Caseinolytic Activity Research Articles

Nattokinase production from Bacillus subtilis using cheese whey: Effect of nitrogen supplementation and dynamic modelling

In this study, cheese whey – a nutrient-rich industrial waste was utilized as a low-cost fermentation medium for the growth of Bacillus subtilis and nattokinase production. The nattokinase yield (caseinolytic activity) using cheese whey (789.93 U ml−1) was 5 %–7 % less compared to glucose- or lactose-based synthetic media (833.44 and 850.84 U ml−1, respectively). Effect of nitrogen supplementation (yeast extract, tryptone, and peptone) on nattokinase yield was tested adopting an L9 (33) Taguchi experimental design, which revealed yeast extract as the best nitrogen source. Yeast extract supplementation (10 g l-1) to cheese whey increased the caseinolytic activity to 833.43 U ml−1, making it comparable to lactose- and glucose-based synthetic media, while lowering the medium cost by 55–60 %. A dynamic model incorporating logistic growth, substrate utilization and Luedeking-Piret product formation kinetics across all carbon-sources offered good agreement (R2 > 0.93). The estimated maximum specific growth rate on lactose-based synthetic medium (0.204 h−1), or yeast extract supplemented cheese whey medium (0.201 h−1) were better than that of the glucose-based synthetic medium (0.122 h−1). A similar observation on the growth associated product formation parameters offers insight on medium selection process. Overall, this study suggested that cheese whey could be utilized as a low-cost medium for nattokinase production.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60°C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

Production and Stability of the Proteinase Complex from Aspergillus ochraceus L-1 with Fibrinolytic and Anticoagulant Activity

A complex of proteolytic enzymes with a specific plasmin-like activity of at least 890 EpNA/mg and a specific protein C activator activity of at least 130 EpNA/mg was isolated from the culture fluid of micromycete Aspergillus ochraceus L-1 by salting out with ammonium sulfate followed by dialysis and depigmentation to phenol-aniline formaldehyde resin at a pH of 8.2. Comparison of the obtained proteinase preparation with caseinolytic, fibrinolytic, fibrinogenolytic, and plasmin-like activities with commercial analogues terrilytin, trypsin, and streptokinase showed its promise for use as a means for thinning purulent burn wounds and fibrin clots. The resulting proteinase complex had high storage stability at low temperatures for up to 9 months.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60°C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Proteolytic parameter changes in the plasma of patients with bladder cancer – depending on tumor stage

Abstract Bladder cancer (BC) is a worldwide common disease with a high mortality rate. Recognizing the dynamic changes in plasma that proteases and their inhibitors undergo might be valuable in understanding the carcinogenesis of invasive bladder cancer and in identifying BC patients with poor prognosis. This study aims to determine the activity of the proteolytic enzyme system and their inhibitors in patients with BC. In this paper, the total proteolytic activity, the activity of matrix metalloproteases (MMPs) and serine proteases was analyzed by the method of caseinolytic activity. For detection of activity of some inhibitors of proteolysis, we used the unified method for determining the activity of alpha-1-antitrypsin (α1A) and alpha-2-Macroglobulin (α2M) in human plasma. The level of medium-mass molecules (MMM) was assessed spectrophotometrically by applying the Nikolaichik method. The activity of MMPs was elevated in all groups of patients. Moreover, the activity of serine proteases was found to be enhanced in patients with Stage I, III and IV BC, while the activity of α1A was up by 1.4 and 1.3 times in patients with Stage I and Stage IV. The most significant increase was observed with regard to the activity of α2M in patients with I and III stages of BC. In addition, the level of MMM correlated with cancer stage. Indeed, the highest increase in the activity of protease inhibitors was observed in Stage I bladder cancer patients, which might signify their protective role at the onset of the bladder carcinogenesis. In contrast, significant growth in activity of α2M in patients with III stage of BC may point at a compensatory mechanism that inhibits tumor growth.

Altered clot formation and lysis are associated with increased fibrinolytic activity in ascites in patients with advanced cirrhosis

Analysis of coagulation disorders and assessment of rebalanced hemostasis with the use of traditional coagulation assays is challenging in cirrhotic patients. Therefore, alternative tests are under investigation for the evaluation of coagulopathy in this specific setting. Aim of this study was to analyze the modifications of clot structure and function in cirrhotic patients with different degrees of severity. Cirrhotic patients referred to our Unit were consecutively enrolled. Global test measurements, including clot and lysis assays, clot lysis time, and determination of other fibrinolytic parameters, were performed. Analyses of clot formation, morphology, and lysis were performed with a turbidimetric clotting and lysis assay (EuroCLOT). Lysis of a tissue factor-induced clot by exogenous tissue plasminogen activator was analyzed by studying the modifications of turbidity during clot formation and the following lysis. We evaluated coagulative and fibrinolytic parameters in both plasma and ascites. Urokinase plasminogen activator (uPA) and gelatinase activity in ascites were also measured. We analyzed data from 33 cirrhotic patients (11 in Child-Pugh class A; 22 in class B or C and with ascites) and 21 healthy subjects (HS). In class B/C patients prolonged latency time, a decline in clotting absorbance, and decreased fibrin formation were observed in comparison with class A and HS. Generated curves and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) progressively declined from HS to class C patients, whereas levels of plasminogen activator inhibitor-1 and tissue plasminogen activator increased. D-dimer levels were markedly increased in ascites, together with significantly smaller levels of TAFI, αlfa2-antiplasmin, and plasminogen. Caseinolytic activity was also present. Class C patients showed smaller amount of uPA and significantly lower levels of matrix metallopeptidases (MMP)2 in ascites in comparison with Class B subjects. Clot formation and lysis are altered in cirrhosis and fibrinolysis is activated in ascites. Ascitic levels of uPA and MMP2 are reduced and inversely related to the severity of liver disease.

Syzygium samarangense Volatile Oil Inhibited Bacteria Growth and Extracellular Protease of Salmonella typhimurium.

Medicinal plants are fast becoming essential pharmaceuticals for disease and infection management. The vast antimicrobial properties of these plants reside in the inhibitory properties of their endogenous secondary metabolites. Therefore, this study aimed to assess if the volatile oil of Syzygium samarangense inhibits enteric bacteria growth and its effect against the caseinolytic activity of the extracellular protease of Salmonella typhimurium. The volatile oil was extracted by hydrodistillation, while the antimicrobial assay was assessed with the microdilution method. The extracellular protease was partially purified by salting out, followed by size-exclusion chromatography. The mode of inhibition of this enzyme was deduced from the enzyme-substrate kinetics using a line-weaver burke plot. The antimicrobial properties of the oil were reported against ten enteric bacteria. Proteus vulgaris has the highest IC50 value of 0.75±0.004% v/v, while S. typhimurium, the most sensitive bacterium, showed the lowest IC50 value of 0.17±0.005% v/v. The extracellular protease of S. typhimurium was partially purified to achieve 3.73 purification fold and 314.2 μmol min-1 mg-1 protein. The optimal caseinolytic activity of this enzyme was found at pH 7.5 and 40 °C. The protease showed significantly higher activity in the presence of Zn2+ (9.3±0.33 U min-1) as compared to the control (7.0±0.58 U min-1) (p<0.05), however, K+, Ca2+, Co2+, Ba2+, Pb2+ and Hg2+ considerably reduced the enzyme activity. The activity of this enzyme was competitively inhibited by the volatile oil as an inhibitor. The volatile oil of S. samarangense inhibited a wide range of enteric bacteria and, therefore proposed as a potential antimicrobial agent. Inhibiting the extracellular protease of S. typhimurium may be one of its modes of action against these pathogens.

Citrobacter diversus-derived keratinases and their potential application as detergent-compatible cloth-cleaning agents.

Currently, poultry farming is one of the sectors that have a significant impact on the global economy. In recent years, there has been an increase in the production of broilers, inflicting this segment of the industry to generate tons of keratin due to huge disposal of chicken feathers. This points to the need to degrade these chicken feathers, as they have emerged as a major threat to the environment. Thus, in this study we aimed to identify keratinases that are produced by the bacterium Citrobacter diversus and further investigate the biochemical characteristics of these keratin-degrading enzymes. In a submerged medium, the bacterium was capable of degrading chicken feathers almost completely after 36h of fermentation. We found a maximum caseinolytic activity at pH9-10.5 and 50-55°C, and keratinolytic activity at pH8.5-9.5 and 50°C. Thus, given its stability at higher temperatures, upon incubation of this enzyme extract for 1h at 50°C, it showed approximately 50% of the keratinolytic and 100% of the caseinolytic activity. Further, under pH stability for 48h at 4°C, the enzyme extract maintained greater residual activity in the pH range 6-8. Caseinolytic activity was inhibited by EDTA and PMSF, whereas the keratinolytic activity was inhibited only by EDTA. Additionally, due to its alkaline activity and detergent compatibility, this enzyme extract could improve washing performance when added to a commercial detergent formulation. Using application tests, we could demonstrate a potential use of this bacterial enzyme extract as an additive in detergents to remove egg stains from cloth.

Activation of the Unfolded Protein Response via Co-expression of the HAC1i Gene Enhances Expression of Recombinant Elastase in Pichia pastoris

The effects of activation of the unfolded protein response (UPR) via co-expression of the HAC1i gene on the production of the recombinant Pseudomonas aeruginosa elastase (rPAE) in Pichia pastoris GS115 were evaluated in this study. The results showed that expression of the HAC1i gene significantly increased the level of Kar2p (a hallmark of UPR activation) in P. pastoris GS115, demonstrating activation of the UPR. This gene did not affect the growth of yeast in the buffered glycerol-complex medium but stimulated its growth in the buffered methanolcomplex medium. Co-expression of the HAC1i gene enhanced the expression level of the heterogeneously Nglycosylated forms of rPAE, as the caseinolytic activity in the supernatant of the various glycoforms of rPAE expressed in P. pastoris GS115/HAC1 was increased 1.8—3.9-fold compared to that in the control strain P. pastoris GS115, respectively. The stimulating effects of co-expression of the gene on rPAE production were observed when 0.5, 1.0, and 2.0% methanol were added every 24 h, as the caseinolytic activity of supernatants of P. pastoris GS115/HAC1 expressing wild-type of rPAE was increased 3.3-, 1.9-, and 1.7-fold at the corresponding methanol concentration. Further, activation of UPR via co-expression of the HAC1i gene enhanced rPAE secretion in P. pastoris at 20, 24, and 28°C, as the caseinolytic activity of supernatants of P. pastoris GS115/HAC1 expressing wild-type rPAE was increased 2.3-, 2.1-, and 2.8-fold over the tested temperatures.

Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.).

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.

ПРОТЕОЛІТИЧНА АКТИВНІСТЬ ПЛАЗМИ КРОВІ ТА ПЕЧІНКИ ЩУРІВ ЗА ХРОНІЧНОЇ АЛКОГОЛЬНОЇ ІНТОКСИКАЦІЇ

Today, the problem of alcohol abuse is quite relevant around the world. According to the World Health Organization, alcohol abuse is one of the three causes of premature death and is one of the main etiological factors that that cause the development of chronic diseases of the liver and other organs. The severity of clinical manifestations and the prognosis of further dynamics of ongoing alcohol intoxication can be investigated by studying the composition of proteins in tissues, as it is known that this pathology has a pronounced protein dystrophy and active proteolysis. Although much is known today about the effects of alcohol on proteolytic systems, many questions still need to be explored, as proteomic studies can be used to find biomarkers and therapeutic targets for ethanol abuse. The aim of the study was: to investigate the proteolytic activity of blood plasma and liver of rats in chronic alcohol intoxication at 11 and 21 days of the experiment. The model of chronic alcohol intoxication was modeled on male rats – weight 180-200g by intragastric administration of 30% ethyl alcohol solution for 10 days on an empty stomach, at the rate of 2 ml per 100 g of animal weight. Total proteolytic activity, activity of metalloproteinases and serine proteinases were determined by the method of determining caseinolytic activity. The protein concentration was determined by the Bradford method. Statistical processing of the study results was performed by conventional methods of variation statistics. It was found that on the 11th day from the beginning of ethanol administration to rats, there was an increase in total proteolytic activity and activity of metetalloproteinases in blood plasma and liver.

First report on Chlorella vulgaris collagenase production and purification by aqueous two-phase system

Collagen is triple helical structured protein, which is undegradable by most proteases and reacts only with collagenases. These enzymes are applied and important in several commercial segments and the interest for new collagenases has only been increasing. Microalgae are not a very prominent source of enzymes but are extremely commercially attractive forms of life due to photosynthetic growth. Yet, the literature is absent on reports about algae collagenases and this work is the first report on Chlorella vulgaris collagenolytic enzyme production, purification and characterization. C. vulgaris was cultivated in autotrophic and mixotrophic conditions and the biomass used to obtain the cell extract. The autotrophic extract displayed a higher collagenolytic activity and was used for ATPS experimental design. Through 24-full factorial design, high collagenolytic activity was observed at pH 8.0, PEG molar mass of 1500 g/mol, PEG concentration of 12.5% (w/w) and phosphate buffer concentration of 15% (w/w). In these conditions, a purification factor of 10.79 was obtained for a partition coefficient of 23.74 with the collagenase retained in top PEG rich phase. Purified collagenase optimum activity was observed under pH 9 and 37 °C, displayed less gelatinolytic and caseinolytic activity, with higher collagenolytic activity with azocoll than crude extract, attesting this enzyme is a true collagenase. The collagenolytic activity was inhibited by EDTA, an indicative of a metallocollagenase.

Improvement in organic solvent resistance and activity of metalloprotease by directed evolution

Improving enzyme stability in the presence of organic solvent is crucial for non-aqueous catalysis. In this study, directed evolution was applied to improve the tolerance of metalloprotease PT121 towards organic solvent. In presence of acetonitrile and acetone, three mutants (T46Y, H224 F, and H224Y) of PT121 showed excellent solvent stability, which increased their half-lives by 1.2–3.5-fold as compared to the wild-type enzyme. Kinetic constants (KM and kcat values) of the caseinolysis reaction presented H224 F and H224Y mutants have higher affinity than the wild-type, but T46Y mutant were similar to those of the wild-type enzyme. Interestingly, combined mutants T46Y/H224 F and T46Y/H224Y mutants presented awesome stability and excellent caseinolytic activity. Molecular dynamic simulation suggest that improved enzyme stability may be attributed to extensive non-covalent bond network resulting in a more compact structure. Disruption of the disulphide bond formation between Cys-30 and Cys-58 residues in the F56 V mutant is possibly the reason behind its low stability among all the selected mutants. Additionally, T46Y/H224 F and T46Y/H224Y showed a higher peptide synthetic activity in the presence of organic solvents than the wild-type, which renders these mutant enzymes as promising biocatalysts for biotechnological applications.

Protein degradation of black carp (Mylopharyngodon piceus) muscle during cold storage

This study investigated the effects of cold storage at different temperatures (4, −0.5, −3, and −20 °C) on protein degradation and its relationship to structural changes of black carp muscle. At −0.5 and 4 °C, major structural changes occurred, including the formation of gaps between myofibers and myofibrils, breakage of myofibrils and myofibers, and degradation of sarcoplasmic reticulum. Gel-based proteomic analysis showed that these structural changes were accompanied by degradation of a series of myofibrillar proteins, including titin, nebulin, troponin, myosin, myomesin, myosin-binding protein, and α-actinin. Loss of extractable gelatinolytic and caseinolytic protease activities was also observed. At −3 and −20 °C, formation of ice crystals was the most noticeable change. The major proteins were degraded at different locations in the black carp muscle, and gelatinolytic and caseinolytic proteases appear to contribute to the degradation of those proteins.

Noni (Morinda citrifolia L.) fruit as a new source of milk-clotting cysteine proteases.

This work reports the characterisation of caseinolytic and milk-clotting activities of proteases extracted from ripe fruits of Morinda citrifolia L., as a potential of their use in cheese production. Noni puree extract (NPE) was obtained by homogenising the fresh puree in 150 mM NaCl/50 mM sodium phosphate buffer (pH 7.0). The resulting protein concentration was of 0.367 ± 0.006 mg/mL, and an electrophoretic profile of the extract revealed protein bands ranging from 14 to 55 kDa. The proteolytic activity of NPE was higher when the extract had been previously incubated at pH 6.0 (8.859 ± 0.216 U/mg), whereas the optimum caseinolytic activity was observed at 50 °C. Noni puree proteases were strongly (98%) inhibited by iodoacetamide and E-64, suggesting the presence of only cysteine proteases in the crude extract. NPE proteases showed a milk-clotting activity (MCA) of 238.80 ± 5.29 U/mL, a specific milk-clotting activity (SMCA) of 9950.17 ± 220.74 U/mg, and an SMCA/PA ratio of 1124.31 ± 24.94, this last being comparable to those of commercial calf rennet. The cheese manufactured using NPE presented brittle and soft texture, high humidity, and showed sanitary conditions compatible with current Brazilian regulations. The product showed a slightly bitter taste, but still good acceptability, rating between 6 and 7 in the hedonic scale for flavour, texture, and overall acceptance. Lastly, there was 60% of positive purchase intent, demonstrating that noni fruit is a promising source of milk-clotting enzymes for the dairy industry.

Comparative compositional and functional analyses of Bothrops moojeni specimens reveal several individual variations.

Snake venoms are complex protein mixtures with different biological activities that can act in both their preys and human victims. Many of these proteins play a role in prey capture and in the digestive process of these animals. It is known that some snakes are resistant to the toxicity of their own venom by mechanisms not yet fully elucidated. However, it was observed in the Laboratory of Herpetology of Instituto Butantan that some Bothrops moojeni individuals injured by the same snake species showed mortalities caused by envenoming effects. This study analyzed the biochemical composition of 13 venom and plasma samples from Bothrops moojeni specimens to assess differences in their protein composition. Application of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed distinct venom protein profiles, but very homogeneous plasma profiles. Western Blotting (WB) was performed with plasma samples, which were submitted to incubation with the respective venom. Some individuals showed an immunorecognized band zone around 25 kDa, indicating interaction between the same individual plasma and venom proteins. Crossed-WB assay using non-self-plasma and venom showed that this variability is due to venom protein composition instead of plasma composition. These venoms presented higher caseinolytic, collagenolytic and coagulant activities than the venoms without these regions recognized by WB. Mass spectrometry analyses performed on two individuals revealed that these individuals present, in addition to higher protein concentrations, other exclusive proteins in their composition. When these same two samples were tested in vivo, the results also showed higher lethality in these venoms, but lower hemorrhagic activity than in the venoms without these regions recognized by WB. In conclusion, some Bothrops moojeni specimens differ in venom composition, which may have implications in envenomation. Moreover, the high individual venom variability found in this species demonstrates the importance to work with individual analyses in studies involving intraspecific venom variability and venom evolution.

Local and systemic effects of BtaMP-1, a new weakly hemorrhagic Snake Venom Metalloproteinase purified from Bothriopsis taeniata Snake Venom

A new weak hemorrhagic metalloproteinase named BtaMP-1 was purified from Bothriopsis taeniata snake venom by molecular exclusion followed by anion exchange chromatographies. This protein showed a molecular mass of 25,968.16 Da and is composed of 218 amino acid residues. The multiple alignments of its partial amino acid sequence showed high structural identity with other P-I class SVMP. BtaMP-1 showed caseinolytic activity that was enhanced by Ca2+ ion, completely inhibited by chelating and reducing agents and can be classified as an α-fibrinogenolytic enzyme. Locally, BtaMP-1 induces hemorrhage and edema, but not myotoxicity. These findings were confirmed by histological analysis of mouse gastrocnemius muscle. “In vitro” studies suggest that BtaMP-1 induce cytotoxicity in myoblast C2C12 but not in the myotubes cell line. BtaMP-1 induced systemic alterations in mice with one MHD and two hours exposure; histological analysis of lungs showed hemorrhagic areas, congestion, and increase the thickness of alveolar septum. Also, this protein induced mild effects on kidney and disruption of coagulation by depletion of fibrinogen plasma levels. This work provides insights into the importance of BtaMP-1 biological effects in envenomation by Bothropsis taeniata snake venom and providing further evidence to understand the role of P-I class SVMP in ophidian envenomation.

Development and characterization of chitosan/polyvinyl alcohol polymer material with elastolytic and collagenolytic activities

The biologically active polymeric material with entrapped peptidase Bacillus thuringiensis var. israelensis with caseinolytic, collagenase and elastase activities was developed as a promising product for medical use. We have evaluated and reported here the following physical-chemical and biochemical characteristics of entrapped enzyme: peptidase/polymer interaction and morphology analyses, film thickness, water content, time of dissolution in water and physiological saline, kinetic of casein hydrolysis and pH- and thermoprofiles of proteolytic activity. Scanning electron microscopy images shows the relative uniformity of the film surface with entrapped peptidase. The released peptidase was characterized by increased proteolytic activity in the acidic (14%–35%) and alkaline (7%–32%) regions. After nine months of storage, peptidase in chitosan/polyvinyl alcohol films retains more than 95% of its initial proteolytic activity. We consider this film as a perspective biotechnological agent in medicine.

Biochemical and immunochemical characterization of venoms from snakes of the genus Agkistrodon.

In the present work, venoms from five species of the genus Agkistrodon were evaluated in terms of their enzymatic (Phospholipase A2 and caseinolytic) and biological (edema forming, hemorrhagic, procoagulant and lethal) effects. Horses were used to produce monovalent hyperimmune sera against each of three venoms (A. bilineatus, A. contortrix and A. piscivorus) and their neutralizing potency, expressed as Median Effective Dose (ED50), was determined against the venoms of all five species. In terms of PLA2 and caseinolytic activities, all venoms are extremely homogeneous. PLA2 activity is high, while caseinolytic activity is low when in contrast with that of the rattlesnake Crotalus simus. On the other hand, biological activities showed marked interspecific differences, particularly between the species from Mexico and those from the United States. Mexican species displayed higher edema-forming, hemorrhagic and lethal effects than US species, while none of the species studied presented procoagulant activity. All three monovalent hyperimmune sera showed good neutralizing potency against the analyzed venoms. Nonetheless, we observed relevant immunochemical differences among the venoms using ELISA and Western Blot assays. We conclude that the venoms of A. piscivorus (USA) and A. bilineatus would be ideal to use as immunogens for the production of a polyvalent antivenom with good neutralizing potency against the venoms of all the species of the genus.

Assessing the toxic potential of enteropathogenic Bacillus cereus

The diarrheal type of food poisoning caused by enteropathogenic Bacillus cereus has been linked to various exotoxins. Best described are the non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), and cytotoxin K (CytK). Due to the ubiquitous prevalence of B. cereus in soil and crops and its ability to form highly resistant endospores, contaminations during food production and processing cannot be completely avoided. Although phylogenetically closely related, enteropathogenic B. cereus strains show a high versatility of their toxic potential. Thus, functional tools for evaluating the pathogenic potential are urgently needed in order to predict hazardous food contaminations. As the diarrheal syndrome is the result of a toxico-infection with enterotoxin production in the intestine, the entire passage of the bacteria within the host, from spore survival in the stomach, spore germination, host cell adherence, and motility, to enterotoxin production under simulated intestinal conditions was compared in a panel of 20 strains, including high pathogenic as well as apathogenic ones. This approach resulted in an overarching virulence analysis scheme. In parallel, we searched for potential toxico-specific secreted markers to discriminate low and high pathogenic strains. To this end, we targeted known exotoxins using an easy to implement immunoblotting approach as well as a caseinolytic exoprotease activity assay. Overall, Nhe component B, sphingomyelinase, and exoproteases showed good correlation with the complex virulence analysis scheme and can serve as a template for future fast and easy risk assessment tools to be implemented in routine diagnostic procedures and HACCP studies.