scholarly journals Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

2012 ◽  
Vol 3 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Abd El Aziem Farouk ◽  
Ralf Greiner ◽  
Anis Shobirin Meor Hussin
Keyword(s):  
Relative Rate ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enterobacter Sakazakii ◽  
Degrading Enzyme ◽  
Purification And Properties ◽  
Sds Page ◽  
Rate Of Hydrolysis

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM).

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

scholarly journals Evidence for N-linked glycosylation in Toxoplasma gondii

1993 ◽  
Vol 291 (3) ◽  
pp. 713-721 ◽  
Author(s):  
M Odenthal-Schnittler ◽  
S Tomavo ◽  
D Becker ◽  
J F Dubremetz ◽  
R T Schwarz
Keyword(s):  
Toxoplasma Gondii ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Common Precursor ◽  
The Common ◽  
Oligosaccharide Structures

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2

1991 ◽  
Vol 274 (3) ◽  
pp. 833-842 ◽  
Author(s):  
H H Locher ◽  
T Leisinger ◽  
A M Cook
Keyword(s):  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Comamonas Testosteroni ◽  
Visible Absorption ◽  
Mononuclear Iron ◽  
Purification And Properties ◽  
A Subunit ◽  
Protein Components

Cell-free extracts of Comamonas testosteroni T-2 grown in toluene-p-sulphonate/salts medium catalyse the conversion of p-sulphobenzoate (PSB) into protocatechuate and sulphite by an NADH-requiring and Fe2(+)-activated dioxygenase. Anion-exchange chromatography of extracts yielded red (A) and yellow (B) protein fractions, both of which were necessary for dioxygenative activity. Further purification of each fraction by hydrophobic interaction chromatography and gel filtration led to two homogeneous protein components (A and B), which together converted 1 mol each of PSB, O2 and NADH into 1 mol each of protocatechuate, sulphite and, presumably, NAD+. The system was named 4-sulphobenzoate 3,4-dioxygenase (PSB dioxygenase system). Monomeric component B (Mr 36,000) was determined to be a reductase that contained 1 mol of FMN and about 2 mol each of iron and inorganic sulphur per mol. This component transferred electrons from NADH to the oxygenase component (A) or to, e.g., cytochrome c. Homodimeric component A (subunit Mr 50,000) of the PSB dioxygenase system contained one [2Fe-2S] centre per subunit and its u.v.-visible-absorption spectrum corresponded to a Rieske-type iron-sulphur centre. The requirement for activation by iron was interpreted as partial loss of mononuclear iron during purification of component A. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. The PSB dioxygenase system displayed a narrow substrate range: none of 18 sulphonated or non-sulphonated analogues of PSB showed significant substrate-dependent O2 uptake. The physical properties of the PSB dioxygenase system resemble those of other bacterial multi-component dioxygenase, especially phthalate dioxygenase. However, it differs from most characterized systems in its overall reaction; the product is a vicinal diphenol, and not a dihydrodiol.

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

Purification and properties of a Meloidogyne-antagonistic chitinase from Lysobacter capsici YS1215

Nematology ◽  
2014 ◽  
Vol 16 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Yong Seong Lee ◽  
Muhammad Anees ◽  
Yun Serk Park ◽  
Sun Bae Kim ◽  
Woo Jin Jung ◽  
...  
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Chitinase Activity ◽  
Exchange Chromatography ◽  
Control Measures ◽  
Agricultural Field ◽  
Glycol Chitosan ◽  
Purification And Properties ◽  
Nematode Eggs

The root-knot nematodes, Meloidogyne spp., cause serious diseases in various plants and their chemical control may lead to environmental problems. Therefore, alternative control measures against the phytopathogenic nematodes are being sought. One of the potential targets against Meloidogyne spp. may be the chitinolysis and degradation of nematode eggs. Therefore, in the present study, a chitinolytic and nematicidal strain of Lysobacter capsici YS1215 was isolated from an agricultural field in Korea. The aim of this study was to purify chitinase secreted by L. capsici YS1215 and investigate its nematicidal role against Meloidogyne incognita. The chitinase secreted by L. capsici YS1215 was purified by protein precipitation with 80% ammonium sulphate, anion-exchange chromatography with DEAE-cellulose and gel-filtration chromatography with Sephadex G-100. By chitinase-active staining of the purified enzyme, a single band was obtained with an estimated molecular mass of 43.6 kDa. The optimal pH and optimal temperature for the highest chitinase activity were 6.0 and 40°C, respectively. The purified chitinase degraded the chitin layer of the eggshells and significantly reduced hatch of second-stage juveniles. The activity of chitinase secreted by L. capsici YS1215 was not affected by CoCl2, MnCl2, MgCl2, CuSO4, CaCl2 or EDTA. The purified enzyme could also hydrolyse swollen chitin, glycol chitin, glycol chitosan and chitin powder. Thus, the role of chitinase secreted by L. capsici YS1215 against Meloidogyne spp. may be useful for further development of a biocontrol agent.

scholarly journals A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.

2011 ◽  
Vol 58 (4) ◽  
Author(s):  
Jian Sun ◽  
Yongchang Zhao ◽  
Hongmei Chai ◽  
Hexiang Wang ◽  
Tzi Bun Ng
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fruiting Bodies ◽  
Wild Mushroom ◽  
Sds Page ◽  
Human Hepatoma Hepg2 Cells

A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

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