Changes In Protease Activity Research Articles

Changes in Protease Activity of Ginger Rhizome during Postharvest Storage

It has been reported that ripe ginger rhizomes contain proteolytic enzymes. In this study, we investigated the fluctuations in protease activity from ginger rhizomes stored after harvest for the utilization of proteases. The juice from ginger rhizomes that had been stored for 4 months after harvest exhibited the highest specific protease activity. Additionally, juices with high specific protease activity could form soymilk and cow milk gels. Electrophoresis experiments revealed that the protein bands in ginger rhizome juice were degraded by the proteases contained in the juice. This degradation was prevented by adding the cysteine protease inhibitor E-64.

A novel approach to produce soy protein isolate: Direct enzymatic hydrolysis of high-temperature soybean meal using proteinase produced by microbial solid-state fermentation

This study proposed a novel extraction method for soy protein isolate, which involved solid-state fermentation of high-temperature soybean meal. The proteinases secreted by microorganisms acted on the high-temperature soybean meal, making the SPI easier to extract. The study concludes that Bacillus amyloliquefaciens subsp. plantarum CICC 10265 could be used for solid-state fermentation of soybean meal, and the fermentation effect was good, with a yield of 41.91 % for SPI. Compared to the direct extraction of SPI from high-temperature soybean meal, the yield had increased by 130.19 %. Meanwhile, we also conducted research on the losses during the SPI extraction process. Through experiments, the study identified the patterns of protease activity changes and microbial colony growth during solid-state fermentation of soybean meal by Bacillus amyloliquefaciens subsp. plantarum CICC 10265. It was concluded that extracting SPI after 8 h of fermentation is more suitable. The experimental results indicated that the total amino acid content of SPI extracted from fermented soybean meal was 2.1 % higher compared to SPI extracted from low-temperature soybean meal. The extracted SPI also met the microbial standards.

Adaptability to the environment of protease by secondary structure changes and application to enzyme-selective hydrolysis

The reactions involving enzymes are significantly influenced by various environmental factors. Clarity of how the activity and structure of proteases impact their function is crucial for more efficient application of enzymes as a tool. The impact of temperature, pH, and ionic strength on changes in protease activity, secondary structure, and protein conformation during enzymatic hydrolysis were investigated in this study. The enzymatic activity and secondary structure of acid-base protease were found to undergo significant modifications under different physical conditions, as demonstrated by UV spectrophotometry and FTIR spectroscopy analysis. Specifically, variations in α-helix and β-fold content were observed to correlate with changes in enzyme activity. Molecular simulation analysis revealed that physical conditions have varying effects on the protease, particularly influencing enzyme activity and secondary structure. Evaluation of the proteases indicated alterations in both enzyme activity and structure. This treatment selectively hydrolyzed β-lactoglobulin and reduced sensitization. These findings offer novel perspectives on the functionalities and regulatory mechanisms of proteases, as well as potential industrial applications.

Population-specific responses to pollution exposure suggest local adaptation of invasive red swamp crayfish Procambarus clarkii along the Mediterranean French coastline

Anthropogenic stressors can have an impact in a broad range of physiological processes and can be a major selective force leading to rapid evolution and local population adaptation. In this study, three populations of the invasive crayfish Procambarus clarkii were investigated. They are geographically separated for at least 20 years, and live in different abiotic environments: a freshwater inland lake (Salagou lake) with no major anthropogenic influence and two other coastal wetlands regularly polluted by pesticides along the Mediterranean coast (Camargue region and Bages-Sigean lagoon). Collected adults were genetically characterized using the mitochondrial COI gene and haplotype frequencies were analyzed for genetic variability within and between populations. Results revealed a higher genetic diversity for these invasive populations than any previous report in France, with more than seven different haplotypes in a single population. The contrasting genetic diversity between the Camargue and the other two populations suggest different times and sources of introduction. To identify differences in key physiological responses between these populations, individuals from each population were maintained in controlled conditions. Data on oxygen consumption rates indicate that the Salagou and Bages-Sigean populations possess a high inter-individual variability compared to the Camargue population. The low individual variability of oxygen consumption and low genetic diversity suggest a specific local adaptation for the Camargue population. Population-specific responses were identified when individuals were exposed to a pesticide cocktail containing azoxystrobin and oxadiazon at sublethal concentrations. The Salagou population was the only one with altered hydro-osmotic balance due to pollutant exposure and a change in protease activity in the hepatopancreas. These results revealed different phenotypic responses suggesting local adaptations at the population level.

An insight into the quality formation of egg-white sufu based on the dynamic changes of protease activity, physicochemical properties and texture

Sufu is a delicious traditional Chinese food, mainly produced by fermentation using soybeans as raw material. Egg white is rich in protein and is theoretically an excellent material for sufu processing, however there are few reports on egg-white sufu (EWS) currently, and no such product is available on the market. In this paper, a novel sufu was processed with egg white as main material, and the formation mechanism of its unique quality was explored by monitoring the dynamic changes in the pre-fermentation and post-fermentation stages. The dynamic change curve of protease activity showed an “S” shape, with a peak of 32.33 U/mL on the 4th day of pre-fermentation stage. After a 4-day pre-fermentation and 90-day post-fermentation, most of the proteins in the EWS had been hydrolyzed, and the sufu was moderately soft and delicate in texture, with a hardness of 950.3 g and a total free amino acid content of 19.863 mg/g. SEM and SDS-PAGE detections suggested that the proteins in EWS had been extensively degraded, which led to the formation of its unique texture and flavor. The EWS was slightly better than soybean sufu in terms of texture, fragrance and mouthfeel, with flavor compounds mainly composed of aldehydes and alcohols, resulting in a slightly higher total sensory evaluation score.

Age-related changes in protease activity as cold stress response by Penicillium strains from different temperature classes

Age-related changes in protease activity as cold stress response by Penicillium strains from different temperature classes

Effect of Bacillus subtilis (Bacillus subtilis subsp.) inoculation on the fermentation characteristics of Penaeus sinensis by-products: Protease activity and volatile property

Protease activity and volatile flavor compounds will determine the overall unique sensory of fermented products. In this study, the changes of protease activity of Penaeus sinensis by-products fermentation inoculated with Bacillus subtilis subsp. (PSB-Bs) for 7 days were evaluated. In addition, the flavor characteristics of PSB-Bs were determined by the combination of electronic nose (E-nose) and gas chromatography-ion mobility spectroscopy (GC-IMS). The results showed that the alkaline protease activity (pH 9) increased significantly in late fermentation stage (5–7 days), and the specific trypsin activity was also significantly higher than that of pepsin. E-nose and GC-IMS distinguished the variations of volatile compounds in PSB-Bs, and 67 volatiles were identified. Through orthogonal partial least squares discriminant analysis and random forest multivariate statistical analysis, 15 flavoring volatiles screened were further determined the correlation with protease activity. Negative relationships were found between the activity of protease (pH 7) or pepsin and pleasant flavors like 2,5-dimethyl furan, hexyl acetate and styrene. Our results suggest that during fermentation, controlling the activities of pH7.0 protease and pepsin might help to improve the production or enrichment of pleasant flavor substances in PSB-Bs, which will help to enhance the acceptability and high-value utilization of shrimp by-products fermentation.

The Potyviral Protein 6K1 Reduces Plant Proteases Activity during Turnip mosaic virus Infection.

Potyviral genomes encode just 11 major proteins and multifunctionality is associated with most of these proteins at different stages of the virus infection cycle. Some potyviral proteins modulate phytohormones and protein degradation pathways and have either pro- or anti-viral/insect vector functions. Our previous work demonstrated that the potyviral protein 6K1 has an antagonistic effect on vectors when expressed transiently in host plants, suggesting plant defenses are regulated. However, to our knowledge the mechanisms of how 6K1 alters plant defenses and how 6K1 functions are regulated are still limited. Here we show that the 6K1 from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and cysteine protease inhibitors when expressed in Nicotiana benthamiana relative to controls. 6K1 stability increased when cysteine protease activity was inhibited chemically, showing a mechanism to the rapid turnover of 6K1 when expressed in trans. Using RNAseq, qRT-PCR, and enzymatic assays, we demonstrate TuMV reprograms plant protein degradation pathways on the transcriptional level and increases 6K1 stability at later stages in the infection process. Moreover, we show 6K1 decreases plant protease activity in infected plants and increases TuMV accumulation in systemic leaves compared to controls. These results suggest 6K1 has a pro-viral function in addition to the anti-insect vector function we observed previously. Although the host targets of 6K1 and the impacts of 6K1-induced changes in protease activity on insect vectors are still unknown, this study enhances our understanding of the complex interactions occurring between plants, potyviruses, and vectors.

Changes in protease activity during fermentation of fish sauce and their correlation with antioxidant activity.

Antioxidant activity has been found in fermented fish sauce. In this experiment, the properties of endogenous protease and antioxidant activity were studied in anchovy sauce during fermentation. The correlation between protease activity and antioxidant activity in fermented anchovy sauce was analyzed using the partial least squares (PLS) method. The results showed that at least four proteases were present in the endogenous enzyme solution, and the optimum pH values were 2.5, 5.5, 9.0, and 12.5, respectively. The maximum inhibition rate of endogenous protease, from high to low, was: serine protease inhibitor > trypsin inhibitor > aspartic protease inhibitor (pepsin inhibitor) > cysteine protease inhibitor > metalloprotease inhibitor. At the sixth month of fermentation, fish sauce had stronger trypsin, pepsin-like activity, and antioxidant activity. At the ninth month of fermentation, the cathepsin activity was greater. A model correlating changes in protease activity with antioxidant activity suggested that the trypsin and serine protease were the main factors affecting antioxidant activity. This study reports a model correlating changes in protease activity with the antioxidant activity of fish sauce. It lays a foundation for further exploration of the formation of antioxidant substances and antioxidant effects during the process of fish sauce fermentation. © 2021 Society of Chemical Industry.

Changes in taste substances during fermentation of fish sauce and the correlation with protease activity

Anchovy sauce shows different taste profiles under different fermentation time. The change rules of free amino acids was measured by amino acid analyzer, and other taste substances, such as nucleotides and organic acids in anchovy sauce under different fermentation time were also investigated. Moreover, the correlation between protease activity and taste substances in anchovy sauce fermentation was analyzed by orthogonal partial least squares. Throughout the fermentation process, the taste substances in anchovy sauce increased during early months and then decreased as time increased. The content of amino acid nitrogen, TCA-soluble peptides, 5′-nucleotides (AMP, GMP, IMP) and organic acids (lactic acid, succinic acid) in anchovy sauce increased by 26%, 33%, (45%, 35%, 68%) and (27%, 2%) respectively in comparison with 6 months fermentation. Total amino acid content reached its maximum after 18 months fermentation. Results of electronic tongue demonstrated that the umami of anchovy sauce after 12 months fermentation increased by 17% in comparison with 6 months fermentation. A model correlating changes in protease activity with taste formation suggested that protease activity impacted the content of Ala, Glu, Lys, Asp, Leu, TCA-soluble peptides and succinic acid. This study can provide empirical evidence to guide the efficient processing of anchovy sauce.

Correction to "Identification of Different Bile Species and Fermentation Times of Bile Arisaema Based on an Intelligent Electronic Nose and Least Squares Support Vector Machine".

Fermentation is one of the most traditionally utilized methods to process the raw materials of traditional Chinese medicine (TCM). Bile Arisaema (BA) is produced by the fermentation of the roots of Arisaema heterophyllum with bile. Fermentation time and bile species are the key factors in producing BA. The study was aimed to develop a new and rapid method for the identification of different fermentation times and bile species of BA. The polysaccharide content (PC), protease activity (PA), and amylase activity (AC) of BA were determined. The changes of PC, PA, and AC were significant indicators for the evaluation of different fermentation times. On the basis of the odor data of BA obtained by electronic nose technology (E-nose), the principal component analysis (PCA) was used to identify bile species. The results were further verified by the least squares support vector machine (LS-SVM). The trained LS-SVM was also used to predict the PC, PA, and AC of the samples to identify fermentation time. The present...

Identification of Different Bile Species and Fermentation Times of Bile Arisaema Based on an Intelligent Electronic Nose and Least Squares Support Vector Machine.

Fermentation is one of the most traditionally utilized methods to process the raw materials of traditional Chinese medicine (TCM). Bile Arisaema (BA) is produced by the fermentation of the roots of Arisaema heterophyllum with bile. Fermentation time and bile species are the key factors in producing BA. The study was aimed to develop a new and rapid method for the identification of different fermentation times and bile species of BA. The polysaccharide content (PC), protease activity (PA), and amylase activity (AC) of BA were determined. The changes of PC, PA, and AC were significant indicators for the evaluation of different fermentation times. On the basis of the odor data of BA obtained by electronic nose technology (E-nose), the principal component analysis (PCA) was used to identify bile species. The results were further verified by the least squares support vector machine (LS-SVM). The trained LS-SVM was also used to predict the PC, PA, and AC of the samples to identify fermentation time. The present study indicated that E-nose combined with LS-SVM could effectively predict the PC, PA, and AC of the samples and identify the bile species and fermentation time of BA, and it was proved to be a useful strategy for quality control of fermented products of TCMs.

Allosteric Modulation of a Serine Protease by Conformationally Selective Nanobodies

Trypsin-like serine proteases regulate many important physiological processes including digestion, blood coagulation, tissue remodelling, complement activation and fibrinolysis. It is well known that ligand binding to allosteric sites modulates the function of the active site region through conformational and/or dynamic changes leading to a change in protease activity and altered substrate specificity. Furthermore, increasing biophysical evidence supports the existence of multiple active and inactive conformational states. However, molecular details about sparsely populated protease conformations, and how ligands modulate the activity of a given protease remains largely elusive. Here we report the X-ray crystal structure of an unappreciated inactive conformational state of the serine protease urokinase-type plasminogen activator (uPA). Next, two distinct uPA conformations were stabilized by different single domain camelid antibodies – an active conformation captured by an active site binding (orthosteric) nanobody (Nb22) and an inactive conformation captured by an allosteric nanobody (Nb7). By combining X-ray crystallography and hydrogen deuterium-exchange mass spectrometry, we identify the mechanism of allostery, which explains at the molecular level how ligand binding regulates uPA activity by changing the dynamics of several surface-exposed loops surrounding the active site region. Collectively, our data supports a model which assumes equilibrium amongst at least three conformational states and shows that ligands modulate the biological activity of the protease by differentially stabilizing a specific protease conformation.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

The role of urinary peptidomics in kidney disease research

Urinary peptidomics focuses on endogenous urinary peptide content. Many studies now show the usefulness of this approach for the discovery and validation of biomarkers in kidney diseases that are as varied as chronic kidney disease, acute kidney injury, congenital anomalies of the kidney and the urinary tract, and polycystic kidney disease. Most studies focus on chronic kidney disease and demonstrate that urinary peptidome analysis can substantially contribute to early detection and stratification of patients with chronic kidney disease. A number of multicenter studies are ongoing that aim further validation in a clinical setting and broaden the applicability of urinary peptides. The association of urinary peptides with kidney disease also starts to deliver information on the pathophysiology of kidney disease with emphasis on extracellular matrix remodeling. Bioinformatic peptide centric tools have been developed that allow to model the changes in protease activity involved in kidney disease, based on the urinary peptidome content. A novel application of urinary peptidome analysis is the back-translation of results obtained in humans to animals for animal model validation and improvement of readout in these preclinical models. In conclusion, urinary peptidomics not only contribute to detection and stratification of kidney disease in the clinic, but might also create a new impulse in drug discovery through better insight in the pathophysiology of disease and optimized translatability of animal models.

Nanoparticle-Based and Bioengineered Probes and Sensors to Detect Physiological and Pathological Biomarkers in Neural Cells

Nanotechnology, a rapidly evolving field, provides simple and practical tools to investigate the nervous system in health and disease. Among these tools are nanoparticle-based probes and sensors that detect biochemical and physiological properties of neurons and glia, and generate signals proportionate to physical, chemical, and/or electrical changes in these cells. In this context, quantum dots (QDs), carbon-based structures (C-dots, grapheme, and nanodiamonds) and gold nanoparticles are the most commonly used nanostructures. They can detect and measure enzymatic activities of proteases (metalloproteinases, caspases), ions, metabolites, and other biomolecules under physiological or pathological conditions in neural cells. Here, we provide some examples of nanoparticle-based and genetically engineered probes and sensors that are used to reveal changes in protease activities and calcium ion concentrations. Although significant progress in developing these tools has been made for probing neural cells, several challenges remain. We review many common hurdles in sensor development, while highlighting certain advances. In the end, we propose some future directions and ideas for developing practical tools for neural cell investigations, based on the maxim “Measure what is measurable, and make measurable what is not so” (Galileo Galilei).

Pathomimetic cancer avatars for live-cell imaging of protease activity.

Proteases are essential for normal physiology as well as multiple diseases, e.g., playing a causative role in cancer progression, including in tumor angiogenesis, invasion, and metastasis. Identification of dynamic alterations in protease activity may allow us to detect early stage cancers and to assess the efficacy of anti-cancer therapies. Despite the clinical importance of proteases in cancer progression, their functional roles individually and within the context of complex protease networks have not yet been well defined. These gaps in our understanding might be addressed with: 1) accurate and sensitive tools and methods to directly identify changes in protease activities in live cells, and 2) pathomimetic avatars for cancer that recapitulate invitro the tumor in the context of its cellular and non-cellular microenvironment. Such avatars should be designed to facilitate mechanistic studies that can be translated to animal models and ultimately the clinic. Here, we will describe basic principles and recent applications of live-cell imaging for identification of active proteases. The avatars optimized by our laboratory are three-dimensional (3D) human breast cancer models in a matrix of reconstituted basement membrane (rBM). They are designated mammary architecture and microenvironment engineering (MAME) models as they have been designed to mimic the structural and functional interactions among cell types in the normal and cancerous human breast. We have demonstrated the usefulness of these pathomimetic avatars for following dynamic and temporal changes in cell:cell interactions and quantifying changes in protease activity associated with these interactions in real-time (4D). We also briefly describe adaptation of the avatars to custom-designed and fabricated tissue architecture and microenvironment engineering (TAME) chambers that enhance our ability to analyze concomitant changes in the malignant phenotype and the associated tumor microenvironment.

Calpains and their endo- and exogenous regulators in various models of neurodegeneration

On the basis of experimental series with murine models there was obtained the evidence on calcium-dependent protease activity changes in rat brain at induced neurodegeneration. The properties of the proteolytic and regulatory components of calpain system under the effect of neurotoxic stimuli--amyloid beta-peptide or glutamate--were characterized; the basic endogenous regulatory mechanisms of calcium-dependent proteolysis modulation were determined as well. Neuroprotective properties of exogenous calpain regulators differing in the mechanisms of action (sex steroids, calcium regulators) were tested on studied neurodegeneration models.

Short chain fatty acids accumulation and microbial community succession during ultrasonic-pretreated sludge anaerobic fermentation process: Effect of alkaline adjustment

Short chain fatty acids (SCFAs) are value-added products from waste activated sludge (WAS) anaerobic fermentation. The effects of alkaline adjustment, in the range of pH 7–10, on SCFAs accumulation and microbial community of ultrasonic-pretreated WAS at 20 ± 2 °C were investigated in this study. Experimental results indicated that optimum conditions for SCFAs accumulation were pH 9 and fermentation time of 7 days. The changes of protease activity showed that on the initial phase, ultrasonic pretreatment and alkaline adjustment rather than biotic effect was the leading cause of SCFAs improvement at high pHs. DGGE analysis revealed that alkaline adjustment had a strong impact on microbial community. Some bacteria belonged to Firmicutes, Proteobacteria and Bacteroidetes could adapt to high alkaline environments and contribute to proteins and carbohydrates degradation and SCFAs production in the later phase of anaerobic fermentation.

Interplay between proteases and protease inhibitors in the sea fan—Aspergillus pathosystem

Environmental conditions greatly impact the dynamics of host–pathogen relationships, affecting outbreaks and emergence of new diseases. In corals, epizootics have been a main contributor to the decline of reef ecosystems and rising sea surface temperatures associated with climate change are thought to exacerbate virulence of pathogens and/or compromise the immune responses of coral hosts. Among pathogens strategies, protease secretion is a common virulence factor that promotes colonization of the host. As such, protease inhibitors play an invaluable role to the host for protection against pathogen invasion. This study describes changes in activity of proteases secreted by the fungal pathogen, Aspergillus sydowii, and protease inhibitors of Gorgonia ventalina under ambient and elevated temperatures and health conditions (disease and healthy). G. ventalina colonies were collected from Florida Keys, Florida (24° 34.138N, 81° 22.905W) or La Parguera, Puerto Rico (17° 56.091N, 67° 02.577W) in 2007 and 2012, respectively. At elevated temperatures, protease activity was significantly higher than at ambient temperatures in A. sydowii. Temperature stress did not induce a change in protease inhibitor activity, but in healthy G. ventalina colonies, inhibitor activity against proteases was higher than in diseased individuals. Healthy colonies appear capable of resisting proteolytic activity, but diseased individuals are still likely affected by pathogen infections. These data suggest that rising sea surface temperatures due to climate change may increase levels of virulence (protease activity) of A. sydowii, while immunity (protease inhibitor) of G. ventalina may not be affected.