Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Abstract

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

“…They have such complicated structures that other factors in addition to chitinases may be required to degrade them efficiently. In fact, other Paenibacillus species, such as Paenibacillus fukuinensis, produce additional degradation enzymes, including glucanase, chitosanase, and protease, which work cooperatively (30,50). Moreover, a unique type of enzyme was recently discovered: surface-active CBM33-type lytic polysaccharide monooxygenase, which acts synergistically with chitinase by splitting the glycoside bond of chitin by an oxidative mechanism (51-53).…”

“…The heart, lungs, liver, kidneys, spleen, and brain were examined. The 50% lethal dose (LD 50 ) was estimated according to the method described by Miller and Tainter (29). Hematological analysis was performed using an automatic hematological analyzer (Beckman Coulter, Indianapolis, IN).…”

Cloning and Characterization of a Gene Coding for a Major Extracellular Chitosanase from the Koji MoldAspergillus oryzae

“…The presence of CBDs has been reported only in the chitosanase of a bacterium, Paenibacillus fukuinensis, to date. 12) Hence we examined whether the R3 domain has binding ability for the insoluble substrate chitosan.…”

Activity Measurement of Chitosanase by an Amperometric Biosensor

“…1 In this work, the catalytic domain of the chitosanase, deficient in the discoidin domains (COOH-terminal 271-amino acid region), 2 was used. The specific activity of the enzyme was determined to be 20.1 units mg -1 by the conventional method described above with glycol chitosan as a substrate.…”

“…1 This enzyme is expected to be a tool for manufacturing chitosan oligosaccharides with potential antibacterial, anticancer, or elicitor activity. 2 So far, the activity of the chitosanase has been assayed by the detection of amino sugars (here, glucosamine and chitosan oligosaccharides) released in the reaction mixture of chitosan and chitosanase after incubation for 10 -120 min at 37°C. The amino sugars have been measured by the conventional Elson-Morgan method, [3][4][5] which consists of the following three steps: (i) amino sugars react with acetylacetone in an alkaline solution for 20 min at 100°C to form a chromogenic material, (ii) the chromogenic material gives chromophores upon a treatment in an acid solution with ethanolic p-dimethylaminobenzaldehyde for 30 min, and (iii) the absorbance of the chromophores at 530 nm, which is proportional to the concentration of the amino sugars in the original solution, is measured.…”