Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001

Abstract

The extracellular chitosanase (34,000 M r) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

Purification of a constitutive chitosanase produced byBacillussp. MET 1299 with cloning and expression of the gene

“…MET 1299 had highest activity on colloidal chitosan 9B and efficiently hydrolyzed colloidal chitosan 8B and 7B. The fact that 85-90% deacetylated chitosan is more susceptible to hydrolysis than 65-85% deacetylated chitosan may suggest that N-acetylglucosamine residues in the chitosan play an important role in the recognition of the substrate by the enzyme [21]. The chitosanase activity of Bacillus sp.…”

“…Enzyme activity was also inhibited by other divalent metal ions (Zn 2+ ,Cu 2+ ). Chitosanase from Matsuebacter chitosanotabidus 3001 is an example of another enzyme inhibited by Zn 2+ ,Cu 2+ ,Hg 2+ and Fe 2+ [21]. This suggest that the metal ions such as Zn 2+ ,Cu 2+ ,Hg 2+ and Fe 2+ are not essential for the catalytic action of the enzyme.…”

Identification and expression of GH-8 family chitosanases from severalBacillus thuringiensissubspecies

“…strain S65 (Su et al , 2006) are GH‐8 enzymes. A few bacterial chitosanases from Mitsuaria chitosanitabida (Park et al , 1999; Yun et al , 2005) and Sphingobacterium multivorum (Matsuda et al , 2001) have also been classified as GH‐80 enzymes. A variety of glycoside hydrolases including chitosanases, cellulases, xylanases, and licheninases are classified as GH‐5 and GH‐8 enzymes.…”

Immunohistochemical and Electron Microscopic Study of the Biodegradation Processes of Chitin and Chitosan Implanted in Rat Alveolar Bone

“…Chitosan is easily hydrolyzed by various chitosanases (25,26), which are completely absent in mammals, and the hydrolization depends on the degree of deacetylation (27,28). Lysozyme, normally produced by macrophages and neutrophils, hydrolyses susceptible modified chitosan to oligomers, which activate macrophages to produce nitric oxide, activated oxygen species, tumor necrosis factor-, interferon and interleukin-1 (16).…”