DEAE-Sepharose CL-6B Research Articles

Evidences of Monomer, Dimer and Trimer of Recombinant Human Cyclophilin A

Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.

수삼추출물 첨가 mushroom complete medium에서 배양된 영지버섯 균사체의 면역증진 효과 및 활성다당류

영지버섯 균사체(Ganoderma lucidum, GL)의 면역활성을 증진시키기 위하여 기본배지인 mushroom complete medium(MCM)에 수삼추출물(GE, 65<TEX>$^{\circ}Bx$</TEX>)을 농도별로 첨가한 혼합 액체배지에서 균사체를 배양한 후 원심분리로 균사체를 회수하여 동결건조하고 열수추출과 에탄올 침전처리를 이용하여 조다당획분(CP)을 분획하였다. MCM 부피의 15% GE가 첨가된 혼합배지에서 배양된 수삼추출물-영지 균사체의 GL-GE-15-CP는 100 <TEX>${\mu}g/mL$</TEX>의 농도에서 5%와 10% GE가 첨가된 GL-GE-5-, 10-CP 및 GE가 첨가되지 않은 일반-영지 균사체의 GL-CP보다 유의적으로 높은 마크로파지 활성을 나타내었으며, Peyer's patch를 경유한 장관면역 활성도 증진되었음을 확인할 수 있었다. 활성획분인 GL-GE-15-CP는 DEAE-Sepharose CL-6B의 분획을 통하여 GL-GE-15-CP의 다른 획분 또는 시료대조군인 GL-CP로부터 분획된 모든 획분보다 유의적으로 높은 마크로파지 활성, IL-12 생산능 및 장관면역 활성(각각 1.75, 5.68, 1.76배)을 갖는 다당획분인 GL-GE-15-CP-II을 분리하였다. 또한, GL-GE-15-CP-II는 동일 NaCl 농도에서 분획된 시료대조군인 GL-CP-II보다 200 <TEX>${\mu}g$</TEX>/마우스의 농도에서 colon 26-M3.1 carcinoma cell에 대하여 유의적으로 높은 암전이 억제활성도 보여주었다(tumor control의 72.8% 억제활성). 한편, 활성 다당획분인 GL-GE-15-CP-II는 주로 중성당(83.00%)과 산성당(9.11%)으로 구성되어 있었으며, 구성당 분석결과에서 Ara, Man, Gal와 Glc의 중성당으로 구성되어져 있음을 확인할 수 있었다(molar ratio; 0.28 : 0.39 : 0.50 : 0.75 : 1.00). 따라서 영지버섯 균사체 배양에 있어서 MCM 기본배지에 수삼추출물의 첨가는 균사체의 면역활성 증진에 중요하게 작용하는 것으로 보이며, 활성 다당획분의 구성당 분포를 비교할 때 중성 다당류가 면역활성 증진에 관여하고 있는 것으로 확인되었다. After Ganoderma lucidum was cultured in mushroom complete medium (MCM) supplemented with ginseng extract (GE), crude polysaccharide (GL-GE-CP) was fractionated from mycelium. Among GL-GE-CP from mycelium in MCM supplemented with 5, 10, and 15% GE (v/v ratio of MCM to GE), GL-GE-15-CP (15% GE) most significantly enhanced macrophage stimulation and intestinal immune system modulating activity compared with GL-CP in MCM without GE. When GL-GE-15-CP was further fractionated on DEAE-Sepharose CL-6B, GL-GE-15-CP-II displayed more potent activity than subfractions from GL-CP on macrophage stimulation, interleukin-12 production, and intestinal immune system modulation (1.75-, 5.68-, and 1.76-fold, respectively). Anti-metastasis effect against colon 26-M3.1 carcinoma cells was also enhanced by GL-GE-15-CP-II (72.8% inhibition). In addition, GL-GE-15-CP-II contained neutral sugar (83.00%) and uronic acid (9.11%), and consisted of Ara, Man, Gal and Glc (molar ratio of 0.39:0.50:0.75:1.00). Furthermore, GE supplementation helped to enhance the immunomodulation in G. lucidum, and it is assumed that neutral polysaccharides play an important role.

Purification and characterization of cathepsin B from the gut of the sea cucumber (Stichopus japonicas)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu2+, Ni2+, and Zn2+ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.

Structure and antitumor (LOVO) activity of Cortex moutan heteroglycan and Curcumin

The Box–Behnken design combined with response surface methodology was used to optimize the extraction of curcumin. The optimized results showed that the highest extraction yield of curcumin could arrive 1.12%. The suitability of the model equation for predicting the optimum response values was tested using the selected optimal conditions. The predicted extraction yield of heteroglycan was 1.13%, which was consistent with the practical extraction yield of heteroglycan of 1.17%. Cortex moutan heteroglycan (500mg) was applied to a column (3.2cm×32cm) of DEAE Sepharose CL-6B (Cl−) that was equilibrated with H2O. The two adsorbed fractions (Fraction I and Fraction II) were obtained as lyophilisate after dialysis The antitumor activities of the Cortex moutan heteroglycan and curcumin were evaluated in vitro. The results indicated that the Cortex moutan heteroglycan and curcumin could inhibit colorectal carcinoma (LOVO) cells growth and stimulate apoptosis. Moreover, antitumor activities of curcumin was stronger than that of Cortex moutan heteroglycan. This indicated that curcumin was useful for therapy of colorectal carcinoma.

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.).

BackgroundWe have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.ResultsA cDNA, named aleh1, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The aleh1 encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (pI) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of aleh1 was produced in Pichia pastoris, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that aleh1 encoded 1-FEH.

PURIFICATION AND CHARACTERIZATION OF AN INTRACELLULAR α-GLUCOSIDASE WITH HIGH TRANSGLYCOSYLATION ACTIVITY FROM A. niger M-1

An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116 kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca2+ enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag+, n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K m values of 4.32 m mol L−1 and V max of 3.10 × 10−2 mol L−1 min−1 were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.

수삼추출물 첨가 혼합배지에서 조제된 상황 균사체 심부배양물 다당획분의 면역활성 증진

면역활성을 증진시키기 위하여 수삼추출물(GE, 65°Bx)이 첨가된 액체배지(mushroom complete medium, MCM)에서 상황(Phellinus linteus, PL) 균사체를 배양하여 배양액과 균사체가 모두 포함된 심부배양물을 조제하고 조다당획분(CP)으로 분획하였다. MCM 부피에 대하여 GE가 15% 첨가된 혼합 액체배지에서 배양된 수삼추출물-상황 심부배양물로부터 분획된 PL-GE-15-CP는 100 μg/mL의 시료농도에서 GE-5와 10%가 첨가된 PL-GE-5와 10-CP 및 GE가 첨가되지 않은 일반-상황 심부배양물의 PL-CP보다 유의적으로 높은 마크로파지(saline 대조군의 1.45배) 및 Peyer's patch를 경유한 장관면역 활성(1.46배)을 확인할 수 있었다. 활성획분인 PL-GE-15-CP는 DEAE-Sepharose CL-6B를 이용한 분획을 통하여 PL-GE-15-CP의 다른 획분 또는 시료대조군인 PL-CP로부터 분획된 모든 획분보다 유의적으로 높은 마크로파지 활성, IL-12 생산능 및 장관면역 활성(각각 1.54, 3.96과 1.56배)을 갖는 다당획분인 PL-GE-15-CP-Ⅱ를 분리할 수 있었다. 또한, PL-GE-15-CP-Ⅱ는 동일NaCl 농도에서 분획된 시료대조군인 PL-CP-Ⅱ보다 시료농도 200 ㎍/mouse에서 colon 26-M3.1 carcinoma cell에 대하여 유의적으로 높은 암전이 억제활성도 보여주었다(대조군의 57.3% 억제활성). 한편, 활성 다당획분인 PL-GE-15-CP-Ⅱ는 주로 중성당(82.45%)과 산성당(12.99%)으로 구성되어 있었으며 구성당 분석결과에서 산성당과 함께 Ara, Man, Gal과 Glc의 중성당으로 구성되어져 있음을 확인할 수 있었다(molar ratio; 0.52:0.97:0.63:1.00:0.54). 결론적으로 GE가 첨가된 상황 심부배양물은 일반배지에서의 상황 심부배양물보다 면역활성이 높아 GE가 균사체 활성의 증진에 영향을 주고 있는 것으로 생각되며, 특히 활성성분으로 다당류가 중요하게 관여하고 있는 것으로 사료된다.

Study on Glycoprotein in Kiwifruit

Two fractions, such as Fr1-3 and Fr2-2, were obtain after separating and purifing crude kiwifruit glycoprotein by using Sepharose Cl-6B gel column and DEAE Sepharose Cl-6B ion exchange column. In the UV and Infrared Spectroscopy of Fr1-3 and Fr2-2, the characteristic absorption peaks of sugar and protein were both found.The molecular weight of Fr1-3 is 30497，the molecular weight of Fr2-2 is 28567. Both of Fr1-3 and Fr2-2 have 17 amino acids, However, the amino acid content of them is different.

Structural characterization of polysaccharides from Zizyphus jujuba and evaluation of antioxidant activity

Fruit of Zizyphus jujuba Mill, a traditional Chinese herb widely consumed in Asian countries, has been reported to possess several vital biological activities. This study intends to develop an appropriate analytical method for isolation of polysaccharides from Z. jujuba fruits and evaluate their antioxidant activity. Initially, powdered Z. jujuba fruits were subjected to hot water extraction, followed by ethanol precipitation, deproteination, dialysis and fractionation in a DEAE-Sepharose CL-6B column. One neutral polysaccharide fraction (ZJPN) and 3 acidic polysaccharide fractions (ZJPa1, ZJPa2 and ZJPa3) were isolated with the average MW ranging from 40,566 to 129,518 Da. GC analysis revealed that 6 monosaccharides, namely, rhamnose, arabinose, xylose, mannose, glucose and galactose were present in polysaccharide fractions. The galacturonic acid content in polysaccharide fractions followed the order: ZJPa3 > ZJPa2 > ZJPa1 > ZJPN. All the 4 polysaccharide fractions were found to be more effective in scavenging superoxide anions than hydroxyl radicals, while acidic polysaccharides showed a more pronounced effect in chelating ferrous ion.

Study of Macrophage Activation and Structural Characteristics of Purified Polysaccharide from the Fruiting Body of Cordyceps militaris

Cordyceps militaris, an entomophathogenic fungus belonging to the class Ascomycetes, has been reported to have beneficial biological activities such as hypoglycemic, anti-inflammatory, anti-tumor, anti-metastatic, hypolipidemic, immunomodulatory, and antioxidant effect. In this study, the crude water-soluble polysaccharides CMP, which was obtained from the fruiting body of C. militaris by hot water extraction and ethanol precipitation, was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This process resulted in three polysaccharide fractions, termed CMP Fr I, CMP Fr II, and CMP Fr III. Of these fractions, CMP Fr II, with an average molecular weight of 127 kDa, was able to upregulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. The chemical property of the stimulatory polysaccharide, CMP Fr II, was determined based on monosaccharide composition, which consisted of glucose (56.4 %), galactose (26.4 %), and mannose (17.2%). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicated that CMP Fr II consisted of the (1-->4) or (1-->2) linked glucopyranosyl or galactopyranosyl residue with a (1-->2) or (1-->6) linked mannopyranosyl, glucopyranosyl or galactopyranosyl residue as a side chain. The configuration of the beta-linkage and random coil conformation of CMP Fr II were confirmed using a Fungi Fluor kit and Congo Red reagent, respectively.

Purification and characterization of the -amylase isolated from Penicillium camemberti PL21

The amylase family of enzymes is a great significance due to its wide area of potential application. α-amylase from Penicillium camemberti PL21 obtained from I. N. A. (The technological laboratory of Agriculture National Institute Paris- France) and using orange waste as substrates was produced under optimum conditions, after 168 h of incubation and subjected for purification and characterization. The enzyme was purified by ammonium sulfate precipitation, dialysis, Sephadex G-100 and DEAE-Sepharose CL-6B column chromatography. A trial for the purification of α-amylase resulted in an enzyme with specific activity of (154.2 units/ml/mg protein) with (38.5 folds) purification .The α-amylase activity increased by enzyme concentration rise. The optimum substrate concentration for soluble starch was 1 % (w/v) while the optimum incubation temperature was 30oC. The purified α-amylase enzyme had a maximum activity at pH 6 and the Km value for solublestarch was 0.92 mg/ml. Analyses of this enzyme for molecular mass was carried out by SDS-PAGE electrophoresis, which revealed one band 60. 5 Kda. Key words: Penicillium camemberti PL21, α-amylase, purification, SDS-PAGE electrophoresis.

Study on macrophage activation and structural characteristics of purified polysaccharide from the liquid culture broth of Cordyceps militaris

The water-soluble crude polysaccharides obtained from the liquid culture broth of Cordyceps militaris by ethanol precipitation were fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This fractionation process resulted in three polysaccharide fractions that were termed CPSN Fr I, CPSN Fr II, and CPSN Fr III. Of the fractions, CPSN Fr II was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide (NO) and expression of cytokines (IL-1β and TNF-α). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography–mass spectrometry (GC–MS). Results indicate that CPSN Fr II was a 1,6-branched-glucogalactomannan that had a molecular weight of 36 kDa. The configuration of the β-linkage and random coil conformation of CPSN Fr II were confirmed using a Fungi-Fluor kit and congo red reagent, respectively.

Macrophage stimulating polysaccharide purified from peels of grape (Vitis labrusca)

To characterize the macrophage stimulating polysaccharide in grape (Vitis labrusca) peels, the active crude polysaccharide (VL-3) has been fractionated from the hot-water extract of grape peels. A macrophage stimulating polysaccharide-rich fraction (VL-3IIb-1-1) was purified from VL-3 by 3 successive column chromatographies on DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephacryl S-300. VL-3IIb-1-1 was eluted as a single peak on high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 194 kDa. VL-3IIb-1-1 consisted mainly of Ara and Gal in addition to uronic acid (GalA+GlcA) (molar ratio 1.00:0.81:0.72). Methylation analysis indicated that VL-3IIb-1-1 consisted mainly of terminal Araf, 4- or 5-linked Ara, 2,4-branched Rha, 6- or 3,4- or 3,6-branched Gal, and 3,4,6-branched Glc. Single radial gel diffusion also indicated that VL-3IIb-1-1 showed an intermediate reactivity with β-glucosyl-Yariv antigen. In addition, oral administration of VL-3IIb enhanced the stimulatory responses of macrophage stimulating activity ex vivo. Therefore, VL-3IIb-1-1 purified from grape peels is suggested to be pectic polysaccharide with arabino-3,6-galactan, and it is assumed that VL-3IIb-1-1 plays an important role for expression of its activity.

Lipases in the castor bean seed of Chinese varieties: Activity comparison, purification and characterization

The lipase activities in the three main Chinese castor bean varieties [ZiBi 05 (ZB 5), TongBi 06 (TB 6), JinBi 02 (JB 2)] were investigated. Two lipases were found in extracts from the endosperms of all the three varieties. One lipase is located in the lipid bodies, showing higher activity at pH 4.5 and exhibiting the highest activity at day 1 of germination. The other lipase is located in the particulates, showing higher activity at pH 9.5 and particularly active at day 4 of germination. The lipid bodies from ZB 5 show the highest lipase activity at day 1 and pH 4.5 among the three varieties during the 8 d of germination. The acid lipase located in the lipid bodies of ZB 5 of germination 1 d was purified to homogeneity by DEAE-sepharose CL-6B and butyl-sepharose CL-4B chromatography after isolation, delipidation and solubilization, with a purification factor of 16.1 and overall activity recovery of 17.7%. The characterizations (such as optimum pH, pH stability, optimum temperature, thermal stability, effect of metal ions, organic solvents stability, substrate specificity) of the purified acid lipase were conducted in detail. A combination of the characterized properties may make this enzyme a useful biocatalyst for industrial application.

Structural characterization of immunostimulating polysaccharide from cultured mycelia of Cordyceps militaris

The water soluble crude polysaccharide obtained from cultured mycelia of Cordyceps militaris (CPM) by hot water extraction followed by ethanol precipitation was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This fractionation process resulted in four polysaccharide fractions that were termed CPMN Fr I, CPMN Fr II, CPMN Fr III, and CPMN Fr IV. Of these fractions, CPMN Fr III was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide (NO) and expression of cytokines (IL-1β and TNF-α). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography–mass spectrometry (GC–MS). Results indicate that CPMN Fr III was a high molecular mass polysaccharide with a random coil conformation of the β-1,4-branched-β-1,6-galactoglucomannan.

Characterization of a glucosyltransferase from Erwinia sp. D12 and the conversion of sucrose into isomaltulose by immobilized cells

Erwinia sp. D12 is able to produce an isomaltulose synthase (EC 5.4.99.11) that converts sucrose into isomaltulose. The enzyme was partially purified using a two-step chromatographic process on DEAE-Sephadex A-50 and DEAE-Sepharose CL-6B. The molecular mass of 63 kDa was estimated by Sephadex G-200 gel filtration, and the K m and V max values determined for the enzyme were 138 mM and 9.81 μmol/min/mg protein with sucrose as the substrate, respectively. Enzyme activity was optimal at pH 6.0 and 40 °C. The glucosyltransferase was completely inhibited by Hg 2+ and Ag +. An experimental design and response surface methodology were used to evaluate the influences of temperature, pH and substrate concentration on isomaltulose production from cells immobilized in chitosan. With the aid of a two-level full factorial design (2 3-FFD), the statistical analysis of the results showed that, in the range studied, the factors had a significant ( p < 0.05) effect on isomaltulose production. The conditions that improved isomaltulose production were: temperature around 35 °C, pH 6.0 and sucrose concentration lower than 40%.

Isolation of carotenoids, flavonoids and polysaccharides from Lycium barbarum L. and evaluation of antioxidant activity

A preparative column chromatography method was developed to isolate carotenoids, flavonoids and polysaccharides, from Lycium barbarum L., possessing vital biological activity, and their antioxidant activity was evaluated. Carotenoids were isolated by a column containing magnesium oxide and diatomaceous earth (1.5:1, w/w), and β-carotene was eluted with hexane, β-cryptoxanthin and neoxanthin with ethyl acetate and zeaxanthin with ethyl acetate–ethanol (80:20, v/v). Flavonoids and phenolic acids were separated using a Cosmosil 140 C18-OPN column, with phenolic acids being eluted with deionized water and neutral flavonoids with methanol. Polysaccharides were fractionated using a DEAE-Sepharose CL-6B column; neutral polysaccharides were eluted with water and acidic polysaccharides with different concentrations of NaCl. For antioxidant activity, the flavonoid fraction was the most effective in scavenging DPPH · and ABTS + free radicals, chelating metal ions and reducing power, while the zeaxanthin fraction and polysaccharides showed the most pronounced effect in scavenging hydroxy free radicals and superoxide anions, respectively.

A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity

An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.

A Glycoprotein Extracted from Golden Oyster Mushroom Pleurotus citrinopileatus Exhibiting Growth Inhibitory Effect against U937 Leukemia Cells

Mushrooms have become popular sources of natural antitumor, antiviral, antibacterial, antioxidative, and immunomodulatory agents. Golden oyster mushroom, Pleurotus citrinopileatus , is a common mushroom in oriental countries for human consumption. We isolated a functional protein (PCP-3A) from the fresh fruiting body of this mushroom. The isolation procedure included ammonium sulfate fractionation, DEAE-Sepharose CL-6B ion exchange chromatography, and Sephacryl S-300 gel filtration. Electrophoresis demonstrated that PCP-3A is a glycoprotein composed of 10 subunits, each approximately 45.0 kDa in size. In vitro cell study showed that PCP-3A at a concentration about 12.5 microg/mL inhibits the proliferation of human tumor cell line U937, in a time- dependent manner (24, 48, and 72 h). It failed to agglutinate rabbit and human erythrocytes, excluding its possibility from being a lectin. Flow cytometry revealed that it is capable of inhibiting the growth of U937 cells by way of S phase arrest and apoptotic induction. We suggest that PCP-3A is worth further investigating for antitumor use.

Structural characterisation and antioxidant properties of polysaccharides from the fruiting bodies of Russula virescens

Water-soluble crude polysaccharide named as RVP was obtained from the fresh fruiting bodies of Russula virescens by boiling-water extraction. DEAE-Sepharose CL-6B column chromatography was used for the fractionation of polysaccharide RVP. Two fractions were obtained, namely RVP-1 and RVP-2. RVP-1 and RVP-2 were composed mainly of glucose, with the estimated equivalent dextran molecular weights of 3.1 × 105 and 4.2 × 105 Da, respectively. Analysis by Periodate oxidation–Smith degradation indicated that RVP-1 was composed of 68.3% (1 →)- or ((1 → 6)-glycosidic linkages and 31.7% (1 → 3)-glycosidic linkages, and RVP-2 7.9% (1 →)- or (1 → 6)-glycosidic linkages, 9.6% (1 → 2)-glycosidic linkages, 35.7% (1 → 4)-glycosidic linkages, and 46.8% (1 → 3)-glycosidic linkages. RVP-1 exhibited equivalent inhibiting power for self-oxidation of 1,2,3-phentriol to vitamin C (Vc), a little higher scavenging activity of superoxide radical and hydroxyl radical than Vc. The reducing power of RVP-1 at 20.0 mg/ml was 0.77, and RVP-1 was a good chelating agent for ferrous ions. Overall, RVP-1 possessed good antioxidant properties and should be developed as a novel potential antioxidant.