Levels Of Proteolytic Activity Research Articles

Скринінг продуцентів протеаз серед представників роду Bacillus, ізольованих з прибережної зони Кінбурнської коси

Protease-producing representatives of Bacillus have been isolated from various environments such as the rhizosphere, soil, sewage, food, waste, and saline environments (sea water, marine sediments, hypersaline lakes, salty food, soda lakes, etc.). However, today, little is known about the characteristics of proteases in representatives of the genus Bacillus, isolated from the coastal and aquatic environments. In this regard, the purpose of this work was to screen for the presence of different types of proteolytic activity in Bacillus cultures isolated from the coastal zone of the Kinburn split. Methods. The objects of the study were 15 cultures (L1-L15), isolated from the dry grass of the coastal zone of the Kinburn spit (Mykolaiv region). The cultures were grown under conditions of deep cultivation at 28 °С, with a mixing speed of the nutrient medium of 230 rpm for 2 days. Methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic, and collagenase) activity in the culture liquid supernatant were used. Results. Study of the spectrum of proteolytic activities of 15 strains of Bacillus sp. showed that L9, L1, L2 and L4 exhibited higher levels of proteolytic activity compared to the other strains tested. Bacillus sp. L9 and L2 showed the highest elastase activity (35.80 and 33.80 U/mL, respectively), L1, L2, and L4 − fibrinogenolytic (12.50, 11.20 and 10.33 U/mL, respectively), and L1 and L4 − fibrinolytic (13.00 and 10.90 U/mL, respectively) activity. Conclusions. According to its catalytic properties, a number of representatives of Bacillus sp. isolated from the dry grass of the coastal zone of the Kinburn split may be promising for further research as producers of enzymes with elastotic, fibrinolytic, and fibrinogenolytic activities.

Molecular basis for the increased activity of ZMS-2 serine protease in the presence of metal ions and hydrogen peroxide

Serine proteases are important enzymes widely used in commercial products and industry. Recently, we identified a new serine protease from the desert bacterium Bacillus subtilis ZMS-2 that showed enhanced activity in the presence of Zn2+, Ag+, or H2O2. However, the molecular basis underlying this interesting property is unknown. Here, we report comparative studies between the ZMS-2 protease and its homolog, subtilisin E (SubE), from B. subtilis ATCC 6051. In the absence of Zn2+, Ag+, or H2O2, both enzymes showed the same level of proteolytic activity, but in the presence of Zn2+, Ag+, or H2O2, ZMS-2 displayed increased activity by 22%, 8%, and 14%, whereas SubE showed decreased activity by 16%, 12%, and 9%, respectively. In silico studies showed that both proteins have almost identical amino acid sequences and folding structures, except for two amino acids located in the protruding loops of the proteins. ZMS-2 contains Ser236 and Ser268, whereas SubE contains Thr236 and Thr268. Replacing Ser236 or Ser268 in ZMS-2 with threonine resulted in variants whose activities were not enhanced by Zn2+ or Ag+. However, this single mutation did not affect the enhancement by H2O2. This finding may be used as a basis for engineering better proteases for industrial uses.

KERATINOLYTIC POTENTIAL OF MICROMYCETE ASPERGILLUS CLAVATUS VKPM F-1593 AND COMPARISON OF ITS ENZYMES WITH THE COMMERCIAL KERATINASE PREPARATION

Animal husbandry is a rapidly developing branch of agriculture, increasing the volume of production annually, and keratin-containing waste (feathers, bristles) make up the majority of all waste from this industry. The development of methods of environmentally friendly processing of such waste to obtain valuable resources (amino acids and oligopeptides) is an important mission of modern science, including biotechnology. One of the ways to dispose of animal husbandry waste that meets modern trends in the development of a green economy is the use of microorganisms and their enzymes. The possibility of obtaining keratinolytic enzymes of Aspergillus clavatus VKPM F-1593 under deep conditions with various sources of nitrogen and carbon, including with the growth of the producer on animal waste, was studied. The highest target activity (96.1 E) was achieved using mixed carbon and nitrogen sources: inorganic - sodium nitrate, easily digestible organic - fi sh meal hydrolysate and hard-toreach organic - ground chicken feather. Varying the content of various substrates in the composition of fermentation media allowed not only to regulate the level of proteolytic activity, but also to reach the peak of producer activity on diff erent days of cultivation. The specifi c keratinase activity of A. clavatus VKPM F-1593 (pI 9.3) to various protein substrates with the activity of a commercial proteinase K. Both enzymes showed a similar level of activity regarding most of the substrates used. However, protease A. clavatus VKPM F-1593 has a greater overall proteolytic activity, which confi rms the prospects of this culture for biodegradation of animal waste

Metabolite profiling of fermented milks as affected by adjunct cultures during long-term storage

The effects of starter strains (Lactiplantibacillus plantarum 156, Lactobacillus casei ATCC 334, Lactobacillus helveticus DPC 4571, and Streptococcus thermophilus 20S4) on the physicochemical and microbiological properties, volatiles, bioactive peptides, and gamma-aminobutyric acid (GABA) contents of fermented milks were evaluated during storage. The use of different adjunct culture combinations had no significant effect on the gross chemical composition. The bacterial strains remained highly viable in the fermented milk during storage. The peptide extracts exhibited a high ACE-inhibitory activity, which was particularly increased in the samples where L. casei ATCC 334 and L. helveticus DPC 4571 strains co-existed. Almost the same levels of antioxidant activity was observed in the peptides extracted from fermented milk. Peptide extracts showed a certain level of antimicrobial activitiy against Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 7644, Salmonella Typhimurium RSHMB 95091, and Escherichia coli O157:H7; however, no inhibitory activity was observed against Bacillus cereus BC 6230. Among the free amino acids, His, Arg, Ala, and Pro were predominant in the samples. Significant differences were observed between samples regarding GABA content, which increased with increasing levels of proteolytic activity. A total of 33 volatile compounds were identified in fermented milks, with acetone, 2,3-butanedione, acetoin, 2-heptanone, acetic and hexanoic acids being the predominant volatiles. These results demonstrate the multifunctional role of peptides and GABA extracted from fermented milk through the action of specific Lactobacillus strains. Therefore, such a functional fermented milk product can be used to reduce the risk of disease or improve specific physiological functions.

Identification, proteolytic activity quantification and biofilm-forming characterization of Gram-positive, proteolytic, psychrotrophic bacteria isolated from cold raw milk.

Psychrotrophic bacteria of raw milk face the dairy industry with significant spoilage and technological problems due to their ability to produce heat-resistant enzymes and biofilms. Despite extensive information about Gram-negative psychrotrophic bacteria in milk, little is known about Gram-positive psychrotrophic bacteria in milk, and their proteolytic activity and biofilm-forming characteristics. In the present study, Gram-positive, proteolytic, psychrotrophic bacteria of cold raw milk were identified, and their proteolytic activity and biofilm-forming capacity were quantified. In total, 12 genera and 22 species were represented among the bacterial isolates, however 50% belonged to three genera, namely Staphylococcus (19.4%), Bacillus (16.7%), and Enterococcus (13.9%). Different levels of proteolytic activity were detected in the identified isolates, even among the strains belonging to the same species. In addition, proteolytic activity was significantly higher at 25°C than at 7°C for all isolates. The crystal violet staining assay in polystyrene microtitre plates revealed a high level of variation in the biofilm-forming capacity at 7°C. After 72 hours of incubation, 11.1% of the strains did not produce a biofilm, while 27.8%, 52.8%, and 8.3% produced low, moderate, and high amounts of biofilm on polystyrene, respectively. The psychrotrophic bacteria were also able to produce biofilms on the surface of stainless steel coupons in ultra-high temperature milk after 72 h of incubation at 7°C; the number of attached cells ranged from 1.34 to 5.11 log cfu/cm2. These results expand the knowledge related to the proteolytic activity and biofilm-forming capacity of Gram-positive psychrotrophic milk bacteria.

Pseudomonas isolates from raw milk with high level proteolytic activity display reduced carbon substrate utilization and higher levels of antibiotic resistance

Milk is an ideal food but can be easily contaminated during milking, transportation and storage by Pseudomonas spp. That secrete proteases resulting in milk spoilage. However, genomic features and functional metabolic diversity of Pseudomonas with different protease activity have not been extensively studied. With this aim, the genomic features and functional metabolic diversity of Pseudomonas with differing proteolytic activity were evaluated using whole genome sequencing and metabolomic approaches, respectively as well as, the presence of genes encoding the aprX protease and antibiotic resistance were screened. Pseudomonas strains with higher protease activity possessed more carbohydrate transport and metabolism, signal transduction, secondary metabolite synthesis, polysaccharide lyase and carbohydrate esterase genes. These isolates were more efficient at carbon source utilization and this phenotype was positively related to incubation temperature. The high protease activity group also displayed a multidrug resistance index that was significantly higher than that for the low protease activity group. This is the first report comparing the gene function and metabolic diversity of Pseudomonas with differing levels of proteolytic activity in raw milk.

The Activity of Metalloproteases and Serine Proteases in Various Organs after Leiurus macroctenus Envenomation.

Scorpion stings may be life-threatening since their venoms are comprised of a wide range of toxins and other bioactive molecules, such as enzymes. At the same time, scorpion envenomation may increase matrix metalloproteases (MMPs) levels, which enhance proteolytic tissue destruction by venom. However, investigations on the impact of many scorpions' venoms, such as those of Leiurus macroctenus, on tissue proteolytic activity and MMP levels have not yet been conducted. The present study aimed to examine the total proteolysis levels in various organs after Leiurus macroctenus envenomation and evaluate the metalloproteases and serine proteases' contributions to the total proteolytic activity. Changes in MMPs and TIMP-1 levels were tested as well. Envenomation led to a significant increase in proteolytic activity levels in all assessed organs, mostly in the heart (by 3.34 times) and lungs (by 2.25 times). Since EDTA presence showed a noticeable decrease in total proteolytic activity level, metalloproteases appeared to play a prominent role in total proteolytic activity. At the same time, MMPs and TIMP-1 levels were increased in all assessed organs, suggesting that Leiurus macroctenus envenomation causes systemic envenomation, which may induce multiple organ abnormalities, mostly because of the uncontrolled metalloprotease activity.

Inflammatory Bowel Disease-Associated Gut Commensals Degrade Components of the Extracellular Matrix.

Extracellular matrix (ECM) remodeling has emerged as a key feature of inflammatory bowel disease (IBD), and ECM fragments have been proposed as markers of clinical disease severity. Recent studies report increased protease activity in the gut microbiota of IBD patients. Nonetheless, the relationship between gut microbiota and ECM remodeling has remained unexplored. We hypothesized that members of the human gut microbiome could degrade the host ECM and that bacteria-driven remodeling, in turn, could enhance colonic inflammation. Through a variety of in vitro assays, we first confirmed that multiple bacterial species found in the human gut are capable of degrading specific ECM components. Clinical stool samples obtained from ulcerative colitis patients also exhibited higher levels of proteolytic activity in vitro, compared to those of their healthy counterparts. Furthermore, culture supernatants from bacteria species that are capable of degrading human ECM accelerated inflammation in dextran sodium sulfate (DSS)-induced colitis. Finally, we identified several of the bacterial proteases and carbohydrate degrading enzymes (CAZymes) that are potentially responsible for ECM degradation in vitro. Some of these protease families and CAZymes were also found in increased abundance in a metagenomic cohort of IBD. These results demonstrate that some commensal bacteria in the gut are indeed capable of degrading components of human ECM in vitro and suggest that this proteolytic activity may be involved in the progression of IBD. A better understanding of the relationship between nonpathogenic gut microbes, host ECM, and inflammation could be crucial to elucidating some of the mechanisms underlying host-bacteria interactions in IBD and beyond. IMPORTANCE Healthy gut epithelial cells form a barrier that keeps bacteria and other substances from entering the blood or tissues of the body. Those cells sit on scaffolding that maintains the structure of the gut and informs our immune system about the integrity of this barrier. In patients with inflammatory bowel disease (IBD), breaks are formed in this cellular barrier, and bacteria gain access to the underlying tissue and scaffolding. In our study, we discovered that bacteria that normally reside in the gut can modify and disassemble the underlying scaffolding. Additionally, we discovered that changes to this scaffolding affect the onset of IBD in mouse models of colitis as well as the abilities of these mice to recover. We propose that this new information will reveal how breaks in the gut wall lead to IBD and will open up new avenues by which to treat patients with IBD.

Effect of the recombinant chymosins of different origins on the quality and shelf life of soft cheeses

The effect of the type (bovine chymosin (Chy-max Extra), camel chymosin (Chy-max M), and modified camel chymosin (Chy-max Supreme)) and applied dose (1500, 2500, or 3500 IMCU/100 kg of milk) of milk-clotting enzyme (MCE) on the proteolysis degree, microstructure, rheological and sensory properties of Crescenza soft cheese was studied. The proteolysis degree of cheeses was directly proportional to the general proteolytic activity (PA) and dose of the MCEs added into milk during the cheese production. With an equal dose of the MCEs added into milk, the highest level of proteolysis was noted in the cheese options produced with recombinant bovine chymosin (Chy-max Extra). There were no statistically significant differences (p > 0.05) in the proteolysis degree between the cheese options made with the same doses of the camel chymosin (Chy-max M) and modified chymosin (Chymax Supreme). After 21 days of storage, the cheeses with Chy-max Extra MCE had the most plastic consistency, while the cheeses with Chy-max Supreme MCE had the hardest and most elastic one. Cheeses produced with Chy-max M MCE occupied an intermediate position in terms of consistency density. Recombinant MCEs based on camel chymosin (Chy-max M) and modified chymosin (Chy-max Supreme) with low level of PA, may be recommended for use in the production of soft cheeses. This will extend the shelf life of cheeses by slowing-down the plasticization rate of their consistency.

Melanoma stimulates the proteolytic activity of HaCaT keratinocytes

BackgroundKeratinocytes constitute a major part of the melanoma microenvironment, considering their protective role towards melanocytes in physiological conditions. However, their interactions with tumor cells following melanomagenesis are still unclear.MethodsWe used two in vitro models (melanoma-conditioned media and indirect co-culture of keratinocytes with melanoma cells on Transwell inserts) to activate immortalized keratinocytes towards cancer-associated ones. Western Blotting and qPCR were used to evaluate keratinocyte markers and mediators of cell invasiveness on protein and mRNA expression level respectively. The levels and activity of proteases and cytokines were analysed using gelatin-FITC staining, gelatin zymography, chemiluminescent enzymatic test, as well as protein arrays. Finally, to further study the functional changes influenced by melanoma we assessed the rate of proliferation of keratinocytes and their invasive abilities by employing wound healing assay and the Transwell filter invasion method.ResultsHaCaT keratinocytes activated through incubation with melanoma-conditioned medium or indirect co-culture exhibit properties of less differentiated cells (downregulation of cytokeratin 10), which also prefer to form connections with cancer cells rather than adjacent keratinocytes (decreased level of E-cadherin). While they express only a small number of cytokines, the variety of secreted proteases is quite prominent especially considering that several of them were never reported as a part of secretome of activated keratinocytes’ (e.g., matrix metalloproteinase 3 (MMP3), ADAM metallopeptidase with thrombospondin type 1 motif 1). Activated keratinocytes also seem to exhibit a high level of proteolytic activity mediated by MMP9 and MMP14, reduced expression of TIMPs (tissue inhibitor of metalloproteinases), upregulation of ERK activity and increased levels of MMP expression regulators-RUNX2 and galectin 3. Moreover, cancer-associated keratinocytes show slightly elevated migratory and invasive abilities, however only following co-culture with melanoma cells on Transwell inserts.ConclusionsOur study offers a more in-depth view of keratinocytes residing in the melanoma niche, drawing attention to their unique secretome and mediators of invasive abilities, factors which could be used by cancer cells to support their invasion of surrounding tissues.52obFQmRyJn1nHi_mM3M7bVideo abstract

Influence of different types of fermentation-produced chymosin on quality of soft cheeses

The disadvantage of soft cheeses is their short shelf life. In soft cheeses with a high moisture content, proteolysis occurs at a high rate, as a result of which the cheeses quickly overripe. The main proteolytic agent in soft cheeses is the milk-clotting enzyme (MCE). Increasing the shelf life of cheeses can be achieved by using MCE types having low proteolytic activity (PA). We have studied the effect of MCE based on different types of fermentation-produced chymosin: Chy-max® Extra (bovine chymosin), Chy-max® M (camel chymosin), Chy-max® Supreme (“modified” chymosin) on the dynamics of proteolysis in soft cheeses and related changes in the structure of cheeses during their storage. All 3 types of studied MCEs have different levels of nonspecific PA. The higher the level of nonspecific PA of the used MCE, the higher the rate of the proteolysis process in the resulting cheeses. Increasing the dose of MCE also increases the rate of proteolysis in cheeses. To increase the shelf life of soft cheeses, which depends on the period of preservation of a dense consistency, it is promising to use MCE with low PA based on camel chymosin (Chy-max® M) and “modified” chymosin (Chy-max® Supreme).

Correlation Between Proteolytic Activity and Abdominal Aortic Aneurysm Wall Morphology with Intraluminal Thrombus Volume

The aim of this study was to examine the influence of intraluminal thrombus (ILT) volume on the level of proteolytic activity and the content of abdominal aortic aneurysm (AAA) wall. The research was designed as a cross-sectional study at the Clinic for Vascular and Endovascular Surgery, Clinical Center of Serbia in the period from April 2017 to February 2018. During this period, a total of 155 patients with asymptomatic AAA underwent open surgical treatment and 50 were included in the study based on inclusion and exclusion criteria. Before surgery, patients included in the study were examined by MRI. During the operation, samples of ILT and AAA wall were taken for biochemical analysis. A statistically significant correlation was found between the volume of the ILT and largest AAA diameter (ρ=0.56; P<0.001). The correlation of the ILT volume on the anterior wall and the concentration of MMP-9, MMP-2 and NE/ELA in the wall did not find statistical significance. Also, no statistically significant association was found between the volume of ILT and the concentration of ECM proteins (collagen type 3, elastin, proteoglycan) in the corresponding part of the wall. The association of ILT volume with MDA was also of no statistical significance. There was a positive statistical significance found in correlation of volume of ILT and catalase activity in the wall of AAA (ρ=0.28, P=0.049). The volume of ILT in the aneurysmal sac seemed not to affect the level of proteolytic activity and the content of the aneurysm wall. However, a positive correlation was found between the ILT and the catalase activity. The effect of ILT on the aneurysm wall and its role in the progression of aneurysmal disease should be examined in future studies.

Polymicrobial synergy stimulates Porphyromonas gingivalis survival and gingipain expression in a multi-species subgingival community

BackgroundDysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket.MethodsGrowth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR.ResultsThe P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis.ConclusionsIn the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community.

Дисбаланс калікреїн-кінінової системи при хронічному панкреатиті у поєднанні з ожирінням і результати його комплексної корекції

Актуальність. Значна роль у виникненні та прогресуванні хронічного панкреатиту, одного з найбільш поширених захворювань у загальнотерапевтичній практиці, належить активації калікреїн-кінінової системи (ККС), яка бере участь у процесах розвитку запалення, згортання крові та фібринолізу, в імунних реакціях, формуванні больового синдрому та ін. Мета. Дослідження динаміки показників ККС при застосуванні препарату метадоксин у складі комплексної терапії хворих на хронічний біліарний панкреатит (ХБП) у поєднанні з ожирінням. Матеріали та методи. Досліджено 75 хворих на ХБП у поєднанні з ожирінням. Хворих було розподілено на 2 групи. Перша група (30 осіб) отримувала загальноприйняте лікування (ЗПЛ). Друга група (45 осіб) отримувала в комплексі до ЗПЛ препарат метадоксин. Результати. Після проведеного лікування позитивна динаміка спостерігалась в обох групах. Проте у пацієнтів другої групи простежувалась більш значуща позитивна динаміка показників ККС: рівень протеолітичної активності знизився на 9,0 %, калікреїну — на 20,0 %, a1-інгібітор протеїназ — на 7,2 %, а показник прекалікреїну зріс на 6,4 %, a2-макроглобулін — на 10,8 %, кініназа II — на 10,7 % у порівнянні з 1 групою. Висновки. Застосування метадоксину у складі комплексної терапії хворих на ХБП із коморбідним ожирінням сприяло вірогідному підвищенню ефективності ЗПЛ.

Protease activity is maintained in Nepenthes ampullaria digestive fluids depleted of endogenous proteins with compositional changes.

Nepenthes ampullaria is a unique carnivorous tropical pitcher plant with the detritivorous capability of sequestering nutrients from leaf litter apart from being insectivorous. The changes in the protein composition and protease activity of its pitcher fluids during the early opening of pitchers (D0 and D3C) were investigated via a proteomics approach and a controlled protein depletion experiment (D3L). A total of 193 proteins were identified. Common proteins such as pathogenesis-related protein, proteases (Nep [EC:3.4.23.12], SCP [EC:3.4.16.-]), peroxidase [EC:1.11.1.7], GDSL esterase/lipase [EC:3.1.1.-], and purple acid phosphatase [EC:3.1.3.2] were found in high abundance in the D0 pitchers and were replenished in D3L samples. This reflects their importance for biological processes upon pitcher opening. Meanwhile, prey-inducible chitinases [EC:3.2.1.14] were found in D0 but not in D3C and D3L samples, which suggests their degradation in the absence of prey. Protease activity assays demonstrated the replenishment of proteases in D3L with similar levels of proteolytic activities to that of D3C samples. This supports a feedback mechanism and signaling in the molecular regulation of endogenous protein secretion, turnover, and activity in Nepenthes pitcher fluids. Furthermore, we also discovered several new enzymes (XTH [EC:2.4.1.207], PAE [EC:3.1.1.98]) with possible functions in cell wall degradation that could contribute to the detritivory habit of N. ampullaria.

Acetate kinase and peptidases are associated with the proteolytic activity of Lactobacillus helveticus isolated from fermented food

Lactobacillus (L.) helveticus is widely used in food industry due to its high proteolytic activity. However, such activity varies greatly between isolates, and the determining factors regulating the strength of proteolytic activity in L. helveticus are unclear. This study sequenced the genomes of 60 fermented food-originated L. helveticus and systemically examined the proteolytic activity-determining factors. Our analyses found that the strength of proteolytic activity in L. helveticus was independent of the isolation source, geographic location, phylogenetic closeness between isolates, and distribution of cell envelope proteinases (CEPs). Genome-wide association study (GWAS) identified two genes, the acetate kinase (ackA) and a hypothetical protein, and 15 single nucleotide polymorphisms (SNPs) that were associated with the strength of the proteolytic activity. Further investigating the functions of these gene components revealed that ackA and two cysteine peptidases coding genes (pepC and srtA) rather than the highly heterogeneous and intraspecific CEPs were linked to the level of proteolytic activity. Moreover, the sequence type (ST) defined by SNP analysis revealed a total of ten STs, and significantly weaker proteolytic activity was observed among isolates of ST2. This study provides practical information for future selection of L. helveticus of strong proteolytic activity.

Gene Expression Analysis of Fruit Bromelain in Ripening of &lt;i&gt;Ananas comosus&lt;/i&gt; Cultivar MD 2

Fruit bromelain is a proteolytic enzyme harbouring cysteine catalytic residue found abundantly in pineapple fruit. The expression of cysteine proteases is usually regulated during fruit ripening. In the present study, we aimed to study the expression and proteolytic activity level of fruit bromelain during the ripening stage of A. comosus cultivar MD 2. The gene expression of fruit bromelain was investigated via relative gene expression analysis using qPCR while the proteolytic activity of fruit bromelain was analysed via enzymatic assay using casein as a substrate. The qPCR analysis revealed that the expression of fruit bromelain was down-regulated 10-fold in ripe pineapple fruits. Besides that, the unripe pineapple fruits [1.9101 ± 0.0831 U/mL] had a higher proteolytic activity than the ripe MD 2 pineapple fruits [1.1333 ± 0.0896 U/mL]. This result showed that the function of fruit bromelain may be related to the protection of young pineapple fruits during the fruit development stage.

The Interplay Between Genetic Risk Factors and Proteolytic Dysregulation in the Pathophysiology of Inflammatory Bowel Disease.

Crohn's disease [CD] and ulcerative colitis [UC] are the two main forms of inflammatory bowel disease [IBD]. Previous studies reported increased levels of proteolytic activity in stool and tissue samples from IBD patients, whereas the re-establishment of the proteolytic balance abrogates the development of experimental colitis. Furthermore, recent data suggest that IBD occurs in genetically predisposed individuals who develop an abnormal immune response to intestinal microbes once exposed to environmental triggers. In this review, we highlight the role of proteases in IBD pathophysiology, and we showcase how the main cellular pathways associated with IBD influence proteolytic unbalance and how functional proteomics are allowing the unambiguous identification of dysregulated proteases in IBD, paving the way to the development of new protease inhibitors as a new potential treatment.

Protease activity in single-chain prekallikrein

AbstractPrekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.

Effect of the Method of Nanocomplex Synthesis on the Proteolytic Activity of Chymotrypsin Immobilized on Silver Nanoparticles

Two methods for the synthesis of hybrid organic-inorganic nanocomplexes comprising chymotrypsin and silver nanoparticles have been developed based on the chemical reduction of silver nitrate by sodium borohydride. In the first method, the enzyme is added to the reaction mixture simultaneously with other reagents. In the second method, it is added to the reaction mixture at certain intervals after the start of the reduction reaction. Chymotrypsin immobilized on nanocomplexes stabilizes silver nanoparticles. As compared to the native enzyme, the immobilized enzyme prepared by the both methods exhibits an increased proteolytic activity in acidic and strongly alkaline media. At the same time, chymotrypsin immobilized on silver nanoparticles by the second method exhibits the highest level of proteolytic activity.