Evaluation of bovine rennets in terms of absolute concentrations of chymosin and pepsin A

1981 ◽  
Vol 48 (3) ◽  
pp. 447-456 ◽  
Author(s):  
Patrice Martin ◽  
Jean-Claude Collin ◽  
Pascaline Garnot ◽  
Bruno Ribadeau Dumas ◽  
Germain Mocquot
Keyword(s):  
Proteolytic Activity ◽  
Skim Milk ◽  
Active Enzyme ◽  
Clotting Time ◽  
Effect Of Ph ◽  
Absorbance Difference ◽  
Absolute Concentrations ◽  
Synthetic Hexapeptide ◽  
Optimal Ph

SummaryA method for determining chymosin and bovine pepsin A in commercial extracts of bovine veils, based on the use of the synthetic hexapeptide (Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe) as reference substrate, is reported. Chymosin and bovine pepsin A were separated chromatographically from extracts and assayed for clotting activity on a reconstituted skim-milk standardized with reference chymosin and bovine pepsin A, themselves standardized in relation to the hexapeptide. The effect of pH on the absorbance difference between the hexapeptide and the Leu-Ser-Phe(NO2) tripeptide resulting from its hydrolysis was studied. It was found that the ‘optimal’ pH for determining the activities of the reference enzyme solutions was 4·7.Six chymosin and 3 bovine pepsin A preparations were assayed on the hexapeptide to define the relation between the proteolytic activity and the amount of active enzyme. At pH 4·7 and 30 °C 1 mg chymosin and 1 mg bovine pepsin A hydrolysed 100 and 2700 μm-peptide/s respectively. The clotting activity of these preparations was assayed on a reconstituted skim-milk to standardize it and thus define the relation between the clotting time and the amount of active enzyme. Chymosin had a specific clotting activity twice that of bovine pepsin A. At equal clotting activities, bovine pepsin A was 55 times more active than chymosin on the hexapeptide at pH 4·7.

scholarly journals Chromatographic Characterization and In Vitro Bioactivity Evaluation of Lactobacillus helveticus Hydrolysates upon Fermentation of Different Substrates

2021 ◽  
Vol 11 (2) ◽  
pp. 811
Author(s):  
Federica Ianni ◽  
Alessandra Anna Altomare ◽  
Beniamino T. Cenci-Goga ◽  
Francesca Blasi ◽  
Luca Grispoldi ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Proteolytic Activity ◽  
Bioactive Peptides ◽  
Biological Activities ◽  
Skim Milk ◽  
Brain Heart Infusion ◽  
Starter Cultures ◽  
Lactobacillus Helveticus ◽  
Food Sources

Among various food sources, milk proteins remain the major vector for functional peptides endowed with several biological activities. Particularly, the proteolytic activity of lactic acid bacteria during milk fermentation has been one of the most followed strategies to produce bioactive peptides. In the present study, the exploration of the activity of several starter cultures, at different fermentation times, was firstly investigated by reversed phase-high performance liquid chromatography. Among the tested strains, Lactobacillus helveticus showed a higher proteolytic activity and it was submitted to further investigations by changing the fermentation substrate (skim milk, brain heart infusion, peptone water) as well as the extraction strategy (trichloroacetic acid vs. glass beads). The chromatographic analyses and the in vitro antioxidant and antihypertensive assays highlighted considerable differences for L. helveticus hydrolysates from different substrates, while a negligible impact by the two extraction protocols emerged. Furthermore, nano-high pressure liquid chromatography coupled with a high resolution mass spectrometry analyzer allowed the preliminary discrimination of fractions from fermented skim milk, likely responsible for the found activity. The obtained results suggest the possibility of varying the fermentation parameters in order to maximize the functional effects of the bioactive peptides.

Effect of pH on rennet clotting properties of CO2-acidified skim milk

2004 ◽  
Vol 14 (5) ◽  
pp. 437-443 ◽  
Author(s):  
C. Guillaume ◽  
E. Gastaldi ◽  
J.-L. Cuq ◽  
S. Marchesseau
Keyword(s):  
Skim Milk ◽  
Effect Of Ph

New insights into the effect of pH on the mechanism of ofloxacin electrochemical detection in aqueous solution

2019 ◽  
Vol 21 (29) ◽  
pp. 16282-16287 ◽  
Author(s):  
Ting Liu ◽  
Qiang Xue ◽  
Jianbo Jia ◽  
Fei Liu ◽  
Shengzhang Zou ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Electrochemical Detection ◽  
Practical Application ◽  
Effect Of Ph ◽  
Optimal Ph

We first found that identifying the protonation status and polarity of the target pollutant helped to rapidly find an optimal pH condition for its electrochemical detection, which promoted practical application.

Influence of chromatographically pure bovine chymosin and pepsin A on cheese curd syneresis

1990 ◽  
Vol 57 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Hans Andersson ◽  
Anders Andrén
Keyword(s):  
Milk Powder ◽  
Skim Milk ◽  
Raw Milk ◽  
Skim Milk Powder ◽  
Clotting Time ◽  
Experimental Conditions ◽  
Cheese Curd ◽  
Light Absorbance

SummaryThe influence of chromatographically pure bovine chymosin and pepsin A on cheese curd syneresis has been studied. The enzymes were compared in experiments using a microscale syneresis assay, where the light absorbance of the released whey was used as a measurement of the syneresis. The following parameters were investigated: reconstituted skim milk powder (RSM), bulk raw milk, CaCl2 addition and cutting time of the curd. The gelling (clotting) time was held constant under all experimental conditions to eliminate its influence on syneresis. The differences in influence on syneresis of the two enzymes were neither obvious nor uniform. As regards the syneresis of RSM, the influence of chymosin and pepsin seemed to be very similar, while the syneresis of bulk raw milk seemed to be more enhanced by chymosin. However, results were not sufficiently clear to prove any distinct differences between the enzymes. As long as the cheese curd is cut at a proper firmness, there are probably no practical problems in using bovine rennets with varying chymosin: pepsin ratio.

scholarly journals THE ENHANCEMENT OF CHLOROFORM-INDUCED PLASMA PROTEOLYTIC ACTIVITY BY EPSILON AMINOCAPROIC ACID

1962 ◽  
Vol 115 (4) ◽  
pp. 695-706 ◽  
Author(s):  
Virginia H. Donaldson ◽  
Oscar D. Ratnoff
Keyword(s):  
Ionic Strength ◽  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Proteolytic Enzyme ◽  
Aminocaproic Acid ◽  
Heat Stability ◽  
Hydrolytic Activity ◽  
Casein Hydrolysis ◽  
Epsilon Aminocaproic Acid ◽  
Optimal Ph

The proteolytic activity in chloroform-treated plasma euglobulins has been attributed to plasmin. Plasmin can digest both casein and fibrin. Epsilon aminocaproic acid, which inhibits the activation of plasminogen, the precursor of plasmin, by streptokinase, urokinase, and tissue activators enhanced the development of casein hydrolytic activity in a mixture of chloroform and plasma euglobulins. Fibrinolytic activity was also enhanced, but this was evident only if the epsilon aminocaproic acid was removed from the chloroform-treated euglobulins prior to assay. The reasons for the paradoxical enhancement of chloroform-induced casein hydrolysis by euglobulins containing epsilon aminocaproic acid are unclear. However, studies of optimal pH, heat stability, and the effect of ionic strength on the activation of the precursor of this proteolytic enzyme do not differentiate it from plasminogen.

scholarly journals Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Bláithín Maunsell ◽  
Claire Adams ◽  
Fergal O'Gara
Keyword(s):  
Pseudomonas Fluorescens ◽  
Proteolytic Activity ◽  
Yeast Extract ◽  
Sigma Factor ◽  
Skim Milk ◽  
Protease Production ◽  
Iron Starvation ◽  
Transcription Analysis ◽  
Skim Milk Agar ◽  
Complex Regulation

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

In Vitro Studies on a Proteolytic Enzyme from Aspergillus Oryzae (Protease I)

1968 ◽  
Vol 19 (01/02) ◽  
pp. 136-144 ◽  
Author(s):  
D Ogston ◽  
C. M Ogston
Keyword(s):  
Proteolytic Activity ◽  
Fibrinolytic Activity ◽  
Proteolytic Enzyme ◽  
Thrombolytic Agent ◽  
Inhibitory Effect ◽  
In Vitro Studies ◽  
Clotting Time ◽  
Proteolytic Action ◽  
Fibrinolytic Action

Summary1. Protease I was found to have potent fibrinolytic activity in concentrations which exceeded the blood inhibitory capacity when tested on fibrin plates and artificial thrombi.2. Plasma inhibited the proteolytic activity of protease I to a greater extent than serum; serum had a greater inhibitory effect on protease I than on plasmin. Trasylol did not inhibit the proteolytic action of protease I.3. Protease I caused the slow formation of fibrin in plasma in concentrations which did not produce fibrinogenolysis; this effect was seen in Al(OH)3-adsorbed plasma, and was not inhibited by heparin. Protease I also shortened the recalcified plasma clotting time.4. The fibrinogenolytic action of protease I was more rapid than its fibrinolytic action both in the presence and absence of plasma inhibitors. No concentration of protease I lysed fibrin in plasma without prior destruction or conversion to fibrin of the surrounding plasma fibrinogen.5. It is concluded from these in vitro studies that protease I does not have the properties necessary for a satisfactory thrombolytic agent.

scholarly journals Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

2009 ◽  
Vol 75 (8) ◽  
pp. 2506-2516 ◽  
Author(s):  
Tanja Waschkowitz ◽  
Stephanie Rockstroh ◽  
Rolf Daniel
Keyword(s):  
Proteolytic Activity ◽  
Signal Sequence ◽  
Skim Milk ◽  
Environmental Dna ◽  
Average Insert Size ◽  
Extracellular Proteases ◽  
Insert Size ◽  
T4 Dna Ligase ◽  
Metagenomic Libraries ◽  
Isolation And Characterization

ABSTRACT Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per μg of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.

Qualitative and quantitative determination of proteolysis in mastitic milks

1982 ◽  
Vol 49 (4) ◽  
pp. 587-596 ◽  
Author(s):  
Olivier de Rham ◽  
Anthony T. Andrews
Keyword(s):  
Proteolytic Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Skim Milk ◽  
Considerable Proportion ◽  
Freezing And Thawing ◽  
Protein Fragments ◽  
Whole Milk ◽  
Proteinase A ◽  
Mastitic Milk ◽  
Induced Changes

SUMMARYProteolytic activity in mastitic skim-milk was often 5–10 fold higher than in normal milk, its level being related to somatic cell count but not precisely correlated with it. In milks with the highest levels of activity plasmin accounted for about one third of the total proteinase. A further third was sedimented with the micellar fraction together with the plasmin, but unlike plasmin, was not inhibited by addition of soyabean trypsin inhibitor (SBTI). The final third remained in the serum phase.Polyacrylamide gel electrophoresis (PAGE) showed that αsl- and β-caseins were degraded at about the same overall rate. The plasmin produced the usual readily identified fragments from β-casein, but incubation of mastitic milk also produced changes in patterns in the γ-casein region differing from plasmin-induced changes which were also apparent when the micellar fraction was incubated. As they were inhibited by SBTI, a second trypsin-like enzyme in addition to plasmin may also have been present. Other proteinase(s) not inhibited by SBTI was also associated with casein micelles and produced at least 3 characteristic protein fragments seen on PAGE. The serum phase proteinase(s) was likewise not inhibited by SBTI, and did not produce any well-defined electrophoretic bands, suggesting a rather non-specific breakdown of caseins. After separation of mastitic whole milk, a considerable proportion of the proteolytic activity was found in the cream phase. The proportion was enhanced by freezing and thawing, and the enzyme appeared to be identical to the SBTI-resistant micellar proteinase.Because of the considerable proteolysis likely to occur under the time and temperature conditions involved, our results may provide some explanation for the problems encountered in cheesemaking with mastitic milks (e.g. yield losses, poor curd strength and off-flavour development).

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