An Automated System for the Quantitative Determination of Proteolytic Enzymes Using Azocasein

Hazen, George G.; Hause, James A.; Hubicki, Joseph A.

doi:10.1111/j.1749-6632.1965.tb12620.x

Cited by 36 publications

(12 citation statements)

References 2 publications

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“…Azocaserin is a suitable substrate for the measurement of protease activity (Hazen et al, 1965). During digestion, the colored components are formed and soluble in trichloroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

“…During digestion, the colored components are formed and soluble in trichloroacetic acid. After removal of the undigested substrate, the color, which is proportional to the proteolytic activity of the enzyme, can be measured by a microplate reader (Hazen et al, 1965). Briefly, 150 μL of sterile supernatant of P. aeruginosa PAO1 and 250 μL of 2% azocasein (Sangon Biotech, China) in 50 mM Tris-HCl were incubated at 37°C for 4 h. The undigested substrate was precipitated with trichloroacetic acid (10%, 1.2 mL) for 20 min and then centrifuged at 12,000 rpm for 10 min, after which the supernatant was supplemented with 1.4 mL of 1 M NaOH.…”

Section: Methodsmentioning

confidence: 99%

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Anti-Biofilm and Antivirulence Activities of Metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa

Zhou

Chen

et al. 2017

Front. Microbiol.

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This study reported the efficacy of the metabolites of Plectosphaerella cucumerina, one phyllosphere fungus from Orychophragmus violaceus, against Pseudomonas aeruginosa quorum sensing (QS) and QS-regulated biofilms. The minimum inhibitory concentration (MIC) of the ethyl acetate (EtOAc) extract from P. cucumerina against P. aeruginosa PAO1 was 1.25 mg mL−1. At sub-MIC concentrations, P. cucumerina extract (0.25–1 mg mL−1) not only inhibited biofilm formation but also disrupted preformed biofilms of P. aeruginosa PAO1 without affecting its growth. Fluorescence and scanning electron microscope (SEM) showed architectural disruption of the biofilms when treated with P. cucumerina metabolites. Further investigation demonstrated that metabolites in P. cucumerina attenuated the QS-dependent virulence factors. LC-MS/MS spectra coupled with experimentally standard samples suggested that patulin and emodin might act as the principal components possessing anti-biofilm and antivirulence activities. This is the first report of (1) the isolation of P. cucumerina from the phyllosphere of O. violaceus and (2) anti-biofilm, antivirulence, and biofilm disruption activities of this fungus. Thus, this study provides fascinating new pathways for screening antipathogenic agents.

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“…Azocaserin is a suitable substrate for the measurement of protease activity (Hazen et al, 1965). During digestion, the colored components are formed and soluble in trichloroacetic acid.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Anti-Biofilm and Antivirulence Activities of Metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa

Zhou

Chen

et al. 2017

Front. Microbiol.

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“…Enzymatic activity was determined using azocasein (Sigma-Aldrich Co., St Louis, United States) as previously described [32]. The reaction mixture contained 100 μL of enzyme preparation and 300 μL of 10 g/L azocasein in 10 mM Tris buffer, pH 7.4.…”

Section: Enzymatic Activitymentioning

confidence: 99%

Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool

Lombardi

Spelzini

Corrêa

et al. 2016

Colloids and Surfaces B: Biointerfaces

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Structural changes of casein micelles and their aggregation induced by a novel enzymatic pool isolated from Bacillus spp. in the presence of calcium, magnesium or zinc were investigated. The effect of cations on milk protein structure was studied using fluorescence and dynamic light scattering. In the presence of cations, milk protein structure rearrangements and larger casein micelle size were observed. The interaction of milk proteins with zinc appears to be of a different nature than that with calcium or magnesium. Under the experimental conditions assayed, the affinity of each cation for some groups present in milk proteins seems to play an important role, besides electrostatic interaction. On the other hand, the lowest aggregation times were achieved at the highest calcium and zinc concentrations (15 mM and 0.25 mM, respectively). The study found that the faster the aggregation of casein micelles, the less compact the gel matrix obtained. Cation concentrations affected milk protein aggregation kinetics and the structure of the aggregates formed.

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“…Proteases (EC 3.4.21-24) activity was carried out using the method of Hanzen et al (1965). The reaction mixture contained 0.75 ml of the culture filtrate and 0.75 ml of 2% sulphanilamide azocasein (Sigma) in 0.2 M acetate buffer, pH 4.5 (acid proteases) or in Tris-HCl buffer, pH 7.5 (neutral proteases).…”

mentioning

confidence: 99%

Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi

Mucha

Dahm

Werner

2007

Antonie van Leeuwenhoek

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The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

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An Automated System for the Quantitative Determination of Proteolytic Enzymes Using Azocasein

Cited by 36 publications

References 2 publications

Anti-Biofilm and Antivirulence Activities of Metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa

Anti-Biofilm and Antivirulence Activities of Metabolites from Plectosphaerella cucumerina against Pseudomonas aeruginosa

Milk protein suspensions enriched with three essential minerals: Physicochemical characterization and aggregation induced by a novel enzymatic pool

Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi

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