Isolation and biochemical characterization of an antifungal peptide from Amaranthus hypochondriacus seeds

An antifungal peptide, Ay-AMP, was isolated from Amaranthus hypochondriacus seeds by acidic extraction and then purified by reverse-phase high-pressure liquid chromatography. The molecular mass of this peptide, as determined by mass spectrometry, is 3184 Da. The peptide belongs to the superfamily of chitin-binding proteins, containing a single cysteine/glycine-rich chitin-binding domain, and it was found that Ay-AMP degrades chitin. Ay-AMP inhibits the growth, at very low doses, of different pathogenic fungi, such as Candida albicans, Trichoderma sp., Fusarium solani, Penicillium chrysogenum, Geotrichum candidum, Aspergillus candidus, Aspergillus schraceus, and Alternaria alternata. Ay-AMP is very resistant to the effect of proteases and heating; however, it showed an antagonistic effect with CaCl2 and KCl.     

Purification and characterization of an isoflavone-conjugates-hydrolyzing beta-glucosidase from endophytic bacterium

An isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) from endophytic bacterium, Pseudomonas ZD-8 was purified to homogeneity by successive ammonium sulfate precipitation, gel filtration on SephadexG-100, DEAE-sephrose CL-6B and DEAE-Sephacel chromatography. The enzyme was a monomeric protein with an apparent molecular mass of 33 kDa as determined by SDS-PAGE and gel filtration. It was optimally active at pH 6.0 and 40 degrees C and had a specific activity of 1485 U mg of protein(-1) against genistin. The ICHG readily hydrolyzed rho-nitrophenyl-beta-glucoside, rho-nitrophenyl-beta-galactoside, genistin, daidzin, with Km values of 1.64, 1.87, 0.012, 0.014 mM, respectively. The ICHG showed a pronounced specificity for glucose in the 7-position of isoflavone and flavone conjugates and hydrolyzed effectively malonyl isoflavone glucosides as well as isoflavone glucosides with similar kinetics. Glucose and glucono-delta-lactone inhibited the enzyme competitively with Ki values of 84 mM and 23 mM, respectively. The enzyme did not require divalent cations for activity, and its activity was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, iodoacetic acid, and N-ethylmaleimide while reducing agents such as beta-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione slightly activated the enzyme.     

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 μM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 μM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 μM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 μM, respectively.     

Purification and characterization of the antitumor protein from Chinese tartary buckwheat (Fagopyrum tataricum Gaertn.) water-soluble extracts

A novel antitumor protein, coded as TBWSP31, was isolated from tartary buckwheat water-soluble extracts and purified by DEAE-Sepharose Fast Flow anion exchange, Sephadex G-100 gel filtration, and Sephacryl S-200 gel filtration column chromatography. TBWSP31 was identified to a homogeneous fraction by native PAGE. The antitumor effect of TBWSP31 against human mammary cancer cell Bcap37 was measured by an MTT assay. TBWSP31 showed higher antitumor activity, and time- and concentration-dependent effects were observed. SDS-PAGE analysis showed that TBWSP31 is composed of a single polypeptide with an approximate molecular weight of 57 kDa. TBWSP31 was rich in Glx (Gln+Glu), Arg, and Asx (Asp+Asn) according to amino acid analysis. Secondary structural analysis by CD spectroscopy revealed that TBWSP31 has the following: alpha-helix (33.9%), beta-sheet (22.8%), beta-turn (11.3%), and random coil (32.0%).     

露地种植大白菜的氮肥效应与氮素损失研究

采用田间小区和微区试验,研究了施用化学氮肥在露地大白菜上的氮肥效应和氮素损失。氮素总损失用15N示踪法测定,氨挥发用通气密闭室法测定,反硝化损失用乙炔抑制原状土柱培养法测定,不加乙炔测定N2O排放。结果表明,施用化学氮肥增产显著,用差值法计算得到的氮肥利用率在25.3%4～7.2%之间,相应的示踪法氮肥利用率为18.1%2～4.6%。化学氮肥显著增加了氨挥发、反硝化和N2O排放等气态氮损失;其中氨挥发占施氮量的0.97%1～7.1%,反硝化占4.33%8～.55%,N2O排放在1.09%1～.63%之间变化。大白菜收获时9.2%～10.9%的标记尿素被淋洗到40.cm以下土层。试验期间尿素的氮素总损失达41.1%4～8.1%,以表观淋洗损失最为严重,其次是氨挥发,而反硝化损失最低。与普通尿素相比,包衣尿素明显降低了氨挥发。     

Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.     

Purification and characterization of lipoxygenase from Pleurotus ostreatus

Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66,000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degrees C, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K(m), V(max), and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 micromol.min(-1).mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z,11E-octadecadienoic acid was found to be the main oxidative product.     

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 an extracellular antimicrobial protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.     

Purification and characterization of a corrinoid compound from Chlorella tablets as an algal health food

Vitamin B(12) content of an algal health food, Chlorella tablets (Chlorella sp.), was determined by both Lactobacillus leichmannii ATCC 7830 microbiological and intrinsic factor-chemiluminescence methods. The values of 200.9-211.6 microg/100 g dry weight determined by the chemiluminescence method were similar to the values (201.3-285.7 microg/100 g dry weight) determined by the microbiological method. A corrinoid compound was purified to homogeneity from the Chlorella tablets and characterized. The purified corrinoid compound was identified as vitamin B12, on the basis of silica gel 60 TLC, C18 reversed-phase HPLC, 1H NMR spectroscopy, and UV-Vis spectroscopy.     

Purification and characterization of bacteriocin from Pediococcus pentosaceus ACCEL

The Pediococcus pentosaceus ACCEL bacteriocin was purified to electrophoretical homogeneity by cell adsorption-desorption and Superose 12 fast performance liquid chromatography (FPLC). The purified bacteriocin, with a molecular mass of 17.5 kDa and an N-terminal sequence of -KYYGNGVTXGKHSXXVDXG-, belongs to class IIa and is designated pediocin ACCEL. It was inactivated by various proteases and stable at pH 2.0-6.0 and <100 degrees C. More than 80% activity was left even after 15 min of heating at 121 degrees C and pH 2.0-4.0. Gram-positive food-borne pathogens were inhibited by this bacteriocin, but Gram-negative ones were not. According to the storage stability study, the purified pediocin was stable at pH <6.0 and low temperature. No significant change in bactericidal activity was observed after freeze-drying and subsequent 1-month storage at room temperature.     

Purification and characterization of polyphenols from chestnut astringent skin

Polyphenolic compounds from chestnut astringent skin (CAS) were purified by dialysis, using Diaion HP-20 and Sephadex LH-20 columns. During purification, specific α-amylase inhibitory activities were increased about 3.4-fold, and the 50% inhibition value was 5.71 μg/mL in the Sephadex LH-20 fraction (SE-fraction). The SE-fraction contained about 67% of the total polyphenols, 57.3% of the flavanol-type tannins, and 51.3% of the procyanidins. Strong antioxidant activity was observed in the SE-fraction. Oral administration of the SE-fraction in rats fed corn starch significantly suppressed an increase in blood glucose levels. The SE-fraction contained gallic acid and ellagic acid. The MALDI-TOF spectrum showed a peak series exhibiting a mass increment of 288 Da, reflecting the variation in the number of catechin/epicatechin units. Our results suggest CAS contains polyphenols with strong α-amylase inhibitory activity. The data also suggest CAS polyphenols might be oligomeric proanthocyanidins with gallic acid and ellagic acid.     

Purification and characterization of polyphenol oxidase from rape flower

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.     

Isolation and characterization of a bioactive mannose-binding protein from the Chinese chive Allium tuberosum

A mannose-binding protein was isolated from two different cultivars of the Chinese chive Allium tuberosum by extraction with 0.2 M NaCl, ammonium sulfate precipitation, and affinity chromatography on mannose agarose and fetuin agarose. It exhibited hemagglutinating activity toward rabbit erythrocytes. The lectin (agglutinin) was adsorbed on the mannose-agarose column, but not on the fetuin-agarose column. This A. tuberosum lectin (ATL) is unglycosylated, and not sialic acid binding. Lectins isolated from the two cultivars exhibited the same molecular mass of 25 kDa on gel filtration (Superose 12) and 12.5 kDa on SDS-polyacrylamide gel electrophoresis, indicating that they might be a dimeric protein composed of two identical subunits. The N-terminal amino acid sequence analysis of the lectin of various cultivars of A. tuberosum revealed that they were identical and showed 50%, or more, homology to the lectins from Galanthus nivalis (family Amaryllidaceae), Narcissus tazetta (family Amaryllidaceae), and Aloe arborescenes (family Liliaceae).     

Structure elucidation and antifungal activity of an anthracycline antibiotic, daunomycin, isolated from Actinomadura roseola

The actinomycete strain Ao108 producing antifungal metabolites active against some plant pathogenic fungi was identified as Actinomadura roseola, based on the analyses of morphological and physiological characteristics. The antibiotic Da2B that showed a strong antifungal activity was isolated from the culture broth and mycelial mats of A. roseola strain Ao108 using various chromatographic procedures. On the basis of (1)H NMR, (13)C NMR, and 2-D NMR correlation data, the antibiotic Da2B was confirmed to have the structure of an anthracycline antibiotic, daunomycin. In vitro antimicrobial spectrum tests showed that the antibiotic Da2B had substantial inhibitory activity (10 microg mL(-)(1) of MICs) against mycelial growth of Phytophthora capsici and Rhizoctonia solani. The antibiotic also showed antiyeast activity against Saccharomyces cerevisiae, but the growth of Candida albicans was not affected. Antibacterial activity was found only against Gram-positive bacteria. In the further evaluation of in vivo efficacy, application of the antibiotic Da2B effectively inhibited the development of Phytophthora blight in pepper plants. However, the control efficacy of the antibiotic against Phytophthora infection was somewhat less than that of metalaxyl. The antibiotic Da2B did not show any phytotoxicity on pepper plants even at 500 microg mL(-)(1).     

Purification, identification, and characterization of peanut isocitrate lyase

Isocitrate lyase (ICL, EC 4.1.3.1) is commonly present in oil-rich seeds in catalyzing the cleavage of isocitrate to glyoxylate and succinate and plays an essential role in lipid metabolism and gluconeogenesis. When peanut kernels (Tainan 14) were germinated at 30 degrees C, the cotyledon ICL activities increased substantially in the initial 4 days, and the 4-day-germinated cotyledons were subjected to ICL purification by Tris-HCl buffer extraction, heat treatment at 55 degrees C for 1 h, (NH4)2SO4 fractionation at 25-35% saturation, DEAE-cellulose chromatography, and Sephacryl S-300 gel filtration. A single 64 kDa SDS-PAGE protein band was obtained with 7.7% recovery and 37.5-fold purity. It was identified as ICL by LC-MS/MS analyses and Mascot Search with 494 as the highest Probability Based Mowse Score (PBMS). On the basis of the sequence of the homologous ICL of Glycine max, 26% of the peptide sequences of the peanut ICL were identified. During gel filtration, separation of peanut catalase (identified by LC-MS/MS and Mascot Search with 405 as the highest PBMS) from peanut ICL was achieved. The highest measured peanut ICL enzymatic activities were obtained at 45 degrees C and pH 7.0-7.8, respectively. The enzyme activities were stable (>80%) as stored for 8 h at 30 degrees C, 15 days at 4 degrees C, or 60 days at -25 degrees C. As affected by the supplements in the reactants for activity determinations, ICL activity was not affected by glucose up to 4%, sucrose up to 5%, or ethanol up to 8.33%.     

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

Purification and characterization of a peroxidase from corn steep water

Three cationic peroxidases have been detected in early, middle, and late corn steep water, with pI values of approximately 8.9, approximately 9.5, and >10.0. The major cationic corn steep water peroxidase (CSWP), with a pI >10, was purified 36400-fold with a 12% recovery from late steep water by a combination of acetone and ammonium sulfate precipitation and sequential chromatography on CM-cellulose, phenyl-Sepharose, and Sephadex G-75. The UV-vis spectrum of purified CSWP is typical of other plant class III peroxidases. The RZ (A(403)/A(280)) of CSWP was between 2.6 and 2.9. It is not glycosylated and exhibited an M(r) of 30662 +/- 7 by MALDI-TOF MS. The pH optimum of CSWP depends on the substrate, and it is active on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), guaiacol, ferulic acid, o-dianisidine, o-phenylenediamine, and pyrogallol but is not active on either syringaldazine or ascorbate. At 75 degrees C and pH 4.5, the enzyme has half-lives of 22.7 min (0 mM Ca(2+)) and 248 min (1 mM Ca(2+)). The enzyme is stable at room temperature (22-25 degrees C), losing <3% of the activity at pH 4.5 and <10% at pH 6.2 over 400 h in the presence of 1 mM Ca(2+).     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.