Abstract
BackgroundChitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures.ResultsThis paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity). The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45°C, but with 30-70% of the maximum activity at 10°C and 30°C, respectively.ConclusionsA novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10°C to 30°C, temperatures that are preferred in transfection and transformation experiments.
Highlights
Chitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated b-(1,4)-linked glucosamine residues
We describe the isolation and characterization of a new chitosanase and we show that the enzyme may be produced heterologously in Escherichia coli resulting in high yields
Isolation of chitosanase enzyme and gene from Janthinobacterium sp Three thousand bacterial isolates were screened for chitosanase activity on agar plates with chitosan
Summary
Chitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated b-(1,4)-linked glucosamine residues. Chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. A polymer of acetylated b-(1,4)-linked glucosamine (GlcNAc) residues, is the second-most abundant polysaccharide in nature, where it constitutes the major structural component in a number of organisms, e.g. crustaceans, insects, nematodes and fungi. Chitosan, which is a partly deacetylated form of chitin, is less abundant but may be found in the cell wall of certain fungi, e.g. Zygomycetes [1,2] and green algae like Chlorella [3].
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