A dye-labelled soluble substrate for the assay of endo-chitosanase activity

Zitouni, Mina; Fortin, Mélanie; Thibeault, Jean-Sébastien; Brzeziński, Ryszard

doi:10.1016/j.carbpol.2009.12.012

Cited by 6 publications

(4 citation statements)

References 20 publications

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“…Chitin agar plate has been used earlier for isolating chitinolytic microorganisms and observing clear zone around the colony of microorganisms ((Wirth & Wolf 1990; Cody 1989); Rojas Avelizapa et al 2001). Previous attempt to synthesize a remazol brilliant blue (RBB) derivative of chitosan resulted in a relatively poorly soluble polymer (Fen et al 2006) which rendered difficult its application to direct screening of microbial colonies on agar media but soluble RBB-chitosan (lower molecular weight derivative) was particularly suitable for the detection of chitosanase positive organisms on agar media (Brzezinski et al 2010). However, these methods have low sensitivity and its results depend on concentration and size of the particles of colloidal chitin, thickness of the media and the amount and kinds of inoculum.…”

Section: Resultsmentioning

confidence: 99%

Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India

2012

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Chitin is the second most abundant polymer in nature after cellulose and plays a major role in fungal cell walls. As a producer of variety of chitinase enzymes Trichoderma has become an important means of biological control of fungal diseases. A simple and sensitive method based on the use of basal medium with colloidal chitin as sole carbon source supplemented with Bromo cresol purple (pH indicator dye) is proposed to evaluate large populations of Trichoderma for chitinase activity. The soluble substrate with pH indicator dye (Bromo cresol purple, BCP) for the assay of chitinase activity on solid media is sensitive, easy, reproducible semi-quantitative enzyme diffusion plate assay and economic option to determine chitinases. Colloidal chitin derived from Rhizoctonia cell wall and commercial chitin included as a carbon source in broth also allowed selection and comparison of chitinolytic and exochitinase activity in Trichoderma spectrophotometrically. Released N-acetyl-β--D-glucosamine (NAGA) ranged from 37.67 to 174.33 mg/ml and 37.67 to 327.67 mg/ml and p-nitrophenol (pNP) ranged from 0.17 to 35.78 X 10-3 U/ml and 0.62 to 32.6 X 10-3 U/ml) respectively with Rhizoctonia cell wall and commercial chitin derived colloidal chitin supplemented broth.

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Section: Resultsmentioning

confidence: 99%

Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India

2012

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“…Chitosanase activity was assayed by two methods. Rapid assay in culture supernatants and fractions eluted from chromatographic columns was performed using the soluble chitosan derivative sRBB-C as substrate (Zitouni et al 2010 ). For all other experiments, chitosanase activity was measured by the release of reducing sugars from chitosan substrate with d -glucosamine as standard by the method of Lever ( 1972 ), using the p -hydroxybenzoic acid (PAHBAH) reagent as modified by Schep et al ( 1984 ).…”

Section: Methodsmentioning

confidence: 99%

Diversity of family GH46 chitosanases in Kitasatospora setae KM-6054

Zitouni

Viens

Ghinet

et al. 2017

Appl Microbiol Biotechnol

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The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid. Kinetic parameters were also determined. These tests unveiled the biochemical diversity among these chitosanases and the peculiarity of Csn3 compared with the other three enzymes. The observed biochemical diversity is discussed based on structural 3D models and sequence alignment. This is a first study of all the GH46 chitosanases produced by a single microbial strain.

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“…Chitosanase activity was measured using the dyed substrate sRBB-C [ 49 ]. Briefly, 50 μl of appropriately diluted culture supernatant were added to 950 μl of soluble Remazol Brilliant Blue chitosan (5 mg/ml in 0.1 M Na-acetate buffer pH 4.5) and the mixture was incubated for 60 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Reaction was stopped with 500 μl of 1.2 N NaOH and cooled on ice for 20 min. After centrifugation, the optical density of supernatant was read at 595 nm and converted into chitosanase activity as described [ 49 ].…”

Section: Methodsmentioning

confidence: 99%

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

2011

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BackgroundChitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous chitosanase gene (csnN106) in Streptomyces lividans.ResultsTwo S. lividans strains were used for induction experiments: the wild type strain and its mutant (ΔcsnR), harbouring an in-frame deletion of the csnR gene, encoding a negative transcriptional regulator. Comparison of chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous chitosanase gene csnN106. Using the ΔcsnR host and a mutated csnN106 gene with a modified transcription operator, substantial levels of chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed.ConclusionsThis new chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.

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A dye-labelled soluble substrate for the assay of endo-chitosanase activity

Cited by 6 publications

References 20 publications

Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India

Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India

Diversity of family GH46 chitosanases in Kitasatospora setae KM-6054

Modification of genetic regulation of a heterologous chitosanase gene in Streptomyces lividans TK24 leads to chitosanase production in the absence of chitosan

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