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Chitinase enzyme is a hydrolytic enzyme that break down glycosidic bonds in chitin. Chitinases are produced by higher plants to defend themselves against pathogenic attacks by degrading chitin in the cell walls of fungi and bacteria.


Master commun: Microbiologie appliquée-Gestion et valorisation des phytoressources, Module 'Phytopathologie et méthodes de lutte'

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Chitinase. An enzyme that breaks down chitin

In this page:

- How is degraded chitin?
- What is chitinase?
- How is tested chitinase
- Chitinase activity produced by muskmelon as example
- Chitinase induction

- Chitinase and biological control towards fungi
- Engineered plants with overexpression of chitinase

Enzymatic degradation of chitin can follow two different paths: a chitinolytic path or via chitosan.

1/ Chitin can be degraded by first being solubilized by deacetylation. This process is carried out by chitin deacetylases, and the derived substrate (chitosan) is hydrolysed by chitosanases.

2/ The chitinolytic process require direct hydrolysis of the beta-1,4 glycosidic bonds between the GlcNAc units by chitinases.

How is degraded chitin?

Chitin

La chitine est un polysaccharide azoté issu de la polymérisation de N-acétylglucosamine liés entre eux par une liaison osidique du type ß-1,4.

What is chitinase?

Chitinases (EC 3.2.1.14, chitodextrinase, 1,4-beta-poly-N-acetylglucosaminidase, poly-beta-glucosaminidase, beta-1,4-poly-N-acetyl glucosamidinase, poly[1,4-(N-acetyl-beta-D-glucosaminide)] glycanohydrolase, (1->4)-2-acetamido-2-deoxy-beta-D-glucan glycanohydrolase) are hydrolytic enzymes that break down glycosidic bonds in chitin.

Chitinases are produced by higher plants, which use the enzymes to defend themselves against pathogenic attacks by degrading chitin in the cell walls of fungi and bacteria. They exist also in fungi and bacteria. Plant chitinases generally range in molecular weight from 25 to 40 kD and may be either acidic or basic.There are two kinds of chitinases: endochitinases and exochitinases.

Chitin, chitinase

Endochitinase cleaves chitin randomly at internal points within the polymer, releasing soluble, low-molecular weight multimers of NAG such as chitotetraose and chitotriose and the dimer, di-acetylchitobiose, which predominates.

Exochitinase hydrolyzes chitin by releasing di-acetylchitobiose, with no mono or oligo-saccharides formed.

Most plant chitinases isolated to date are endochitinases.

However, several studies have shown that some plants have exochitinases. All plant chitinases are relatively small proteins with molecular weights ranging from 25 to 40 kD.

Chitinase could be induced by Aabiotic and biotic agents

How is tested chitinase?

The activity could be assayed by gel diffusion test (agarose, polyacrylamide gels)

Chitinase test

- Agar diffusion test: Paper discs are coated with chitinase enzyme and placed onto a chitin agar plate. Chitinolytic zones around the discs could be observed after 12-24 h of incubation. Chitin agar plate has been used earlier for isolating chitinolytic microorganisms and observing clear zone around the colony of microorganisms.



Livre 'Sciences de la vie. Protéines et Enzymes' (+ DVD), Baaziz, 2013: QCM corrigés, Exercices corrigés, Contrôles corrigés en Biologie cellulaire et Biochimie pour S1, S3, S4 et S5.

Protéines et Enzymes, Baaziz 2013


Glossaire trilingue (Fr, Ar, Eng) des Sciences de la vie. A apparaître bientôt dans les librairies:

Sciences de la vie. Glossaire


- Principle of detecting chitinase activity after electrophoresis using glycol-chitin as substrate.

1/ Preincubate the electrophorezid native gel in 150 Mm sodium acetate buffer (pH 5.0) for 5 min.

2/ Cover the electrophorized gel with another gel containing substrate and incubate à 37°C for 1 h.

3/ Transfer the two gels in a freshly prepared solution of 0.015% calcofluor white M2R in 0.5 M Tris-HCl (pH 8.9) for 5 min.

4/ Wash the gels in distilled water for 1-2 h to destain zones of chitin lysis.

5/ Observe dark (nonfluorescent) bands of chitinase on a light (fluorescent) background in a long-wave UV light.

Calcofluor-white is a special fluorescent stain that binds strongly to structures containing cellulose and chitin.

Calcofluor staining

Article analysis
Chitinase activity produced by muskmelon

Muskmelon (melon, البطيخ، الشمام), Cucumis melo L., is different from watermelon (Citrullus lanatus var. lanatus, pastèque, الدلاع). They belong to Cucurbitaceae family.

Gel diffusion assay of chitinase activity

Muskmelon

Chitinases accumulate in seeds of several species as part of their developmental programme, while others can be induced in response to microbial attack. Seed chitinases may protect against chitin-containing pathogenic fungi, because substrates for chitinase are found in some fungal cell walls, but not in plants.

Chitinase test by gel diffusion

When compared with embryo, endosperm shows high chitinase activities. Chitinase diffuses from the well and catalyzes the cleavage of glycol chitin leaving a clear non-fluorescent zone in the gel, the diameter of which is proportional to enzyme activity.

Separation of chitinase isoenzymes by electrophoresis

Native polyacrylamide gel electrophoresis (native-PAGE) was performed in a 7.5% (w/v)
polyacrylamide gel containing 0.5% glycol chitin as the substrate.

Protein extracts (20 µL) were separated by electrophoresis at either pH 8.8 for acidic chitinases or pH 4.3 for basic chitinases. After electrophoresis, the gels were equilibrated in 0.1 M. citric acid/0.2 M sodium phosphate buffer (pH 5.0) at
28°C for 20 h and then stained with 0.1% calcofluor. After washing the gel with distilled water, the activity of chitinase isoforms was visualized under UV light (Trudel and Asselin, 1989).

One acidic chitinase isoform, AD1, was detected in young muskmelon seeds at least 40 days after anthesis, and its activity increased with further maturation.

Three basic chitinase isoforms, BD1, BD2 and BD3, were detected in developing muskmelon seeds beginning at 40 days after anthesis. The activity of basic isoform BD3 was greater than that of either BD1 or BD2.

some chitinase isoforms appear to be synthesized during seed development and remain active during germination, while other isoforms are induced in association with, or soon after, radicle emergence.

Native gel PAGE

Acidic isoenzymes of chitinase


Native acidic gel

Basic isoenzymes of chitinase

Basic and acidic chitinases

1/ Both acidic and basic chitinase isoforms were detected in endosperm tissue during seed imbibition and after radicle emergence.

2/ Basic chitinase isoforms, but not acidic isoforms, were detected in embryo tissue.


Preparation and cloning chitinase cDNAs using RT-PCR

Using total RNA isolated from developing seeds at 10, 20 and 30 days after anthesis or mature seeds imbibed for 16, 24, 32 or 48 h, as templates, reverse transcriptase (RT) reactions were performed. Polymerase chain reaction (PCR) was performed using the degenerate primers designed from conserved domains within chitinases from several different plant species ( RT-PCR technique). The PCR products with expected sizes were cloned.

DNA electrophoresis

Chitinase induction

Abiotic agents such as chitosan, ethylene, ozone, wounding, polysaccharides, salicylic acid, salt solution, and UV light can induce higher expression of chitinases in plants.

Biotic agents such as bacteria, insects, fungi, viruses, and fungal cell wall fragments can also induce the expression of chitinases in plants. Studies of chitinases from bean, cucumber, pea, potato, sugar beet, tomato, and tobacco have shown that the expression of chitinases is induced dramatically after infections. Induction usually occurs strongly at the point of infection and drops rapidly as the distance from the infection site increases.

The induction of chitinase can spread to adjacent tissues, resulting in a systemic acquired resistance that may enable the plant to protect itself from secondary infection.


Chitinase and biological control towards fungi

Biological control towards soilborne fungal pathogens Phytophora parasitica and Fusarium oxysporum, Rhizoctonia solani, Phytophora capsici, and Fusarium oxysporum f. sp. radicis-lycopersici.

Example of bacteria with hydrolytic enzymes:

Study 1 : Antifungal activity of chitinase obtained from Paenibacillus ehimensis MA2012 against conidial of Collectotrichum gloeosporioides in vitro, Dong-Jun Seo et al. (2016), Microbial Pathogenesis 96, 10-14

Paenibacillus species are a well-known producer of hydrolytic enzymes such as chitinase and beta-1,3-glucanase. Paenibacillus is a genus of facultative anaerobic, endospore-forming bacteria, originally included within the genus Bacillus and then reclassified as a separate genus in 1993.

Paenibacillus ehimensis

Bacteria belonging to this genus have been detected in a variety of environments, such as soil, water, rhizosphere, vegetable matter, forage and insect larvae, as well as clinical samples.

Preparation of crude chitinase
Paenibacillus ehimensis MA2012 was incubated in a 500 mL Erlenmeyer flask with 200 mL gelatin-chitin medium

Antifungal activity of partial purified chitinase from Paenibacillus ehimensis

Paenibacillus ehimensis, Colletotrichum

The Colletotrichum gloeosporioides conidia were incubated with and without purified chitinase of Paenibacillus ehimensis MA2012 for 12 h at 30°C. After incubation, a little conidial development of C. gloeosporioides was observed in the purified chitinase (100 µg/100 mL) treatment. Inhibition rate of conidial germination was 87% in partially purified chitinase treatment compared with control.


To evaluate the active staining of chitinase, the culture filtrate was saturated with 80% ammonium sulfate and then centrifuged. The precipitate was dissolved in a small volume of sterile distilled water and dialyzed distilled water overnight at 4°C.

Chitinase activity staining on SDS-PAGE

SDS-PAGE 12% (w/v) was carried out on 12% acrylamide separating gels containing 0.01% glycol chitin. Aliquots of crude protein (2 µg-20 µg) were loaded onto SDS-PAGE
gels. After electrophoresis, the gel was incubated for 2 h at 37°C with shaking in 100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100 and 1% skim milk. Subsequently, the gel was again incubated at 37°C overnight with shaking in 100 mM sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100. The gel was immersed into 500 mM Tris-HCl (pH 8.9) solution containing 0.01% Calcofluor white M2R (Sigma F3397). The zones lysed were visualized and photographed on a UV transilluminator. Also, the SDS-PAGE gel was stained with silver staining.

Chitinase isozymes in gelatin-chitin medium show as Ch1, Ch2, Ch3, Ch4, Ch5, Ch6, Ch7, and Ch8 bands on SDS-PAGE gel (Figure A). Six major bands (Ch3, Ch4, Ch5, Ch6, Ch7, and Ch8) of chitinase isozymes appeared on SDS-PAGE gel for incubation period.

Chitinase. Electrophoresis SDS-PAGE

The pI value for each chitinase isoenzyme was determined by IEF

Molecular weights and isoelectric points of each chitinase isoform could be determined by using 2D-electrophoresis like as in proteomic studies.


Engineered plants with overexpression of chitinase


Plant genes encoding cell wall degrading enzymes, especially chitinases, have been used to alter plant resistance to fungal pathogens, but no single genes have produced an adequate level of resistance, and bring resistance to multiple pathogens. From many reasons for this may be that plant chitinases usually affect only the hyphal tip and are unable to effectively degrade harder chitin structures.

In terms of antifungal activity, chitinase genes from biocontrol fungi such asTrichoderma are clearly an improvement over corresponding plant genes. These fungal genes encode for chitinolytic enzymes that can reach the antifungal activity level of some chemical fungicides. Furthermore, extensive testing in vitro has shown that there are virtually no chitinous pathogens resistant to Trichoderma chitinases. Therefore, it is expected that the transgenic use of these enzymes should produce a high level of resistance in crop plants against a variety of fungal pathogens. See an example study as article analysis.

Links: Master exam June 10, 2016 (chitinase) ------- Contrôle S6 sur transformation génétique du tabac et de la pomme de terre par le gène de la chitinase de Trichoderma harzianum ----- Inhibiteurs de la polygalacturonase chez la betterave sucrière (Master contrôle).


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