A Colorimetric Method for the Determination of the Proteolytic Activity of Duodenal Juice

Charney, Jesse; Tomarelli, R. M.

doi:10.1016/s0021-9258(17)41059-3

Cited by 616 publications

(63 citation statements)

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“…The total activity of the degrading proteins in the cultivation medium was analyzed by azocasein assay. The measurement method was adapted from Charney and Tomarell ( 1947 ) and applied by Baur et al, 2015 . In detail, 100 µl of cell‐free supernatant was mixed with an equal volume of a pre‐warmed (40℃ for 5 min) azocasein solution (5 g/L, pH 7, dissolved in H 2 O dd ) and subsequently incubated for 1 h at 37℃ and 1.07 g .…”

Section: Methodsmentioning

confidence: 99%

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

et al. 2021

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Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B . subtilis strains JABs24 and DSM10 T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho‐transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B . subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14‐fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B . subtilis JABs24, a lipopeptide‐producing derivative of strain 168, and B . subtilis wild‐type strain DSM10 T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ , a deletion mutant was constructed for DSM10 T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain‐specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10 T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10 T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.

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Section: Methodsmentioning

confidence: 99%

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

et al. 2021

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“…The proteolytic activity was quantified using azocasein (Sigma, St. Louis, MO) as substrate. 18 Thus, 500 μl of crude extract containing the proteases and 500 μl of 6 mg/ml azocasein were mixed in 50 mM Tris-HCl buffer (pH 7.5) at 37 C for 20 min. Then, 500 μl of 10 wt%/vol% trichloroacetic acid was added to stop the reaction.…”

Section: Quantification Of Enzymatic Activitymentioning

confidence: 99%

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates

Pillaca-Pullo

Intiquilla

Santos

et al. 2020

Biotechnology Progress

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Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 2 3 + 4 were performed to evaluate the following parameters: molar mass of PEG (M PEG ), concentration of PEG (C PEG ), concentration of citrate buffer (C Cit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of M PEG = 10,000 g/mol, C PEG = 22 wt%, C Cit = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercialprotease Alcalase ® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase ® (1.40 and 3.47 μmol Trolox equivalent/mg protein, for 2,2 0 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 μmol Trolox/mg protein). Nevertheless, the IC 50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

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“…The proteolytic activity was determined by the method described by Charney and Tomarelli (1947) with some modifications, using the azocasein as substrate. For this, 500 μL of the crude extract was incubated with 500 μL of azocasein solution (0.5%) (Sigma Aldrich) in 50 mM sodium acetate buffer, pH 5.0, for 40 min.…”

Section: Proteolytic Activitymentioning

confidence: 99%

Production of fibrinogenolytic and fibrinolytic enzymes by a strain of <i>Penicillium</i> sp. isolated from contaminated soil with industrial effluent

Baggio

Panagio

Gasparin

et al. 2019

Acta Sci. Health Sci.

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Cardiovascular diseases associated with thrombosis are one of the main causes of death all around the world. Urokinase, streptokinase, and tissue plasminogen activator are the major thrombolytic agents used to treat thrombosis. However, the fact that these agents have several side effects and high prices has driven the search for safer and more economically viable compounds for the treatment of cardiovascular diseases. Thus, the aim of this study was to evaluate the potential of fungi isolated from industrial effluents to produce fibrino(geno)lytic enzymes. The selection of the protease-producing strains showed that only the BF20 strain was able to produce proteolytic halos in milk agar. This strain identified as belonging to the genus Penicillium was cultivated in submerged fermentation. Different media composition were tested to evaluate proteolytic activity, and the results showed that the medium containing 0.1% yeast extract and 1% skim milk, pH 5.0, present higher azocaseinolytic activity (0.24 U mL-1 min.-1). This sample also showed the ability to degrade fibrinogen and fibrin after 15 and 120 min. of incubation, respectively. These results indicate that the BF20 strain has a thrombolytic potential, effectively degrading fibrinogen and fibrin, having great application in the health area.

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A Colorimetric Method for the Determination of the Proteolytic Activity of Duodenal Juice

Cited by 616 publications

References 0 publications

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates

Production of fibrinogenolytic and fibrinolytic enzymes by a strain of <i>Penicillium</i> sp. isolated from contaminated soil with industrial effluent

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