DEAE-Sepharose Ion Exchange Chromatography Research Articles

Purification and biochemical characterization of two laccase isoenzymes isolated from Trichoderma harzianum S7113 and its application for bisphenol A degradation

Two laccase isoenzymes (LacA and LacB) were isolated from a novel Trichoderma harzianum S7113 isolate employing ammonium sulfate precipitation, Sephadex G100, and DEAE Sepharose ion exchange chromatography. The molecular weights of the purified LacA and LacB laccases were estimated to be 63 and 48 kDa, respectively. The two isoenzymes had their optimum activities at the same temperature (50 °C), but at slightly different pH values (pH 3.0 for LacA and pH 2.5 for LacB). LacA and LacB had the same thermal stability at 40 °C and pH stability at pH 9.0. The two isoenzymes also showed a high level of specific activity toward ABTS, where the Km values of LacA and LacB were 0.100 and 0.065 mM, whereas their Vmax values were 0.603 and 0.182 µmol min−1, respectively. LacA and LacB catalytic activity was stimulated by Mg2+, Zn2+, K+, and Ni2+, whereas it was inhibited by Hg2+ and Pb2+, β-mercaptoethanol, EDTA, and SDS, and completely inhibited by sodium azide. Our findings indicate that purified laccase has a promising capacity for bisphenol A (BPA) bioremediation across a broad pH range. This finding opens up new opportunities for the commercialization of this technique in a variety of biotechnology-based applications, particularly for removing endocrine chemicals from the environment.

Reprint of: Purification and Characterization of an Extracellular Mn(ll)-Dependent Peroxidase from the Lignin-Degrading Basidiomycete, Phanerochaete chrysosporium

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N3− readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 μM. The enzyme is also stimulated to varying degrees by α-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl- 6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.© 1985 Academic Press, Inc.

In silico and biochemical analysis on a newly isolated Trichoderma asperellum l-asparaginase

Microbial l-asparaginase enzyme has been considered as a good source of potential anticancer drugs. The enzyme purified from different microbes has been shown different biochemical and kinetics properties. Thus, our study aims to investigate l-asparaginase from Trichoderma asperellum (MN960163), including in silico characterization of the l-asparaginase encoding genes as well as the biochemical characters of the purified enzyme. In silico, analysis of the T. asp genome database revealed the presence of six protein encoding genes with N-terminal asparaginase domain that has asparaginase production potential as indicated from their physico-chemical properties, verification of three of them was confirmed. T. asperellum l- asparaginase with a specific activity of 200 U/mg and 61.7 fold purification was achieved by ammonium sulphate precipitation (60%) followed by DEAE-sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The purified enzyme has a molecular mass of 65 kDa and exhibited its maximum activity at pH 8 and 50 °C. The purified enzyme showed high thermal stability up to 50 °C and a high affinity to l- asparagine with a Km of 0.35 mM. The activity was significantly enhanced by Cu, K and Ca ions while Fe and EDTA was completely inhibited l-asparginase activity. In conclusion, the current data highlight new possible genes that could be employed as a promising source in asparaginase production for medical purposes. Additionally, the feature of the purified asparaginase enzyme could help to elucidate the unique properties of T. asp asparaginase.

Isolation, characterization and regulation of moonlighting proteases from Candida glabrata cell wall

Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10–50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.

Purification of chitosanase from Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 for chitooligosacharide production

Stenotrophomonas maltophilia KPU 2123 and Micromonospora sp. T5a1 are bacterial strains isolated from shrimp waste capable of hydrolyzing chitosan to produce chitooligosaccharides (COSs). Their COS products showed antimicrobial activity. The aim of this study was to purify chitosanase from those bacteria and used for COSs production. Chitosanase from both strains were produced using 0.5% colloidal chitin as inducer. The chitosanase was then purified by ultrafiltration, DEAE Sepharose ion exchange and Separacyl S-300 gel filtration chromatography. The specific activity and the yield of chitosanase KPU 2123 increased 2.35-fold and 30.90% after three steps of purifications, respectively, whereas chitosanase T5a1 increased 3.32-fold and 22.79%, respectively. The molecular weight of both chitosanases KPU 2123 and T5a1 were about 76 and 20 kDa, respectively. The COS products generated by chitosanase KPU 2123 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine, Tri-N-acetyl-D-glucosamine, Tetra-acetyl-D-glucosamine and Penta-N-acetyl-D-glucosamine while by chitosanase T5a1 contained N-acetyl-D-glucosamine, Di-N-acetyl-D-glucosamine. Based on their COS products, chitosanase KPU2123 can be categorized as endo-type chitosanase. Further study is needed to analysis the bioactivity of COSs obtained from the pure of chitosanases KPU 2123 and T5a1.

Purification and characterization of two pathogenesis-related class 10 protein isoforms with ribonuclease activity from the fresh Angelica sinensis roots

In this study, two pathogenesis-related (PR) class 10 protein isoforms, ASPR-1 and ASPR-2, were purified from fresh roots of the Chinese medicinal plant Angelica sinensis (A. sinensis) using 80% ammonium sulfate precipitation, Sephadex G50 gel filtration chromatography, and DEAE-Sepharose ion-exchange chromatography. The molecular masses of ASPR-1 and ASPR-2 were estimated to be 16.66 kDa and 16.46 kDa, respectively, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoforms are both glycoproteins containing glycosyl contents of 1.8% (ASPR-1) and 3.4% (ASPR-2). The two isoforms were predominantly present as monomers, but they partially dimerized in solution. The 15 N-terminal amino acids of ASPR-1 were determined to be GIQKTEVEAPSTVSA, with significant sequence homology to certain PR-10 proteins. ASPR-2 was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis to be a PR-10 protein. The isoforms both exhibited ribonuclease (RNase) activity, with ASPR-2 having higher specific activity (128.85 U mg−1) than ASPR-1 (68.67 U mg−1). The isoforms had the same optimal temperature of 50 °C but different optimal pH values of 5.0 (ASPR-1) and 6.0 (ASPR-2). The RNase activities of the isoforms were both stable for 30 min at 50 °C, rapidly decreasing at higher or lower processing temperatures. However, ASPR-1 retained higher residual activity (89.4%–80.9%) than ASPR-2 (74.3%–67.9%) at temperatures from 40 °C to 60 °C. These results provide additional information to enrich the current knowledge of poorly annotated A. sinensis proteins.

Antioxidant and antidiabetic activities of vanadium binding proteins purified from the sea squirt Halocynthia roretzi.

Sea squirts accumulate vanadium compounds with potent antidiabetic activity, which are involved in immune defense. In this study, vanadium concentrations of fresh blood plasma, intestine, and muscle of the sea squirt Halocynthia roretzi were 6.3, 3.7 and 2.1mg/kg respectively. Two vanadium binding proteins (VBPs) from blood plasma and intestine were purified through (NH4)2SO4 precipitation, and DEAE-Sepharose ion exchange and Sephacryl S-200 HR gel filtration chromatography, in that order. The purity and yield of the intestine and blood plasma vanadium binding proteins, VBPintestine and VBPblood plasma, were 13.4 folds and 7.1%, and 20.9 folds and 6.8%, respectively. There were two protein bands on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with molecular weights of 24.3 and 68.8kDa and one with 96.7kDa on the native-PAGE of VBPblood plasma, whereas only one protein band of VBPintestine on the SDS-PAGE with 26.5kDa. Antioxidant activities of VBPs were lower than that of ascorbic acid. Both VBPs exerted strong inhibitory activity against Saccharomyces cerevisiae and mild against Bacillus stearothermophilus and rat intestinal α-glucosidase. IC50 values of VBPintestine and VBPblood plasma against S. cerevisiae α-glucosidase were 28.34 and 12.60μg/ml, respectively. The Km , Vmax , kcat , and kcat/Km values of VBPintestine and VBPblood plasma were 4.29, 0.036, 6.58 and 1.53 × 103, and 7.63mM, 0.057mM/min, 10.41s-1 and 1.36 × 103 (Msec)-1, respectively. There was a synergistic interaction between VBPblood plasma and VBPintestine on rat intestinal α-glucosidase inhibitory activity.

Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

Objective: The intact human Parathyroid hormone (hPTH) is one of the biopharmaceutical drug produced by biotechnology, this hormone can be provided in a good amount using simple batch culture, and therefore the main purpose of this study was the production of purified bioactive intact hPTH.Methods: A cloning BL21(DE3) strain (which already cloned in our Lab. by synthetic human Parathyroid hormone (shPTH) gene) was grown in LB medium and the cloning gene expression was induced by addition of IPTG to the medium. The recombinant fused protein was purified from the bacterial cell lysate by affinity chromatography and underwent proteolysis by Enterokinase enzyme to obtain the target hPTH, and it's further purified by DEAE and SP Sepharose ion exchange chromatography. RP-HPLC analysis and biological activity on the MCF-7 breast cancer cells were done for both the standard and produced hPTH. Furthermore, the haemolysis ability of the latter was tested on the human RBC.Results: 225ng/ml of hPTH was produced after SP chromatography which detected by specific PTH ELISA kit, standard and produced hPTH showed the same peaks in the same retention time when analyzed by HPLC. The produced hPTH haemolysis assay showed the ability of the produced hPTH to partially haemolyze the RBC in a concentration above 200µg/ml. The bioactivity assay for the produced and standard rhPTH demonstrated that both compounds had a biological activity on the MCF-7 cells with IC50 of about 84.4 and 75.7 µg/ml for standard and produced hPTH respectively, and no significant difference was detected between them.Conclusions: Via suitable purification processes, the biologically active hPTH with structure similar to the standard ones as detected by RP-HPLC and bioactivity assay, can be obtained by using already cloned isolate with shPTH gene for hormone production. The hormone showed haemolysis effect on the RBC similar to the normal secreted hPTH.

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters KM and Vmax for free and immobilized cutinase were found to be 0.55mM and 1410μmolmin−1mg−1 protein, 2.99mM and 996μmolmin−1mg−1 protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.

Purification and characterization of a surfactant-compatible lipase from Aspergillus tamarii JGIF06 exhibiting energy-efficient removal of oil stains from polycotton fabric.

An extracellular lipase with 23,666.66 U/ml/min activity was produced by Aspergillus tamarii JGIF06 under submerged fermentation in mineral salt medium containing coconut oil (2.5 % v/v), tryptone (2 % w/v) and ammonium chloride (2 % w/v), with initial pH of 5 ± 0.2, incubated at 25 °C for 7 days on a rotary shaker at 120 rpm. A 7.9-fold increase in lipase-specific activity was recorded after purification by DEAE Sepharose ion exchange and Sephadex G200 column chromatography. The apparent molecular mass of this enzyme was revealed as 50 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimal lipase activity was recorded at pH 4 and 37 °C. The enzyme revealed broad specificity towards different vegetable oils. The K m and V max of the lipase on olive oil was found to be 330.4 mg and 53,690 U/ml/min, respectively. The lipase activity was stable in the presence of surfactants such as cetrimonium bromide, sodium dodecyl sulphate and Tween 80, and metal ions and reagents such as Ca2+, Ba2+ and 2-mercaptoethanol. However, the activity was greatly reduced in the presence of organic solvents such as chloroform. The stain removal potential of the crude lipase was determined on polycotton fabric pieces stained with peanut oil. Lipase added to cold water alone significantly enhanced the removal of stain by 152 %. The addition of lipase also improved the stain removal efficiency of a commercially available detergent in the presence of either cold (25 ± 2 °C) or hot (65 ± 2 °C) water. The current findings suggest the potentiality of this enzyme for energy-efficient biocatalytic application.

Crude extract of maggots: Antibacterial effects againstEscherichia coli, underlying mechanisms, separation and purification

To investigate the antibacterial effects of a crude extract of maggots against Escherichia coli (E. coli) and the underlying mechanisms, and to separate and purify the crude extract of maggots to assess the antibacterial effects of the active ingredients in the crude extract. Different concentrations of the crude extract of maggots were incubated with E. coli (O157:H7) and cultured. The optical density (OD) was measured at different time points to plot the OD-T curve. The effects of different concentrations of the crude extract on bacterial membrane permeability were determined by fluorescence probe technique. The effects of different concentrations of the crude extract on plasmid DNA replication were determined by agarose gel electrophoresis. DEAE-Sepharose ion exchange chromatography and Sephacryls-200HR gel filtration chromatography were used to separate and purify the crude extract of maggots. The molecular weight of proteins in the purified crude extract was determined by SDS-PAGD electrophoresis, and its antibacterial effects were determined by turbidimetric method. The antibacterial effects of the crude extract of maggots at concentrations > 0.5 mg/mL were significant. The antibacterial effects of the crude extract at concentrations of 1.0, 1.5 and 2.0 mg/mL did not differ significantly. Fluorescence probe analysis showed that the rate of membrane permeability change was 1223.1% in bacteria incubated with 2 mg/mL of the crude extract, and 1300.0% in those incubated with 80 mg/mL of the crude extract. Plasmid DNA was undetectable in E. coli incubated with 2 and 80 mg/mL of the crude extract. A low molecular weight protein band (about 15 kDa) was detected in the crude extract of maggots and eluent, but not in eluant, from DEAE-Sepharose ion exchange chromatography. The antibacterial effects of the crude extract of maggots and eluent were superior to those of eluant, with the antibacterial effects of eluents being better than those of the crude extract of maggots. Of 24 tubes of filtrates, the antibacterial effects of filtrates in tubes 4, 5 and 11 were significantly higher than those of the control. The molecular weight of the protein in filtrates in tubes 4, 5 and 11 was about 15 kDa. The crude extract of maggots exhibits obvious, dose-dependent antibacterial effects. The crude extract exerts antibacterial effects by changing the bacterial membrane permeability and inhibiting plasmid DNA replication. The protein that has antibacterial effects in the crude extract of maggots has a molecular weight of about 15 kDa.

Amylase production by Preussia minima, a fungus of endophytic origin: optimization of fermentation conditions and analysis of fungal secretome by LC-MS

BackgroundEnvironmental screening programs are used to find new enzymes that may be utilized in large-scale industrial processes. Among microbial sources of new enzymes, the rationale for screening fungal endophytes as a potential source of such enzymes relates to the hypothesised mutualistic relationship between the endophyte and its host plant. There is a need for new microbial amylases that are active at low temperature and alkaline conditions as these would find industrial applications as detergents.ResultsAn α-amylase produced by Preussia minima, isolated from the Australian native plant, Eremophilia longifolia, was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE-Sepharose ion exchange chromatography. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L-asparagine as the nitrogen source. Bioreactor studies showed that enzyme activity was comparable to that obtained in shaker cultures, which encourages scale-up fermentation for enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9-mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae.ConclusionsThe findings of the present study indicate that the purified α-amylase exhibits a number of promising properties that make it a strong candidate for application in the detergent industry. To our knowledge, this is the first amylase isolated from a Preussia minima strain of endophytic origin.

Isolation and Characterization of Chelonia mydas Myoglobin

Green sea turtle – Chelonia mydas is a lung respiration animal that is able to dive and stay uder sea water for hours without needing to surface for oxygen. As oxygen supply in muscles is assured by myoglobin, we propose to study some characteristic of this muscle protein in green sea turtles. To achieve this objective, pure green sea turtle myoglobin has to be made available. Therefore, our first task is to purify this muscle protein from green sea turtles. Skeletal muscles from 3 green sea turtle hatchlings were studied microscopically and biochemically. Microscopy observation showed a general structure of striated muscle. Biochemical studies revealed that green sea turtle myoglobin could be more purely isolated to a certain degree by Sephadex G-75 gel filtration and purified by immunoaffinity gel chromatography rather than direct purification by DEAE-Sepharose ion exchange chromatography technique. SDS-PAGE analysis showed that green sea turtle myoglobin ran together with horse myoglobin as 17 kDa molecular weight proteins.

Interaction of superoxide dismutase with the glycine zipper regions of β-amyloid peptides: Is there an implication towards Alzheimer’s disease and oxidative stress?

Not only are β-amyloid peptides and senile plaque deposits characteristics in Alzheimer’s disease but there is growing evidence to suggest that oxidative stress also plays a role with a decrease in levels of brain superoxide dismutase (SOD), an enzyme that catalyses the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide. We show through kinetic and fluorescence analysis that β-amyloid peptides, in the glycine zipper region [Aβ29–33 and Aβ25–37] of Aβ1–40 interact with, and inhibit, SOD directly. The enzyme was purified 15.7-fold from bovine brain by DEAE-Sepharose ion exchange chromatography in a yield of 68.8% and specific activity of 3.66 U.mg−1. The subunit structure of the enzyme was monomeric with a molecular mass of 13 kDa, as estimated by SDS-PAGE. Inhibitor constants (Ki) and dissociation constants (Kd) were calculated as 14.44, 13.16 and 11.72 µM and 9.38, 15.7 and 12.13 for Aβ25–37, Aβ29–33 and Aβ1–40, respectively; the number of binding sites on the enzyme for the peptides was 1.

Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

Purification and characterization of cold-active endo-1,4-β-glucanase produced by Pseudoalteromonas sp. AN545 from Antarctica

A bacterium hydrolyzing carboxymethylcellulose, isolated from Antarctic sea ice, was identified as Pseudoalteromonas sp. based on 16S rDNA gene sequences and named as Pseudoalteromonas sp. AN545. The extracellular endo-1,4-β-glucanase AN-1 was purified successively by ammonium sulfate precipitation, DEAE-Sepharose ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The molecular mass of AN-1 was estimated to be 47.5 kDa utilizing SDS-PAGE and gel chromatography analysis. AN-1 could hydrolyze caboxymethylcellulose, avicel and β-glucan, but not cellobiose, xylan and p-Nitrophenyl-β-D-glucopyranoside. The optimal temperature and pH for the β-glucanase activity of AN-1 were determined to be at 30°C and pH 6.0, respectively. AN-1 was stable at acidic solutions of pH 5.0–6.5 and temperatures below 30°C for 1 h. Moreover, the specific activity was enhanced by Ca2+ and Mg2+, and inhibited by Cu2+. The kinetic parameters Michaelis constant (K m) and maximum velocity (V max) of AN-1 were 3.96 mg/mL and 6.06×10−2 mg/(min·mL), respectively.

Selenium-Containing Allophycocyanin Purified from Selenium-Enriched Spirulina platensis Attenuates AAPH-Induced Oxidative Stress in Human Erythrocytes through Inhibition of ROS Generation

Both selenium and allophycocyanin (APC) have been reported to show novel antioxidant activities. In this study, a fast protein liquid chromatographic method for purification of selenium-containing allophycocyanin (Se-APC) from selenium-enriched Spirulina platensis and the protective effect of Se-APC on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress have been described. After fractionation by ammonium sulfate precipitation, and separation by DEAE-Sepharose ion-exchange and Sephacryl S-300 size exclusion chromatography, Se-APC with purity ratio (A652/A280) of 5.30 and Se concentration of 343.02 μg g(-1) protein was obtained. Se-APC exhibited stronger antioxidant activity than APC by scavenging ABTS (2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid) and AAPH free radicals. The oxidative hemolysis and morphological changes induced by AAPH in human erythrocytes were effectively reversed by coincubation with Se-APC. Lipid oxidation induced by the pro-oxidant agent cupric chloride in human plasma, as evaluated by formation of conjugated diene, was blocked by Se-APC. The accumulation of malondialdehyde, loss of reduced glutathione, and increase in enzyme activities of glutathione peroxidase and reductase induced by AAPH in human erythrocytes were effectively suppressed by Se-APC. Furthermore, Se-APC significantly prevented AAPH-induced intracellular reactive oxygen species (ROS) generation. Taken together, our results suggest that Se-APC demonstrates application potential in treatment of diseases in which excess production of ROS acts as a casual or contributory factor.

Purification and characterisation of polyphenol oxidase from leaves of Cleome gynandra L.

A polyphenol oxidase was purified and characterised from leaves of the common spiderflower. Purification sequentially with ammonium sulphate, dialysis, DEAE-Sepharose ion-exchange chromatography and Sephadex G-75 gel filtration chromatography resulted in 37.8-fold enrichment in the specific activity and 44.3% recovery of the total activity. Purified PPO is a monomeric protein of 52.6kDa revealed by Coomassie and active staining and Western blot. It was optimally active at pH 8.0 and 60°C, and stable from pH 3.0 to 9.0 and below 60°C. It displayed enzymatic activity towards monophenols, diphenols and triphenols, especially towards diphenols, and substrate specificity towards methylated and methoxylated substrates. Its activity was slightly increased by 0.1% SDS, heavily inhibited by Hg2+ and Pb2+, and completely inhibited by 1.0mM of ascorbic acid, l-cysteine, β-mercaptoethanol, sodium diethyldithiocarbamate and thiourea, and by 10mM of dithioerythritol, sodium metabisulphite and sodium sulphite.

Purification and Characterization of a Protease from Korean Pear (Pyrus serotina L.) as Meat Tenderizer

This study was conducted for the isolation, purification, and characterization of a protease from Korean pear, to see its proteolytic activity on chicken actomyosin and to find the optimum pH and temperature of activity on chicken actomyosin. The protease was isolated from crude extract of Korean pear by ammonium sulfate precipitation. Further purification was done by DEAE-Sepharose ion-exchange chromatography, Mono-Q and Mini-Q column chromatography. The purified enzyme gave a single protein band on SDS polyacrylamide gel electrophoresis and the molecular weight was found to be 38 kDa. The specific activity of purified enzyme was 34,907 unit/mg with 25 fold purification and the yield was 2%. The purified enzyme incubated with chicken actomyosin showed high activity. The optimum pH and temperature for enzyme activity on chicken actomyosin were 6.5 and <TEX>$70^{\circ}C$</TEX>, respectively. A protease was purified from Korean pear for the first time and characterized. It was found to be promising for meat tenderization.

Purification of phycoerythrin from Porphyra yezoensis Ueda (Bangiales, Rhodophyta) using expanded bed absorption

R-phycoerythrin was purified by means of phenyl-sepharose expanded bed absorption and DEAE-sepharose ion-exchange chromatography from Porphyra yezoensis, one of the largest and important aquaculture species in China. Final R-phycoerythrin preparation was characterized by purity ratio above 4 and the homogeneity in native PAGE, respectively. The results of absorption spectrum, fluorescence spectrum and SDS-PAGE were in agreement with previous reports on R-PE. The yield of R-phycoerythrin was 0.82 mg g−1 wet leafy gametophyte of P. yezoensis. This method is a high-protein recovery technology while reducing processing time, and is suitable for the large batch production of R-phycoerythrin, which will enhance the value of P. yezoensis in China, especially the inferior P. yezoensis which can not be used for flake processing.