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Extracellular hydrolytic activity (phospholipase, protease and hemolysin production) was evaluated in 178 strains of potentially pathogenic ascomycetous (Candida parapsilosis, Candida tropicalis) and basidiomycetous (Rhodotorula mucilaginosa) yeasts isolated from the excreta of Mew Gulls. Two bird colonies, one nesting in a natural habitat and the other in an urban habitat at the landfill, were studied simultaneously during their 7-month breeding season. Significant differences in phospholipase and protease production were found between natural and anthropophized strains. Both virulent activities were higher in strains of potentially pathogenic yeast species isolated from the excreta of Mew Gulls nesting in the anthropogenic habitat near the landfill. No significant differences were found in hemolysin production. Thus, synanthropic migratory birds nesting in an urban area could be involved in the wide spread of yeast virulence. It seems very important to advocate that landfills in urban areas are nowadays closed and "relocated" to large distances from cities and that biowaste is treated before dumping in order to prevent and minimize its negative impact on the environment (water, soil, air) and human health., Competing Interests: Declarations. Ethics approval: The study was approved by the Ethics Committee of Lomonosov Moscow State University (№ 144-5/3/2008-2020, https://bioethics.msu.ru ). Additional ethical review and approval were waived for this study because the study did not involve the capturing or manipulation of birds, only their observation. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

Leather industry is traditionally characterized by the use of large amounts of chemical agents, some of which are toxic to human health and the environment. However, during the last years, many efforts have been made with the aim of successfully implement enzymes as agents for different leather production stages. The lipopeptides produced by the Bacillus spp. genus have excellent surfactants and antibacterial properties and may collaborate in the soaking stage of leather processing as well as in leather preservation. Moreover, Bacillus sp. proteases and lipopeptides can be co-produced in one culture medium, saving the production costs. In the present work, a screening of enzymatic activities was performed on 11 strains of the Bacillus sp. genus that have been isolated from samples of an artisan tannery from Salta, Argentina. In particular, the ability of B. amyloliquefaciens B65 to degrade α-type (nails, hair, wool) and β-type (feathers) keratin was demonstrated by scanning electron microscopy (SEM). The co-production of proteases, keratinases, glycosidases, and lipopeptides of this strain was conducted at 37 °C in mineral media supplemented with chicken feathers. In these nutrient-deficient media, the strain secreted amylases, pectinases, proteases, keratinases, and collagenases. A MALDI-TOF study also revealed that the strains secreted homologues of kurstakins, iturins, surfactins, and fengycines lipopeptides families. Therefore, B. amyloliquefaciens B65 presents great industrial potential applications, not only for tanneries but also for other industries such as pharmaceuticals, food, textiles, and detergents, among others., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Conflict of interest: No., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

A Gram-positive, moderately halophilic, rod-shaped and facultatively anaerobic strain T66 T , was isolated from the rhizosphere soil of mangrove Acanthus ebracteatus in Guangxi, China. Strain T66 T was observed to grow at 15-40 ℃ (optimum 30 ℃), pH 5-8 (optimum 6-7) and with 2-20% (w/v) NaCl (optimum 6%). The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain T66 T to be closely related to Halobacillus litoralis SL-4  T (98.72%), Halobacillus dabanensis D-8  T (98.70%) and Halobacillus campisalis ASL-17  T (98.66%). The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain T66 T belonged to the genus Halobacillus and formed a separate branch. The digital DNA-DNA hybridisation and average nucleotide identify values between strain T66 T and its related species were 17.80-19.80% and 70.78-73.22%, respectively, which were lower than the threshold recommended for species delineation. The genomic DNA G + C content was 40.9%. The predominant isoprenoid quinone was MK-7, the cell-wall peptidoglycan contained meso-diaminopimelic acid, and the major fatty acids that accounted for more than 10.0% were anteiso-C 15:0 (68.4%) and anteiso-C 17:0 (13.4%). Phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and an unidentified glycolipid were found in the polar lipid extraction. Strain T66 T could hydrolyse casein and contains several genes (e. g. vanY, vpr, pbpG and elpC) involved in protease. Therefore, strain T66 T is salt-tolerant and produces protease, and it may have development and utilisation value in flavouring agents and food curing. Based on phenotypic, chemotaxonomic data and genotypic characteristics, strain T66 T represents a novel species of the genus Halobacillus, for which the name Halobacillus rhizosphaerae sp. nov. is proposed. The type strain is T66 T (= JCM 36534  T  = MCCC 1K08701 T )., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Consent for publication: The authors are consent to publication. Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Aquatic ecosystems are increasingly affected by anthropogenic pollution, including heavy metals like mercury, which accumulate in organisms and cause harmful effects. At the same time, human activities such as industrial operations and the use of electric power lines also alter the magnetic background in natural water bodies. However, the interaction between mercury exposure and magnetic fields remains poorly understood. This study aimed to investigate the combined effects of mercury and magnetic fields on the digestive enzyme activity of common carp (Cyprinus carpio), focusing on the relevance of magnetic fields due to their increasing presence in industrialized environments. Two groups of fish were fed diets with low (0.02 mg/kg) or high (0.27 mg/kg) mercury content for 6 months and exposed to extremely low-frequency magnetic fields or hypomagnetic conditions. Results showed significant differences in mercury accumulation between groups, with higher levels in carps fed with high-mercury content diets. These fish also exhibited increased body length and weight compared to those on a low-mercury diet. The amylolytic activity (total activity of enzymes hydrolyzing starch: α-amylase, glucoamylase, and maltase) and proteolytic activity (total activity of serine proteinases) in the fish intestine were assessed. Magnetic exposure had contrasting effects on enzyme activity, depending on mercury levels in the diet. Fish fed the low-mercury diet exhibited decreased amylolytic activity following magnetic field exposure, while fish on the high-mercury diet showed increased activity. Proteolytic activity followed a similar pattern, with opposite effects observed between the two dietary groups. These findings suggest that mercury accumulation alters the biological response to magnetic fields, possibly through compensatory biochemical mechanisms. Understanding the interactions between toxic substances and magnetic fields is critical for improving environmental risk assessments., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Gloriosa superba L., a medicinally important plant, is often affected by leaf blight disease caused by Alternaria alternata, which compromises its productivity. This study explores the protective effects of Bacillus australimaris endophyte (NBRI GS34), demonstrating that its inoculation not only inhibits the disease but also promotes plant growth and increases the concentrations of bioactive metabolites. Co-culturing NBRI GS34 with A. alternata significantly boosts protease (30-50%) and chitinase (6-28%) activities, evidencing a synergistic interaction. Scanning electron microscopy and GC-MS analysis confirm NBRI GS34's antagonistic action and reveal antifungal compounds like undecanoic acid and benzene carboxylic acid in treatments. Greenhouse experiments show a 78% reduction in disease incidence with NBRI GS34 treatment, enhancing vegetative growth and upregulating defense-related genes. Additionally, HPLC analysis reveals increased gloriosine and colchicine concentrations by 52% and 33%, respectively. These findings suggest NBRI GS34 could serve as a sustainable fungicide alternative, enhancing the production of medically valuable compounds and highlighting its potential pharmaceutical applications., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The increasing global consumption of poultry meat has led to the generation of a vast quantity of feather keratin waste daily, posing significant environmental challenges due to improper disposal methods. A growing focus is on utilizing keratinous polymeric waste, amounting to millions of tons annually. Keratins are biochemically rigid, fibrous, recalcitrant, physiologically insoluble, and resistant to most common proteolytic enzymes. Microbial biodegradation of feather keratin provides a viable solution for augmenting feather waste's nutritional value while mitigating environmental contamination. This approach offers an alternative to traditional physical and chemical treatments. This review focuses on the recent findings and work trends in the field of keratin degradation by microorganisms (bacteria, actinomycetes, and fungi) via keratinolytic and proteolytic enzymes, as well as the limitations and challenges encountered due to the low thermal stability of keratinase, and degradation in the complex environmental conditions. Therefore, recent biotechnological interventions such as designing novel keratinase with high keratinolytic activity, thermostability, and binding affinity have been elaborated here. Enhancing protein structural rigidity through critical engineering approaches, such as rational design, has shown promise in improving the thermal stability of proteins. Concurrently, metagenomic annotation offers insights into the genetic foundations of keratin breakdown, primarily predicting metabolic potential and identifying probable keratinases. This may extend the understanding of microbial keratinolytic mechanisms in a complex community, recognizing the significance of synergistic interactions, which could be further utilized in optimizing industrial keratin degradation processes., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The presence of infective larvae (L 3 ) of gastrointestinal nematode (GIN) parasites in pastures directly contributes to the constant recurrence of infections in ruminant herds. This study aimed to evaluate the nematophagous fungus Duddingtonia flagrans (AC001) (proteolytic crude extract and/or conidia) in the in vitro control of GIN L 3 in coprocultures. To produce the proteolytic crude extract, a suspension (10 7 conidia/mL) of D. flagrans was inoculated into a liquid medium. After 6 days, the medium was filtered, centrifuged, and its proteolytic activity was measured. For the experimental assay, fecal samples were collected directly from the rectal ampulla of naturally infected sheep, and egg counts per gram of feces (EPG) were performed. Coprocultures were prepared using 10 g of fecal material with the groups defined as follows: control group G1 (1.0 mL of denatured proteolytic crude extract); treated group G2 (1.0 mL of active proteolytic crude extract); treated group G3 (1.0 mL of active proteolytic crude extract + 1.0 mL of AC001 conidia). The coprocultures were maintained at room temperature (25ºC), for 7 days, and then the L 3 larvae were recovered. The results demonstrated that AC001 successfully produced protease (56.34 U/mL). The treatments with active proteolytic crude extract (G2) and active proteolytic crude extract + AC001 conidia (G3) were significantly different (p < 0.01) from the control group with denatured proteolytic crude extract (G1). AC001 and its proteolytic crude extract acted concomitantly on helminths directly in the fecal environment, suggesting potential future applications in the field., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 o C with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. K m and V max of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

MALT1 has been implicated as an upstream regulator of NF-κB signaling in immune cells and tumors. This study determined the regulatory mechanisms and biological functions of MALT1 in non-small cell lung cancer (NSCLC). In cell culture and orthotopic xenograft models, MALT1 suppression via gene expression interference or protein activity inhibition significantly impaired malignant phenotypes and enhanced radiation sensitivity of NSCLC cells. CSN5, the core subunit of COP9 signalosome, was firstly verified to stabilize MALT1 via disturbing the interaction with E3 ligase FBXO3. Loss of FBXO3 in NSCLC cells reduced MALT1 ubiquitination and promoted its accumulation, which was reversed by CSN5 interference. An association between CSN5/FBXO3/MALT1 regulatory axis and poor prognosis in NSCLC patients was identified. Our findings revealed the detail mechanism of continuous MALT1 activation in NF-κB signaling, highlighting its significance as predictor and potential therapeutic target in NSCLC., (© 2024. The Author(s).)

Protein hydrolysates might display diverse bioactivities with potential relevance to human and animal health and food technology. Enzymatic hydrolysis of agro-industrial by-products is increasingly focused. In this study, a crude protease from Bacillus sp. CL18 was applied to obtain antioxidant protein hydrolysates from porcine, bovine, poultry, and fish by-products. The crude enzyme hydrolyzed all the twelve investigated by-products, as detected by increased soluble protein contents after 4 h of proteolysis. Hydrolysates exhibited higher radical-scavenging, Fe 2+ -chelating and reducing power capacities than non-hydrolyzed by-products. Hydrolysis times (0-8 h) and enzyme-to-substrate (E/S) ratios (384, 860, and 1,400 U/g) were assessed to produce antioxidant bovine lung hydrolysates. The highest E/S ratio accelerated both hydrolysis and increases in antioxidant activities; however, it did not result in bioactivities higher than hydrolysates obtained with the intermediate E/S ratio. Optimal antioxidant activities could be reached after 6 h of hydrolysis using 860 U/g. Animal by-products are interesting sources of bioactive protein hydrolysates, which could be produced with a non-commercial bacterial protease. This might represent a promising strategy for the valorization of animal by-products generated in large amounts by the agri-food sector., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322-330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection., (© 2024. The Author(s).)

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC., (© 2024. The Author(s).)

Feather, horn, hoof, and other keratin waste are protein-rich but limited by natural keratinase synthesis, activity, pH, and temperature stability. It is challenging to realize its large-scale application in industries. Bacillus subtilis spores are a safe, efficient, and highly resistant immobilized carrier, which can improve target proteins' resistance. In this research, KERQ7, the keratinase gene of Bacillus tequilensis strain Q7, was fused to the Bacillus subtilis genes coding for the coat proteins CotG and CotB, respectively, and displayed on the surface of B. subtilis spores. Compared with the free KERQ7, the immobilized KERQ7 showed a greater pH tolerance and heat resistance on the spore surface. The activity of CotG-KERQ7 is 1.25 times that of CotB-KERQ7, and CotG-KERQ7 is more stable. When the flexible linker peptide L3 was used to connect CotG and KERQ7, the activity was increased to 131.2 ± 3.4%, and the residual enzyme activity was still 62.5 ± 2.2% after being kept at 60 ℃ for 4 h. These findings indicate that the flexible linker and CotG were more effective for the spore surface display of keratinase to improve stress resistance and promote its wide application in feed, tanning, washing, and other industries., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Keratinases have drawn increasing attention in recent decades owing to their catalytic versatility and broad applications from agriculture to medicine. In the present study, we isolated a highly keratinolytic and fibrinolytic bacterium from the campus soil and named it Stenotrophomonas sp. LMY based on genetic information. To identify the potential keratinase genes, the genome sequence of the strain was obtained and analyzed. Sequence alignment and comparison revealed that the protein 1&#95;737 (KerZJ) had the highest sequence homology to a reported keratinase KerBL. We recombinantly expressed KerZJ in Escherichia coli Origami™ (DE) pLysS and purified it to homogeneity. KerZJ showed the highest activity at 40 °C and pH 9.0, and metal ions exhibited no significant effects on its activity. Although reducing agents would break the disulfide bonds in KerZJ and reduce its activity, KerZJ still exhibited the ability to hydrolyze feather keratin in the presence of β-ME. KerZJ could efficiently digest human prion proteins. In addition, KerZJ showed fibrinolytic activity on fibrin plates and effectively eliminated blood clots in a thrombosis mouse model without side effects. Our results suggest that KerZJ is a versatile keratinase with significant potential for keratin treatment, decontamination of prions, and fibrinolytic therapy., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4 + T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Although aspartic intramembrane-cleaving proteases (I-CLIPs) are crucial switches of multiple signaling pathways and involved in several devastating diseases, little is known about their physiological regulation. We have recently identified Frey regulator of sperm-oocyte fusion 1 (Frey1) as an inhibitory protein of Signal Peptide Peptidase-like 2c (SPPL2c), a member of this protease family. Employing structure modeling along with cell-based inhibition and interaction studies, we identify a short motif within the Frey1 transmembrane domain essential for inhibition of SPPL2c. Intriguingly, this motif can be transplanted to the SPPL2c substrate PLN, thereby transforming it into an inhibitor of this enzyme. It can be adopted for the generation of Notch1-based γ-Secretase inhibitors demonstrating its versatile use among aspartic I-CLIPs. In summary, we describe a mechanism of aspartic I-CLIP inhibition which allows the targeted generation of specific inhibitors of these enzymes and might enable the identification of endogenous negative regulators of these enzymes., (© 2023. The Author(s).)

The objective of the present study was to evaluate the effects of increasing doses of protease on broilers from 1 to 42 days of age. A total of 1290 Ross AP broilers were used, distributed among five treatments: positive control diet, negative control diet (NC), NC + 50 ppm of protease, NC + 100 ppm of protease, and NC + 200 ppm of protease. Each treatment contained six replicates of 43 animals each. The inclusion of proteases in the diet had effects (P < 0.05) on body weight, feed intake, weight gain, and feed conversion in the 12 to 21 day period; body weight, weight gain, and feed intake in the 29 to 42 day period; nutrient digestibility (energy metabolizability coefficient and crude protein at 28 days); and intestinal parameters (crypt and muscle width of jejunum and ileum at 28 days and villus length, crypt length, and jejunum thickness muscle layer at 42 days). These results indicate that the inclusion of protease in broiler feed can improve production parameters when the amount of crude protein in the diet is reduced., (© 2023. The Author(s).)

A method of dehairing of goat skins using oxidative chemicals and protease enzymes has been attempted. The dehairing process is one of the important and essential steps in leather making, where hair is removed by lime and sodium sulphide in the conventional process. This conventional dehairing system generates a higher amount of pollution problem as compared to the other unit operations and unit processes. In this work, dehairing of the goat skins through oxidative agents namely magnesium peroxide and protease enzyme has been attempted. For this, protease has been produced from Bacillus sp. at the laboratory level and the activity was found. The dehairing of goat skins takes place for the duration of 14-16 h. The leather produced with the experimental sample showed comparable organoleptic and strength properties with the conventional sample. This method paved the way for the reduction of pollution loads especially BOD, COD, and TDS to the level of 59, 27, and 77%, respectively, in comparison with the control sample. The reaction kinetics for the formation of the ligand-macromolecular complex is found in the isothermal titration calorimetry (ITC) experiment and a mathematical model has been formulated. The dyed crust leather showed comparable colour properties. In addition to that, there is a reduction in processing time for leather making through skipping reliming and deliming processes which are said to be another advantage of this method. The physical strength properties of the experimental leather were also comparable with conventionally produced leather., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Intervertebral disc degeneration (IVDD) mainly manifests as an imbalance between the synthesis and degradation of cellular and extracellular matrix (ECM) components. The cytokine interleukin (IL)-1β-induced inflammatory response of intervertebral discs causes ECM degradation. The aim of this study was to investigate the effects of a 970-nm diode laser therapy (DLT) on inflammatory cytokine IL-1β and ECM degradation proteinases in nucleus pulposus (NP) tissues in a puncture-induced rabbit IVDD model. Thirty-six New Zealand white rabbits were randomly divided into six groups: the normal group, IVDD group, laser group, sham laser group, IVDD + anisomycin (p38MAPK signaling pathway agonist), and laser + anisomycin group. Effects of laser on IVDD progression were detected using radiographic and magnetic resonance imaging. Hematoxylin and eosin, Alcian blue, safranin O-fast green staining, western blotting, and immunohistochemistry staining were performed for the histological analysis and molecular mechanism underlying protection against puncture-induced matrix degradation in NP tissues by DLT. DLT reduced the degree of disc degeneration in the gross anatomy of the disc and increased the T2-weighted signal intensity of NP. Inflammatory cytokine IL-1β levels in the disc were significantly reduced after DLT suppressed the matrix-degrading proteinases MMP13 and ADAMTS-5 and upregulated the protein expression of collagen II and aggrecan. Moreover, it inhibited the p38MAPK signaling pathway in NP tissues in a puncture-induced rabbit IVDD model. DLT reduced puncture-induced overexpression of inflammatory cytokines, mainly IL-1β, thus inhibiting matrix degeneration of NP tissues and ameliorating IVDD. This may be related to inhibition of the p38 MAPK signaling pathway., (© 2023. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Feather biodegradation is an important premise for efficient resource development and utilization, in which keratinase plays an important role. However, there are few keratinases that combine the high activity, thermal stability, and organic solvent tolerance required for industrialization. This paper reported an efficient feather-degrading Pseudomonas aeruginosa 4-3 isolated from slaughterhouses. After 48 h of fermentation by P. aeruginosa 4-3 in a feather medium at 40 °C, pH 8.0, keratinase was efficiently produced (295.28 ± 5.42 U/mL) with complete feather degradation (95.3 ± 1.5%). Moreover, the keratinase from P. aeruginosa 4-3 showed high optimal temperature (55 °C), good thermal stability, wide pH tolerance, and excellent organic solvent resistance. In addition, P. aeruginosa 4-3-derived aminopeptidases also exhibit excellent thermal stability and organic solvent tolerance. Encouragingly, the reaction of crude keratinase and aminopeptidase with feathers for 8 h resulted in a 78% degradation rate of feathers. These properties make P. aeruginosa 4-3 keratinase and aminopeptidase ideal proteases for potential applications in keratin degradation, as well as provide ideas for the synergistic degradation of keratin by multiple enzymes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To elucidate how Shewanella putrefaciens survives and produces spoilage products in response to cold conditions, the metabolic and protease activity of S. putrefaciens DSM6067 cultured at three different temperatures (30 °C, 10 °C, and 4 °C) was studied by determining the bacterial growth, total volatile basic nitrogen (TVB-N), biogenic amines, extracellular protease activity, as well as the differential expressed proteins via Label-free quantitative proteomics analysis. The lag phase of the strain cultured at 10 °C and 4 °C was about 20 h and 120 h longer than at 30 °C, respectively. The TVB-N increased to 89.23 mg N/100 g within 28 h at 30 °C, and it needed at least 72 h and 224 h at 10 °C and 4 °C, respectively. Cold temperatures (10 °C and 4 °C) also inhibited the yield factors and the extracellular protease activity per cell at the lag phase. However, the protease activity per cell and the yield factors of the sample cultivated at 10 °C and 4 °C well recovered, especially at the mid and latter stages of the log phase. The further quantitative proteomic analysis displayed a complex biological network to tackle cold stress: cold stress responses, nutrient uptake, and energy conservation strategy. It was observed that the protease and peptidase were upregulated, so as to the degradation pathways of serine, arginine, and aspartate, which might lead to the accumulation of spoilage products. This study highlighted the spoilage potential of S. putrefaciens still should be concerned even at low temperatures., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Since the dawn of century, tons of keratin bio-waste is generated by the poultry industry annually, and they end up causing environmental havoc. Keratins are highly flexible fibrous proteins which exist in α- and β- forms and provide mechanical strength and stability to structural appendages. The finding of broad-spectrum protease, keratinase, from thermophilic bacteria and fungi, has provided an eco-friendly solution to hydrolyze the peptide bonds in highly recalcitrant keratinous substances such as nails, feathers, claws, and horns into valuable amino acids. Microorganisms produce these proteolytic enzymes by techniques of solid-state and submerged fermentation. However, solid-state fermentation is considered as a yielding approach for the production of thermostable keratinases. This review prioritized the molecular and biochemical properties of microbial keratinases, and the role of keratinases in bringing prodigious impact for the sustainable progress of the economy. It also emphasizes on the current development in keratinase production with the focus to improve the biochemical properties related to enzyme's catalytic activity and stability, and production of mutant and cloned microbial strains to improve the yield of keratinases. Recently, multitude molecular approaches have been employed to enhance enzyme's productivity, activity, and thermostability which makes them suitable for pharmaceutical industry and for the production of animal feed, organic fertilizers, biogas, clearing of animal hides, and detergent formulation. Hence, it can be surmised that microbial keratinolytic enzymes are the conceivable candidates for numerous commercial and industrial applications., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dengue fever has been a global health concern. Mitigation is a challenging problem due to non-availability of workable treatments. The most difficult objective is to design a perfect anti-dengue agent capable of inhibiting infections caused by all four serotypes. Various tactics have been employed in the past to discover dengue antivirals, including screening of chemical compounds against dengue virus enzymes. The objective of the current study is to investigate phytocompounds as anti-dengue remedies that target the non-structural 2B and non-structural 3 protease (NS2B-NS3 pro ), a possible therapeutic target for dengue fever. Initially, 300 + antiviral phytocompounds were collected from Duke's phytochemical and ethnobotanical database and 30 phytocompounds with anti-dengue properties were identified from previously reported studies, which were virtually screened against NS2B-NS3 pro using molecular docking and toxicity evaluation. The top five most screened ligands were naringin, hesperidin, gossypol, maslinic acid and rhodiolin with binding affinities of - 8.7 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol and - 8.1 kcal/mol, respectively. The finest docked compounds complexed with NS2B-NS3 pro were subjected for molecular dynamics (MD) simulations and binding free energy estimations through molecular mechanics generalized born surface area-based calculations. The results of the study are intriguing in the context of computer-aided screening and the binding affinities of the phytocompounds, proposing maslinic acid (MAS) as a potent bioactive antiviral for the development of phytocompound-based anti-dengue agent., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Myocardial ischemia-reperfusion injury (MI/RI) is a leading cause of death globally. Whereas some long noncoding RNAs (lncRNAs) are known to participate in the progression of MI/RI, the role of urothelial carcinoma associated 1 (UCA1) in conjunction with sevoflurane treatment remains largely unknown. H9C2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) to establish an in vitro MI/RI model, and sevoflurane was then added. Cell viability, apoptosis, SOD activity, and MDA levels were measured. Levels of inflammatory cytokines and methylation of apoptosis protease-activating factor 1 (APAF1) were determined. Interactions among lncRNA UCA1, enhancer of zeste homologue 2 (EZH2), DNA methyltransferase-1 (DNMT1), and APAF1 were analyzed. After H/R treatment, the viability of H9C2 cardiomyocytes decreased and apoptosis rate, oxidative stress factor levels, inflammatory cytokine levels, and apoptosis-related protein levels all increased. Sevoflurane treatment reversed these changes. LncRNA UCA1 knockdown attenuated the therapeutic effect of sevoflurane on H/R-treated cardiomyocytes, and silencing of APAF1 reversed this role of UCA1 knockdown. Moreover, lncRNA UCA1 recruited DNMT1 through EZH2, thus promoting methylation of the APAF1 promoter region. LncRNA UCA1 recruits DNMT1 to promote methylation of the APAF1 promoter through EZH2, thus strengthening the protective effect of sevoflurane on H/R-induced cardiomyocyte injury., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Since the appearance of SARS-CoV-2 and the COVID-19 pandemic, the search for new approaches to treat this disease took place in the scientific community. The in silico approach has gained importance at this moment, once the methodologies used in this kind of study allow for the identification of specific protein-ligand interactions, which may serve as a filter step for molecules that can act as specific inhibitors. In addition, it is a low-cost and high-speed technology. Molecular docking has been widely used to find potential viral protein inhibitors for structural and non-structural proteins of the SARS-CoV-2, aiming to block the infection and the virus multiplication. The papain-like protease (PLpro) participates in the proteolytic processing of SARS-CoV-2 and composes one of the main targets studied for pharmacological intervention by in silico methodologies. Based on that, we performed a systematic review about PLpro inhibitors from the perspective of in silico research, including possible therapeutic molecules in relation to this viral protein. The neurological problems triggered by COVID-19 were also briefly discussed, especially relative to the similarities of neuroinflammation present in Alzheimer's disease. In this context, we focused on two molecules, curcumin and glycyrrhizinic acid, given their PLpro inhibitory actions and neuroprotective properties and potential therapeutic effects on COVID-19., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Chikungunya virus (CHIKV) is the etiological agent of the Chikungunya fever which has spread worldwide. Clinically, this disease may lead to prolonged incapacitating joint pain that can compromise remarkably the patients' quality of life. However, there are no licensed vaccines or specific drugs to fight this infection yet, making the search for novel therapies an imperative need. In this scenario, the CHIKV nsP2 protease emerged as an attractive therapeutic target once this protein plays a pivotal role in viral replication and pathogenesis. Hence, we investigated the structural basis for the inhibition of this enzyme by using molecular docking and dynamics simulations. Compounds with inhibitory activities against CHIKV nsP2 protease determined experimentally were selected from the literature. Docking studies with a set of stereoisomers showed that trans isomers, but not cis ones, bound close to the catalytic dyad which may explain isomerism requirements to the enzyme's inhibition. Further, binding mode analyses of other known inhibitors revealed highly conserved contacts between inhibitors and enzyme residues like N1011, C1013, A1046, Y1079, N1082, W1084, L1205, and M1242. Molecular dynamics simulations reinforced the importance of some of these interactions and pointed to nonpolar interactions as the main forces for inhibitors' binding. Finally, we observed that true inhibitors exhibited lower structural fluctuation, higher ligand efficiency and did not induce significant changes in protein correlated motions. Collectively, our findings might allow discerning true inhibitors from false ones and can guide drug development efforts targeting the nsP2 protease to fight CHIKV infections in the future., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The information about toxic effects of thiamethoxam on non-target aquatic organisms is still incomplete. The semi-static toxicity test method was used to investigate the acute toxic effects of thiamethoxam on Eriocheir sinensis. The results showed that the median lethal concentration (LC50) of thiamethoxam to E. sinensis at 96 h was 510 μg/L, and the safety concentration (SC) was 51 μg/L. After 96 h exposure to thiamethoxam, the survival rates of crabs at concentrations of 0, 151.11, 226.67, 340, and 510 μg/L were 100%, 76.19%, 64.29%, 61.91%, and 46.43%, respectively. A significant (P < 0.05) decrease of the number of hemocytes was observed in thiamethoxam groups. With the increase of thiamethoxam concentration, the phagocytic activity of hemocytes, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and the activities of protease, amylase, and lipase of crabs increased firstly and then decreased, and the above indexes reached the maximum in 151.11 μg/L thiamethoxam group. Collectively, a high concentration of thiamethoxam (510 μg/L) had a great effect on the gene expression of immune metabolism-related factors in hepatopancreas and gill of crabs. These findings indicated that thiamethoxam exposure had the ability to impair immune and metabolic systems and resulted in the reduction of survival rate of crabs., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Viral-cell entry and cell-cell viral spreading processes of SARS-CoV-2 are subjected to fast evolutionary optimization because of its worldwide spreading, requiring the need for new drug developments. However, this task is still challenging, because a detailed understanding of the underlying molecular processes, mediated by the key cellular proteases TMPRSS2 and furin, is still lacking. Here, we show by large-scale atomistic calculations that binding of the ACE2 cell receptor at one of the heteromers of the SARS-CoV-2 spike leads to a release of its furin cleavage site (S1/S2), enabling an enhanced furin binding, and that this latter process promotes the binding of TMPRSS2 through the release of the TMPRSS2 cleavage site (S2') out of the ACE2-binding heteromer. Moreover, we find that, after proteolytic cleavage, improved furin binding causes that parts of the S2 subunit dissociate from the complex, suggesting that furin promotes the fusion of the S2 subunit with the cell membrane before transfer of the viral RNA. Here we show by computational means that binding of the ACE2-cell receptor at one of the heteromers of the SARS-CoV-2 spike leads to an enhanced binding of the protease furin, promoting the binding of the protease TMPRSS2. Moreover, we show that, after proteolytic cleavage, improved furin binding causes that parts of the heteromer dissociate from the spike., (© 2022. The Author(s).)

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2 -/- ) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2 +/+ but not of Habp2 -/- mice. However, Habp2 -/- mice were not protected from sodium retention compared to nephrotic Habp2 +/+ mice. Western blot analysis revealed that in nephrotic Habp2 -/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2 +/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice., (© 2021. The Author(s).)

Deubiquitinylases (DUBs) are central regulators of the ubiquitin system involved in protein regulation and cell signalling and are important for a variety of physiological processes. Most DUBs are cysteine proteases, and few other proteases are metalloproteases of the JAB1/MPN +/MOV34 protease family (JAMM). STAM-binding protein like 1 (STAMBPL1), a member of the JAMM family, cleaves ubiquitin bonds and has a function in regulating cell survival, Tax-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and epithelial-mesenchymal transition. However, the molecular mechanism by which STAMBPL1 influences cell survival is not well defined, especially with regard to its deubiquitinylation function. Here, we show that reactive oxygen species (ROS) induced by chemotherapeutic agents or the human microbial pathogen Helicobacter pylori can induce cullin 1-RING ubiquitin ligase (CRL1) and 26S proteasome-dependent degradation STAMBPL1. Interestingly, STAMBPL1 has a direct interaction with the constitutive photomorphogenic 9 (COP9 or CSN) signalosome subunits CSN5 and CSN6. The interaction with the CSN is required for the stabilisation and function of the STAMBPL1 protein. In addition, STAMBPL1 deubiquitinylates the anti-apoptotic protein Survivin and thus ameliorates cell survival. In summary, our data reveal a previously unknown mechanism by which the deubiquitinylase STAMBPL1 and the E3 ligase CRL1 balance the level of Survivin degradation and thereby determine apoptotic cell death. In response to genotoxic stress, the degradation of STAMBPL1 augments apoptotic cell death. This new mechanism may be useful to develop therapeutic strategies targeting STAMBPL1 in tumours that have high STAMBPL1 and Survivin protein levels., (© 2022. The Author(s).)

Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12-302 ss] was not further active which could be due to the large truncation. However, T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25-90 °C and pH 6-9.5. Interestingly, remaining enzyme activity of T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8-339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Proteases are ubiquitous in prokaryotes and eukaryotes. Plant proteases are key regulators of various physiological processes, including protein homeostasis, organelle development, senescence, seed germination, protein processing, environmental stress response, and programmed cell death. Proteases are involved in the breakdown of peptide bonds resulting in irreversible posttranslational modification of the protein. Proteases act as signaling molecules that specifically regulate cellular function by cleaving and triggering receptor molecules. Peptides derived from proteolysis regulate ROS signaling under oxidative stress in the plant. It degrades misfolded and abnormal proteins into amino acids to repair the cell damage and regulates the biological process in response to environmental stress. Proteases modulate the biogenesis of phytohormones which control plant growth, development, and environmental stresses. Protein homeostasis, the overall balance between protein synthesis and proteolysis, is required for plant growth and development. Abiotic and biotic stresses are major factors that negatively impact cellular survivability, biomass production, and reduced crop yield potentials. Therefore, the identification of various stress-responsive proteases and their molecular functions may elucidate valuable information for the development of stress-resilient crops with higher yield potentials. However, the understanding of molecular mechanisms of plant protease remains unexplored. This review provides an overview of proteases related to development, signaling, and growth regulation to acclimatize environmental stress in plants., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel protease-producing actinobacterium, designated strain NEAU-A11 T , was isolated from soil collected from Aohan banner, Chifeng, Inner Mongolia Autonomous Region, China, and characterised using a polyphasic approach. The hydrolytic enzymes, such as proteases, played critical roles in destruction of fungi by degrading the protein linkages to disrupt integrity in the cell wall. This suggested that the isolate could be a good biocontrol candidate against pathogens to control fungal diseases. On the basis of 16S rRNA gene sequence analysis, strain NEAU-A11 T was indicated to belong to the genus Actinoplanes and was most closely related to Actinoplanes rectilineatus JCM 3194  T (98.9%). Cell walls contained meso-diaminopimelic acid as the diagnostic diamino acid and the whole-cell sugars were arabinose, xylose and glucose. The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. The predominant menaquinones were MK-9(H 4 ), MK-9(H 6 ) and MK-9(H 8 ). The major fatty acids were C 18:0 , C 16:0 , C 18:1 ω9c, C 17:0 and C 15:0 . Genome sequencing revealed a genome size of 10,742,096 bp, a G + C content of 70.5% and 9,514 protein-coding genes (CDS), including 102 genes coding for protease. Moreover, Genome analysis showed that strain NEAU-A11 T contained 255 glycoside hydrolases (GHs), 152 glycosyl transferases (GTs), 40 carbohydrate esterases (CEs), 26 polysaccharide lyases (PLs), and 12 auxiliary activities (AAs) genes. Genome mining analysis using antiSMASH 5.0 led to the identification of 20 putative gene clusters responsible for the production of diverse secondary metabolites. Phylogenetic analysis using the 16S rRNA gene sequences showed that the strain formed a stable clade with A. rectilineatus JCM 3194  T in the genus Actinoplanes. Whole-genome phylogeny showed strain NEAU-A11 T formed a stable phyletic line with Actinoplanes lutulentus DSM 45883  T (97.6%). However, whole-genome average nucleotide identity value between strain NEAU-A11 T and its reference strains A. rectilineatus JCM 3194  T and A. lutulentus DSM 45883  T were found to be 81.1% and 81.6%, respectively. The levels of digital DNA-DNA hybridization between them were 24.6% (22.2-27.0%) and 24.8% (22.5-27.3%), respectively. The values were well below the criteria for species delineation of 70% for dDDH and 95-96% for ANI, suggesting that the isolate differed genetically from its closely related type strain. The content of G + C in genomic DNA was 70.5%, within the range of 67-76%. In addition, evidences from phenotypic, chemotaxonomic and genotypic studies indicated that strain NEAU-A11 T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes aureus sp. nov. is proposed, with NEAU-A11 T (= CCTCC AA 2019063  T  = JCM 33971  T ) as the type strain., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Bacillus licheniformis HJ4 showing strong fibrinolytic activity was isolated from Hwangseokae jeotgal. aprEHJ4, a major fibrinolytic gene, was cloned by PCR, and an ORF consisting of 379 amino acids was located. The mature enzyme was expected to be 27 kDa in size after processing, but a 24-kDa protein was observed by SDS-PAGE and fibrin zymography, indicating additional processing. RT-qPCR showed that expression level of aprEHJ4 in culture with 0% salt (control) was the highest followed by culture with 8% salt (89.7% of control) and 5% salt (74.2%) at 84 h. The expression level in culture with 15% salt was 46.9%. The results matched with the fibrinolytic activity measurements of cultures and indicated that AprEHJ4 maintained significant activity in the presence of salt up to 15% (w/v). AprEHJ4 was overproduced in Escherichia coli, and mature 27 kDa protein was purified after in vitro renaturation. The optimum pH and temperature of AprEHJ4 were pH 8 and 40 ℃, respectively., (© 2021. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

The diversity of actinobacteria associated with marine ascidian Phallusia nigra from Andaman Islands was investigated. A total of 10 actinobacteria were isolated and based on the biochemical and molecular characterization, the isolates were assigned to 7 different actinobacterial genera. Eight putatively novel species belonging to genera Rhodococcus, Kineococcus, Kocuria, Janibacter, Salinispora and Arthrobacter were identified based on 16S rDNA sequence similarity with the NCBI database. The organic extracts of ten isolates displayed considerable bioactivity against test pathogens, which were Gram-positive and Gram-negative in nature. PCR-based screening for type I and type II polyketide synthases (PKS-I, PKS-II) and nonribosomal peptide synthetases (NRPS) revealed that, 10 actinobacterial isolates encoded at least one type of polyketide synthases biosynthesis gene. Majority of the isolates found to produce industrially important enzymes; amylase, protease, gelatinase, lipase, DNase, cellulase, urease, phosphatase and L-asparaginase. The present study emphasized that, ascidians are a prolific resource for novel bioactive actinobacteria with potential for novel drug discovery. This result expands the scope to functionally characterize the novel ascidian associated marine actinobacteria and their metabolites could be a source for the novel molecules of commercial interest.

Amylases and proteases are among the industrially most important enzymes for food processing, animal feed, brewing, starch processing, detergents, healthcare, leather processing, and biofuel production. In this study, we investigated the growth kinetics and statistically optimized the co-production of amylase and protease in a phylogenetically novel haloalkaliphilic actinomycete, Streptomyces lopnurensis KaM5 of seawater. The Plackett-Berman design using Minitab 14.0 software was employed to assess the impact of the nutritional factors, temperature, pH, and incubation time. Further, starch, yeast extract, NaCl concentrations, and incubation time were optimized by Box-Behnken design at their three levels. The Pareto charts, contour, surface plots, and individual factorial analysis expressed the variability and levels for the optimal enzyme production. ANOVA analysis admitted the statistical fitness and significance level among the variables. A two-fold increase in enzyme production was achieved by cost-effective co-production media. The study was further extended to growth kinetics associated with enzyme production. Specific growth rate (μ), maximal cell mass (X max ), volumetric product formation (P max ), rate of product formation (Q p ), and generation time (g) were computed and analyzed. These parameters significantly improved when compared with the pre-optimized conditions, and the production economics of the enzyme was industrially viable. The initial studies on the characteristics of the enzymes suggested its ability to function under the combination of alkaline pH and high salt concentrations. The co-production of enzymes from extremophiles can be a potentially viable option for large-scale production and applications.

Mast cells (MC) are immune cells that produce a variety of mediators, such as proteases, that are important in the body's immune responses. MC proteases have pronounced multifunctionality and in many respects determine the biological characteristics of the organ-specific MC population. Although, increased numbers of MC are one of the objective mastocytosis signs, a detailed assessment of the proteases biogenesis and excretion mechanisms in the bone marrow (BM) has not yet been carried out. Here, we performed an analysis of the expression of proteases in patients with various forms of systemic mastocytosis. We presented data on intracellular protease co-localization in human BM MCs and discussed their implication in secretory pathways of MCs in the development of the disease. Systemic mastocytosis, depending on the course, is featured by the formation of definite profiles of specific proteases in various forms of atypical mast cells. Intragranular accumulation of tryptase, chymase and carboxypeptidases in the hypochromic phenotype of atypical mast cells is characterized. Characterization of MC proteases expression during mastocytosis can be used to refine the MC classification, help in a prognosis, and increase the effectiveness of targeted therapy.

S-RNAse-based self-incompatibility (SI) in petunia (Petunia hybrida L.) is a self-/non-self-recognition system underlying the pistil rejection of self-pollen. Using different methods, including a TUNEL assay, we have recently shown that programmed cell death (PCD) is a factor of the SI in petunia. Here, we show that the growth of self-incompatible pollen tubes in the style tissues during 4 h after pollination is accompanied by five-sixfold increase in a caspase-like protease (CLP) activity. Exogenous cytokinin (CK) inhibits the pollen tube growth and stimulates the CLP activity in compatible pollen tubes. The actin depolymerization with latrunculin B induces a sharp drop in the CLP activity in self-incompatible pollen tubes and its increase in compatible pollen tubes. Altogether, our results suggest that a CLP is involved in the SI-induced PCD and that CK is a putative activator of the CLP. We assume that CK provokes acidification of the cytosol and thus promotes the activation of a CLP. Thus, our results suggest that CK and CLP are involved in the S-RNAse-based SI-induced PCD in petunia. Potential relations between these components in PCD signaling are discussed.

Keratinases are proteolytic enzymes with a particular ability to cleave peptide bonds in keratin, and in other proteins. Due to their broad-spectrum of activity, keratinases are considered viable substitutes for chemical and thermal treatments of protein-rich industrial by-products. Among these protein residues, special attention has been given to keratinous materials (feathers, hair, horns, etc.), which disposal through harsh conditions methods, such as acid/alkaline hydrolysis or incineration, is not considered ecologically safe. Microbial keratinolytic enzymes allow for keratin degradation under mild conditions, resulting in keratin hydrolysates containing undamaged amino acids and peptides. In this review article, we offer perspectives on the relevance of these unique biocatalysts and their revolutionary ascent in industries that generate keratin-rich wastes. Additionally, we share insights for applications of keratinases and protein hydrolysates in agriculture, animal feed, cosmetics, phamaceuticals, detergent additives, leather processing, and others. Due to the scientific importance of keratinases and their potential use in green technologies, searching for bacterial and fungal species that efficiently produce these enzymes may contribute to the sustainability of industries.

Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH 4 ) 2 SO 4 , Na 2 SO 4 and MgSO 4 , were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl 2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.

Polyurethanes (PU) are multifunctional polymers, used in automotive industry, in coatings, rigid and flexible foams, and also in biomimetic materials. In the same way as all plastic waste, the incorrect disposal of these materials leads to the accumulation of polyurethanes in the environment. To reduce the amount of waste as well as add value to degradation products, bioremediation methods have been studied for waste management of PU. Enzymes of the hydrolases class have been experimentally tested for enzymatic degradation of PU, with very promising results. In this work, two enzymes that can degrade polyurethanes were studied by molecular dynamics simulations: a protease and an esterase, both from Pseudomonas. From molecular dynamics simulations analysis, it was observed the stability of the structures, both in the simulations of the free enzymes and in the simulations of the complexes with a PU monomer. Hydrogen bonds were formed with the monomer and the enzymes throughout the simulation time, and the interaction free energy was found to be strongly negative, pointing to strong interactions in both cases.

Transmembrane substrate cleavage by the small Escherichia coli rhomboid protease GlpG informs on mechanisms by which lipid interactions shape reaction coordinates of membrane-embedded enzymes. Here, I review and discuss new work on the molecular picture of protein-lipid interactions that might govern the formation of the substrate-enzyme complex in fluid lipid membranes. Negatively charged PG-type lipids are of particular interest, because they are a major component of bacterial membranes. Atomistic computer simulations indicate POPG and DOPG lipids bridge remote parts of GlpG and might pre-occupy the substrate-docking site. Inhibition of catalytic activity by PG lipids could arise from ligand-like lipid binding at the active site, which could delay or prevent substrate docking. Dynamic protein-lipid H-bond networks, water access to the active site, and fluctuations in the orientation of GlpG suggest that GlpG has lipid-coupled dynamics that could shape the energy landscape of transmembrane substrate docking.

This study was conducted to study the effect of protease as well as three dietary crude protein levels on growth and nutrient digestibility in broilers. The broiler chickens (n = 540) were assigned to six dietary treatments with six replicates. Three dietary crude protein (CP) levels (17, 19, and 21%) along with two levels of protease (0 and 30,000 IU/kg) were investigated in 3 × 2 factorial arrangement during the starter phase. Body weight was higher (P < 0.05) in group fed with CP-21; however, feed intake was significantly (P < 0.05) lower in CP-19 and CP-21. Resultantly, FCR was significantly (P < 0.05) improved in birds fed with CP-21. Similarly, body weight, feed intake, and FCR were significantly (P < 0.05) improved in enzyme-treated birds compared to the untreated birds. For practical purpose, birds fed CP-19 with protease enzyme resulted in improved FCR compared to birds fed with CP-21. The results revealed that CP and apparent metabolizable energy (AME) were significantly (P < 0.05) higher in birds fed with CP-21 along with supplementation of enzyme while abdominal fat was significantly (P < 0.05) lower in the same group compared to birds fed with CP-17. These results indicated that a diet having 21% CP supplemented with 30,000 IU/kg protease enzyme was superior in improving the performance and digestibility of nutrients in broiler during the starter phase.

A novel coronavirus (SARS-CoV-2) identified in Wuhan state of China in 2019 is the causative agent of deadly disease COVID-19. It has spread across the globe (more than 210 countries) within a short period. Coronaviruses pose serious health threats to both humans and animals. A recent publication reported an experimental 3D complex structure of the S protein of SARS-CoV-2 showed that the ectodomain of the SARS-CoV-2 S protein binds to the peptidase domain (PD) of human ACE2 with a dissociation constant (Kd) of ~ 15 nM. In this study, we focused on inhibitors for ACE2: S protein complex using virtual screening and inhibition studies through molecular docking for over 200,000 natural compounds. Toxicity analysis was also performed for the best hits, and the final complex structures for four complexes were subjected to 400 ns molecular dynamics simulations for stability testing. We found two natural origin inhibitors for the S protein: human ACE2 complex (Andrographolide and Pterostilbene) which displayed better inhibition potential for ACE2 receptor and its binding with the S protein of SARS-CoV-2. Comparative studies were also performed to test and verify that these two drug candidates are also better than hydroxychloroquine which is known to inhibit this complex. However, we needed better potential drug candidates to overcome the side effects of hydroxychloroquine. Supplementary experimental studies need to be carried forward to corroborate the viability of these two new inhibitors for ACE2: S protein complex so as to curb down COVID-19.

Microbial and enzymatic degradation of keratin waste is more preferred over various conventional approaches which are costly and not environmentally suitable. Diverse niches are auspicious for the discovery of new microorganisms. To discover novel keratinolytic bacteria, 60 isolates from different poultry dumping sites were initially screened, and among these found a potent keratinolytic isolate (NKSP-7) that displayed higher feather-degrading ability. The selected isolate was identified as Bacillus sp. NKSP-7 based on 16S rDNA sequencing as well as physiochemical and morphological characteristics. The strain NKSP-7 showed complete hydrolysis of native chicken feathers (10 g/L) in nutrient medium after 24 h of incubation at 37 °C under agitation (150 rev/min) and produced thermostable extracellular keratinase. The crude enzyme displayed maximal keratinolytic activity (34.7 U/mL) in phosphate buffer of pH 7.0, and at 60 °C using keratin azure as a substrate. Keratinolytic enzyme showed stability at 20-65 °C for 4 h over the pH range of 5.5-8.0. No obvious inhibitory influence was perceived by cations, organic solvents, EDTA, and detergents. Whereas, enzyme activity was enhanced by adding β-mercaptoethanol, Na + , Cd 2+ , and Mn 2+ . All these notable features of keratinase make it a promising candidate for various industrial applications especially for dehairing process in leather industry, bioconversion of poultry waste, and in detergents formulations.

Halophilic bacterias from saline soil from former Lake Texcoco were isolated, identified based on 16 rRNA and tested to produce glucolytic, nucleolytic, proteolytic and lipolytic exoenzymes. The Bacillus, Virgibacillus, Kocuria, Salinicoccus, Gracilibacillus, Halobacillus, Tenuibacillus and Nesterekonia genera where identified. Lipase/eserases and proteases from Nesterenkonia sp. and Nesterenkonia aethiopica showed halotolerant characteristics and were selected to synthesize the oleochemical n-butyl oleate and antioxidant peptides from muscle protein of common carp (Cyprinus carpio), respectively. In organic media (2,2,4-Trimethylpentane), the lipase/esterases from Nesterenkonia sp. (0.6 U/mL) and N. aethiopica (1.2 U/mL) achieved a 62.7% and 53.2% of n-butyl oleate conversion, respectively. The protein hydrolysis from muscle of common carp (C. carpio) showed a degree of hydrolysis of 4.5 ± 0.2% and 2.8 ± 0.1% when proteases from Nesterenkonia sp. and N. aethiopica were used, respectively. Three peptidic fractions ranging molecular masses between 254 and 1002 Da [M + H] show antioxidant scavenging activity, and the principal fraction with a peptide of 547.3 Da [M + H] showed an inhibition of 37.7 ± 1.8% and 16.3 ± 0.6%, when 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were used, respectively. These findings showed that the enzymatic battery of the halophilic bacteria from former lake Texcoco can be used in hydrolysis and synthesis of molecules with applications in different fields as food technology or bioenergy.

A new keratinase producer, Bacillus sp. BK111, isolated from a poultry feather was identified as Bacillus zhangzhouensis, which is the first report for its keratinolytic activity. The keratinase production was optimized, followed by the enzyme purification and characterization using biochemical assays. A 2.34-fold increase was observed in the enzyme production under optimized conditions. The enzyme was characterized as a serine protease with 42 kDa molecular weight, stable in a wide range of temperature and pH with maximum keratinolytic activity at 60 °C and pH 9.5. The enzyme had a wide range of different substrates with the best performance on the feather meal substrate. Metal ions of Ca 2+ , K + , Na + and Mn 2+ enhanced the enzyme activity. The enzyme showed a great deal of stability in the presence of ethanol, methanol, acetone, 2-propanol, dimethyl sulfoxide, Tween-80 and Triton X-100. Dithiothreitol (DTT), as a reducing agent, caused a twofold increase in keratinolytic activity. The half-life of the enzyme at optimum temperature was calculated to be 125 min and the ratio of keratinolytic:caseinolytic for the enzyme was 0.8. Our results showed the remarkable features of the enzyme that make it suitable for biotechnological usages.

Background: Obtaining tumor-free margins is critical to prevent recurrence after lumpectomy for breast cancer. Unfortunately, current approaches leave positive margins that require second surgeries in 20-40% of patients. We assessed the LUM Imaging System for real-time, intraoperative detection of residual tumor., Methods: Breast lumpectomy cavity walls and excised specimens were assessed with the LUM Imaging System after 1 mg/kg intravenous LUM015, a protease-activatable fluorescent agent. Fluorescence at potential sites of residual tumor in lumpectomy cavity walls was evaluated intraoperatively with a sterile hand-held probe, with real-time predictive results displayed on a monitor intraoperatively, and later correlated with histopathology., Results: In vivo lumpectomy cavities and excised specimens were imaged after LUM015 injection in 45 women undergoing breast cancer surgery. Invasive ductal and lobular cancers and intraductal cancer (DCIS) were included. A total of 570 cavity margin surfaces in 40 patients were used for algorithm development. Image analysis and display took approximately 1 s per 2.6-cm-diameter circular margin surface. All breast cancer subtypes could be distinguished from adjacent normal tissue. For all imaged cavity surfaces, sensitivity for tumor detection was 84%. Among 8 patients with positive margins after standard surgery, sensitivity for residual tumor detection was 100%; 2 of 8 were spared second surgeries because additional tissue was excised at sites of LUM015 signal. Specificity was 73%, with some benign tissues showing elevated fluorescent signal., Conclusions: The LUM015 agent and LUM Imaging System allow rapid identification of residual tumor in the lumpectomy cavity of breast cancer patients and may reduce rates of positive margins.