Action Of Proteases Research Articles

Effects of High Pressure on In Vitro Bioavailability of Curcumin Loaded in Whey Protein Isolate/Carrageenan Composite Emulsion Gel: In Vitro Digestion Coupled with Cell Culture Model

The oral bioavailability of curcumin is inherently low, which significantly limits its application in food systems. The objective of this study was to evaluate the impact of high-pressure processing on the stability and bioaccessibility of curcumin within an emulsion gel during simulated gastrointestinal transit and to assess its cellular uptake. Our findings suggest that increasing pressure levels and high κ-carrageenan concentrations can enhance the stability of the curcumin delivery system. Elevated κ-CG concentrations were found to retard the action of proteases on dissociating protein molecules from the gel network. The emulsion gel effectively slowed the release of free fatty acids and reduced the curcumin release rate during the gastric phase. Scanning electron microscopy images revealed that higher pressures induced the formation of a more uniform and dense network structure in the gel. While the gel network structures were well-preserved after gastric digestion, they were disrupted into smaller particles following intestinal digestion, with particle size increasing with higher applied pressures. Cytotoxicity assays indicated that the digesta from the intestinal phase was highly toxic to Caco-2 cells. Among the tested samples, the emulsion gel prepared with 1.0% κ-CG at 600 MPa demonstrated the highest curcumin bioavailability, reaching 63.82 ± 7.10%. These findings underscore the potential of HPP-induced emulsion gels as a viable delivery system for enhancing curcumin bioaccessibility and cellular uptake.

Antioxidant activities of Saudi honey samples related to their content of short peptides

This study explored the effect of geographical and floral origins on the antioxidant activities of Saudi honey samples related to their content of short peptides originated from honeybee proteins. The studied antioxidants were the total protein concentration, catalase activity, phenolic acids and flavonoids. The antioxidant activity assays included were the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, the ferric reducing antioxidant power (FRAP) assay and Ascorbic acid Equivalent Antioxidant Capacity (AEAC). The studied honey samples were obtained from the southwestern region of Saudi Arabia, namely Asir (65) and Jazan (25). The floral origins of the honey samples were Acacia (51), Ziziphus (4) and polyfloral (35). The LC/MS technique was used to detect the short peptides and the mascot database was used to identify the short peptides, their precursor proteins and the protease enzymes that produce them. Jazan honey was characterized by high number of short peptides. The short peptides were originated from honeybee proteins by the action of proteases from the honeybees and bacteria. The antioxidant activity of the honey samples increase with the increase of their content of short peptides and proteins. The amino acids type and sequence of the short peptides qualify them to act as antioxidant, antimicrobial, anti-diabetic, anti-hypertension, immunomodulatory and cholesterol lowering peptides.

SUMOylation modulates mitochondrial dynamics in an in vitro rotenone model of Parkinson's disease

SUMOylation is a post-translational modification essential for various biological processes. SUMO proteins bind to target substrates in a three-step enzymatic pathway, which is rapidly reversible by the action of specific proteases, known as SENPs. Studies have shown that SUMOylation is dysregulated in several human disorders, including neurodegenerative diseases that are characterized by the progressive loss of neurons, mitochondrial dysfunction, deficits in autophagy, and oxidative stress. Considering the potential neuroprotective roles of SUMOylation, the aim of this study was to investigate the effects of SENP3 knockdown in H4 neuroglioma cells exposed to rotenone, an in vitro model of cytotoxicity that mimics dopaminergic loss in Parkinson's disease (PD). The current data show that SENP3 knockdown increases SUMO-2/3 conjugates, which is accompanied by reduced levels of the mitochondrial fission protein Drp1 and increased levels of the mitochondrial fusion protein OPA1. Of high interest, SENP3 knockdown prevented rotenone-induced superoxide production and cellular death. Taken together, these findings highlight the importance of SUMOylation in maintaining mitochondrial homeostasis and the neuroprotective potential of this modification in PD.

New Proline-Specific Endoprotease for Improved Colloidal Beer Stability and Gluten Reduction

Chill-haze is a well-known concern for brewers and most commonly involves polyphenols that interact with proline-rich hordein proteins in beers. An effective haze-suppressing effect has previously been established by a proline-specific endoprotease (PEP). In the present study, the protease action of a new Trichoderma reesei expressed PEP enzyme, available as the product BCLEAR™ was studied by assessing its impact on colloidal as well as foam stability and gluten reduction in beer. All malt beers treated with the BCLEAR™ enzyme or beers produced using BCLEAR™ at low dosage levels during fermentation, showed improved colloidal and foam stability when compared to the benchmark PEP investigated. In addition, gluten reduction was investigated using the BCLEAR™ enzyme and was found to have a very good correlation with the observed haze reduction, suggesting that the PEP enzyme dosage can accurately be determined from the calculated gluten content of the given raw materials. Thus, for optimal PEP enzyme use, the brew processes may most efficiently be controlled by following the gluten content, which was demonstrated with use of Lateral Flow Device (LFD) analysis technology.

Quercetin and taxifolin inhibits TMPRSS2 activity and its interaction with EGFR in paclitaxel-resistant breast cancer cells: An in silico and invitro study.

Transmembrane protease/serine (TMPRSS2), a type II transmembrane serine protease, plays a crucial role in different stages of cancer. Recent studies have reported that the triggering epidermal growth factor receptor (EGFR) activation through protease action promotes metastasis. However, there are no reports on the interaction of TMPRSS2 with EGFR, especially in triple-negative triple negative (TNBC). The current study investigates the unexplored interaction between TMPRSS2 and EGFR, which are key partners mediating metastasis. This interaction is explored for potential targeting using quercetin (QUE) and taxifolin (TAX). TMPRSS2 expression patterns in breast cancer (BC) tissues and subtypes have been predicted, with the prognostic significance assessed using the GENT2.0 database. Validation of TMPRSS2 expression was performed in normal and TNBC tissues, including drug-resistant cell lines, utilizing GEO datasets. TMPRSS2 was further validated as a predictive biomarker for FDA-approved chemotherapeutics through transcriptomic data from BC patients. The study demonstrated the association of TMPRSS2 with EGFR through in silico analysis and validates the findings in TNBC cohorts using the TIMER2.0 web server and the TCGA dataset through C-Bioportal. Molecular docking and molecular dynamic simulation studies identified QUE and TAX as best leads targeting TMPRSS2. They inhibited cell-free TMPRSS2 activity like clinical inhibitor of TMPRSS2, Camostat mesylate. In cell-based assays focused on paclitaxel-resistant TNBC (TNBC/PR), QUE and TAX demonstrated potent inhibitory activity against extracellular and membrane-bound TMPRSS2, with low IC50 values. Furthermore, ELISA and cell-based AlphaLISA assays demonstrated that QUE and TAX inhibit the interaction of TMPRSS2 with EGFR. Additionally, QUE and TAX exhibited significant inhibition of proliferation and cell cycle accompanied by notable alterations in the morphology of TNBC/PR cells. This study provides valuable insights into potential of QUE and TAX targeting TMPRSS2 overexpressing TNBC.

Effect of different temperature–time combinations on the quality and protein digestive properties of stewed beef tendons

This study investigated the effects of sous vide cooking and traditional high-temperature cooking on the texture, microstructure, protein structure and digestive properties of home made beef tendon using transmission electron microscopy, in vitro digestion and peptidomics. The adverse effects of heating on the muscle fiber structure and protein conformation of beef tendon gradually expanded with increasing temperature. In general, the degree of muscle fiber contraction, protein denaturation, and protein aggregation increases with temperature and time, with a concomitant decrease in protease action. The extent of protein digestion, and the yield of small fragments such as amino acids also decreased with increasing heating temperature and time. However, the solubilization of collagen makes the beef collagen more digestible after heating at 80°C, which indirectly leads to higher digestibility at 80°C. The combined peptide release and bioactive peptide results showed that the beef tendon heated at 60°C for 8 h produced the most peptides after digestion, and also produced four specific bioactive peptides, which were advantageous in terms of the number of digested products and bioactivity. The higher proportion of antioxidant amino acids in the product also gave the tendon heated at 60°C for 8 h a greater potential antioxidant capacity. As a result, beef tendon heated at 60°C for 8 h is more favorable for human digestion. This work may provide guidance for home cooking and dietary health.

Rapid screening and isolation of potential xanthine oxidase inhibitors in Ganoderma lucidum

Given the abundance and intricate nature of the chemical components found in natural food ingredients, functional food research has long grappled with two significant technical challenges: rapid and precise screening of active substances and targeted preparation. To enhance the efficiency of fungal food preparation, novel methodologies for screening and the production of xanthine oxidase inhibitors from G. lucidum have been developed. The xanthine oxidase inhibitors were rapidly screened using AUF-LC-MS. Molecular docking and molecular dynamics simulation were used to verify the anti-gout activity of active compounds. Subsequently, with activity screening as the guide, HSCCC and SPLC were employed to separate the active ingredients. Simultaneously, the mechanism of action of xanthine oxidase inhibitors was analyzed by enzymatic reaction kinetics. Finally, the anti-gout activity of chemical components in G. lucidum was verified by network pharmacology, and its mechanism of action was discussed. Four potential xanthine oxidase inhibitors, namely ganoderic acid C2, B, A, and D2, were screened from G. lucidum. Their docking binding energies with xanthine oxidase were −6.29, −6.65, −4.29, and −5.96 kcal/mol, respectively. The purity of the four triterpenoid active ingredients isolated from G. lucidum was more than 90 %. These triterpenoid active ingredients showed a reversible inhibitory effect on xanthine oxidase. Network pharmacological studies revealed 71 common targets of active ingredients and gout, obtained by screening and intersecting the two targets, and identified the main metabolic pathways. The results indicate that G. lucidum extract can be employed for the prevention and treatment of gout disease. We enhance the efficiency and purity of active compounds preparation by incorporating enzyme inhibition assays and computerized virtual screening with compound chromatography. With this approach, we seamlessly incorporate fast discovery, screening, and focused preparation of active ingredients, as well as delving into the mechanisms of action of active monomers and proteases, and the molecular-level interactions between active ingredients and proteases. This study providing more opportunities to discover and develop new potential therapeutics from health food resources.

Aprotinin (I): Understanding the Role of Host Proteases in COVID-19 and the Importance of Pharmacologically Regulating Their Function.

Proteases are produced and released in the mucosal cells of the respiratory tract and have important physiological functions, for example, maintaining airway humidification to allow proper gas exchange. The infectious mechanism of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), takes advantage of host proteases in two ways: to change the spatial conformation of the spike (S) protein via endoproteolysis (e.g., transmembrane serine protease type 2 (TMPRSS2)) and as a target to anchor to epithelial cells (e.g., angiotensin-converting enzyme 2 (ACE2)). This infectious process leads to an imbalance in the mucosa between the release and action of proteases versus regulation by anti-proteases, which contributes to the exacerbation of the inflammatory and prothrombotic response in COVID-19. In this article, we describe the most important proteases that are affected in COVID-19, and how their overactivation affects the three main physiological systems in which they participate: the complement system and the kinin-kallikrein system (KKS), which both form part of the contact system of innate immunity, and the renin-angiotensin-aldosterone system (RAAS). We aim to elucidate the pathophysiological bases of COVID-19 in the context of the imbalance between the action of proteases and anti-proteases to understand the mechanism of aprotinin action (a panprotease inhibitor). In a second-part review, titled "Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions", we explain in depth the pharmacodynamics, pharmacokinetics, toxicity, and use of aprotinin as an antiviral drug.

The barley dehydrin 4 and stress tolerance: From gene to function

Dehydrins constitute a major group among Late Embryogenesis Abundant Proteins, and are known to play a crucial role in water stress tolerance in many plant species. However, little is known regarding their functional relevance in cereals such as barley. In this study, we investigated the function and the expression of the barley DHN4 in response to stress. The DHN4 transcription was strongly induced by salinity, cold and dehydration, in Rihane compared to another barley landrace Manel. Interestingly, we found that CBF1 and CBF6 transcription factors can bind to the RhDHN4 promoter and promote its transcription in yeast cells and in planta. A recombinant RhDHN4 protein was purified and shown to be heat stable and sensitive to protease action, a common feature of intrinsically disordered protein. Moreover, RhDHN4 protects in vitro Lactate Dehydrogenase (LDH) against cold, heat and desiccation and is able to prevent the heat-induced aggregation of leaf’s proteome. RhDHN4 exhibited also in vitro radical scavenging activity, suggesting that the RhDHN4 protein may protects DNA and enzymes during stress. On another hand, the overexpression of RhDHN4 conferred stress tolerance in yeast, an effect that seems to occur via a self dimerization of the protein as it was confirmed by yeast-two hybrid and GST-pull down assays. Our results are in favor of a potential role of RhDHN4 in enhancing stress tolerance in barley.

Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEβ7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.

Lactiplantibacillus plantarum enhances the texture of fermented milk by facilitating protein gelatinization through the action of a novel coagulation-promoting protease

Enzymatic modification can effectively improve the gel properties of protein-based food matrices. However, how bacterial proteases influence milk gels has been rarely reported. In the current study, the proteases isolated from two strains of Lactiplantibacillus plantarum, namely YM13–8.1 and YN22–3.1, demonstrated significant improvement in the textural properties of acid-induced gels characterized by increased hardness and a more compact, dense and fine-pored structure. The inclusion of milk-coagulating proteases resulted in a significant augmentation of hydrophobic interactions and disulfide bonds within the protein gels. Furthermore, both proteases exhibited rennet-like characteristics through κ-casein hydrolysis, with key hydrolysis sites identified as Tyr25-Ile26, Ala65-Ala66 and Lys112-Asn113. By utilizing MALDI-TOF-MS, whole genome sequencing and RT-qPCR analysis, we successfully identified the gene responsible for encoding the proteases as prtA, which has not been previously characterized. In conclusion, our findings demonstrate that the newly characterized protease prtA exerts a positive influence on milk gelling.

Exploitation of Selected Fungal Endophytes of Andrographis paniculata (Burm. f.) Wall. ex Nees for the Production and Optimisation of Tannase and Screening for their Associated Hydrolysing Enzymes; Amylase, Protease, Lipase, and Laccase

Endophytes represent a diverse domain of microorganisms with immense biotechnological potentialities. Metabolites from endophytes, especially fungi, are useful in industrial as well as pharmaceutical aspects. Here, endophytic fungal isolates of Andrographis paniculata collected from the Tapobon, Junglemahal region of West Bengal, India, have been studied for their hydrolyzing enzyme production abilities. Out of eighty-one isolates, twenty-one were positive for amylase, protease, lipase, laccase, and tannase action. Microscopic features of positive isolates revealed that the enzyme producers were Aspergillus sp., Fusarium sp., Alternaria sp., Trichoderma sp., Exerohilum sp., Nigrospora sp., Curvularia sp., Cladosporium sp., Cochliobolus sp., Tricothecium sp., Penicillium sp., Verticillium sp., and Cephalosporium sp. The amylolytic activity was remarkable in the case of endophytic Aspergillus sp. and Fusarium sp. Also, Aspergillus sp. and Alternaria sp. had proteolytic activity. Aspergillus sp., Mycelia sterilia-2, and Trichoderma sp. were potent lipase producers. Along with Exerohilum sp. endophytic Aspergillus sp. had positive laccase activity. The tannic acid degrading activity was highest for Aspergillus sp. APL11 followed by Helicosporium sp. and Fusarium sp. Tannase producing ability of Aspergillus sp. was optimized and an incubation time- 96h, incubation temperature- 29°C, initial medium pH- 6.5, carbon source- glucose, and nitrogen source- NaNO3 exhibited a two-fold scale-up of (2.83±0.05 u mL-1) in tannase action. These enzymes offer eco-friendly and efficient solutions across a wide range of industrial processes including food production, textile processing, and pharmaceutical synthesis. Fungal endophytes of medicinal plants act as an alternative source for hydrolysing enzymes.

Modulation of Angiotensin-II and Angiotensin 1-7 Levels Influences Cardiac Function in Myocardial Ischemia-reperfusion Injury.

The angiotensin-converting enzyme-2 (ACE-2) alters the pathophysiology of various fatal cardiovascular diseases, including ischemic heart disease, whereas angiotensin 1-7 (Ang 1-7) exerts a wide range of actions. The effects of ischemia-reperfusion (IR) injury include damage to myocardial tissue that initiates protease action, causing cardiac cell death. Angiotensin- II (Ang-II) contributes through the renin-angiotensin system (RAS) to the IR injury, whereas Ang 1-7 paradoxically exerts a protective effect through the same. Thus, the myocardial ischemic reperfusion injury (MIRI) may be altered by the RAS of the heart. This review paper focuses on ACE-2, angiotensin-converting enzyme (ACE), and Ang 1-7 regulation in the RAS of the heart in the pathophysiology of MIRI. The treatment in such conditions using ACE-2 activator, ACE inhibitor, and Ang-II antagonists may promote vascular functions as well as cardio- protection.

Biodiversity and suppression of wood-decaying fungi in marine environment, Alexandria, Egypt by selected natural products: Control the fungal growth/contamination

The current study investigated the biodiversity and control of wood-decaying fungi isolated from marine environment, at Alexandria, Egypt. The fungal growth was in vitro control via selected plant natural products (seaweeds and aromatic herbs). Among 45 marine fungal isolates from decaying-wood samples were detected. At 85% similarity level, 5 clusters (A, B, C, D, and E) were obtained. According to numerical classification, one strain from each cluster was identified based on the sequences of ITS-PCR. The species level because their best matched references from BLAST search were higher than 96% of similarities with ITS sequences obtained from GenBank (four Aspergillus and one Penicillium species as the most abundant isolates). Additionally, the enzymatic action of cellulase, amylase, protease, and lipase was detected by fungal isolates. The highest enzymatic degradation coefficient (EDC) was of 1.38 for lipase secreted by P. chrysogenum strain HGM3. Only three strains (A. sydowi strain HGM1, P. chrysogenum strain HGM3 and A. amstelodami strain HGM5) produced cellulase enzyme with EDC value (1.075, 1.27 and 1.08), respectively. Moreover, the methanolic extracts of Ulva lactuca and Corallina officinalis collected from the Egyptian coasts besides aromatic plant; Salvia. officinalis purchased from herbal market were qualitatively analyzed using Gas chromatographic mass spectra. Various organic crude extracts exhibited that there were different types of high and low molecular weight bioactive compounds. Most of them are basically biologically important. Thus the identification of a good number of compounds in various crude extracts of seaweeds and herbal plants might have potent ecological role.

Unraveling the Mechanism of Action of Ubiquitin-Specific Protease 5 and Its Inhibitors in Tumors.

Ubiquitin-specific protease 5 (USP5), a member of the ubiquitin-specific proteases (USPs) family, functions by specifically removing ubiquitin chains from target proteins for stabilization and degrading unbound polyubiquitin chains to maintain a steady-state monoubiquitin pool. Ubiquitin-specific protease 5 regulates various cellular activities, including DNA double-strand break repair, transmission of neuropathic and inflammatory pain signals, immune response, and tumor cell proliferation. Furthermore, USP5 is involved in the development of multiple tumors such as liver, lung, pancreatic, and breast cancers as well as melanoma. Downstream regulatory mechanisms associated with USP5 are complex and diverse. Ubiquitin-specific protease 5 has been revealed as an emerging target for tumor treatment. This study has introduced some molecules upstream to control the expression of USP5 at the levels of transcription, translation, and post-translation. Furthermore, the study incorporated inhibitors known to be associated with USP5, including partially selective deubiquitinase (DUB) inhibitors such as WP1130, EOAI3402143, vialinin A, and chalcone derivatives. It also included the ubiquitin-activating enzyme E1 inhibitor, PYR-41. These small molecule inhibitors impact the occurrence and development of various tumors. Therefore, this article comprehensively reviews the pivotal role of USP5 in different signaling pathways during tumor progression and resumes the progress made in developing USP5 inhibitors, providing a theoretical foundation for their clinical translation.

Assessment of optimized FRET substrates as universal corona- and picornavirus main protease substrates for screening assays.

Coronaviral infections are an important cause of enteric and respiratory diseases in humans and animals that are generally associated with a high level of morbidity and mortality. Similarly, picornavirus infections can lead to various illnesses that severely impact human and animal health. Despite belonging to different virus families, viral replication in all of these pathogens relies on the action of a central cysteine protease called 3C/3CL or main protease (Mpro). Due to the high functional and structural conservation of this enzyme among viral species and robustness against mutation it is considered a good target for antiviral inhibitor development. The evaluation of inhibitor potency, expressed as IC50, in many studies is achieved by measuring the inhibition of cleavage of a fluorogenic substrate in a Fluorescence Resonance Energy Transfer (FRET)-type assay. The FRET substrate is engineered after common recognition sequences of each viral Mpro, resulting in different sequences and limited comparability of IC50 between species. Our aim was to overcome this inconsistency by identifying common recognition motives of coronavirus and picornavirus Mpros to develop a unique FRET substrate that can be used universally for FRET assay tests of these enzymes. We synthesized a variety of FRET substrates with common recognition sequences and compared their cleavage kinetics towards main proteases from different species to determine the optimal sequence for universal application in FRET assays.

Advances in DNA walking nanomachine‐based biosensors

AbstractThe field of DNA nanotechnology has evolved beyond the realm of controllable movements and randomly shaped nanostructures, now encompassing a diverse array of nanomachines, each with unique nanostructures and biofunctional attributes. These DNA nanostructures boast exceptional characteristics such as programmability, integrability, biocompatibility, and universality. Among this variety, DNA walking nanomachines have emerged as one of the most prominent nanomotors, distinguished by their ingenious design and comprehensive functionality. In recent times, these DNA walkers have witnessed remarkable advancements in areas ranging from nanostructural designs to biological applications, including the creation of sophisticated biosensors capable of efficiently detecting tumor‐related biomarkers and bioactive substances. This review delves into the operational mechanisms of DNA walking nanomachines, which are driven by processes such as protease and DNAzyme action as well as strand displacement and photoactivated reactions. It further provides a comprehensive overview of DNA walking nanomachines with different dimensional (1D, 2D, and 3D) walking tracks. A subsequent section introduces the biosensing applications of DNA walking nanomachines including electrochemical, optical, and other biosensors. The review concludes with a forward‐looking perspective on the novel advancements and challenges in developing DNA walking nanomachine‐based biosensors.

Roles of Dietary Supplementation of Exogenous Protease in Low Fishmeal Aquafeed − A Mini Review

Abstract The replacement of fishmeal (FM) with economical and nutritious FM alternatives including plant- and animal-based protein ingredients has become a global research priority. However, the presence of several anti-nutritional factors (ANFs) in these alternatives may impair nutrient utilization in fish. The supplementation of exogenous protease as feed additives could be an effective approach to improve the nutrient digestibility of these alternative proteins. Proteases are protein-digesting enzymes that catalyze the hydrolysis of complex protein macromolecules into simpler amino acids. Exogenous protease supplementation stimulates the activities of endogenous proteolytic enzymes for better nutrient digestion and absorption in fish. This review article summarizes the optimum protease supplementation levels in various fish species and its beneficial effects on growth performance, nutrient digestibility, intestinal morphology, digestive enzyme activities, whole-body composition, physiochemical parameters, immunity, and gene expression of fish. Moreover, the interactive effects of exogenous protease with organic acids and probiotics are also discussed. Overall, the supplementation of exogenous protease in fish feed is a viable solution for poor nutrient utilization of FM alternatives. Important gaps, however, exist in the current knowledge, particularly with regard to the effect of protease supplementation in animal-based proteins as most of the studies have focused on the effect of protease supplementation on plant-based proteins as FM alternatives. Moreover, additional studies focused on the effect of protease supplementation on the digestive enzyme activities, immunity, gut microbes and antioxidant parameters of fish will provide the basis for a better understanding of the mode of action of protease. The scarce knowledge in these areas limits the scope of protease supplementation as a feed additive in the aquaculture sector.

Post-translational secretion stress regulation in Bacillus subtilis is controlled by intra- and extracellular proteases

The Gram-positive bacterium Bacillus subtilis is a prolific producer of industrial enzymes that are effectively harvested from the fermentation broth. However, the high capacity of B. subtilis for protein secretion has so far not been exploited to the full due to particular bottlenecks, including product degradation by extracellular proteases and counterproductive secretion stress responses. To unlock the Bacillus secretion pathway for difficult-to-produce proteins, various cellular interventions have been explored, including genome engineering. Our previous research has shown a superior performance of genome-reduced B. subtilis strains in the production of staphylococcal antigens compared to the parental strain 168. This was attributed, at least in part, to redirected secretion stress responses, including the presentation of elevated levels of the quality control proteases HtrA and HtrB that also catalyse protein folding. Here we show that this relates to the elimination of two homologous serine proteases, namely the cytosolic protease AprX and the extracellular protease AprE. This unprecedented posttranslational regulation of secretion stress effectors, like HtrA and HtrB, by the concerted action of cytosolic and extracellular proteases has important implications for the biotechnological application of microbial cell factories. In B. subtilis, this conclusion is underscored by extracellular degradation of the staphylococcal antigen IsaA by both AprX and AprE. Extracellular activity of the cytosolic protease AprX is remarkable since it shows that not only extracellular, but also intracellular proteases impact extracellular product levels. We therefore conclude that intracellular proteases represent new targets for improved recombinant protein production in microbial cell factories like B. subtilis.

APPLICATION OF BACTERIAL SOURDOUGH AND ENZYMES IN THE PRODUCTION OF BREAD FROM A MIXTURE OF RYE AND WHEAT FLOUR

Interest in sourdough rye-wheat bread with addition of enzymes is actively demonstrated by various manufacturers. Enzymes associated with the metabolic activity of sourdough microorganisms and exogenous enzymes deliberately added to the dough recipe are used in the production of these bakery products. Such combinations contribute to the improvement of both dough structure and the final product. The research investigated the influence of the combined application of the IPROVIT sourdough and enzymes α-amylase, glucoamylase and protease on the structural-mechanical properties of the dough and the quality of the resulting rye-wheat bread. Optimal concentrations of individual enzymes and their combinations were established: α-amylase – 0.35 mL, protease - 0.05 mL, glucoamylase - 3 mL, α-amylase+glucoamylase and α-amylase+protease in a 1:1 ratio. Adding enzymes at rational concentrations positively affected on the physical properties of the dough, making it elastic, shaping well into a ball and preventing spreading. Increased enzyme concentrations led to stickiness and eventual spreading of the dough. It was observed that the combined application of Lactobacillus plantarum, L. brevis, Lactobacillus paracasei ssp. paracasei bacteria and enzymatic preparations (α-amylase, protease, glucoamylase) of different actions positively affected the physical properties of the dough. The dough mixed well, did not stick to hands, maintained its shape, faster increased of volume and had moisture levels within the control range of 41–43%. The acidity of the resulting bread met the requirements of DSTU 4583:2005 and amounted to 8.9–10 degrees for all tested samples. Reduced acidity in the sample with the enzyme complex α-amylase+protease is explained by the fact that the amino acids formed as a result of the protease action were used by lactic acid bacteria as a source of nutrients. The combined application of lactic acid bacteria and enzymes positively contributed to intensifying fermentation processes, indicated by higher dough rising indicators, which were 3–9 min higher than the control. Through the use of enzymes with different actions, it was proven that the organoleptic characteristics of the bread improved, acquiring an attractive color, distinct taste and pleasant specific aroma with hints of nutmeg. The porosity of the experimental bread samples with the enzyme mixture was at the control level. Thus, the use of rye-wheat flour, sourdough and enzymes has a positive effect on the quality of bread and contributes to increasing its nutritional and biological value.