Total proteolytic, elastolytic and mucolytic activities were measured using fecal supernatants from mice. Non-specific proteolytic activity was determined using the substrate azocasein (Sigma-Aldrich) as previously described.13 (link) Fecal supernatants were incubated in 50 mmol/L Tris-HCl buffer pH 8.2 supplemented with 1 mmol/L CaCl2, 50 mmol/L NaCl, and Triton 0.25% (weight/volume (w/v)), and 0.5% (w/v) azocasein at 37ºC. The reaction was stopped by adding 10% trichloroacetic acid, and absorbance was measured at 366 nm. Elastolytic activity was analyzed using the substrate Suc-Ala3-pNa (Sigma-Aldrich). Fecal supernatants were incubated in the same buffer at 37ºC with the substrate. Absorbance at 410 nm was measured at several timepoints. Enzyme activity was determined by using a standard curve of pancreatic porcine elastase (Sigma-Aldrich). Mucolytic activity was determined by a bioassay using Brain Heart Infusion (BHI) media enriched with 0.05% (w/v) of mucin from porcine stomach type III (Sigma-Aldrich). Fecal supernatant was spotted into agar wells and incubated for 24 h. Zones of clearance were measured after staining agar plates with 10% amido black for 24 h. A standard curve was used to determine mucin-degrading activity of the samples using collagenase type I (0.25–1.0 FALGPA units/mg) from Clostridium histolyticum (Sigma-Aldrich).
Azocasein
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, China, Japan, Poland, Brazil
Azocasein is a colorimetric substrate used for the quantitative determination of protease activity. It is a modified casein protein that undergoes a color change when cleaved by proteolytic enzymes, allowing for the measurement of protease levels in a sample.
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Lab products found in correlation
Trichloroacetic acid, by Merck Group (11 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Elastin congo red, by Merck Group (7 mentions)
Casein, by Merck Group (7 mentions)
Trypsin, by Merck Group (5 mentions)
Bradford reagent, by Merck Group (4 mentions)
Bovine fibrinogen, by Merck Group (4 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Dithiothreitol (dtt), by Merck Group (4 mentions)
T4 dna ligase, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Ficin, by Merck Group (3 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (3 mentions)
Acrylamide, by Merck Group (3 mentions)
Gelatin, by Merck Group (3 mentions)
Glycine, by Merck Group (3 mentions)
O phenylenediamine, by Merck Group (3 mentions)
Synergy ht, by Agilent Technologies (3 mentions)
Bromelain, by Merck Group (3 mentions)
183 protocols using azocasein
1
Fecal Enzyme Activity Assays
2
Measuring SpeB Protease Activity
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SpeB protease activity was determined as previously described (Collin and Olsen, 2000 (link)). Briefly, overnight GAS cultures were diluted 1:50 into fresh THB media and cultured for 17 h at 37°C to early stationary phase. Cultures were centrifuged at 3,200 × g, and supernatants were filter sterilized. Equal volumes of filtered supernatant and activation buffer (1 mM EDTA, 100 mM sodium acetate, and 20 mM freshly prepared DTT) were mixed and incubated at 40°C for 30 min. 2% azocasein (Sigma) was dissolved in activation buffer and added to the activated supernatant in a 1:1 (v/v) ratio. The mixture was then incubated 1 additional h at 40°C. Excess azocasein was precipitated with the addition of trichloroacetic acid (Sigma) to a final concentration of 15% (w/v) and centrifuged for removal. Supernatants were transferred into a 96-well plate. Absorbance was measured at 366nm and normalized to wild-type levels to determine relative protease activity.
3
Azocasein Assay for Protease Activity
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Azocasein assay was performed to determine the protease activity of the bacterial isolates as described earlier (Syngkon et al., 2010).75 (link) Briefly, the overnight grown culture supernatants were mixed with 0.7% Azocasein (prepared in 100 mM Tris-HCl; pH 8.0) (Sigma, Cat# A2765) followed by incubation at 37° C for 1 h. The reaction was stopped using 10% Tri Chloro Acetic Acid (Sigma; Cat# T6399) and kept at 4° C for 30 min. Precipitate was removed by centrifugation (12,000 rpm for 10 min). NaOH (500 mM) was added to the supernatant and absorbance was measured at 440 nm. Nutrient broth and purified haemagglutinin protease (HAP) from Vibrio cholerae C6709 were used as negative and positive controls respectively.
4
Ficin Catalytic Activity Evaluation
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Ficin (F4165) from Ficus latex purchased by Sigma, Burlington, MA, USA was used as the research object. Azocasein and N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) supplied by Sigma Aldrich, Munich, Germany, were used as a hydrolysis substrate in the catalytic activity evaluation assays. Carboxymethyl cellulose sodium salt with Mw ~90 kDa and degree of substitution of 0.7 (Sigma Aldrich, Munich, Germany) was used as a carrier and in the graft copolymer synthesis. These products were utilized without any purification. N,N-dimethyl amino ethyl methacrylate (Sigma Aldrich, Munich, Germany) was previously distilled in a vacuum (Tb = 62–65 °C/11 mm Hg; n20D = 1.4395) before the carrier synthesis.
5
Quantifying Extracellular Protease Activity
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The Azocasein method (Andrejko et al., 2013 (link)) was utilized to demonstrate total extracellular protease production. Here, 100 μl SN was added to 100 μl of Azocasein (Sigma-Aldrich) solution (5 mg/ml in 0.1 M Tris–HCL, pH 8.8) and incubated at 37°C for 3 h. The reaction was stopped with 10% (v/v) trichloroacetic acid (25 μl per tube) and samples were centrifuged at 14,000 × g for 15 min at RT. To each well of a 96-well plate, 50 μl of 0.5 M NaOH was added to 50 μl of Azocasein SN in triplicate. NaOH was used as a blank, bacterial protease (Sigma-Aldrich) was included as a positive control, and absorbance was measured at 405 nm. For the Azocasein assay, one protease activity unit was defined as an absorbance increase (OD405 nm) of 0.02 per hour (Andrejko et al., 2013 (link)).
6
Protease Activity Assay Protocols
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Azocasein, phenyl methyl-sulfonyl fluoride (PMSF), tosyl phenylalanyl chloromethyl ketone (TPCK), tosyl lysine chloromethylketone (TLCK), E-64 (10 μM), ethylenediaminetetraacetic acid (EDTA), N-α-benzoyl-Arg p-nitroanilide (BApNA), N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), leucine p-nitroanilide (LpNA), p-Glu-Phe-Leu-pNA (pGPLpNA), Z-Arg-Arg-pNA (AApNA), calf thymus DNA and baker’s yeast RNA were purchased from Sigma-Aldrich, St. Louis, MO, (USA).
7
Enzymatic Activity Assay Protocol
8
Quantifying Protease Activity in Biofilms
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The proteases activity caused by extracellular enzymes produced by biofilm cells together with cells dispersed from the biofilm was evaluated using colorimetric assay based on digestion of chromogenic azocasein according to Das et al. (2016) (link). By the digestion, azopeptides are created which are soluble in trichloroacetic acid and can be measured spectrophotometrically. The cell suspension taken from the vicinity of the biofilm was centrifuged for 5 min at 10,000 g and the supernatant in a volume of 150 µl was mixed with 1 ml of 3 mg/ml azocasein (Sigma Aldrich, Czech Republic) dissolved in 50 mM Tris-HCl (pH 8 in demineralized water). The tubes were mixed and incubated at 37 °C and 150 rpm for 30 min. The enzymatic reaction was terminated by addition of 500 µl of 10% trichloroacetic acid. The tubes were mixed and centrifuged. The 100 µl aliquot of each supernatant was measured spectrophotometrically at 400 nm. The experiments were carried out in three independent repetitions.
9
Enzymatic Hydrolysis of Azocasein
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Bromelain (B4882), papain (P4762), and ficin (F4165) were purchased from Sigma-Aldrich, Germany and were used without any treatments. Azocasein (Sigma-Aldrich, Munich, Germany) was used as a hydrolysis substrate in catalytic activity evaluation experiments. Chitosan with molecular weights of 200, 350, and 600 kDa and a degree of deacetylation of 0.73–0.85 (Bioprogress, Shchelkovo, Russia), 4-acetylsulfanilyl chloride (Sigma-Aldrich, Munich, Germany), dimethyl sulfoxide, and acetone (both analysis grade, ReaKhim, Moscow, Russia) were applied for 2-(4-acetamido-2-sulfanilamide) chitosan synthesis.
10
Azocasein Assay for LasA Protease
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LasA protease production in the supernatants of baicalin-treated or untreated samples was evaluated using an azocasein assay [32 (link)]. For each sample, 150 μL of filtered supernatant was mixed with 250 μL of 2% azocasein (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM Tris-HCl buffer (pH = 7.8). The mixture was incubated at 4°C for 4 h. The reaction was terminated by the addition of 1.2 mL of 10% trichloroacetic acid followed by incubation at 4°C for 15 min and centrifugation at 10,000 rpm for 10 min. The supernatant was collected and mixed with 1.4 mL of 1 M NaOH. The relative protease activity was measured as the OD440 of the supernatant.
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