scholarly journals The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein

2003 ◽  
Vol 369 (2) ◽  
pp. 417-427 ◽  
Author(s):  
Piku BASU ◽  
Bettina KATTERLE ◽  
K. Kristoffer ANDERSSON ◽  
Howard DALTON
Keyword(s):  
Specific Activity ◽  
Copper Ion ◽  
Exchange Chromatography ◽  
Paramagnetic Species ◽  
Methylococcus Capsulatus ◽  
Intracytoplasmic Membrane ◽  
Ionic Detergent ◽  
Unidentified Component ◽  
Solubilized Enzyme ◽  
High Yields

A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMOm), using the non-ionic detergent dodecyl-β-d-maltoside, to yield solubilized enzyme (pMMOs). Further purification gave rise to an active complex (pMMOc) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24kDa subunits) and the pMMOR (63 and 8kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with 63Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe3+ ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.

scholarly journals Production of High-Quality Particulate Methane Monooxygenase in High Yields from Methylococcus capsulatus (Bath) with a Hollow-Fiber Membrane Bioreactor

2003 ◽  
Vol 185 (20) ◽  
pp. 5915-5924 ◽  
Author(s):  
Steve S.-F. Yu ◽  
Kelvin H.-C. Chen ◽  
Mandy Y.-H. Tseng ◽  
Yane-Shih Wang ◽  
Chiu-Feng Tseng ◽  
...  
Keyword(s):  
Growth Medium ◽  
Hollow Fiber ◽  
Specific Activity ◽  
Hollow Fiber Membrane ◽  
Methane Monooxygenase ◽  
Copper Ion ◽  
Ion Concentration ◽  
Methylococcus Capsulatus ◽  
Particulate Methane Monooxygenase ◽  
High Yields

ABSTRACT In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 μM. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is ∼13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl β-d-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an αβγ protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.

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

2012 ◽  
Vol 10 (1) ◽  
Author(s):  
Ismat Bibi ◽  
Haq Nawaz Bhatti
Keyword(s):  
Lignin Peroxidase ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Gel Filtration ◽  
Veratryl Alcohol ◽  
Exchange Chromatography ◽  
Different Temperatures ◽  
Scale Experiment ◽  
Menten Kinetic

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.

scholarly journals Acetylcholinesterase aus Rindererythrozyten. Reinigung und Eigenschaften des mit und ohne Triton X-100 solubilisierten Enzyms / Acetylcholinesterase from Bovine Erythrocytes. Purification and Properties of the En­zyme Solubilized in the Presence and the Absence of Triton X-100

1979 ◽  
Vol 34 (9-10) ◽  
pp. 721-725 ◽  
Author(s):  
Heinz Großmann ◽  
Manfred Liefländer
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Multiple Forms ◽  
Terminal Amino Acid ◽  
Enzyme Preparations ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Terminal Amino ◽  
Triton X ◽  
Bovine Erythrocytes

Abstract Acetylcholinesterase was released from bovine erythrocytes by Triton X-100 treatment and pu­rified by twofold affinity chromatography. The detergentfree enzyme was obtained with a specific activity of 4130 U /mg (303 000-fold purification) and a 25% yield. Alternatively, the commercial available crude enzyme was purified. The latter preparation has an uniform molecular weight (Mr 175 000). The Triton-solubilized enzyme, however, can be resolved after removal of the detergent in eight multiple forms (Mr 175 000 and multiple values), in the presence of Triton there exists only one form (Mr 338 000). The amino acid composition of the two enzyme preparations differs significantly. No differences were observed with respect to other properties: SDS gel electrophore­sis revealed two protein bands (Mr 166 000 and 86 000) with both preparations. The enzyme is a glycoprotein with a pI value of 4.3 and contains strongly bound phosphatidylethanolamine. The N-terminal amino acid has been found to be Glu (or Gin).

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

scholarly journals Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

2021 ◽  
Vol 9 (2) ◽  
pp. 24-30
Keyword(s):  
Metal Ions ◽  
Sugarcane Bagasse ◽  
Specific Activity ◽  
Gel Filtration ◽  
Value Added ◽  
Cost Effective ◽  
Fermentation Medium ◽  
Exchange Chromatography ◽  
Sds Page ◽  
The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15.

2014 ◽  
Vol 61 (4) ◽  
Author(s):  
Mohd Adilin Yaacob ◽  
Wan Atiqah Najiah Wan Hasan ◽  
Mohd Shukuri Mohamad Ali ◽  
Raja Noor Zaliha Raja Abdul Rahman ◽  
Abu Bakar Salleh ◽  
...  
Keyword(s):  
Molecular Dynamic ◽  
Conformational Changes ◽  
Specific Activity ◽  
3D Structure ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Molecular Dynamic Simulations ◽  
Type I ◽  
Enzymatic Reactions ◽  
Dynamic Simulations

Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.

scholarly journals Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

2013 ◽  
Vol 10 (3) ◽  
pp. 844-853
Author(s):  
Baghdad Science Journal
Keyword(s):  
Aspergillus Flavus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Stability ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Original Activity ◽  
A Value ◽  
Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

scholarly journals Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A

1993 ◽  
Vol 295 (3) ◽  
pp. 813-819 ◽  
Author(s):  
A Varro ◽  
G J Dockray
Keyword(s):  
Ion Exchange ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Specific Activity ◽  
Brefeldin A ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Gastrin Cells ◽  
Tryptic Fragment ◽  
C Terminus

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.

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