteinase. The SSP was responsible for hydrolyzing myosin heavy chain (MHC) and inducing modori phenomenon in the threadfin bream surimi gel. Thus, the SSP was considered as a modori-inducing proteinase., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A 2 (PLA 2 ), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA 2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

In recent years, skin reactions such as phytophotodermatitis, contact dermatitis, and other inflammatory responses after contact with chemicals from various plants, e.g., Heracleum mantegazzianum or Hippomane mancinella , are one of the hot topics in phytobiology. Occupational skin inflammation after contact with latices of plants from Euphorbiaceae are common among people who work with plants of this family. Activation of protein kinase C by G protein-coupled receptors such as protease-activated receptors is associated with skin inflammation. In this study, we focused on the inflammatory modulation potential of proteases combined with diterpenes on human skin. Because of its role as a proinflammatory cytokine, we concentrated on the release of IL-8 by fibroblasts and keratinocytes. Therefore, primary human dermal fibroblasts and the HaCaT keratinocytes cell line were used as a model. The results indicated that the combination of the protease mauritanicain from Euphorbia mauritanica and phorbol-12-myristate-13-acetate induced a significantly increased IL-8 release in HaCaT keratinocytes compared to single treatments. The obtained results also suggest that mauritanicain has an anti-inflammatory effect on primary human dermal fibroblasts., Competing Interests: The authors declare no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The T m of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345 K, and the MD data at 345 K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour., (Copyright © 2018. Published by Elsevier B.V.)

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bβ chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

A new ascomycete fungus X5, a hyperproducer (9000 U/mL) of a serine alkaline protease (SAPTEX) was identified as Penicillium chrysogenum. The experimental purification protocol comprises three steps: heat treatment (10 min at 80 °C) followed by an ammonium sulfate precipitation (30-50%)-dialysis, and a UNO Q-12 anion exchange chromatography using the FPLC system. The chemical characterizations performed include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 43,074.11 Da. The 25 residue NH 2 -terminal sequence of the enzyme showed high homology with Penicillium proteases. The optimum pH and temperature values for protease activity were pH 10 and 80 °C, respectively. Compared to other proteases (SPTC; Flavourzyme® 500 L; Proteinase, type XXIII; Proteinase K; and Alcalase® 2.4 L), SAPTEX has the highest catalytic efficacy, hydrolysis degree, and a powerful stability toward some commercial detergents. According to morphological, physico-chemical [scanning electron microscopy (SEM), energy dispersive X-Ray analysis (EDX), and FTIR-Fourier transform infrared spectroscopy], and mechanical evaluation, SAPTEX has no destructive impact on fibers after the enzyme treatment and a very slight effect on textile support. Obtained results suggested that SAPTEX may be considered as a potential candidate as a protein stain removal product for textile supports., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg ++ and Ca ++ , but inhibited by Zn ++ , and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and V max  = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60 °C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg 2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA₂). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA₂s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-l-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respectively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co 2+ ions stimulated protease activity very strongly. Cu 2+ , Hg 2+ , Cd 2+ and Mn 2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions., (Copyright © 2018. Published by Elsevier B.V.)

A new set of applications can be achieved when using high stability proteases. Industrially, high costs can be related to production medium and purification process. Magnetic nanoparticles have been successfully used for rapid and scalable purification. In this work, azocasein were immobilized on magnetite nanoparticles and applied in a single step purification of protease produced by Penicillium aurantiogriseum using soybean flour medium, and the new purified enzyme was characterized. Glutaraldehyde activated nanoparticles were used in azocasein immobilization and then incubated with dialyzed 60-80% saline precipitation fraction of crude extract for purification. Adsorbents were washed 7 times (0.1 M NaCl solution) and eluted 3 times (1 M NaCl solution), these final elutions contained the purified protease. This protease was purified 55.68-fold, retaining 46% of its original activity. Presented approximately 40 kDa on SDS-PAGE and optimum activity at 45 °C and pH 9.0. Maintained over 60% of activity from pH 6.0 to 11.0. Kept more than 50% activity from 15 to 55 °C, did not lose any activity over 48 h at 25 °C. Inhibitors assay suggested a serine protease with aspartic residues on its active site. Results report a successful application of an alternative purification method and novel broad pH tolerant protease., (Copyright © 2018. Published by Elsevier Inc.)

The characterisation of a serine protease isolated from tamarillo (Solanum betaceum) fruit and its milk casein hydrolysis activity were investigated. Compared with calf rennet, a crude extract from tamarillo exhibited wider caseinolytic activity on sodium caseinate. The purified protease was named "tamarillin" and revealed proteolytic activity toward purified α-, β- and κ-casein. Similar to calf rennet, tamarillin preferably hydrolysed κ-casein, but, unlike calf rennet, it also displayed high proteolytic activity toward both α- and β-casein. The major peptide generated from κ-casein by tamarillin was analysed by gel electrophoresis and liquid chromatography mass spectrometry to confirm its molecular mass as 14,290 Da. The cleavage site was confirmed by in-gel tryptic digestion and time-of-flight mass spectrometry analysis to be at Asn 123 -Thr 124 . This was in contrast to the Phe 105 -Met 106 cleavage site of rennet hydrolysis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This study demonstrates the purification and characterization of a fibrinolytic serine metalloprotease from the marine Serratia marcescens subsp. sakuensis (KU296189.1). The purified enzyme (1033 U/mg) had a molecular weight of 43 KDa, with optimum pH and temperature being 7 and 55 °C. The in vitro half-life of the fibrinolytic enzyme at 37 °C was found to be 19 h. The kinetic constants, K m and V max of the purified enzyme determined using fibrin as substrate was 0.66 mg/mL and 158.73 U/mL. The K cat and catalytic efficiency of the enzyme was found to be 12.21 min -1 and 18.32 mL/(mg min) respectively. The fibrinolytic enzyme did not show any proteolytic activity towards blood plasma proteins like haemoglobin, γ-globulins and transferrin. In vitro studies revealed that the fibrinolytic enzyme displayed 38% clot lysis for a period of 3 h which was higher than that displayed by streptokinase and heparin. A total of seven peptide sequences were obtained after the LC-MS/MS-TOF analysis, out of which only four sequences showed 67% homology with the sequences of the other proteases. All these results suggest its novelty and potential application in thrombolytic therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The harnessing of medicinal plants containing a plethora of bioactive molecules may lead to the discovery of novel, potent and safe therapeutic agents to treat thrombosis-associated cardiovascular diseases. A 35 kDa (m/z 34747.5230) serine protease (lunathrombase) showing fibrin(ogen)olytic activity and devoid of N- and O- linked oligosaccharides was purified from an extract of aqueous leaves from L. indica. The LC-MS/MS analysis, de novo sequencing, secondary structure, and amino acid composition determination suggested the enzyme's novel characteristic. Lunathrombase is an αβ-fibrinogenase, demonstrating anticoagulant activity with its dual inhibition of thrombin and FXa by a non-enzymatic mechanism. Spectrofluorometric and isothermal calorimetric analyses revealed the binding of lunathrombase to fibrinogen, thrombin, and/or FXa with the generation of endothermic heat. It inhibited collagen/ADP/arachidonic acid-induced mammalian platelet aggregation, and demonstrated antiplatelet activity via COX-1 inhibition and the upregulation of the cAMP level. Lunathrombase showed in vitro thrombolytic activity and was not inhibited by endogenous protease inhibitors α 2 macroglobulin and antiplasmin. Lunathrombase was non-cytotoxic to mammalian cells, non-hemolytic, and demonstrated dose-dependent (0.125-0.5 mg/kg) in vivo anticoagulant and plasma defibrinogenation activities in a rodent model. Lunathrombase (10 mg/kg) did not show toxicity or adverse pharmacological effects in treated animals.

A new intracellular serine protease gene of Bacillus megaterium, ispK, encoding a protein composed of 332 amino acid residues with a predicted pI of 4.7 was cloned into Escherichia coli. The deduced amino acid sequence of IspK showed 49-56% similarity with the other microbial intracellular serine proteases described in the literature. The enzyme was effectively purified by one-step chromatography after heat-treatment, and showed a homogeneous band corresponding to 35kDa by SDS-PAGE analysis. Amino acid analysis showed that 16 amino acids of the N-terminus of IspK were removed by post-translational protease activity. The optimum pH and temperature of IspK were 6.0-7.0 and 50°C, respectively. In the presence of 2mM of Ca 2+ ion, the optimum temperature was increased to 65°C and thermostability (t 1/2 ) increased 32.9-fold from 3.3min to 108.5min at 60°C. The enzyme was activated by Ca 2+ and Mg 2+ , almost completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and EDTA, but tolerant to nonionic surfactants, such as, Triton X-100 or Tween 80. IspK efficiently hydrolyzed natural proteins, such as, casein and hemoglobin, and improved blood stain removal. These results suggest IspK can be used as a useful additive for detergent formulations and for deproteinizations., (Copyright © 2017 Elsevier B.V. All rights reserved.)