Effects of the degree of deacetylation (DDA) and the molecular mass of chitosan oligosaccharides (CTS-OS), obtained from the enzymatic hydrolysis of high molecular weight chitosan (HMWC), on antitumor activity was explored. The DDA and molecular weights of CTS-OS were determined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF MS) analysis. The CTS-OS were found to be a mixture of mainly dimers (18.8%), trimers (24.8%), tetramers (24.9%), pentamers (17.7%), hexamers (7.1%), heptamers (3.3%), and octamers (3.4%). The CTS-OS were further fractionated by gel-filtration chromatography into two major fractions: (1) COS, consisting of glucosamine (GlcN)n, n = 3–5 with DDA 100%; and (2) HOS, consisting of (GlcN)5 as the minimum residues and varying number of N-acetylglucosamine (GlcNAc)n, n = 1–2 with DDA about 87.5% in random order. The cytotoxicities, expressed as the concentration needed for 50% cell death (CC50), of CTS-OS, COS, and HOS against PC3 (prostate cancer cell), A549 (lung cancer cell), and HepG2 (hepatoma cell), were determined to be 25 μg·mL−1, 25 μg·mL−1, and 50 μg·mL−1, respectively. The HMWC was approximately 50% less effective than both CTS-OS and COS. These results demonstrate that the molecular weight and DDA of chitosan oligosaccharides are important factors for suppressing cancer cell growth.
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.
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