Protease Preparation Research Articles

Mucoadhesive polymers in peroral peptide drug delivery. V. Effect of poly(acrylates) on the enzymatic degradation of peptide drugs by intestinal brush border membrane vesicles

The purpose of the study was to evaluate the inhibitory effect of the mucoadhesive poly(acrylates) polycarbophil and carbomer on the activity of proteolytic enzymes bound to the intestinal brush border. To that end, the degradation of a number of peptide drugs in the presence or absence of the poly(acrylates) was investigated, using rat brush border membrane vesicles (BBMV) as the protease preparation. Both carbomer and polycarbophil in concentrations of 0.25 and 0.5% (w/v) reduced rather weakly the enzymatic degradation of the peptide 9-desglycinamide, 8-arginine vasopressin (DGAVP), and only 0.5% (w/v) carbomer inhibited metkephamid degradation, but not polycarbophil. More pronounced inhibitory effects on DGAVP breakdown were found following a 30 min preincubation of the BBMV suspension with 0.5% (w/v) carbomer. However, the poly(acrylic acid) derivatives were unable to inhibit the degradation of buserelin at all. On the other hand, the polypeptide hormone insulin was remarkably stable in the BBMV preparations. In conclusion, the poly(acrylic acid) derivatives polycarbophil and carbomer show rather weak inhibitory effects on enzymes of the intestinal brush border cell membranes responsible for DGAVP and metkephamid degradation.

Prediction ofin situ Rumen Escape Protein fromin vitroIncubation with Protease fromStreptomyces griseus

Research was performed to determine in vitro the amount of rumen escape protein in mainly concentrate feedstuffs by incubation with a proteolytic enzyme preparation of Streptomyces griseus, with 24 feed samples with known in situ crude protein degradation characteristics. A close correlation (R 2 = 0.75) was found between the percentage of escape protein, calculated from the in situ and the in vitro methods. However, the percentage escape protein in situ could be estimated with a greater accuracy (R 2 = 0.80) using the undegraded fraction determined in vitro after 24 h incubation. Expressing escape protein as g kg -1 DM instead of a percentage, further improved the correlation with the amount of escape protein in situ (R 2 = 0.95). The amount of escape protein in situ could be estimated with similar accuracy from the undegraded crude protein fraction determined in vitro after 24 h incubation (R 2 = 0.92). Estimation of the amount of escape protein in situ did not improve with pre-incubation of the samples with amylase and carbohydrate degrading enzymes.

The effects of protease and carbohydrase supplementation on the nutritive value of canola meal for poultry: In vitro and in vivo studies

An in vitro incubation system was used to assess a variety of enzyme preparations regarding activities toward protein and carbohydrate components in canola meal with the objective of identifying those preparations that possessed potential for improvement of the nutritive value of canola meal. Promising preparations were evaluated further in 2-week growth trials utilizing 4-day-old broiler chickens. Enhanced protein hydrolysis was demonstrated for several of the protease enzyme preparations studied in the in vitro incubation system. Protein hydrolysis was most effective when either pancreatin or pronase were included in the incubation medium along with the protease enzyme and for the most effective protease preparations values for percent of total protein hydrolyzed exceeded those for pancreatin or pronase acting alone. The most effective protease preparation also resulted in improved broiler chick growth performance when added to semipurified canola meal diets. While some carbohydrase enzyme preparations were shown to be effective in the in vitro test of cell wall polysaccharide solubilization, only a trend toward improved growth performance was noted when broiler chickens were fed semipurified canola meal diets containing these enzyme preparations. A synergistic response in growth of young (4–11 days of age) broiler chickens was noted when phytase, carbohydrase and protease enzymes were added to a wheat/canola meal based diet deficient in available phosphorus.

Debridement of porcine burns with a highly purified, ananain-based cysteine protease preparation.

A novel enzymatic debriding agent was evaluated on experimental full-thickness porcine contact burns. This agent consists of a highly purified, ananain-based, cysteine protease preparation formulated in a hydrophilic cream vehicle. Debridement of full-thickness burns was found to be dependent on several factors including the concentration of enzyme in the vehicle, the duration of treatment, and the hydration status of the burn wound before treatment. With an optimized debridement regimen, burns were consistently debrided of all gelatinized tissue with two 5-hour treatments. Histologic evaluation of the debrided wounds revealed an acellular deeper dermis that was debrided of necrotic cellular debris; however, the collagen matrix of the deeper dermis remained intact. This observation was consistent with a demonstrated in vitro specificity of the ananain-based protease for gelatin over collagen. A direct comparison of debridement efficacy with sutilains ointment, showed the ananain-based, debriding enzyme preparation to provide more rapid debridement of gelatinized tissue. Enzymatically debrided wounds exhibited graft take only after surgical excision of approximately 1 mm of the remaining acellular, avascular dermis. This highly purified enzyme preparation offers the potential for rapid nonsurgical debridement of gelatinized burn tissue, but required additional surgical debridement for graft take in this porcine model.

Stable expression of vicilin fromVicia faba with eight additional single methionine residues but failure of accumulation of legumin with an attached peptide segment in tobacco seeds

Two different attempts have been undertaken to improve the amino acid composition of storage proteins from field bean (Vicia faba) by genetic engineering. First, legumin was modified to generate a new peptide sequence at the C-terminus containing 4 methionine residues. Second, vicilin was modified by generating 8 single methionine residues distributed over the peptide sequence. The genes were expressed in different systems includingin vitro transcription and translation and stable transformation into tobacco. The modified legumin was found to be unstable when expressed in tobacco seeds. Although specific mRNA was detected on RNA gel blots, no protein could be found by using protein gel blotting and ELISA. Furthermore, a protease preparation able to process the original legumin precursorin vitro degraded the modified legumin precursor. Contrary, the modified vicilin was accumulated in seeds of tobacco transformed with the gene under the control of the seed specific USP promoter. Both the original and the modified vicilin could be detected on protein gel blots at the expected position. Two-dimensional electrophoresis was employed to analyse the expression of original vicilin. Three vicilin-specific products of almost equal size were observed, indicating a slight modification leading to a change of pI. Quantitative determination using competitive ELISA showed that there is no significant difference in accumulation between original and modified vicilin. In both cases, three plants were found with vicilin amounts in the range of 1–3% of total globulin.

Proteins in white wine, II: Their resistance to proteolysis is not due to either phenolic association or glycosylation

Soluble wine proteins were separated by anion exchange chromatography. When the fractionated proteins were treated in model wine solution with an active peptidase preparation over six weeks at 15°C, all but one of the separated proteins were totally resistant to proteolysis. Because all the separated proteins contained no associated phenolic compounds and because, from neutral sugar analysis, most contained less than one mole of carbohydrate per mole of protein, neither phenolic association nor glycosylation are responsible for their proteolytic resistance.

Evaluation of digestive proteinases from the Antarctic krill Euphasia superba as potential chemonucleolytic agents. In vitro and in vivo studies.

Chemonucleolysis is a therapeutic procedure whereby a degradative enzyme is injected intradiscally to reduce disc height/width by depolymerisation of extracellular matrix components. This process is considered to diminish disc pressure on inflamed nerve roots, resulting in the alleviation of sciatic pain. In the present study two krill (Euphasia superba) enzyme preparations, a proteinase and an esterase preparation, were evaluated for their potential as chemonucleolytic agents. Initially, their ability to degrade several protein (azocoll, casein, proteoglycans, PGs) and peptide (CBZ-arg-4-nitroanilide, CBZ-lys-thiobenzyl ester) substrates was assessed in vitro. The krill proteinase preparation rapidly converted azocoll, casein and PGs to small peptides. Furthermore, when this degradative enzyme preparation was evaluated in vivo, a relatively low intradiscal dose (0.54 mg/disc) was found to reduce intervertebral disc widths in beagles to 48% +/- 10.5% (mean +/- SEM) of their pre-injection values within 2 weeks of administration. Moreover, the discs injected with this proteinase had reconstituted up to 80% +/- 9% (mean +/- SEM) of their pre-injection widths at the termination of the experiment (32 weeks). These data suggest that the krill protease preparation has potential as a chemonucleolytic agent which would allow disc matrix reconstitution. Conversely, the krill esterase preparation also degraded PGs, but into relatively large fragments. This limited digestion of PGs indicates that the krill esterase would be a less effective chemonucleolytic agent than the corresponding proteinase.

Zymogen Activation in Pancreatic Endoproteolytic Preparations and Influence on Some Whey Protein Hydrolysate Characteristics

ABSTRACTA proteolytic preparation from porcine pancreas was isolated. Trypsin, chymotrypsin and elastase were characterized and their time‐dependent stability at 37°C was studied. The supematant of a 30% (w/v) saturated ammonium sulfate precipitation of a pancreatic extract (30S) was developed to pilot‐scale level. The influence of zymogen activation time on molecular characteristics of whey protein hydrolysates produced by 30S and the commercial pancreatic preparation Corolase PP were compared. Amino acid analysis and gel permeation chromatography were used to characterize lactalbumin hydrolysates produced. Physicochemical characteristics of whey protein hydrolysates could be altered by manipulation of zymogen activation conditions in pancreatic proteinase preparations to be used during subsequent protein hydrolysis.

The properties of subtilisin, Novo type, immobilized on porous glass

AbstractStudies of the properties of subtilisin, Novo type, immobilized on porous glass with the aid of hexamethylene diisocyanate were carried out. The immobilized proteinase preparation shows optimum activity at a pH value of 10.7 and at a temperature between 60–65°C. It was stable in a wider range of pH and temperature values than the native subtilisin. The KM values for hemoglobin and BAEE were 9.2 × 10−5 [M] and 139 × 10−5 [M], respectively. Under relatively non‐aqueous conditions, immobilised subtilisin was able to synthesize phenylacetic acid ethyl ester.

Proteolytic and Peptidolytic Activities in Commercial Pancreatic Protease Preparations and Their Relationship to Some Whey Protein Hydrolyzate Characteristics

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProteolytic and Peptidolytic Activities in Commercial Pancreatic Protease Preparations and Their Relationship to Some Whey Protein Hydrolyzate CharacteristicsMargaret M. Mullally, Daniel M. O'Callaghan, Richard J. FitzGerald, W. J. Donnelly, and John P. DaltonCite this: J. Agric. Food Chem. 1994, 42, 12, 2973–2981Publication Date (Print):December 1, 1994Publication History Published online1 May 2002Published inissue 1 December 1994https://doi.org/10.1021/jf00048a062RIGHTS & PERMISSIONSArticle Views488Altmetric-Citations57LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Preliminary characterisation of antigens recognised by monoclonal antibodies raised to Porphyromonas gingivalis and by sera from patients with periodontitis.

A panel of 15 monoclonal antibodies (MAbs) was raised to Porphyromonas gingivalis and used to characterise the antigens which they recognise by ELISA, immunofluorescence (IF), Western blotting and patient serum inhibition studies. All the MAbs were specific for P. gingivalis and did not recognise 24 other species. Eight MAbs gave bright ring-like staining patterns against the majority of serotypes of P. gingivalis tested by IF. The antibodies could be grouped into 5 different antigen recognition profiles on Western blotting, and ELISA indicated that 8 of them bound to a capsular extract. Five antibodies bound to antigenic determinants recognised by sera from patients with periodonitis and 4 of these (1A1, 2B/H9, 3B1, 7D5) bound to a P. gingivalis 47kDa protease preparation on Western blotting. These antibodies are potentially useful for the purification and characterisation of biologically active components of P. gingivalis that are recognised by sera from patients with periodontitis.

The Use of Lipolytic and Proteolytic Enzymes in the Manufacture of Manchego Type Cheese from Ovine and Bovine Milk

The objective was to study the effect of two fungal lipases (Palatase 200 from Mucor miehei and Palatase 750L from Aspergillus niger), alone and combined with a fungal protease from Aspergillus oryzae (MKC fungal protease), and a bacterial neutral protease from Bacillus subtilis (Neutrase) on the maturation of Manchego type cheese to determine the practical application of these enzymes in cheese manufacture. The addition of both lipases, alone or in combination with the proteases, increased lipolysis. The M. miehei lipase affected mainly the longchain fatty acids of cheese, and the A. niger lipase affected the short- and long-chain fatty acids equally. The B. subtilis protease preparation increased lipolysis significantly, either by contamination of the enzymatic solution or by a synergetic effect with the added A. niger lipase. The protease from A. oryzae had the opposite effect on lipolysis, significantly decreasing the release of fatty acids by either partial proteolytic digestion or inhibition of the lipase. The combination of proteases and lipases increased proteolysis as measured by indexes of soluble N and HPLC of free amino acids. The PAGE of the casein fractions showed the intense breakdown of β-caseins but not of αs-caseins. The exclusive addition of lipases decreased proteolysis. Sensory analysis showed a slight but significant acceleration of ripening (16%) for the cheeses treated with A. niger lipase at the beginning of aging. The M. miehei lipase induced the appearance of soapy flavor in the cheeses because of the excessive release of long-chain fatty acids. The cheeses containing the B. subtilis protease developed bitterness and sticky and crumbly texture because of the intense breakdown of β-casein.

Cleavage of pseudomonas exotoxin and diphtheria toxin by a furin-like enzyme prepared from beef liver.

Pseudomonas exotoxin (PE) is cleaved within mammalian cells between Arg279 and Gly280 to generate an enzymatically active COOH-terminal fragment of 37 kDa which translocates to the cytosol and ADP-ribosylates elongation factor 2. A protease, with toxin cleaving activity, was prepared from beef liver and subsequently characterized. After achieving a 500-fold enrichment in several chromatographic steps, a soluble form of this protease was identified as a furin-like enzyme. It cleaved PE on the COOH-terminal side of the sequence of RQPR (amino acids 276-279) producing the same fragments as those generated within cells. Cleavage had a pH optimum of 5.0-5.5, was inhibited by EDTA or p-hydroxymercuribenzoate but not by O-phenanthroline,N-ethylmaleimide, trans-epoxysuccinyl-L-leukcylamido-(4-guanidino)-butane, or PMSF (or other well known inhibitors of serine proteases). The beef protease cleaved PE with an apparent Km of 7 microM. A mutant form of PE, PEala281, was cleaved at the same site, with the same pH optimum, a similar Km (9 microM) but with a Vmax 150 times faster than was seen with the native toxin. Mutational analysis of the amino acids located just before the site of cleavage, confirmed the importance of arginines at P-1 and P-4. It was also noted that the introduction of a dibasic pair at 278-279 did not increase toxicity or appreciably improve the rate of cleavage. Unnicked diphtheria toxin (DT) was also cleaved by the beef protease; cleavage was on the COOH-terminal side of the sequence RVRR (amino acids 190-193), was seen at pH values ranging from 5.5 to 8.5 and had an optimum at pH 8.0. Recombinant furin cleaved PE, PEala281, and DT with the same characteristics as the beef protease. In addition, Western blot analysis revealed that anti-furin antibodies reacted specifically with components in the beef protease preparation. Immunodepletion experiments showed that all toxin-cleavage activity could be removed from the beef protease using anti-furin antibodies. The relevance of furin-mediated cleavage was further assessed by adding nicked toxins to intact cells. Nicked PE and DT both killed cells at a faster rate than their unnicked counterparts.

A Radiometric Assay for Ras-Processing Peptidase Using an Enzymatically Radiolabeled Peptide

A simple and sensitive radiometric assay for the peptidase involved in the post-translational processing of p21ras proteins at the carboxy-terminal Cys-aliphatic-aliphatic-any amino acid (CAAX) motif is described. An isoprenylated tetrapeptide substrate, N-acetyl-S-[3H]farnesyl-Cys-Val-Ile-Ser-OH (22-27 Ci/mmol), was synthesized from N-acetyl-Cys-Val-Ile-Ser-OH and commercial [3H]farnesyl pyrophosphate via farnesyltransferase. The isoprenylated tetrapeptide was then used at a concentration (0.3 μM) well below Km (6 μM) in assays with a microsomal preparation of Ras-processing peptidase from bovine liver. Under assay conditions, the peptidase reaction followed first order kinetics with respect to the substrate, allowing the IC50 values for alternative substrates and inhibitors to approximate Km and Ki values, respectively. In a further simplification, substrate and N-acetyl-S-[3H]farnesyl-Cys-OH product were separated by thin-layer chromatography on silica gel plates using chloroform:acetic acid:methanol:acetone (60:5:10:20, v/v) as solvents. The assay does not require costly, specialized equipment and provides easy means for screening potential substrates and inhibitors of Ras-processing peptidase.

Porphyromonas gingivalis Trypsin-Like Protease: A Possible Natural Ligand for the Neutrophil Formyl Peptide Receptor

Porphyromonous gingivalis is a periodontopathic Gram-negative anaerobe associated with chronic adult periodontitis. P. gingivalis proteases are considered important virulence factors in the pathogenesis of periodontal diseases. In addition, defective bactericidal activity of neutrophils has also been observed in periodontitis. In this report we describe the effects of trypsin-like protease(s) secreted from P. gingivalis cells on the ligand binding of FMLP receptor on neutrophils. It was observed that trypsin-like protease(s) from P. gingivalis stimulate neutrophils by means of superoxide anion production. Subsequently, the proteases were found to cleave the FMLP receptor protein as evident by direct labeling of the FMLP receptor molecule. These results suggest that typsin-like protease(s) secreted from P. gingivalis cells contribute to attenuate the bactericidal activity of neutrophils by cleaving the polypeptide chain of the FMLP receptor molecule. The finding that neutrophils after the incubation with P. gingivalis released protease preparation fail to respond to further stimulation by FMLP suggests that P. gingivalis trypsin-like protease(s) may be a possible ligand for the FMLP receptor.

Review of the Problems Involved in Using Enzymes in Blood Group Serology - Provision of Freeze- Dried ICSH/ISBT Protease Enzyme and Anti-D Reference Standards

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Investigation on Assay for the Proteolytic Activity of Protease and Papain Preparations for Food Manufacturing

Investigation on Assay for the Proteolytic Activity of Protease and Papain Preparations for Food Manufacturing

Ultrafiltration of technical proteolytic enzymes

The possibility of using an ultrafiltration technique to improve the quality of the commercial, technical proteolytic enzyme preparation, Proteopol BP-S was studied. Membranes synthesized from polyacrylonitrile in the shape of tubes were selected and the optimum or limiting vlaues of the operational parameters of the process, namely transmembrane pressure, flow velocity of the solution tangentially to the membrane and temperature, were determined.

鶏卵黄からの酸性プロテアーゼ製剤処理による黄卵油製法

鶏卵黄からの酸性プロテアーゼ製剤処理による黄卵油製法

Safety evaluation of esperase

Esperase is a proteolytic enzyme preparation that can be used as a processing aid in the food industry. The following studies were performed to establish safety for the consumer: oral toxicity study (13 wk) in the rat; teratogenicity study in the rat; gene mutation assays in Salmonella typhimurium and mammalian cells in vitro, and chromosome aberration assay in vitro. General toxicity was low; the effects seen were attributed to proteolytic activity and the loading with sodium chloride. Neither of these factors will be relevant to consumers of the processed food. There was no evidence of effects on pregnancy outcome or mutagenic potential. When these results are considered together with existing knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, they indicate that Esperase will be safe for its intended application in food processing.