Mixture Of Proteases Research Articles

Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Involvement of Xyr1 and Are1 for Trichodermapepsin Gene Expression in Response to Cellulose and Galactose in Trichoderma reesei.

The secretome of Trichoderma reesei contains a mixture of cellulases, hemicellulases, amylases, proteases, and lipases that synergistically degrade plant biomass. Trichodermapepsin (TrAsP), the most prominent protease of T. reesei, affects the stability of cellulases. Similar to cellulase production, TrAsP production also depends on carbon and nitrogen sources. Unlike the cellulase mechanism, the regulatory mechanism of TrAsP remains unknown. Therefore, this study aimed to determine the effect of the main cellulase regulator Xyr1 and nitrogen regulator Are1 on trasp regulation. Cellulase inducer Avicel and TrAsP inducer galactose were used as carbon sources. qRT-PCR analysis revealed that Xyr1 and Are1 acted as a repressor and an activator for trasp expression, respectively. Compared to Avicel, relative expression was higher in galactose. The binding motifs of Xyr1 and Are1 were located in upstream of the trasp promoter. From promoter deletant analysis using the β-glucuronidase reporter gene, the area from - 870bp to - 670bp was identified as the only region for positive regulation and there were both binding motifs of Xyr1 and Are1. Reporter assay of mutants confirmed functions of downregulation of Xyr1 and upregulation of Are1. Electrophoretic mobility shift assay demonstrated the binding ability of Xyr1 and Are1 to the particular binding motifs and their functionality was confirmed. Further, this study demonstrated that Cre1, Xpp1, and Pac1 downregulate trasp expression similar to that in cellulase regulation mechanism. These results demonstrate that transcriptional regulators of cellulase control trasp expression and suggest the possibility of the existence of specific protease regulators in T. reesei.

Development and validation of an analytical assay for electrochemical detection and quantification of protein‐bound 3‐nitrotyrosine in biological samples and comparison with classical, antibody‐based methods

BackgroundReactive oxygen species (ROS) cause oxidative damage which is associated with endothelial dysfunction and cardiovascular disease. Majority of ROS are formed by mitochondria and NADPH oxidase. Overproduction of ROS leads to oxidative damage and disease progression, therefore, accurate determination of ROS and reactive nitrogen species (like nitric oxide, superoxide and peroxynitrite) is of great importance but also represents a great challenge, since these species are short‐lived and methods that are usually used relay on indirect detection. Here we compared 3 different methods for the detection of 3‐nitrotyrosine, a footprint of peroxynitrite formation in vivo, in biological samples.Methods and ResultsNitrated proteins were generated by incubation with peroxynitrite or 3‐morpholino sydnonimine (SIN‐1) and digested using pronase, a mixture of different proteases. The free 3‐nitrotyrosine was then separated by HPLC and quantified by a coulometric method using the CoulArray system. Concentration‐response‐curves of 3‐nitrotyrosine standards were highly linear (detection limit 50 nM). Concentration‐response‐curves of free 3‐nitrotyrosine obtained from digested nitrated bovine serum albumin standards were also highly linear before reaching a plateau, probably representing nitration of all available tyrosine residues. The presence of 3‐nitrotyrosine in nitrated bovine serum albumin standards was validated by other methods (ELISA and dot blot analysis using specific 3‐nitrotyrosine antibodies), showing a similar pattern as compared to HPLC/ECD detection. Ex vivo nitration of isolated mitochondrial or membranous fractions as well as serum/plasma yielded a concentration‐dependent 3‐nitrotyrosine signal using the HPLC/ECD method. Ongoing studies are dedicated to the quantification of 3‐nitrotyrosine in tissue samples of nitrate‐tolerant, diabetic, hypertensive and septic mice and rats. A major draw‐back seems to be the separation of the 3‐nitrotyrosine peak in complex biological samples, which is currently our main focus.ConclusionsBased on our ex vivo data, the CoulArray quantification method for 3‐nitrotyrosine seems to have some advantages regarding sensitivity and selectivity. In the future, we hope to be able to establish a reliable automated HPLC assay for the routine quantification of 3‐nitrotyrosine in biological samples of cell culture, animal and human origin. Our most recent data show that protein‐bound 3‐nitrotyrosine not only represents a footprint of peroxynitrite formation and marker of oxidative stress but also correlates with increased mitochondrial superoxide formation rates pointing towards self‐propagating oxidative stress vicious circles.Support or Funding InformationVascular biology research grant from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”

Effect of Protease and Prebiotic mixtures with Free Fishmeal Diets on Physiological Responses and Histological Examinations of the Red Tilapia, Oreochromis sp.

Effect of Protease and Prebiotic mixtures with Free Fishmeal Diets on Physiological Responses and Histological Examinations of the Red Tilapia, Oreochromis sp.

Proteolytic enzymes positively modulated the physicochemical and antioxidant properties of spent yeast protein hydrolysates

The spent brewer yeast (SBY) is a by-product with great potential for the production of peptides. Protein hydrolysates of SBY with improved physicochemical and antioxidant properties were produced by a system containing mixtures of proteases (Alcalase™, Brauzyn® and Protamex™). The amount of solids and crude proteins recovered from the raw material after the protein hydrolysis was, in average, 57% and 70% (w/w), respectively. Hydrolysis resulted in intermediate degrees of hydrolysis (15%), promoting the release of hydrophobic residues (8% higher related to the control) with antioxidant properties. The presence of all three enzymes influenced the degree of hydrolysis but the darkening and browning of the hydrolysates were mainly affected by Protamex™. Most of the hydrolyzed proteins presented a molecular weight (MW) of 35 kg mol-1, as confirmed after membrane separation and electrophoresis. The membrane fractionation step using 30 kg moL-1 membranes effectively produced peptide-enriched fractions of 30-35 kg moL-1. In addition, it was possible to correlate the degree of hydrolysis, solids release, color parameters L*, Browning Index and antioxidant properties by FRAP and DPPH in function of the hydrolysis conditions, showing the potential use of SBY protein hydrolysates as a source of peptides in value-added functional foods.

Characterization of Female Reproductive Proteases in a Butterfly from Functional and Evolutionary Perspectives.

Molecules that mediate reproductive interactions are some of the most rapidly evolving traits. Researchers have often suggested that this is due to coevolution at key physiological interfaces. However, very few of these interfaces are well understood at the functional level. One such interface is the digestion of the spermatophore in Lepidoptera. Female Lepidoptera have a specialized reproductive organ called the bursa copulatrix that receives and processes the male spermatophore, a complex proteinaceous ejaculate. In the cabbage white butterfly, Pieris rapae, the bursa secretes a mixture of proteases hypothesized to digest the spermatophore. However, these proteases remain biochemically uncharacterized. Using a zymogram approach, we identified six proteases in bursal extracts at sufficiently high concentrations to characterize their in vitro activity. We assessed the modes of action of these bursal enzymes by quantifying their activity following exposure to diagnostic protease inhibitors. A serine protease-specific inhibitor failed to reduce bursal protease digestion of casein. However, a cysteine protease-specific inhibitor did decrease the activity of some proteases. To explore the possible molecular mechanisms responsible for these responses, we created protease homology models. The models mirrored the results of our in vitro experiments, indicating that protease homology models may offer insight into underlying functional mechanisms. Whether the observed bursal protease resistance to known inhibitors is important in the context of spermatophore digestion remains to be tested. However, our results suggest the exciting possibility that bursal protease specificity may have evolved in response to interactions with various proteins and inhibitors present within the female tract during the reproductive process.

Effects of dietary mixture of protease and probiotics on growth performance, blood constituents, and carcass characteristics of growing-finishing pigs.

This study was conducted to evaluate the effects of dietary mixture of protease and probiotics on growth performance, blood constituents, and carcass characteristics of growing-finishing pigs. A total of 48 growing pigs were randomly allotted into 2 dietary (6 pigs/pen; 4 replicates/treatment). The treatments were a diet based on corn and soybean meal (CON) and CON supplemented with 0.01% of dietary mixture of protease and probiotics (MULTI). No differences were found on growth performance (average daily gain, ADG; overall, 874.06 vs. 881.14 g/d; p > 0.05), blood constituents (white blood cell, WBC; phase I, 17.51 vs. 19.96 × 103 /μL; phase II, 19.65 vs. 21.95 × 103 /μL; p > 0.05), and carcass characteristics during overall experimental period between CON and MULTI. In conclusion, the addition of dietary mix-ture of protease and probiotics in growing-finishing pig diet did not have any beneficial effects.

A comparative study on properties of fish meat hydrolysates produced by an enzymatic process at high pressure.

Fish meat hydrolysates (FMHs) were produced from nine fishes at a high pressure of 300MPa using Flavourzyme 500MG and a protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E. The electropherograms of the FMHs showed major far-migrating peptide bands in the vicinity of 5kDa. The total soluble solids (TSS) and soluble nitrogen content in the FMHs were species-specific and were mostly higher in the case of four-enzyme hydrolysis. Most of the HPLC peptide peaks of the rockfish meat hydrolysates appeared within 10min of elution, and total free amino acids in the hydrolysate increased abruptly as a result of four-enzyme hydrolysis. The FMHs, which were high in TSS and soluble nitrogen, may be applicable for use in food as seasoning, and could be produced efficiently via the enzymatic process used in this study.

Low Cost, Enriched Collagenase-Purified Protease Enzyme Mixtures Successfully Used for Human Islet Isolation

Background: Purified bacterial collagenase and protease enzymes are commonly used to recover human islets from clinical grade pancreata for subsequent clinical islet transplantation. The high cost of purified enzymes can be prohibitive for recovering human islets from research pancreata used for translational research or pre-clinical studies. In this study, we successfully isolated islets from human research pancreata using enriched collagenase products supplemented with BP Protease (DE Collagenase 800 or Collagenase Gold-BP Protease mixtures) that cost significantly less than purified collagenase-protease enzymes used for clinical islet isolation. Materials & methods: Defined, enriched collagenase products (DE Collagenase 800 and Collagenase Gold) with a purity comparable to purified clinical collagenase blends (> 85% purity) were prepared from Clostridium histolyticum culture supernatants. These products contained primarily intact class I (C1) and class II (C2) at about a 75:25 C1:C2 ratio as determined by analytical anion exchange chromotography Enriched collagenase-BP Protease mixtures were used at either 19,000-48,000 or 32,500-78,800 neutral protease U (NP U) per g pancreas, and hereafter referred to as low or high protease enzyme mixtures. Each enzyme mixture was diluted up to 350 mL of HBSS and used in a modified Ricordi procedure with with isolations performed at three different islet processing centers. Results: Comparison of the biochemical characteristics of enriched collagenase to the purified collagenase products showed the enriched product is comparable and in some cases superior to those higher priced purified collagenase products. The mean post-purification islet yields from three different centers were 4444 and 4261 IEQ/g pancreas when using the high and low protease enzyme mixtures, respectively. The mean stimulation indices calculated after analysis of glucose-stimulated insulin release were 2.68 and 2.11 when using the high and low protease enzyme mixtures, respectively. Conclusions: The use of a low cost enriched collagenase products with biochemical characteristics comparable to purified collagenase enzymes were shown to be as effective in human islet isolation as higher priced products. These data indicate that using enzyme mixtures with high Wunsch activity may not be required for successful human islet isolation. The amount of Wunsch activity/g tissue can be reduced to 25-50% of the amount currently used by some islet transplant centers for isolating human islets. Further studies are needed to validate enzyme compositions required for successful human islet isolation.

084 Bromelain Induces Relaxation of Human Corpus Cavernosum Tissue in Vitro Independent Nitric Oxide-cGMP Pathway: Possible Significance for Erectile Dysfunction

Bromelain (BRM) is a mixture of proteolytic enzymes found in several parts of the pineapple (Ananas comosus) plant. BRM is widely administered for its well-recognized properties, mainly anti-inflammatory, antithrombotic and fibrinolytic, anticancer, and immunomodulatory. BRM gained universal acceptance as a phytotherapeutic agent due to its extensive dietary usage and lack of side effects; however, the complete molecular mechanism of action of BRM has not been completely identified. This exploratory study focuses on the erectile responses of human cavernosal strips on isometric tension measurements. To evaluate the effectiveness of (a) pure BRM (from pineapple stem, Sigma-Aldrich, St Louis, MO), (b) BRM dietary supplement (BG, Natural Brand Bromelain 500mg tablet, GNC, Pittsburgh, PA) and (c) fresh pineapple juice (PAJ) on human corpus cavernosum (HCC). HCC tissues were obtained from men undergoing penile prosthesis implantation (n =8-10) and prepared for organ bath experiments as per our published procedures. BRM-induced relaxation response (10−8-10−3 M) was performed in vitro using HCC strips in an organ bath. The effects of inhibitors [NG-nitro-L-arginine methyl ester (L-NAME, 100μM), 1H-[1,2,4]-oxadiazolole-[4,3-a] quinoxalin-10one (ODQ, 30μM), and tetraethylammonium (TEA, 100 μM, blocker Ca2+ - activated K+ channels)] on BRM-mediated relaxation responses were evaluated. The relaxation effects of BRM, BG, and PAJ on phenylephrine (Phe)-induced contraction were also compared. The protein expression of endothelial (e) and neuronal (n) nitric oxide synthase (NOS) markers and vascular endothelium growth factor (VEGF) were determined using Western blotting and immunohistochemistry.

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix.

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17%±7.5%) and higher postpurification islet recovery (83.8%±5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

A response of the imbibed dormant red rice caryopsis to biotic challenges involves extracellular pH increase to elicit superoxide production

AbstractSeeds often survive in the soil in a dormant state, but their persistence is endangered by micro-organisms that could feed on them. Seed–microbe interactions in the soil are, however, poorly understood. We used dormant caryopses of red rice to study the defence response induced by pronase, a mixture of proteases secreted byStreptomyces griseus, a non-pathogenic bacterium. Pronase was shown to activate the plant immune reaction, indicating that its activity was recognized as a potential microbial attack. The defence reaction included extracellular alkalinization and superoxide production, and the former was necessary to activate the latter, since buffering at pH 6 inhibited the oxidative burst. Alkalinization was sufficient to trigger the oxidative burst, as superoxide production increased when caryopses were incubated in buffered solutions of increasing pH without pronase. Release of proanthocyanidins was observed, with or without pronase. These diverse mechanisms are hypothesized to cooperate in reinforcing seed protection. Finally, time profiles of superoxide production by dormant and non-dormant red rice caryopses during imbibition did not support a relationship between extracellular superoxide and dormancy breaking or germination. Thus, the role of this reactive oxygen species in red rice imbibed caryopses appears to be essentially aimed at defence against attacks by challenging micro-organisms.

Blocking negative effects of senescence in human skin fibroblasts with a plant extract

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

Effects of Eco-friendly Multi-enzyme on Diarrhea and Immune Response of weaned Pigs

This study was designed to investigate the effects of multi-enzyme on diarrhea and immune responses of weaned pigs. A total 36 weaned pigs (5.92 ± 0.48 kg BW; 28 d old) were randomly allotted to 2 dietary treatments (3 pigs/pen, 6 replicates/ treatment) in a randomized complete block design. The dietary treatments were a typical diet based on corn and soybean meal (CON) and CON with 0.1% multienzyme (Multi; mixture of β-mannanase, xylanase, α-amylase, protease, β-glucanase, and pectinase). Pigs were fed their respective diets for 6 wk. Frequency of diarrhea, levels of packed cell volume (PCV), white blood cells (WBC), immunoglobulins, cortisol, tumor necrosis factor-α (TNF-α), transforming growth factor- β (TGF-β), and C-reactive protein (CRP) were measured. Multi group tended to decrease (p<0.1) diarrhea frequency than CON group during 2 wk after weaning. Lower values of PCV on d 3 (p<0.05) and d 7 (p<0.1) were found in Multi group compared with CON group. There were no significant differences on WBC number and immunoglobulin (Ig) M and A between Multi and CON groups. However, Multi group tended to increase (p<0.1) Ig G on d 7 than CON group. Moreover, Multi group showed modulated immune responses, indicated by decreased levels of cortisol (p<0.05) on d 7 and 14, TNF-α on d 3 (p<0.05) and d 7 (p<0.10), TGF- β on d 2 (p<0.05) and d 7 (p<0.10), and CRP (p<0.10) on d 3 and 7 after weaning compared with CON group. Consequently, inclusion of multi-enzyme in diets for weaned pigs improved gut health and modulated immune responses of weaned pigs.

Study of Non Starch Polysaccharide Enzymes in Relation to Performance and Commercial Utilization of Low Energy Cereal Based Diet in Broiler Chickens

This experiment was performed to evaluate the effect of Non starch polysaccharide (NSP) enzymes product (a mixture of xylanase, cellulase, hemicellulase, β-glucanase, α-amylase, protease, pectinase and β-mannanase), supplemented in cereal based diet on broiler performance. Total 120 day old broiler birds were randomly divided in two experimental groups. Control group received normal cereal based diet and treatment group received NSP enzyme mixed diet (1g/kg of diet). The results showed that NSP supplemented diet improved body weight gain by 5.1% (p

Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

Eschar removal by bromelain based enzymatic debridement (Nexobrid®) in burns: An European consensus

Early debridement and/or eschar removal is regarded as a significant step in the treatment of deep partial and full thickness burns. It aims to control wound bioburden and allows early wound closure by conservative treatment or skin grafting. Preservation of viable dermis accompanied by early wound closure, is regarded as a necessary step to reduce scar related complication, e.g. functional limitations and/or unaesthetic scar formation. Aside from the classical techniques of surgical excision as tangential excision for eschar removal, hydro-surgery, maggot therapy, laser, enzymatic debridement have been described as additional techniques in the burn surgeon’s armamentarium. It is widely accepted that early eschar removal within 72h improves the outcome of burn wound treatment by reducing bacterial wound colonization, infection and length of hospital stay. In contrast, the right technique for eschar removal is still a matter of debate. There is increasing evidence that enzymatic debridement is a powerful tool to remove eschar in burn wounds, reducing blood loss, the need for autologous skin grafting and the number of wounds requiring surgical excision. In order to assess the role and clinical advantages of enzymatic debridement by a mixture of proteolytic enzymes enriched in Bromelain (Nexobrid®) beyond the scope of the literature and in view of users’ experience, a European Consensus Meeting was scheduled. The aim was to provide statements for application, based on the mutual experience of applying enzymatic debridement in more than 500 adult and pediatric patients by the consensus panelists. Issues to be addressed were: indications, pain management and anesthesia, timing of application, technique of application, after-intervention care, skin grafting after enzymatic debridement, blood loss, training strategies and learning curve and areas of future research needs. Sixty-eight (68) consensus statements were provided for the use of enzymatic debridement. The degree of consensus was remarkably high, with a unanimous consensus in 88.2% of statements, and lowest degree of consensus of 70% in only 3 statements. This consensus document may serve as preliminary guideline for the use of enzymatic debridement with user-oriented recommendations until further evidence and systematic guidelines are available.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Binary mixture of proteases increases the antioxidant properties of white bean (Phaseolus vulgaris L.) protein-derived peptides obtained by enzymatic hydrolysis

Abstract In this study, hydrolysates from white bean protein concentrate were obtained using the commercial proteases Flavourzyme® 500 L, Alcalase® 2.4 L and Neutrase® 0.8 L and a statistical mixture design. The antioxidant properties of the protein hydrolysates, the modulation of the protease activities by substrates and products and the antioxidant activities of fractionated samples by ultrafiltration were investigated. The results showed that the antioxidant activities of the white bean protein increased from 45% to 70% after enzymatic hydrolysis. The use of the binary mixture of Flavourzyme® 500L and Alcalase® 2.4 L showed a significant synergistic effect and resulted in maximum antioxidant activity of the protein hydrolysates when evaluated by DPPH-radical scavenging and reducing power assay. It was observed that proteins from white bean decreased the t1/2 and D values of the enzymes and their combinations during enzymatic hydrolysis. For the fractionated samples, a tendency was observed to show less antioxidant activities with a reduction in molecular weight, reaching the lowest values in the 3 kDa permeate fraction. This study demonstrated the usefulness of statistical mixture design in the production of protein hydrolysates with improved antioxidant properties.

Application of Pineapple Juice in the Fish Digestion Process for Carcinogenic Liver Fluke Metacercaria Collection

Pepsin is common digestive enzyme used for fish digestion in the laboratory to collect trematode metacercariae. In a field study, to survey the infected fish is needed a huge yield of pepsin and it is very expensive. Therefore, our purpose of this study was to investigate the candidate enzyme from pineapple juice which has a digestive enzyme called bromelain, a mixture of proteolytic enzymes, to digest fish in order to harvest metacercariae. Fish were divided into 2 groups: one group in which metacercariae were harvested using acid pepsin as a control and other groups in which the fish was digested using fresh pineapple juices. The results showed that pineapple juice is able to digest fish similarly to pepsin. The Pattavia pineapple juice had the highest number of metacercariae similar to the control. For Trat Si Thong pineapple juice, we found the number of metacercariae was less than control. This result suggests that the Pattavia pineapple juice was optimal juice for fish digestion to metacercaria collection and can be used instread of pepsin acid.