Exogenous Protease Research Articles

Uremic pruritus

Uremic pruritus

Apparent and standardised ileal amino acid digestibility of wheat distillers dried grains with solubles with or without exogenous protease in broilers and turkeys

The apparent (AIAAD) and standardised ileal amino acid digestibility (SIAAD) of wheat distillers dried grains with solubles (wheat-DDGS) was determined for broilers and turkeys at 28 d using two experiments.A total of 84 male Ross 308 broiler chicks (Experiment 1) or 84 male BUT 10 turkey poults (Experiment 2) were offered a nutrient-adequate diet from d 1 to 23. On d 23, birds in each experiment were allocated to 4 treatments consisting of a nitrogen-free diet (NFD) (with or without protease) and an assay diet containing wheat-DDGS as the only source of amino acids (with or without protease) in a randomised complete block design.In Experiments 1 and 2, the coefficient of AIAAD or SIAAD of Lys and Asp were low irrespective of protease. In Experiment 1, the coefficient of AIAAD ranged from 0.35 (Ala) to 0.75 (Pro) without protease, whereas the range was 0.42 (Thr) to 0.82 (Pro) with protease. Protease improved the coefficient of AIAAD for Arg and Pro and tended to improve the coefficient of AIAAD for Met. Without protease, the coefficient of SIAAD ranged from 0.51 (Ala) to 0.84 (Pro), whereas the range was from 0.65 (Ala) to 0.93 (Pro) with protease. Protease improved the coefficient of SIAAD of Arg, Leu, Phe, Met, Val and Pro.In Experiment 2, the coefficient of AIAAD was lower than 0.50 for all amino acids except for Glu (0.70) and Pro (0.81) without protease. On the other hand, the coefficient of SIAAD ranged from 0.41 (Thr) to 0.89 (Pro) without protease, whereas the range was from 0.56 (Arg) to 0.88 (Pro) with protease supplementation. With the exception of Cys and Pro, protease improved the coefficient of AIAAD and SIAAD of all other amino acids by 0.05–0.19.It was concluded that the AIAAD and SIAAD of wheat-DDGS are variable and are generally greater in broilers compared to turkeys at 28 d of age. Protease improved the ileal digestibility of a large number of amino acids in wheat-DDGS for broilers and turkeys.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.

Effects of a Monocomponent Protease on Performance Parameters and Protein Digestibility in Broiler Chickens

Abstract The impact of dietary fortification of an exogenous mono component protease enzyme with or without its matrix values (ME 31.5 Kcal, CP 0.84%, methionine 0.018%, lysine 0.05%, threonine 0.034%) on growth performance traits, feed digestibility, serum parameters, selected immune parameters, total counts of ileal clostridia species, carcass traits as well as duodenal histomorphological picture was studied. A total of 300- day old chicks (Cobb500) were randomly assigned to five separate floor pens equal experimental groups with three replicates (20 birds /replicate). Birds were fed either on corn-soybean meal basal diets (starter- grower and finisher) without any supplementation (G1) and served as control group, or basal diets to which protease enzyme was added on top at a rate of 200 ppm (G2). Birds in (G3) were fed on the basal diets to which protease was added at the same inclusion level but subtracting the whole matrix value of the enzyme. Birds in (G4) were fed on the fortified basal diets after subtracting only the metabolizable energy matrix value of the enzyme. Birds in (G5) were reared on the fortified basal diets after subtracting only the crude protein as well as amino acids matrix value of the enzyme. The experimental period extended for five weeks. Birds in groups 2 and 4 significantly (P≤ 0.05) surpassed all groups in weight gain, feed consumption and FCR, also these groups were significantly (P≤ 0.05) better in dressing %. Also G2 (protease on the top) and G4 (Low ME values) showed a significant elevation of CP digestibility and showed a significant (P≤0.05) reduction of total ileal clostridia species counts. In addition G2 showed an increase in villus height and villus to crypt ratios compared to other experimental groups. In conclusion, broiler chickens respond positively to supplementation of protease enzyme and it can be used safely on the top of their diets.

Protease-activated receptors and itch.

Protease-activated receptors (PARs) have been implicated in a variety of physiological functions, as well as somatosensation and particularly itch and pain. Considerable attention has focused on PARs following the finding they are upregulated in the skin of atopic dermatitis patients. The present review focuses on recent studies showing that PARs are critically involved in itch and sensitization of itch. PARs are expressed by diverse cell types including primary sensory neurons, keratinocytes, and immune cells and are activated by proteases that expose a tethered ligand. Endogenous proteases are also released from diverse cell types including keratinocytes and immune cells. Exogenous proteases released from certain plants and insects contacting the skin can also induce itch. Increased levels of proteases in the skin contribute to inflammation that is often accompanied by chronic itch which is not predominantly mediated by histamine. The neural pathway signaling itch induced by activation of PARs is distinct from that mediating histamine-induced itch. In addition, there is evidence that PARs play an important role in sensitization of itch signaling under conditions of chronic itch. These recent findings suggest that PARs and other molecules involved in the itch-signaling pathway are good targets to develop novel treatments for most types of chronic itch that are poorly treated with antihistamines.

A Dual-enzyme Product Containing Protease in Broiler Diet: Efficacy and Tolerance

AbstractExogenous enzymes are commonly added to poultry diets to improve nutrient utilisation, reduce excretion of nutrients into the environment as well as to improve zootechnical performance and reduce the cost of production. A series of four experiments were conducted in broilers to determine the efficacy and tolerance to an enzyme product (Vegpro™) containing protease and xylanase activities in wheat-soybean meal (SBM) diets fed to broilers from 1 to 39 or 42 d of age. Collectively, four experiments tested the following exogenous protease activity levels in wheat-SBM diet at levels of 0, 2,500, 5,000, 10,000, 20,000, 30,000, 40,000 or 100,000 HUT/kg of feed. Analysis of the four experiments together confirmed that Vegpro™ improved average daily gain and feed conversion ratio, especially in birds up to 21 d of age. Overall, inclusion of Vegpro™ to broiler diets at 10,000 HUT protease per kg of feed was shown to provide the maximum performance benefit (i.e. weight gain and feed conversion ratio). An exceptionally high protease activity (100,000 HUT/kg, ten times the manufacturer's recommended inclusion rate) was well tolerated and did not adversely affect the measured health and performance indicators from 1 to 42 d of age.

Effects of exogenous protease supplementation on endogenous trypsin activity and gene expression in broilers.

Two hundred and forty one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets with 0, 40, 80, or 160 mg/kg of an acid protease preparation from 1 to 42 days of age, in order to investigate the effects of an exogenous enzyme on growth performance, pancreatic trypsin activity and mRNA expression. Average daily gain (ADG) was greatest in the 160-mg/kg treatment group at 1-21 days, which was significantly higher than that in the control. After 42 days, average daily feed intake (ADFI) and ADG had significantly increased in the 80- and 160-mg/kg treatments (P < 0.05), with the 80-mg/kg treatment group having the highest ADFI and ADG values. The feed conversion ratio was not affected. Dietary acid protease at 80 and 160 mg/kg significantly decreased trypsin activity in the pancreas (relative to the control group) by 35.71 and 47.29%, respectively (P < 0.05). After 42 days, trypsin mRNA expression in the pancreas had significantly decreased by 19.5% in the 80-mg/kg treatment group relative to the control (P < 0.05). After 21 and 42 days, the diet supplemented with 160 mg/kg acid protease significantly decreased pancreatic trypsin mRNA by 19.6 and 37.7%, respectively, compared to the control. There were the 40-mg/kg treatment group and the control. Our results suggest that the amount of acid protease in the diet significantly affects trypsin activity and mRNA expression in broilers.

Validation of an Analytical Method for the Determination of the Activity of Protease in Animal Feed Additives and in Animal Feedingstuffs

SummaryThe nutrient availability in animal feeds can be improved by including exogenous enzymes to the feed, either by helping breakdown anti-nutritional factors or by increasing digestibility of complex ingredients thereby releasing more nutrients for utilisation. This process can improve the efficiency of meat and egg production, increase animal health, decrease feeding costs and reduce nutrients in animal waste. Proteases are protein-digesting enzymes that are used in animal nutrition to break down storage proteins in various plant materials and proteinaceous anti-nutrients in vegetable proteins. The analysis of exogenous proteases in feed additives and after they have been added to feed has proven technically challenging. Accordingly, the purpose of this work was to validate a method for the determination of the activity of protease in animal feed additives and supplemented animal feed. The approach used for the assay was to adapt an assay based on the hydrolysis of haemoglobin. The method validations examined a range of parameters including; linearity &amp; range; uncertainty, sensitivity, accuracy and studies designed to highlight any possible matrix effects on various types of supplemented feed. The assay method described herein is convenient and inexpensive and could be applied to the analysis of proteases in animal feeds during quality control and in investigating fraudulent adulteration of feed to ensure the authenticity and traceability of the product.

Role of a Bacillus subtilis Direct-Fed Microbial on Digesta Viscosity, Bacterial Translocation, and Bone Mineralization in Turkey Poults Fed with a Rye-Based Diet.

Rye contains high concentrations of non-starch polysaccharides (NSPs), leading to reduced digestibility. Since poultry have little or no endogenous enzymes capable of hydrolyzing these NSP, exogenous carbohydrases as feed additives are used in an attempt to reduce the anti-nutritional effects of these polysaccharides. Previously, an in vitro study conducted in our laboratory showed that inclusion of certain Bacillus direct-fed microbial (DFM) candidates that produce exogenous phytase, lipase, protease, cellulase, and xylanase in high-NSP diets significantly reduced both digesta viscosity and Clostridium perfringens proliferation. In the present study, rye-based turkey starter diets with or without Bacillus-DFM were administered ad libitum to day-of-hatch turkey poults in two independent experiments. In both experiments, day-of-hatch turkey poults were randomly assigned to either a control diet (CON) or a DFM treated diet (n = 25 birds/group). At 10 days-of-age, all turkey poults from experiments 1 and 2 were weighted and 12 turkey poults/group were randomly selected and humanely killed. Liver samples were aseptically collected to evaluate bacterial translocation, and intestinal digesta samples were individually collected to evaluate viscosity. Additionally, in experiment 2 both tibias were removed for assessment of bone parameters. In both experiments, the treated group showed a reduction in the total number of coliforms in the liver and a reduced digesta viscosity when compared to the CON group (P < 0.05). Turkey poults fed the Bacillus-DFM candidate had increased tibia diameter, breaking strength, ash content, calcium content, and phosphorus content when compared with CON turkey poults. In summary, turkey poults fed with a rye-based diet without DFM showed an increase in bacterial translocation and digesta viscosity, accompanied by a reduction in bone mineralization; however, these adverse effects can be prevented by the inclusion of selected a Bacillus-DFM candidate in high-NSP diets.

Extraction of aggrecan-peptide from cartilage by tissue autolysis.

Aggrecan is a cartilage specific proteoglycan containing chondroitin sulfate (CS) and keratan sulfate (KS). CS is an acidic polysaccharide having wide range of applications in pharmaceutical, cosmetic, and food industries. CS is extracted from cartilage by tissue proteolysis with an exogenous proteinase or by activating endogenous proteinases (autolysis) to release aggrecan-peptides from the tissue. This review is focused on the latter technique. Bovine nasal and tracheal cartilages, and broiler chicken sternum cartilage have been used for autolysis studies. To extract aggrecan-peptide, cartilage tissues are cut into small pieces, and incubated in a monovalent or divalent salt solution (e.g., 0.1 M sodium or calcium acetate) at pH 4.5 and 37 &#176;C for 7 - 24 h. Most (~80% or more) of total tissue uronic acid, a constituent sugar of aggrecan, is extracted and released into the salt solution during incubation. Reextraction of the tissue residue results in release of a small amount of uronic acid. Aggrecan-peptides purified using anion exchange chromatography are large compounds containing CS and KS. On gel chromatography, they are excluded from the column of Sephacryl S-300. Chemical composition analysis demonstrated that aggrecan-peptides from either bovine or chicken cartilage contain >90% CS with small amount (< 10%) of either KS or peptide. Patent information included production of aggrecan-peptide substantially free of DNA. The bovine aggrecan-peptide prepared by tissue autolysis has been used as a plate coating antigen in enzyme- linked immunosorbent assay (ELISA) to determine KS.

A cockroach derived serine protease allergen activates airway epithelial cells via PAR2 receptors

Both endogenous and exogenous proteases have been implicated in allergic airway diseases. Though recent studies have demonstrated the role of airway epithelial cells in pathophysiology of allergic diseases, the mechanisms of epithelial activation remain largely unknown. Recently published study (Kale and Arora, 2014) focused on elucidating the role of serine protease activity of Per a 10 on airway epithelial activation and to gain an insight into possible mechanism. We found that airway epithelial activation by Per a 10 is dependent on its serine protease activity. Further we showed that this activation is PAR2 dependent and leads to Ca 2+ mobilisation. This Research Highlight discusses the findings of our recent study and active research endeavors.

EndoProteoFASP: A novel FASP approach to profile salivary peptidome and disclose salivary proteases

The salivary peptidome, which can represent up to 20% of total secreted proteins in human saliva, is highly influenced by proteolytic events. However, the development of strategies to understand the dynamics underlying the generation of salivary peptides has been a challenging task. In order to disclose in more detail the proteolytic events taking place in saliva, we aimed to characterize salivary peptidome and predict salivary proteases by applying, for the first time, a filter-aided sample preparation (FASP) approach to saliva. Thus, as a proof-of-concept of this application, harvested saliva samples from healthy individuals were incubated in 30kDa cut-off spin filters for 18 or 115h, at 37°C, to promote saliva autolysis and the attained peptidome was characterized and compared with the naturally occurring one. In ex vivo conditions, proline-rich proteins, P-B peptide, histatin 1 and statherin were found to be the most susceptible salivary proteins to proteolysis. Peptide fragments were mainly attributed to the activity of cathepsin L1 and K at 18h, whereas at 115h, the attained peptide fragments were attributed to the activity of cathepsins K and L1, and MEP1A. Overall, the described endoProteoFASP approach makes the most of saliva׳s own protease pool and avoids the use of synthetic peptides and exogenous proteases to understand the proteolytic events occurring in the oral fluid. Hence, it could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from different pathophysiological conditions.

Increasing and decreasing the ultrastability of bacterial chemotaxis core signaling complexes by modifying protein-protein contacts.

The chemosensory signaling array of bacterial chemotaxis is composed of functional core units containing two receptor trimers of dimers, a homodimeric CheA kinase, and two CheW adaptor proteins. In vitro reconstitutions generate individual, functional core units and larger functional assemblies, including dimers, hexagons, and hexagonal arrays. Such reconstituted complexes have been shown to have both quasi-stable and ultrastable populations that decay with lifetimes of 1–2 days and ∼3 weeks at 22 °C, respectively, where decay results primarily from proteolysis of the bound kinase [Erbse, A. H., and Falke, J. J. (2009) Biochemistry 48, 6975–6987; Slivka, P. F., and Falke, J. J. (2012) Biochemistry 51, 10218–10228]. In this work, we show that the ultrastable population can be destabilized to the quasi-stable level via the introduction of a bulky tryptophan residue at either one of two essential protein–protein interfaces within the core unit: the receptor–kinase contact or kinase–adaptor interface 1. Moreover, we demonstrate that the quasi-stable population can be made ultrastable via the introduction of a disulfide bond that covalently stabilizes the latter interface. The resulting disulfide at least doubles the functional lifetime of the ultrastable population, to ≥5.9 weeks at 22 °C, by protecting the kinase from endogenous and exogenous proteases. Together, these results indicate that the ultrastability of reconstituted core complexes requires well-formed contacts among the receptor, kinase, and adaptor proteins, whereas quasi-stability arises from less perfect contacts that allow slow proteolysis of the bound kinase. Furthermore, the results reveal that ultrastability, and perhaps the size or order of chemosensory complexes and arrays, can be increased by an engineered disulfide bond that covalently cross-links a key interface. Overall, it appears that native ultrastability has evolved to provide an optimal rather than maximal level of kinetic durability, suggesting that altered selective pressure could either increase or decrease the functional lifetime of core complexes.

Adaptive evolution and divergence of SERPINB3: a young duplicate in great Apes.

A series of duplication events led to an expansion of clade B Serine Protease Inhibitors (SERPIN), currently displaying a large repertoire of functions in vertebrates. Accordingly, the recent duplicates SERPINB3 and B4 located in human 18q21.3 SERPIN cluster control the activity of different cysteine and serine proteases, respectively. Here, we aim to assess SERPINB3 and B4 coevolution with their target proteases in order to understand the evolutionary forces shaping the accelerated divergence of these duplicates. Phylogenetic analysis of primate sequences placed the duplication event in a Hominoidae ancestor (∼30 Mya) and the emergence of SERPINB3 in Homininae (∼9 Mya). We detected evidence of strong positive selection throughout SERPINB4/B3 primate tree and target proteases, cathepsin L2 (CTSL2) and G (CTSG) and chymase (CMA1). Specifically, in the Homininae clade a perfect match was observed between the adaptive evolution of SERPINB3 and cathepsin S (CTSS) and most of sites under positive selection were located at the inhibitor/protease interface. Altogether our results seem to favour a coevolution hypothesis for SERPINB3, CTSS and CTSL2 and for SERPINB4 and CTSG and CMA1. A scenario of an accelerated evolution driven by host-pathogen interactions is also possible since SERPINB3/B4 are potent inhibitors of exogenous proteases, released by infectious agents. Finally, similar patterns of expression and the sharing of many regulatory motifs suggest neofunctionalization as the best fitted model of the functional divergence of SERPINB3 and B4 duplicates.

Effect of sorghum type and malting on production of free amino nitrogen in conjunction with exogenous protease enzymes.

Sorghum types suitable for brewing and bioethanol production are required. The effect of sorghum type (white non-tannin versus white type II tannin) on free amino nitrogen (FAN) production from sorghum grain and malt using exogenous protease enzymes was investigated over extended incubation at moderate temperature (45 °C). With grain in the absence of exogenous proteases, white non-tannin sorghum produced substantially higher levels of FAN than white type II tannin sorghum, due to the tannins in the latter. Incubating sorghum grain with neutral proteinase and amino-peptidase in combination improved FAN production. The two sorghum types produced similar FAN levels when malted and incubated in the absence of the exogenous proteases. When both sorghums were malted and incubated with neutral proteinase alone substantially more FAN yield (124-126 mg 100 g(-1)) occurred than with grains (61-84 mg 100 g(-1)). The combination of amino-peptidase and proteinase did not improve FAN further. Neither, did malting influence wort free amino acid profile. Group B amino acids constituted the highest percentage (42-47%). With grain, white non-tannin sorghum plus proteinase and amino-peptidase yields the highest FAN, with malt both white non-tannin and white type II tannin sorghums plus proteinase yield the highest FAN.

Pseudomonas aeruginosa elastase disrupts the cortisol-binding activity of corticosteroid-binding globulin.

The serine protease inhibitor (SERPIN) family member corticosteroid-binding globulin (CBG) is the main carrier of glucocorticoids in plasma. Human CBG mediates the targeted release of cortisol at sites of inflammation through cleavage of its reactive center loop (RCL) by neutrophil elastase. The RCLs of SERPIN family members are targeted by diverse endogenous and exogenous proteases, including several bacterial proteases. We tested different bacteria for their ability to secrete proteases that disrupt CBG cortisol-binding activity, and characterized the responsible protease and site of CBG cleavage. Serum CBG integrity was assessed by Western blotting and cortisol-binding capacity assay. Effects of time, pH, temperature, and protease inhibitors were tested. Proteolytically active proteins from bacterial media were purified by fast protein liquid chromatography, and the active protease and CBG cleavage sites were identified by mass spectrometry. Among the bacteria tested, medium from Pseudomonas aeruginosa actively disrupted the cortisol-binding activity of CBG. This proteolytic activity was inhibited by zinc chelators and occurred most efficiently at pH 7 and elevated physiological temperature (ie, 41°C). Mass spectrometric analysis of a semi-purified fraction of P. aeruginosa media identified the virulence factor LasB as the responsible protease, and this was confirmed by assaying media from LasB-deficient P. aeruginosa. This metalloprotease cleaves the CBG RCL at a major site, distinct from that targeted by neutrophil elastase. Our results suggest that humoral responses to P. aeruginosa infection are influenced by this pathogen's ability to secrete a protease that promotes the release of the anti-inflammatory steroid, cortisol, from its plasma transport protein.

Increasing transcriptome response of serpins during the ontogenetic stages in the salmon louse Caligus rogercresseyi (Copepoda: Caligidae)

Serine protease inhibitors, or serpins, target serine proteases, and are important regulators of intra- and extracellular proteolysis. For parasite survival, parasite-derived protease inhibitors have been suggested to play essential roles in evading the host's immune system and protecting against exogenous host proteases. The aim of this work was to identify serpins via high throughput transcriptome sequencing and elucidate their potential functions during the lifecycle of the salmon louse Caligus rogercresseyi. Eleven putative, partial serpin sequences in the C. rogercresseyi transcriptome were identified and denoted as Cr-serpins 1 to 11. Comparative analysis of the deduced serpin-like amino acid sequences revealed a highly conserved reactive center loop region. Interestingly, P1 residues suggest putative functions involved with the trypsin/subtilisin, elastase, or subtilisin inhibitors, which evidenced increasing gene expression profiles from the copepodid to adult stage in C. rogercresseyi. Concerning this, Cr-serpin 10 was mainly expressed in the copepodid stage, while Cr-serpins 3, 4, 5, and 11 were mostly expressed in chalimus and adult stages. These results suggest that serpins could be involved in evading the immune response of the host fish. The identification of these serpins furthers the understanding of the immune system in this important ectoparasite species.

Effects of an exogenous protease on the fermentation and nutritive value of corn silage harvested at different dry matter contents and ensiled for various lengths of time

The objective of this experiment was to evaluate the effects of adding an experimental protease to corn plants harvested at different maturities on silage fermentation and in vitro ruminal starch digestibility (IVSD). Corn plants were harvested at maturities resulting in plants with 31 or 40% dry matter (DM). Plants were chopped, kernel processed, and treated with (1) only a 0.1 M phosphate buffer (pH 5.5, 5% vol/wt of fresh forage), (2) buffer with protease to obtain a final concentration of 20mg of protease/kg of wet forage, and (3) buffer with protease to obtain a final concentration of 2,000mg of protease/kg of wet forage. Treated forages (about 500g) were ensiled in nylon-polyethylene pouches and stored between 21 and 23°C for 0, 45, 90, and 150d. Data were analyzed as a 2×3×4 factorial arrangement of treatments, with the main effects of harvest DM, dose of protease, days of ensiling, and their interactions. The treatment with the highest dose of protease resulted in more robust fermentations across harvest DM with higher concentrations of lactic and acetic acids compared with untreated silage. Concentrations of soluble protein (% of crude protein) increased with time of ensiling, regardless of DM content at harvest. However, averaged over both harvest DM contents, it increased by 37% for silages treated with the high dose of protease compared with an average 11% increase for untreated silages and silage treated with the low dose of protease, between d 0 and 45. Averaged over both harvest DM contents, the concentration of soluble protein peaked in silages treated with the high dose of protease after 45d of ensiling, whereas it peaked at d 90 in untreated silages and silage treated with the low dose of protease. Similar changes occurred in the concentration of NH3-N due to length of ensiling and treatment with protease. In fresh forages, the concentration of starch for early- and late-harvested forages was similar, but IVSD was lower in the latter. After 45d of ensiling, IVSD was highest in both early- and late-harvested silages that were treated with the high level of protease. After 150d of ensiling, IVSD was similar among silages treated with protease, regardless of DM at harvest. Treating corn plants with a high dose of an experimental protease at harvest accelerated proteolysis during ensiling, resulting in corn silages with levels of IVSD after 45d of ensiling that were only obtained in untreated corn silages after 150d of ensiling.

Proteolytic enzymes in embryonated chicken eggs sustain the replication of egg-grown low-pathogenicity avian influenza viruses in cells in the absence of exogenous proteases

Low pathogenic influenza viruses grow readily in embryonated chicken eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that the influenza viruses propagated previously in eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in vitro pathogenicity index should be performed.

The effect of an exogenous protease on the fermentation and nutritive value of high-moisture corn

The objective of this study was to determine if treating high-moisture corn at harvest with an exogenous protease could accelerate the increase in in vitro ruminal starch degradation that is normally found with advancing times of ensiling. Ground high-moisture corn (HMC; 73% dry matter) was untreated or treated with an exogenous protease to achieve a final concentration of 2,000mg of protease/kg of fresh corn. Corn was ensiled in laboratory-scale bags (approximately 500g) that were evacuated of air, heat-sealed, and stored at 22 to 23°C for 70 and 140d. Samples of freshly treated corn samples were collected to represent d 0 samples. Treatment of HMC with protease did not affect the gross populations of lactic acid bacteria or yeasts throughout the ensiling period. Treatment of HMC with protease resulted in higher concentrations of lactic acid and ethanol after 70 but not 140d of ensiling. Concentrations of crude protein, water-soluble carbohydrates, and starch were unaffected by treatment with protease within each sampling day. After 70 or 140d of ensiling, HMC that was treated with protease had higher concentrations of soluble protein (as a % of crude protein) and NH3-N, and had lower concentrations of prolamin protein, compared with untreated corn. In vitro rumen degradability (7-h incubation) of starch was greater in protease-treated versus untreated corn at all sampling days but the difference was more pronounced after 70 and 140d compared with d 0. Concentrations of soluble protein and NH3-N were positively correlated with in vitro starch degradation. Conversely, the concentrations of prolamin protein in HMC were negatively correlated with in vitro starch degradation. Treating HMC with an exogenous protease could be a method to obtain greater potential for ruminal starch fermentation after a relatively short period of ensiling.