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.

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.

Characterization of an acidic chitinase from seeds of black soybean (Glycine max (L) Merr Tainan No. 3).

Using 4-methylumbelliferyl-ß-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 Km 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.

Cinnamomum cassia essential oil inhibits α-MSH-induced melanin production and oxidative stress in murine B16 melanoma cells.

Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for the development of aromatherapy for complementary and alternative medicine. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties; however, little information is available regarding its anti-tyrosinase and anti-melanogenic activities. In this study, 16 compounds in the CC-EO have been identified; the major components of this oil are cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%). CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities; however, cis-2-methoxycinnamic acid did not demonstrate tyrosinase inhibitory activity. In murine B16 melanoma cells stimulated with α-melanocyte-stimulating hormone (α-MSH), CC-EO and cinnamaldehyde not only reduced the melanin content and tyrosinase activity of the cells but also down-regulated tyrosinase expression without exhibiting cytotoxicity. Moreover, CC-EO and cinnamaldehyde decreased thiobarbituric acid-reactive substance (TBARS) levels and restored glutathione (GSH) and catalase activity in the α-MSH-stimulated B16 cells. These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase and anti-melanogenic activities that are coupled with antioxidant properties. Therefore, CC-EO may be a good source of skin-whitening agents and may have potential as an antioxidant in the future development of complementary and alternative medicine-based aromatherapy.

Chemical composition and tyrosinase inhibitory activity of Cinnamomum cassia essential oil.

BACKGROUND: Essential oils extracted from aromatic plants exhibit important biological activities and have become increasingly important for scientific research. The essential oil extracted from Cinnamomum cassia Presl (CC-EO) has various functional properties, however, little information is available regarding the tyrosinase inhibitory activity. Therefore, the objectives of this study were to investigate the chemical composition and tyrosinase inhibitory activity of the CC-EO. RESULTS: cis-2-methoxycinnamic acid (43.06%) and cinnamaldehyde (42.37%) were found to be the two major components of the CC-EO identified by gas chromatography-mass spectrometry (GC-MS). The inhibitory activities of CC-EO and its major constituents were further evaluated against mushroom tyrosinase. The results showed that CC-EO and cinnamaldehyde exhibited anti-tyrosinase activities with IC50 values of 6.16 ± 0.04 mg/mL and 4.04 ± 0.08 mg/mL, respectively. However, cis-2-methoxycinnamic acid did not show any anti-tyrosinase activity. The inhibition kinetics were analyzed by Lineweaver-Burk plots and second replots, which revealed that CC-EO and cinnamaldehyde were mixed-type inhibitors. The inhibition constants (Ki) for CC-EO and cinnamaldehyde were calculated to be 4.71 ± 0.09 mg/mL and 2.38 ± 0.09 mg/mL, respectively. CONCLUSION: These results demonstrate that CC-EO and its major component, cinnamaldehyde, possess potent anti-tyrosinase activities and may be a good source for skin-whitening agents.

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk.

An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4 kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate-phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50°C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a K(m) value of 2.8mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H(2)O(2) in milk, the lower limit of determination was 0.1 µM, and the recoveries of added H(2)O(2) were 96-109%.

Aristolochic acid-induced accumulation of methylglyoxal and Nε-(carboxymethyl)lysine: an important and novel pathway in the pathogenic mechanism for aristolochic acid nephropathy.

Aristolochic acid, found in the Aristolochia species, causes aristolochic acid nephropathy (AAN) and can develop into renal failure. Methylglyoxal (MGO) is a highly cytotoxic compound generated from the metabolic process of glucose or fatty acids. It binds to proteins and forms N(ε)-(carboxymethyl)lysine (CML), which contributes to aging and diabetes mellitus complications. However, no relevant literature explores the relationship of MGO and CML with AAN. By injecting AA (10mg/kg BW) into C3H/He mice for 5 consecutive days, we successfully developed an AAN model and observed tubular atrophy with decreased renal function. Creatinine clearance also decreased from 10.32 ± 0.79 ml/min/kg to 2.19 ± 0.29 ml/min/kg (p<0.01). The concentration of MGO in kidney homogenates increased 12 × compared to the control group (from 18.23 ± 8.05 µg/mg of protein to 231.16 ± 17.57 µg/mg of protein, p<0.01), and CML was observed in the renal tubules of the mice by immunohistochemistry. Furthermore, compared to the control group, GSH levels decreased by 0.32 × (from 2.46 ± 0.41 µM/µg of protein to 0.78 ± 0.15 µM/µg of protein, p<0.01), whereas intra-renal antioxidant capacity decreased by 0.54×(from 6.82 ± 0.97 U to 3.71 ± 0.25 U; unit is equivalent to µM Trolox/mg of protein, p<0.01). In this study, we found that serious kidney damage induced by AA is related to an increase and accumulation of MGO and CML.

Purification and characterization of two chitosanase isoforms from the sheaths of bamboo shoots.

Two thermally stable chitosanase isoforms were purified from the sheaths of chitosan-treated bamboo shoots. Isoforms A and B had molecular masses of 24.5 and 16.4 kDa and isoelectric points of 4.30 and 9.22, respectively. Using chitosan as the substrate, both isoforms functioned optimally between pH 3 and 4, and the optimum temperatures for the activities of isoforms A and B were 70 and 60 °C, respectively. The kinetic parameters K(m) and V(max) for isoform A were 0.539 mg/mL and 0.262 µmol/min/mg, respectively, and for isoform B were 0.183 mg/mL and 0.092 µmol/min/mg, respectively. Chitosans were susceptible to degradation by both enzymes and could be converted to low molecular weight chitosans between 28.2 and 11.7 kDa. Furthermore, the most susceptible chitosan substrates were 50-70 and 40-80% deacetylated for isoforms A and B, respectively. Both enzymes could also degrade chitin substrates with lower efficacy. N-Bromosuccinimide and Woodward's reagent K strongly inhibited both enzymes.

Zanthoxylum ailanthoides Sieb and Zucc. extract inhibits growth and induces cell death through G2/M-phase arrest and activation of apoptotic signals in colo 205 human colon adenocarcinoma cells.

The effects of 50% ethanolic stem extracts of Zanthoxylum ailanthoides Sieb and Zucc. (ZASZ) on the cell viability, cell cycle and apoptosis were investigated in a human colon adenocarcinoma cell line (colo 205). The results demonstrated that ZASZ induced morphological changes and decreased the cell viability. ZASZ promoted Wee1, checkpoint kinase 2 (CHK2), p21 and p53 levels, decreased cyclin B and cdc25c associated with that led to G(2)/M phase arrest. ZASZ-triggered apoptosis was confirmed by 4' -6-diamidino-2-phenylindole (DAPI) staining and DNA gel electrophoresis. ZASZ increased the levels of glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153), and promoted an increase of reactive oxygen species (ROS) and Ca(2+) release, and loss of mitochondrial membrane potential (ΔΨ(m)) accompanied by cytochrome c release that was due to the decrease of Bcl-2 and increase of Bax levels in the colo 205 cells. ZASZ also induced the protein levels of apoptosis-inducing factor (AIF) and endonuclease G (Endo G), increased the levels of caspase-3, -7 and -9, and stimulated the levels of fatty acid synthase (Fas) and Fas ligand in the colo 205 cells. ZASZ contains phenolic compounds, including flavone, chlorogenic acid and isofraxidin, among which, flavone was found to be the most effective in reducing cell viability and proliferative responses in the colo 205 cells. ZASZ induces cytotoxicity and apoptosis in colo 205 cells which provides the rationale for studies in animal models on the utilization of ZASZ as a potential cancer therapeutic compound.

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 yeast's 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.

Isolation and characterization of superoxide dismutase from wheat seedlings.

Two major superoxide dismutases (SODs; SODs I and II) were found in the crude enzyme extract of wheat seedlings after heat treatment, ammonium sulfate fractionation, anionic exchange chromatography, and gel permeation chromatography. The purification fold for SODs I and II were 154 and 98, and the yields were 11 and 2.4%, respectively. SOD I was further characterized. It was found that SOD I from wheat seedlings is a homodimer, with a subunit molecular mass of 23 kDa. Isoelectric focusing electrophoresis (IEF) and zymogram staining results indicated that the isoelectric point of SOD I is 3.95. It belongs to the MnSOD category due to the fact that it was insensitive to KCN or hydrogen peroxide inhibitor. This MnSOD from wheat seedlings was found to be stable over pH 7-9, with an optimum pH of 8, but was sensitive to extreme pH, particularly to acidic pH. It was stable over a wide range of temperatures (5-50 degrees C). Thermal inactivation of wheat seedling MnSOD followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy for thermal inactivation of wheat seedling MnSOD in the temperature range of 50-70 degrees C was found to be 150 kJ/mol. HgCl2 and SDS at a concentration of 1.0 mM significantly inhibited enzyme activity. Chemical modification agents, including diethyl pyrocarbonate (2.5 mM) and Woodward's reagent K (50 mM), significantly inhibited the activity of wheat seedling SOD, implying that imidazole groups from histidine and carboxyl groups from aspartic acid and glutamic acid are probably located at or near the active site of the enzyme.

Biochemical properties of genetic recombinant xylanase II.

The aim of this study was to overexpress the xylanase II gene of Trichoderma reesei in Escherichia coli and determine the characteristics of the recombinant enzyme. Recombinant xylanase II gene was constructed by ligating the cDNA of xylanase, obtained from reverse transcriptase-polymerase chain reaction, and fused with NusA protein of pET-431b plasmid. An Ni2+-NTA affinity column was used to further purify the recombinant xylanase II. The molecular mass of the recombinant enzyme measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approx 76 kDa (including 55 kDa of NusA and 21 kDa of xylanase II), and the isoelectric point and specific activity were 7.5 and 225 U/mg, respectively. The optimal reaction temperature and pH for the recombinant enzyme were 50 degrees C and 4.0, respectively. The recombinant enzyme was stable at a pH range of 5.0-10.0 and maintained 95% residual activity after incubating at 30-35 degrees C for 30 min. The kinetic parameters KM and Vmax of the recombinant xylanase II were 13.8 mg/mL and 336 micromol/(mg.min), respectively, using birchwood xylan as the substrate.

Catalytic characteristics of peroxidase from wheat grass.

The crude enzyme extract of wheat grass was heated at 60 degrees C for 30 min, followed by ammonium sulfate fractionation and isoelectric chromatofocusing on Polybuffer exchanger (PBE 94) for purification. The purified peroxidase was then characterized for its catalytic characteristics. It was found that AgNO3 at a concentration of 0.25 mM and MnSO4 and EDTA at concentrations of 5 mM significantly inhibited the activity of wheat grass peroxidase. However, KCl, NaCl, CuCl2, CaCl2, ZnCl2, and MgCl2 at concentrations of 5.0 mM and HgCl2 at a concentration of 0.25 mM enhanced enzyme activity. Chemical modification significantly influenced the activity of wheat grass peroxidase. Particularly, N-bromosuccinimide (5 mM) inhibited 16% of the enzyme activity, whereas N-acetylimidazole (2.5 mM), diethyl pyrocarbonate (2.5 mM), and phenylmethanesulfonyl fluoride (2.5 mM) enhanced by 18-29% of the enzyme activity. Such results implied that tryptophan, histidine, tyrosine, and serine residues are related to enzyme activity. The pH optima for wheat grass peroxidase to catalyze the oxidation of o-phenylenediamine (OPD), catechol, pyrogallol, and guaiacol were 5.0, 4.5, 6.5, and 5.0, respectively. The apparent Km values for OPD, catechol, pyrogallol, and guaiacol were 2.9, 18.2, 2.5, and 3.8 mM, respectively. Under optimal reaction conditions, wheat grass peroxidase catalyzed the oxidation of OPD (an aromatic amine substrate) 3-11 times more rapidly than guaiacol, catechol, and pyrogallol (phenolic substrates containing one to three hydroxy groups in the benzene ring).

Characterization of a chitosanase isolated from a commercial ficin preparation.

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).

Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes.

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.

Effects of Taiwanese yam (Dioscorea japonica Thunb var. pseudojaponica Yamamoto) on upper gut function and lipid metabolism in Balb/c mice.

OBJECTIVE: This study investigated the effects of a Taiwanese yam, Dioscorea japonica Thunb var. pseudojaponica Yamamoto, on upper gut function and lipid metabolism in adult Balb/c mice. METHODS: Mice were randomly allocated to consume the control, 25%, or 50% yam diet in which yam in an uncooked lyophilized form was incorporated into the diet for 21 d. RESULTS: Growth rates were similar among groups, even though the apparent protein absorption rate was decreased by the 50% yam diet. Both yam diets decreased gastric villous width but did not significantly modulate other morphologic and proliferative indices. Brush-border leucine aminopeptidase activities in the small intestine were increased approximately 30% by the 25% and 50% yam diets, respectively. In contrast, sucrase activity was decreased 40% by the 25% yam diet and 50% by the 50% yam diet. The 50% yam diet consistently improved the cholesterol profile in the plasma and liver, whereas the 25% yam diet reduced only the level of low-density lipoprotein cholesterol in plasma. Changes in blood lipid levels were associated with reduced fat absorption. CONCLUSION: A 25% uncooked yam diet may benefit upper gut function and prevent hypercholesterolemia in humans, but the 50% yam diet negatively affected protein absorption.

Identification of essential histidine residues in a recombinant alpha-amylase of thermophilic and alkaliphilic Bacillus sp. strain TS-23.

To understand the structure-function relationships of a truncated Bacillus sp. strain TS-23 alpha-amylase, each of His-137, His-191, His-239, His-269, His-305, His-323, His-361, His-436, and His-475 was replaced with leucine. The molecular masses of the purified wild-type and mutant enzymes were approximately 54 kDa. The specific activity of His323Leu and His436Leu was decreased by more than 52%, while His239Leu, His305Leu, and His475Leu showed activity similar to that of the wild-type enzyme. As compared with the wild-type enzyme, His323Leu and His436Leu exhibited a 62% decrease in the value of k(cat)/ K(m). Alterations in His-191, His-239, His-305, and His-475 did not cause a significant change in the K(m) or k(cat) values. At 70 degrees C, a decreased half-life was observed in His436Leu. These results indicate that His-137, His-269, and His-361 of Bacillus sp. strain TS-23 alpha-amylase are important for proper catalytic activity and that His-436 may contribute to the thermostability of the enzyme.

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation.

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.