Detection of extracellular proteases from microorganisms on agar plates

Vermelho, Alane Beatriz; Meirelles, Maria de Nazareth Leal de; Lopes, Angela H.; Petinate, Simone Dias Gonçalves; Chaia, Andre Adriano; Branquinha, Marta H.

doi:10.1590/s0074-02761996000600020

Cited by 103 publications

(60 citation statements)

References 24 publications

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“…In this study, gelatin was used as a substrate as it is the most effective protease inducing substrate, have a high molecular weight protein, induces an increase in the protease production to degrade the substrate to an available form for the microorganism. Laila et al (1986) and Vermelho et al (1996) also detected the presence of extracellular proteases from microorganisms on agar plates. Michael et al (1980) recorded that protease activity was present in 98% of the clinical isolates of P. aeruginosa.…”

Section: Advances In Animal and Veterinary Sciencesmentioning

confidence: 98%

See 1 more Smart Citation

Extracellular Enzymes and Toxins of Pseudomonas aeruginosa Strains Isolated from Clinically Diseased Egyptian Cows

Younis¹

2015

Adv. Anim. Vet. Sci.

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Section: Advances In Animal and Veterinary Sciencesmentioning

confidence: 98%

“…After inoculation, the plates were incubated at 37°C and observed daily for ten days. Extracellular protease detection was done after staining Coomassie blue (0.25%, w/v) in methanol-acetic acid-water 5:1:4 (v/v/v) for 1 h at 28°C (Vermelho et al, 1996).…”

Section: Advances In Animal and Veterinary Sciencesmentioning

confidence: 99%

Extracellular Enzymes and Toxins of Pseudomonas aeruginosa Strains Isolated from Clinically Diseased Egyptian Cows

Younis¹

2015

Adv. Anim. Vet. Sci.

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“…To investigate the role of E. festucae VibA in the production of proteases, the ability of the E. festucae strains to produce extracellular proteases was detected on PDA medium, with gelatin as the substrate. Extracellular protease activity was detected as a visible halo around each colony of endophyte isolates (35). Within 10 days after inoculation, a visible halo (cleared area) around the colony was obvious for wild-type E437, while no halo was detected around the colony of the vibA mutant.…”

Section: Resultsmentioning

confidence: 99%

“…Extracellular protease activity of the epichloae isolates was detected as follows (35). A 3-mm mycelial plug from each isolate was separately placed in the center of a PDA plate supplemented with 1% (wt/vol) gelatin (Becton, Dickinson) and incubated at 23°C.…”

Section: Methodsmentioning

confidence: 99%

VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae

Niones

Takemoto

2015

Eukaryot Cell

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Symbiotic association of epichloae endophytes (Epichloë/Neotyphodium species) with cool-season grasses of the subfamily Pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. While the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little is known about the antibiosis mechanism against grass pathogens by the mutualistic endophyte. In this study, an Epichloë festucae mutant defective in antimicrobial substance production was isolated by a mutagenesis approach. In an isolated mutant that had lost antifungal activity, the exogenous DNA fragment was integrated into the promoter region of the vibA gene, encoding a homologue of the transcription factor VIB-1. VIB-1 in Neurospora crassa is a regulator of genes essential in vegetative incompatibility and promotion of cell death. Here we show that deletion of the vibA gene severely affected the antifungal activity of the mutant against the test pathogen Drechslera erythrospila. Further analyses showed that overexpressing vibA enhanced the antifungal activity of the wild-type isolate against test pathogens. Transformants overexpressing vibA showed an inhibitory activity on test pathogens that the wildtype isolate could not. Moreover, overexpressing vibA in a nonantifungal E. festucae wild-type Fl1 isolate enabled the transformant to inhibit the mycelial and spore germination of D. erythrospila. These results demonstrate that enhanced expression of vibA is sufficient for a nonantifungal isolate to obtain antifungal activity, implicating the critical role of VibA in antifungal compound production by epichloae endophytes. E pichloae endophytes (holomorphic Epichloë and anamorphic Neotyphodium) are clavicipitaceous fungi that maintain a systemic and constitutive symbiotic relationship with a broad spectrum of cool-season grasses of the subfamily Pooideae (1). In the mutualistic relationship between epichloae endophytes and coolseason grasses, the reported benefits include protection of the host plant from insect and vertebrate herbivores (2-5), resistance to diseases (6-9), and an increase in tolerance to abiotic stresses, such as drought (10, 11). The protective ability of some epichloae species makes them suitable agents of biological plant protection against economically important grass diseases and insect and small animal herbivores (12). This encouraged breeders to develop and eventually led to the release of "endophyte-enhanced" turfgrass and perennial ryegrass cultivars (13).The production and release of anti-insect metabolites are among the known mechanisms for how epichloae endophytes protect their hosts from insect herbivory. Epichloae endophytes in association with their grass hosts are noted to synthesize bioprotective alkaloids, such as peramine and lolines, and another class of compound, the janthitrems, which increase resistance of the plant hosts to insect feeding (2, 14-16). Moreover, the genetic basis of the biosynthesis of endophyte-derived anti-in...

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“…These methods usually vary in their simplicity, rapidity, sensitivity, range of detection and cost per test. The routinely used quantitative assay methods for protease activity involves the use of natural or synthetic substrates by employing spectrophotometric, fluorimetric, radiometric and enzyme-linked immunosorbent assay (ELISA) (Sumantha et al, 2006). An absorption spectroscopy is widely used for protease assay (Mokashe et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Protease Assay by Substrate-Agarose Plate Method

Mokashe¹,

Patil²

2016

J microb biotech food sci

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Proteases are unique class of industrial biocatalyst; occupy a key chair with respect to wide range of utility in both physiological and commercial sector. Repertoire of various enzyme assays were suggested for determining enzyme units of protease. Majority of currently reported assays are expensive, time consuming and cumbersome. In this direction, a development of speedy and sensitive quantitative enzyme assay for the characterization of proteases is highly desirable. The present investigation reported a quick method for characterization of proteases using single substrate-agarose plate method. A trypsin (obtained from bovine pancreas) and bacterial protease (serine protease of Bacillus circulans MTCC 7942) on subsequent interaction with proteinaceous substrate gives proteolytic zone on substrate-agarose plate. The clear zone diameter of proteolysis was directly proportional to enzyme units of protease. Furthermore, enzyme activities of protease treated with various chemical activator/inhibitor gives vis-à-vis response and yielded significant data. The biochemical characteristics of proteases are documented using rapid single substrate-agarose plate method. A rapid, sensitive and reliable improved quantitative protease assay using single substrate-agarose plate method could be used at academic, research and commercial level.

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Detection of extracellular proteases from microorganisms on agar plates

Abstract: We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin)

Cited by 103 publications

References 24 publications

Extracellular Enzymes and Toxins of Pseudomonas aeruginosa Strains Isolated from Clinically Diseased Egyptian Cows

Extracellular Enzymes and Toxins of Pseudomonas aeruginosa Strains Isolated from Clinically Diseased Egyptian Cows

VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae

Quantitative Protease Assay by Substrate-Agarose Plate Method

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