Chitinase assay

Chitinase assay was determined by a modified Schales procedure using colloidal and/or soluble chitin as the amount of enzyme that liberated 1 pmol of reducing sngar per minute (Imoto and Yagishita 1971). This method was nsed as the standard assay. To measnre the activity toward other substrates, powdered chitin, colloidal chitin, carboxymethyl chitin, ethylene glycol chitin, chitosan 7B, chitosan 8B, chitosan 9B, chitosan lOB, p-nitrophenyl-P-o-A-acetyl glucosaminide (pNP-Gl-cNAc), and several chitin/chitosan oligomers were used instead of solnble chitin. 

The simple definition of chitinase activity, EC 3.2.1.14, "hydrolysis of iV-acetyl-D-glucosaminide (l-4)-P-linkages in chitin and chitodextrins", belies the complexity and diversity of this group of enzymes. When chitinolytic organisms are investigated in detail, they are found to produce a range of chitinase activities. Usually these can be separated readily by chromatography or electrophoresis. However, it is more difficult to define their precise activities, chiefly because of the uncertain nature of the available assays for chitinases, with non-linear time courses... 

An antibiotic inhibition zone often appears around Trichoderma spp. interacting with other fungi. The genus contains many species which produce secondary metabolites. Claydon et al. (23) have identified an antibiotic from T. harzianum as a volatile, 6-n-pentyl-2H-pyran-2-one this was recently shown to be an active antibiotic from T. koningii (24). The volatile appeared to be the factor responsible for the coconut smell of some biocontrol-effective strains of T. harzianum (25). However, in a Petri-plate assay, it can be difficult to be certain that antibiosis is involved. As well as competitive growth, lytic enzymes could also contribute to the action and Trichoderma has been shown to produce / -l,3-glucanase and chitinase (26-29). 

Aguilera, B., Ghauharali-van der Vlugt, K., Helmond, M.T. et al. 2003. Transglycosidase activity of chi-totriosidase Improved enzymatic assay for the human macrophage chitinase. J. Biol. Chem. 278 40911 0916. 

A rapid and sensitive assay for chitinase using [ H]chitin has been developed. ... 

Radioactive chitin (reacetylated chitosan) has been used as the substrate for the sensitive assay of chitinase. The procedure is based on the insolubility of chitin and solubility of the reaction product A, A -diacetylchitobiose. Re-ac tylated chitosan proved to be a more satisfactory adsorbent during the puri fication of chitinase than did colloidal chitin. 

The dyeing of polysaccharides has been used for three purposes (1) the formation of dyefast fabrics, (2) the visible identification of polysaccharides for study in electrophoresis and gel chromatography [32], and (c) to provide substrates for identification of enzymes in enzyme screening tests and in assaying of specific enzymes such as a-amylase [33], cellulase [34], chitinase [35], dextranases [36], and so forth. 

The assay for chitinase activity used vas that of Molano et al. 

Chitinase hydrolysis of radiolabeled substrate was determined by liquid scintillation counting of trichloracetic acid-soluble (TCA) products (Smucker and Wright, 1984). Sediment (20 grams) was homogenized along with 60 ml sterile-filtered seawater (10% salinity). Feces were homogenized in the sterile-filtered seawater. Aliquots were removed for replicate experimental and control samples. For each assay, reactants were added in the following proportions 500 y 1... 

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