Assay Of Proteolytic Activity Research Articles

Application of Prebiotics and Probiotics to Improve Oral and Dental Health by Inhibiting Streptococcus mutans

Background: The most common method to combat microorganisms that cause tooth decay is the use of antibiotics. However, despite the benefits of antibiotics, their use has significant disadvantages, such as the increased resistance of microorganisms to these chemicals. Objectives: A promising and safer alternative is the use of probiotics and prebiotics, which can reduce or inhibit the growth of disease-causing oral microorganisms. Streptococcus salivarius is one such probiotic, while inulin and nisin are the prebiotics commonly recommended for this purpose. Methods: Several assays, including agar bilayer interference, agar well penetration, and microdilution, were used to assess the antibacterial activities of S. salivarius K12 and S. salivarius M18 probiotics, as well as those of the prebiotics nisin and inulin. Additionally, bile salt and low pH tolerance assessments were conducted for the probiotics, and the synthesis of bacteriocins by the probiotics was verified using proteolytic and enzymatic activity assays. Results: The minimum inhibitory concentration (MIC) assay revealed that Streptococcus mutans growth was inhibited by S. salivarius K12 and S. salivarius M18 at a concentration of 75%, and by nisin and inulin at 200 IU/mL. In the well penetration assay, S. salivarius K12 exhibited significantly better results compared to S. salivarius M18. Nisin had significantly greater inhibitory effects on S. mutans compared to inulin. A combination of all four components (S. salivarius K12, S. salivarius M18, nisin, and inulin) resulted in maximum antibacterial activity, indicating a synergistic effect. Based on the agar bilayer assay and the growth inhibition zone diameters, nisin showed the highest antibacterial activity against S. mutans, followed by S. salivarius K12, inulin, and S. salivarius M18. Enzymatic digestion and proteolytic activity assays confirmed the synthesis of bacteriocins by S. salivarius K12 and S. salivarius M18. Both probiotics demonstrated bile salt tolerance at a concentration of 0.3% and surviving rates of 36% and 33%, respectively, at pH 2. Conclusions: The most important finding of the current study is the synergistic effect between the highlighted prebiotics and probiotics, which may offer a novel and effective combination for preventing tooth decay.

Alkaloids from Siparuna (Siparunaceae) are predicted as the inhibitors of proteolysis and plasma coagulation caused by snake venom and potentially counteract phospholipase A2 activity of Bothrops jararaca

Ethnopharmacological relevanceSnakebite envenomation (SBE) is the world's most lethal neglected tropical disease. Bothrops jararaca is the species that causes the greatest number of SBEs in the South and Southeastern of Brazil. The main symptoms are local (inflammation, edema, hemorrhage, and myonecrosis) and systemic (hemorrhage, hemostatic alterations with consumptive coagulopathy, and death) effects. Species of the genus Siparuna, Siparunaceae, are used in folk and traditional medicine to treat SBE. However, limited information is available concerning Brazilian Siparuna species against SBE. Aim of the studyTo investigate the correlation between the compounds present in the extracts of five Siparuna species as potential agents against proteolytic activity, plasma coagulation, and phospholipase A2 (PLA2) activity caused by B. jararaca venom, using data obtained by UHPLC-MS/MS, biological activity, and multivariate statistics. Materials and methodsThe ethanol extracts from leaves of S. ficoides, S. decipiens, S. glycycarpa, S. reginae, and S. cymosa were fractionated by liquid-liquid extraction using different solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and n-butanol), affording their respective extracts, totaling 25 samples that were assayed through in vitro plasma coagulation and proteolytic activity assays. Moreover, the extracts were analyzed by UHPLC-MS/MS, using electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) in negative and positive ionization modes. The data was processed in MZmine v. 2.53 and evaluated by multivariate statistical tests (PLS) using the software UnscramblerX v. 10.4. These data were also used to build molecular networks (GNPS), and some ions of interest could be annotated using the library of molecules on the GNPS platform. ResultsA total of 19 extracts inhibited B. jararaca-induced plasma coagulation, with emphasis on S. cymosa and S. reginae (800 s). The inhibition of the proteolytic activity was also promising, ranging from 16% (S. glycycarpa) to 99% (S. cymosa, S. decipiens, and S. reginae). In addition, most extracts from S. cymosa and S. reginae inhibited 70–90% of PLA2 activity. Based on data from positive mode APCI analyses, it was possible to obtain a statistic model with reliable predictive capacity which exhibited an average R2 of 0.95 and a Q2 of 0.88, indicating a robust fit. This process revealed five ions, identified as the alkaloids: coclaurine (1), stepholidine (2) O-methylisopiline (3), nornantenine (4) and laurolitsine (5). This is the first study to evidence the potential antivenom of alkaloids from Siparuna species. ConclusionsAltogether, our results give support to the popular use of Siparuna extracts in SBE accidents, suggesting their potential as an alternative or complementary strategy against envenoming by B. jararaca venom. The predicted ions in the chemometric analysis for the assayed activities can also be correlated with the blocking activity and encourage the continuation of this study for possible isolation and testing of individual compounds on the used models.

Exploring Host-Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR.

The DegP protease-chaperone operates within the periplasm of Gram-negative bacteria, where it assists in the regulation of protein homeostasis, promotes virulence, and is essential to survival under stress. To carry out these tasks, DegP forms a network of preorganized apo oligomers that facilitate the capture of substrates within distributions of cage-like complexes which expand to encapsulate clients of various sizes. Although the architectures of DegP cage complexes are well understood, little is known about the structures, dynamics, and interactions of client proteins within DegP cages and the relationship between client structural dynamics and function. Here, we probe host-guest interactions within a 600 kDa DegP cage complex throughout the DegP activation cycle using a model α-helical client protein through a combination of hydrodynamics measurements, methyl-transverse relaxation optimized spectroscopy-based solution nuclear magnetic resonance studies, and proteolytic activity assays. We find that in the presence of the client, DegP cages assemble cooperatively with few intermediates. Our data further show that the N-terminal half of the bound client, which projects into the interior of the cages, is predominantly unfolded and flexible, and exchanges between multiple conformational states over a wide range of time scales. Finally, we show that a concerted structural transition of the protease domains of DegP occurs upon client engagement, leading to activation. Together, our findings support a model of DegP as a highly cooperative and dynamic molecular machine that stabilizes unfolded states of clients, primarily via interactions with their C-termini, giving rise to efficient cleavage.

Genomic characterization and probiotic potential of lactic acid bacteria isolated from feces of guinea pig (Cavia porcellus).

Presently, there exists a growing interest in mitigating the utilization of antibiotics in response to the challenges emanating from their usage in livestock. A viable alternative strategy encompasses the introduction of live microorganisms recognized as probiotics, exerting advantageous impacts on the immune system and nutritional aspects of the host animals. Native lactic acid bacteria, inherently possessing specific properties and adaptive capabilities tailored to each animal, are deemed optimal contenders for probiotic advancement. In the current investigation, microorganisms exhibiting probiotic potential were isolated, characterized, and identified from the fecal samples of guinea pigs (Cavia porcellus) belonging to the Peruvian breed. The lactic acid bacteria isolated on Man, Rogosa, and Sharpe agar underwent Gram staining, catalase testing, proteolytic, amylolytic, and cellulolytic activity assays, low pH tolerance assessment, hemolytic evaluation, antagonism against Salmonella sp., determination of autoaggregation and coaggregation capacity, and genotypic characterization through sequencing of the 16S rRNA gene. A total of 33 lactic acid bacteria were isolated from the feces of 30 guinea pigs, also 10 isolates were selected based on Gram staining and catalase testing. All strains exhibited proteolytic activity, while only one demonstrated amylolytic capability, and none displayed cellulase activity. These bacteria showed higher tolerance to pH 5.0 and, to a lesser extent, to pH 4.0. Furthermore, they exhibited antagonistic activity against Salmonella sp. Only two bacteria demonstrated hemolytic activity, and were subsequently excluded from further evaluations. Subsequent assessments revealed autoaggregation capacities ranging from 4.55% to 23.19%, with a lesser degree of coaggregation with Salmonella sp. ranging from 3.53% to 8.94% for the remaining eight bacterial isolates. Based on these comprehensive tests, five bacteria with notable probiotic potential were identified by molecular assays as Leuconostoc citreum, Enterococcus gallinarum, Exiguobacterium sp., and Lactococcus lactis. The identified bacteria stand out as promising probiotic candidates, deserving further assessment in Peruvian breed guinea pigs. This exploration aims to enhance production outcomes while mitigating the adverse effects induced by pathogenic microorganisms.

Fibrinolytic Protease Activity of Crude Enzyme from Fermented Sunflower (Helianthus annuus) and Common Bean (Phaseolus vulgaris) seeds by Rhizopus microsporus var. oligosporus FNCC 6010 in Solid State Fermentation

In the entire world, cardiovascular diseases (CVDs) are the main cause of death. For the treatment of CVDs, microbial fibrinolytic enzymes are highly regarded as novel therapeutic candidates. This study was purposed to determine the fibrinolytic protease activity produced by fungus source, which is Rhizopus microsporus var. oligosporus FNCC 6010 in fermented sunflower (Helianthus annuus) seed and common bean (Phaseolus vulgaris) seed. Fermentation was carried out by solid-state fermentation method at an initial pH of 5, incubation temperature of 33±1°C, and incubation time of 24 hours. The fermented seed was extracted to obtain supernatant as the crude enzyme. The proteolytic activity assay was done by the skimmed milk agar (SMA) plate method to obtain the proteolytic index, and the fibrinolytic activity assay was conducted by the fibrin-agarose plate method to get the fibrinolytic index. The results show that crude enzymes from fermented H. annuus and P. vulgaris seeds by R. microsporus have fibrinolytic protease activity with proteolytic index 2.64 ± 0.01 and 2.23 ± 0.04, respectively. The fibrinolytic index is 2.40 ± 0.06 and 1.64 ± 0.06, respectively. Therefore, the crude enzyme has the potential to be further researched as a candidate for thrombolytic agents. The purification, characterization, and in-depth research are needed to develop enzymes into preparations for preventing and treating CVDs.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

BackgroundStreptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase.MethodsBacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry.ResultsHere, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation.ConclusionsOur findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

Electrochemical biosensor for trypsin activity assay based on cleavage of immobilized tyrosine-containing peptide

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min−1, 198 ± 24 nM and 0.0016 min−1 nM−1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.

Insulin mimetic potential of Hylocereus undatus from extracted myo-inositol and proteins

Diseases are spreading like a trend and victimising every other individual globally. Here, we are referring to one such most common disease that has not even spared young lives i.e., diabetes. Annually, several people lose their lives and loved ones because of this dangerous disease. This has compelled the researchers to think and work on some life saving treatment. People suffering from hyperglycaemic conditions have insulin resistance which can be improved by intake of myo-inositol. Myo-inositol has potential to regulate this insulin hormone which can prevent and control diabetes mellitus. In this research, we have used a natural source (fruit) Hylocereus undatus; it has proved to be a good source of myo-inositol and some proteins that help in insulin regulation naturally. Several techniques and tests were performed such as extraction, purification, crystallisation, proteolytic activity assay, protein estimation, etc. Positive results of myo-inositol were observed through crystallisation together with decent amount of protein concentrations by Folin Lowry test and SDS-PAGE analysis.

Carboxymethyl Cellulose-Based Polymers as Promising Matrices for Ficin Immobilization.

The present work is devoted to research on the interaction between carboxymethyl cellulose sodium salt and its derivatives (graft copolymer of carboxymethyl cellulose sodium salt and N,N-dimethyl aminoethyl methacrylate) with cysteine protease (ficin). The interaction was studied by FTIR and by flexible molecular docking, which have shown the conjugates' formation with both matrices. The proteolytic activity assay performed with azocasein demonstrated that the specific activities of all immobilized ficin samples are higher in comparison with those of the native enzyme. This is due to the modulation of the conformation of ficin globule and of the enzyme active site by weak physical interactions involving catalytically valuable amino acids. The results obtained can extend the practical use of ficin in biomedicine and biotechnology.

Metabolism-independent phenomenon in ethanolic propolis inhibitory capacity towards enterococcus spp proteolytic activity

Background: Root canal bacteria produce many virulence factors which are responsible for endodontic pathological states. Bacteria are assumed to utilize energy from bacterial cell metabolism activity for producing these virulence factors. Propolis extracts are commonly reported to have antibacterial abilities against dental pathogens. The purpose of this study is to investigate the possible correlation between bacterial proteolytic and metabolism activities under the treatment of ethanolic extract of propolis (EEP).Method: The 0.00125%; 0.05%; 0.1%; 0.2%; 0.4%; and 0.8% ethanolic propolis were prepared for recovery rate confirmative procedure, proteolytic, and metabolism activity assay, with 2% of chlorhexidine gluconate (CHX) was used as a positive control. The bacteria were cultured in brain heart infusion (BHI) media after EEP treatment. Bacterial suspension was initially prepared in broth culture dilution with BHI media, followed by the gelatin liquefaction measurement for proteolytic assay. Phenol-red and arginine dehydrogenase enriched media for observing both carbohydrate and arginine metabolism activities, respectively, in the clinical Enterococcus spp. and E. faecalis ATCC 29212.Result: The recovery rate of the bacteria was not terminated after several EEP treatments. Proteolytic activity of the bacteria was likely decreased in several EEP treatments. EEP tended to affect the carbohydrate and arginine metabolism of the bacteria in certain fashions.Conclusion: This study suggested that the EEP treatment affected both proteolytic and metabolism activity in negative regulation tendencies.

Physiotherapeutic Protocol and ZnO Nanoparticles: A Combined Novel Treatment Program against Bacterial Pyomyositis.

Simple SummaryBacterial infections are among the widely disturbing diseases and leading cause of mortality around the world. Some bacterial strains can invade the skeletal muscles by producing proteolytic enzymes, causing pyomyositis. The aim of the present investigation was to evaluate ZnO nanoparticles (ZnO-NPs) as an effective treatment for bacterial pyomyositis in combination with a physiotherapeutic program (consisting of swimming and a low-level laser treatment (LLLT)). Results revealed the significant and synergistic effect of both ZnO-NPs along with the physiotherapeutic program in enhancing the histoarchitecture, immunity and hemostasis, and some vital physiological biomarkers. This emphasizes the urgency of spreading more awareness about the importance of the combined effect between nanotechnology and physiotherapeutic programs in muscle regeneration after pyomyositis caused by virulent infection with proteolytic bacteria.Myositis tropicans or pyomyositis is a muscle inflammation resulting from a bacterial infection of skeletal muscle (commonly caused by Staphylococcus aureus) that usually leads to hematogenous muscle seeding. The present study was designed to estimate the role of ZnO-NPs and a physiotherapeutic program in the management of induced biceps femoris atrophy in rats through histological, biochemical, and radiological examinations at different time intervals. At the beginning, several bacterial strains were evaluated through a proteolytic enzyme activity assay and the highest activity was recorded with the Staphylococcus aureus strain. ZnO-NPs were synthesized with the arc discharge method with an average size of 19.4 nm. The antibacterial activity of ZnO-NPs was investigated and it was revealed that the prepared ZnO-NPs showed a minimum inhibitory concentration of 8 µg/mL against the tested bacterium. The cytotoxicity of the prepared ZnO-NPs was tested in C2C12 myoblast cells, and it was elaborated that CC50 was 344.16 µg/mL. Biceps femoris pyomyositis was induced with a potent strain (Staphylococcus aureus); then, a physiotherapeutic program combined with the prepared ZnO-NPs treatment protocol was applied and evaluated. The combined program claimed antibacterial properties, preventing muscle atrophy, and resulted in the most comparable value of muscle mass.

Production of a murine mAb against Bothrops alternatus and B. neuwiedi snake venoms and its use to isolate a thrombin-like serine protease fraction

Accidents with snakes from the genus Bothrops represent ~90 % of all snakebites in Brazil. Monoclonal antibodies (mAbs) targeting venom components can be important assets for treating envenoming syndromes, for developing diagnostic tests and for research purposes. Therefore, in this study, we aimed to generate murine mAbs against the antigenic mixture of Bothropic venoms traditionally used as immunogen to produce Bothropic antivenoms in Brazil. ELISA showed that one of the produced mAbs recognizes B. alternatus and B. neuwiedi venoms (mAb anti-Ba/Bn) specifically and Western Blot revealed that this mAb binds to a single protein band of molecular mass of ≈50 kDa. MAb anti-Ba/Bn inhibited the coagulant activity but was unable to neutralize hemorrhagic and phospholipase A2 activities caused by the B. neuwiedi venom. MAb anti-Ba/Bn was immobilized to Sepharose beads and used for immunoaffinity chromatography of B. neuwiedi venom. Proteolytic activity assays indicated that the immunoaffinity-purified fraction (BnF-Bothrops neuwiedi fraction) has a serine protease thrombin-like profile, which was supported by coagulability assays in mice. Bottom-up proteomic analysis confirmed the prevalence of serine proteases in BnF using label-free quantification. In conclusion, this work characterized a mAb with neutralizing properties against B. neuwiedi coagulant activity and demonstrates that immunoaffinity chromatography using mAbs can be a useful technique for purification of bioactive toxic proteins from Bothrops spp. snake venoms.

Development and validation of a label-free method for measuring the collagen hydrolytic activity of protease.

Collagen is the most abundant fibrous structural protein, and therefore, the quantitative evaluation of the effect of protease on collagen has a profound influence on enzyme application. In this research, unlabeled native bovine hide powder was utilized to detect collagen hydrolytic activity of the protease. The optimum conditions of the determination method were as follows: 30mg/mL substrate concentration, 30min reaction time, and 2-9 U/mL enzyme concentration. Then, several typical industrial protease preparations were chosen to measure collagenolytic activities at different temperatures and pH values, whose change trends were quite distinct from those of proteolytic activity assay method based on casein or dye-labeled hide powder substrate. Especially, in the pH 5-7, casein hydrolytic activities of these proteases showed sharper peaks with relative activity from 6% to 100%, whereas, their collagen hydrolytic activities based on native hide powder exhibited 30-100% with broader peaks. And collagen hydrolytic activities resulted from using dye-labeled substrate reached a lower optimum pH value than that of other methods. Besides, the results of these measurements displayed a moderate degree of reproducibility. This method is more reasonable than the protease assay method using casein or labeled hide powder as the substrate in many fields.

Discovery of novel indolin-2-one compounds as potent inhibitors of HsClpP for cancer treatment

Human caseinolytic protease proteolytic subunit (HsClpP) is a highly conserved serine protease that plays an essential role in cell homeostasis through removal of the damaged and/or misfolded proteins. Recently, due to its critical role in cancer proliferation and metastasis, HsClpP has been considered as a promising target for the cancer treatment. In this paper, through a random screening toward a library of 2086 bioactive chemicals, a novel compound I, 3-(3,5-dibromo-4-hydroxybenzylidene) -5-iodoindolin-2-one, was identified as a potent suppressor of HsClpP. Herein, a series of compound I derivatives were synthesized, and evaluated for their anti-cancer activities on a variety of cancers cells. Through the preliminary biological assay in vitro, including MTT assay and proteolytic activity assay, compound I was identified as the most potent inhibitor. Treatment with compound I impaired the migration of Hela cells. In addition, compound I disrupted the mitochondrial function, and reduced the level of the SDHB and induced the production of the ATF4. In general, compound I is a promising probe of HsClpP for cancer treatment, and is a good lead compound for the development of novel anti-cancer agent.

Ribavirin shows antiviral activity against SARS-CoV-2 and downregulates the activity of TMPRSS2 and the expression of ACE2 in vitro.

Ribavirin is a guanosine analog with broad-spectrum antiviral activity against RNA viruses. Based on this, we aimed to show the anti-SARS-CoV-2 activity of this drug molecule via invitro, insilico, and molecular techniques. Ribavirin showed antiviral activity in VeroE6 cells following SARS-CoV-2 infection, whereas the drug itself did not show any toxic effect over the concentration range tested. Insilico analysis suggested that ribavirin has a broad-spectrum impact on SARS-CoV-2, acting at different viral proteins. According to the detailed molecular techniques, ribavirin was shown to decrease the expression of TMPRSS2 at both mRNA and protein levels 48h after treatment. The suppressive effect of ribavirin in ACE2 protein expression was shown to be dependent on cell types. Finally, proteolytic activity assays showed that ribavirin also showed an inhibitory effect on the TMPRSS2 enzyme. Based on these results, we hypothesized that ribavirin may inhibit the expression of TMPRSS2 by modulating the formation of inhibitory G-quadruplex structures at the TMPRSS2 promoter. As a conclusion, ribavirin is a potential antiviral drug for the treatment against SARS-CoV-2, and it interferes with the effects of TMPRSS2 and ACE2 expression.

Biological protein precipitation: A green process for the extraction of cucumisin from melon (Cucumis melo L. inodorus) by-products

Cucumisin (CUC) from industrial melon by-products was separated for the first time through biological precipitation using carrageenan (CRG). This approach could represent a cost-effective and environmentally friendly process for the industries, avoiding the use of expensive equipment and toxic salts or solvents, such as butanol and ethanol. In this study, biological precipitation of proteins from melon by-products using CRG was studied and compared with conventional precipitation with ammonium sulphate. Different methods were applied for the identification and characterization of isolated proteins, including SDS-PAGE gel, FPLC and proteolytic activity assays. The isolated CUC confirmed a molecular weight of 68 kDa and showed highly stable proteolytic (PA) and milk-clotting (MCA) activities in a wide range of CaCl2 (20–60 mM), pH (5–7) and temperatures (30–85 °C). Melon peel extract demonstrated to possess significant PA (4.24 U/mg protein) and MCA (191.50 MCU/mg protein), but such values were increased by ammonium sulphate precipitation (1.60 and 2.06-folds, respectively), and specially a noticeable increment was observed by biological precipitation with 2.11 and 17.65-folds, respectively, demonstrating the capability to be an effective strategy to isolate and purify CUC, allowing a yield of 0.17 g CUC/100 g of by-products and keeping its biological properties.

Generation of Lactose- and Protease-Positive Probiotic Lacticaseibacillus rhamnosus GG by Conjugation with Lactococcus lactis NCDO 712.

Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L. rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L. lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L. rhamnosus LAB49 effectively degraded β-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GMOs), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture.IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.

Dextran-Functionalized Semiconductor Quantum Dot Bioconjugates for Bioanalysis and Imaging.

The prerequisites for maximizing the advantageous optical properties of colloidal semiconductor quantum dots (QDs) in biological applications are effective surface functionalization and bioconjugation strategies. Functionalization with dextran has been highly successful with some nanoparticle materials, but has had very limited application with QDs. Here, we report the preparation, characterization, and proof-of-concept applications of dextran-functionalized QDs. Multiple approaches to dextran ligands were evaluated, including performance with respect to colloidal stability across a range of pH, nonspecific binding with proteins and cells, and microinjection into cells and viability assays. Multiple bioconjugation strategies were demonstrated and applied, including covalent coupling to develop a simple pH sensor, binding of polyhistidine-tagged peptides to the QD for energy transfer-based proteolytic activity assays, and binding with tetrameric antibody complexes (TACs) to enable a sandwich immunoassay and cell immunolabeling and imaging. Our results show that dextran ligands are highly promising for the functionalization of QDs, and that the design of the ligands is tailorable to help optimally meet the requirements of applications.

Investigation of the potential regulator proteins associated with the expression of major surface protein and dentilisin in Treponema denticola

ABSTRACT Objective Treponema denticola is involved in ‘chronic’ periodontitis pathogenesis. The mechanism underlying the regulation of the expression of its virulence factors, such as major surface protein (Msp) and prolyl-phenylalanine specific protease (dentilisin) is yet to be clarified. We determined the gene expression profiles of Msp- and dentilisin-deficient mutants of T. denticola to identify the regulation network of gene expression concomitant with the inactivation of these virulence genes. Methods Gene expression profiles of T. denticola ATCC 35405 (wild type), dentilisin-deficient mutant K1, and msp-deficient mutant DMSP3 were determined using DNA microarray analysis and quantitative real-time reverse transcription PCR (qRT-PCR). Msp and dentilisin protein levels were determined by immunoblotting and proteolytic activity assays. Results In addition to several differentially expressed genes, dentilisin expression was reduced in DMSP3; msp expression was significantly reduced in K1 (p < 0.05), both at the gene and protein levels. To identify the regulatory system involved, the expression levels of the potential regulators whose expression showed changes in the mutants were evaluated using qRT-PCR. Transcriptional regulators TDE_0127 and TDE_0814 were upregulated in K1, and the potential repressor, TDE_0344, was elevated in DMSP3. Conclusions Dentilisin and Msp expression were interrelated, and gene expression regulators, such as TDE_0127, may be involved in their regulation.

Characterization of buffalo native pregnancy-associated glycoprotein: mass spectrometry-based glycan composition analysis, sugar-binding characteristics and proteolytic activity assay

Pregnancy-associated glycoproteins constitute a large family of extensively glycosylated secretory proteins which are specifically expressed in outer epithelial cell layer (chorion/trophectoderm) of placenta. The present study reports purification and characterization of buffalo pregnancy-associated glycoprotein (BuPAG) from foetal cotyledons during early pregnancy (< 3 months). Native BuPAG was purified by anion-exchange and Vicia villosa affinity chromatography. Three distinct bands were observed in SDS-PAGE and Western blot with higher molecular weight range of ~ 55–70 kDa in comparison to the calculated one (~ 40 kDa) which may be attributed to the presence of glycosylation in BuPAG. Glycan analysis of purified BuPAG by high-resolution mass spectrometry (ESI-qTof) revealed the presence of nine N-glycan structures and three O-glycan structures. Interaction analysis of various sugars with purified BuPAG using fluorescence quenching assay showed decrease in fluorescence intensity without any shift in emission maxima. BuPAG exhibited proteolytic activity within pH range of 2.0–5.5 with highest activity at pH 3.0. In the presence of inhibitor pepstatin A, proteolytic activity reduced by 84%. The glycan structures and sugar-binding characteristics of BuPAG are identified for the first time in this study. The activity of BuPAGs as active peptidases indicates their possible role in various molecular pathways required for sustenance of pregnancy.