Water-soluble Chitin Derivative Research Articles

Purification and Characterization of Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase (EC 3.5.1.41), the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetyl-D-glucosamine in chitin, has been purified to homogeneity from the culture filtrate of the fungus Colletotrichum lindemuthianum and further characterized. The enzyme is a glycoprotein, and its apparent molecular mass was determined to be approximately 150 kDa. The glycosylation pattern of the enzyme is consistent with a mixture of N-linked glycans including oligomannosidic hybrid and/or complex type, and its carbohydrate content is approximately 67% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives, is not considerably inhibited by carboxylic acids, especially acetic acid, and exhibits a remarkable thermostability. The enzyme requires at least two N-acetyl-D-glucosamine residues (chitobiose) for catalysis. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be 50 degrees C, and the optimum pH was approximately 8.5.

Synthesis of 6-O-carboxymethylchitin immobilizing doxorubicins through tetrapeptide spacer groups and its enzymatic release behavior of doxorubicin in vitro

Chitin is a non-toxic biodegradable polysaccharide. 6-O-Carboxymethylchitin (CM-chitin) is a water-soluble chitin derivative. In order to provide a water-soluble macromolecular prodrug of doxorubicin (DXR) reducing the side-effects and exhibiting high antitumor activity, the fixation of DXRs to CM-chitin through covalent bonds was carried out. Especially, a lysosomally digestible tetrapeptide (Gly-Phe-Leu-Gly) was used as spacer groups between CM-chitin and DXRs. In this paper, two kinds of conjugates, CM-chitin/Gly-Phe-Leu-Gly/DXR conjugate 5 having lysosomally digestible tetrapeptide spacer groups and CM-chitin/C5/DXR conjugate 6 having pentamethylene spacer groups, were synthesized. The effect of introduction of the spacer groups on the release behavior of DXR from the conjugate was investigated. The conjugate 5 having tetrapeptide spacer groups showed a distinct increase in the release rate of DXR in the presence of the lysosomal enzyme cathepsin B at 37°C in vitro; however, the conjugate 6 did not show such specific release behavior.

Research report: Elicitation of chitinases and anthraquinones inMorinda citrifoliacell cultures

Abstract The induction of stress metabolites by endogenous and exogenous elicitors found in plant‐microorganism interactions has been studied in Morinda citrifolia suspension cultures. Chitin 50, a water soluble chitin derivative, and chitosans as constituents of microbial cell walls were used as models for an exogenous elicitor. Addition of commercial pectic acid or pectins derived from Morinda citrifolia plant cell walls to plant cell culture media simulated the presence of endogenous elicitors. Chitin 50 was found to induce chitinase and anthraquinone synthesis, and Morinda citrifolia derived pectins were highly active in stimulating plant stress response. The elicitor effect of the polysaccharides applied was also found to be time dependent. The initial defense mechanism was induced by pectins and resulted in increasing chitinase and lysozyme activities. The next step of defense mechanism was predominantly induced by pectins causing enhanced production of anthraquinones initiated by pectins, chitosans and especially by Chitin 50.

Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii.

Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized. The enzyme exhibits a low pI (approximately 3). Its apparent molecular mass was determined to be approximately 75 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and approximately 80 kDa by size-exclusion chromatography, suggesting that the enzyme exists as a monomer. Carbohydrate analysis of purified chitin deacetylase revealed that the enzyme is a high-mannose glycoprotein and that its carbohydrate content is approximately 30% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives. The enzyme requires at least four N-acetylglucosamine residues (chitotetraose) for catalysis, and it is inhibited by carboxylic acids, particularly acetic acid. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be approximately 50 degrees C and the optimum pH was approximately 4.5.

Antimetastatic activity of neocarzinostatin incorporated into controlled release gels of CM-chitin

6- O-carboxymethyl chitin (CM-chitin), a water-soluble chitin derivative, was gelled in the presence of 15 to 30 m m iron (III) chloride. At the time of gel formation, a peptidic anticancer drug neocarzinostatin (NCS), was efficiently (>50%) incorporated into the gel in the co-presence of 25 to 50 m m calcium chloride and iron (III) chloride. CM-chitin gel containing NCS was digested by lysozyme in vitro and NCS was released from the gel in both a time- and dosedependent manner. In-vivo studies in mice showed that free NCS was rapidly cleared from the circulation, but NCS released from the gel was detectable in plasma even 48 h after the subcutaneous injection of the gel. Antimetastatic effects of the CM-chitin gel containing NCS were studied in the spontaneous pulmonary metastasis model using Bl6-BL6 melanoma; these results suggest that CM-chitin gels are useful as a sustained-release drug carrier.

Activation of a limulus coagulation factor G by (1 → 3)-g⊝ d-glucans

Various oligo- and poly-saccharides differing in sugar compositions and types of linkage were examined for their ability to activate a limulus coagulation factor G, the first protease in an alternative coagulation cascade of the horseshoe crab, Tachypleus tridentatus, whose amebocytes have originally been known to contain a lipopolysacchaide(LPS)-driven pathway leading to the formationof coagulin gel. Linear and branched (1 → 3)-β- d-glucans and mixed linkage (1 → 3),(1 → 4)-β- d-glucans were found to exhibit the ability to activate factor G at concentrations of 10 −8–10 −10 g/mL as assayed by amidolytic activity of the clotting enzyme. Laminaran oligosaccharides, laminaran dextrins (number-average mol. wt. ≦ 5800), and other polysaccharides including carboxymethylcellulose, amylose, starch, d-fructans, α- l-arabinan, β- d-xylans, (1 → 3)-β- d-galactan, water-soluble chitin derivatives, chondroitin, chondroitin sulfates, hyaluronan, keratan sulfate, heparins, heparan sulfate, and LPS's were virtually inactive as activators even at a concentration of 10 −7 g/mL. The activating ability increased with increasing number-average mol. wt. (6800–216 000) of linear (1 → 3)-β- d-glucans. The apparent activating ability of gyrophoran, nigeran, and yeast α- d-mannan was largely abolished by digestion with a highly purified Arthrobacter luteus endo-(1 → 3)-β- d-glucanase, which provided supportive evidence for the activation to be ascribed to contaminating (1 → 3)-β- d-glucan(s). Possible participation of ordered structures of (1 → 3)-β- d-glucans in the activation of factor G is discussed.

Preparation and characterization of water-soluble chitin phosphate

New water-soluble chitin derivatives, chitin phosphate of various degrees of substitution, were successfully prepared by the reaction of chitin with phosphorus pentoxide in methanesulphonic acid. These materials behaved hydrodynamically as typical polyelectrolyte, and showed high ability to adsorb metal ions.

Studies on Chitin VIII. Some Properties of Water Soluble Chitin Derivatives

Preparation de carboxymethyl- et dihydroxypropylchitine. Etude des sites d'alkylation par spectrometrie RMN

Studies on Chitin and Glucosamine. (III). : Degradation of Chitin and Its Related Derivatives by Snail Enzyme.

Enzymatic degradation of chitin and its derivatives was attempted by means of snail digestive fluid and its acetone-dried powder, which was made from snail gastrointestine and liver. N-acetylglucosamine could be detected by paper chromatography in the degradation process of purified chitin, colloidal chitin, regenerated chitin, acetylated chitosan, carboxymethylchitin or glycolchitin. Glucosamine could be also characterized from degradation product of chiosan by the conversion to arabinose and N-acetylglucosamine. To investigate the above degradation process, viscosity reduction of water-soluble chitin derivatives were measured, and production of N-acethylglucosamine or glucosamine was determined by colorimetric method. In case of carboxymethylchitin and glycolchitin, the hexosamine was produced remarkably by the degradation and viscosity reduced distinctly, but in case of chitosan the yield of glucosamine was small.