Chen-Tien Chang

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells.

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeasts culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.

Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters K m and kcat were 245 µM and 10.31 min−1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.

In vitro and in vivo safety evaluation of low molecular weight chitosans prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase.

Our previous study demonstrated that the oral administration of low molecular weight chitosans (LMWC), prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase in an appropriate dose, reduced aristolochic acid-induced renal lesions in mice. The objectives of this study were to evaluate the safety of LMWC using genetic and animal toxicity assays. Two assays for genotoxicity were performed: the chromosomal aberration of Chinese hamster ovary cells (CHO-K1 cells) (in vitro) and micronucleus assays in mice (in vivo). Acute oral toxicity and 28-day repeated feeding toxicity tests were performed via the oral gavage method in Sprague-Dawley (SD) rats. LMWC did not induce an increase in micronucleus ratios in vivo, and the chromosome aberration assay indicated that the LMWC was safe in terms of clastogenicity in doses up to 5.0 mg/ml. No acute lethal effect at a maximum tested dose of 5.0 g LMWC/kg body weight (bw) was observed in rats. The results of the 28-day study revealed no adverse effects on the body weight, feed consumption, hematology, blood biochemical parameters, organ weights or pathology. The no observed adverse effect level (NOAEL) of LMWC in rats was 1.0 g/kg bw for the subacute toxicity study.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50–80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 μmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21–94%). The enzyme hydrolyzed 70–84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Protective effect of medicinal fungus Xylaria nigripes mycelia extracts against hydrogen peroxide-induced apoptosis in PC12 cells

Xylaria nigripes (XN) is a medicinal fungus that was used traditionally as a diuretic, nerve tonic, and for treating insomnia and trauma. In this study, we elucidated possible mechanisms of neuroprotective effects of XN mycelia extracts. XN mycelia were produced by fermentation. Hot water extract and 70% ethanol extract of XN mycelia were evaluated on hydrogen peroxide (H2O2)-induced apoptosis in PC12, a rat pheochromocytoma cell line. Both XN extracts effectively protected PC12 cells against H2O2-induced cell damage by inhibiting release of lactate dehydrogenase, decreasing DNA damage, restoring mitochondrial membrane potential, and arresting abnormal apoptosis through upregulation of Bcl-2 and downregulation of Bax and caspase 3. Compared to water extract, ethanol extract showed not only greater neuroprotective effects but also a higher antioxidant activity by scavenging DPPH radicals, inhibiting lipid peroxidation, and reducing power. High phenolic content and antioxidant activity may provide the neuroprotective properties of XN ethanol extract.