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Chitinase. An enzyme that breaks down chitin
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How is degraded 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.
Chitinases (EC 22.214.171.124, 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.
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.
- 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.
- Principle of detecting chitinase activity after electrophoresis using glycol-chitin as substrate.
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.
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
polyacrylamide gel electrophoresis (native-PAGE) was performed in
a 7.5% (w/v)
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
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.
Molecular weights and isoelectric points of each chitinase isoform could be determined by using 2D-electrophoresis like as in proteomic studies.
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).