scholarly journals Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Morphological, Molecular, Biochemical and Nutritional Characterization of Three Major Thais Species from the Sindh Coast of Pakistan

2018 ◽  
Vol 10 (1) ◽  
pp. 33-45
Author(s):  
Syed Abid Ali ◽  
Fozia Humayun ◽  
Iqra Munir ◽  
Shakil Ahmad ◽  
Zarrien Ayub ◽  
...  
Keyword(s):  
Total Protein ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Nutritional Composition ◽  
The Body ◽  
Size Exclusion ◽  
High Concentration ◽  
Sds Page

Objective: The present study was conducted to investigate the biomass assessment, morphological and molecular identification, nutritive status and biochemical characterization of three major Thais species (T. bufo, T. hippocastanum and T. rudolphi) from the Sindh Coast, Pakistan. Methods: Samples were collected from Buleji and Paradise Point at the Sindh Coast. Species were identified morphologically as well as genetically by amplifying two mitochondrial 16S rDNA & Cytochrome Oxidase I (COI) and one nuclear (Histone H3) genes. Shell microstructure and chemistry were also studied by scanning electron microscopy and Energy Dispersive X-ray spectrometry (EDX). The body muscle was dissected and used for nutritional composition determination such as estimation of total protein, carbohydrates, lipids, protein fingerprinting by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Size-Exclusion - Fast Protein Liquid Chromatography (SEC-FPLC), amino acid and fatty acid analysis. Results: Nutritionally, the total protein was found to be the major content followed by carbohydrate and lipid in the three Thais sp. The presence of medicinally important hemocyanin as abundant hemolymph protein was confirmed via SDS-PAGE and SEC FPLC. Nine different types of fatty acids and a high concentration of essential amino acids were also determined. Conclusion: Our findings suggest that Thais sp. are nutritionally rich and can be consumed as a valuable marine resource to overcome the malnutrition problem in developing countries.

scholarly journals Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

2001 ◽  
Vol 67 (8) ◽  
pp. 3746-3749 ◽  
Author(s):  
Yu-Huan Liu ◽  
Ying-Cheng Chung ◽  
Ya Xiong
Keyword(s):  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Degrading Enzyme

ABSTRACT A dimethoate-degrading enzyme from Aspergillus nigerZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (K m ) andV max for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively.

scholarly journals Purification and Characterization of Thermostable Endo-1,5-α-l-Arabinase from a Strain of Bacillus thermodenitrificans

2002 ◽  
Vol 68 (4) ◽  
pp. 1639-1646 ◽  
Author(s):  
Makoto Takao ◽  
Kana Akiyama ◽  
Takuo Sakai
Keyword(s):  
Sugar Beet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Thermophilic Bacterium ◽  
Size Exclusion ◽  
Purification And Characterization ◽  
Optimal Activity ◽  
End Products ◽  
Beet Pulp

ABSTRACT A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60°C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75°C. The enzyme had optimal activity at 70°C and pH 6.0. The enzyme had apparent Km values of 8.5 and 45 mg/ml and apparent V max values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (α-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for l-arabinose production from sugar beet pulp at high temperature.

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme

2002 ◽  
Vol 69 (3) ◽  
pp. 419-431 ◽  
Author(s):  
SUBHADRA PRIYADARSHINI ◽  
VINOD K. KANSAL
Keyword(s):  
Antibacterial Activity ◽  
Amino Acid ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Buffalo Milk ◽  
Size Exclusion ◽  
Sds Page ◽  
Wide Range

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39·3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56·5% homology with bovine milk lysozyme and 30·4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

Expression of an abundant α-class glutathione S-transferase in bovine and human adrenal cortex tissues

1992 ◽  
Vol 132 (1) ◽  
pp. 83-92 ◽  
Author(s):  
I. Meikle ◽  
J. D. Hayes ◽  
S. W. Walker
Keyword(s):  
Adrenal Cortex ◽  
Catalytic Properties ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gene Families ◽  
Glutathione S Transferase ◽  
Purification And Characterization ◽  
Sds Page ◽  
Highperformance Liquid Chromatography

ABSTRACT Bovine adrenal cortex tissue expresses high levels of glutathione S-transferase (GST) from each of the α, μ and π gene families. We describe the purification and characterization of an abundant α-class GST from this tissue that has not been identified previously because of its failure to bind to S-hexylglutathione–Sepharose 6B (S-hexG-Ag). This enzyme has been affinity purified on glutathione–Sepharose 6B (GSH-Ag) and was obtained in a highly purified form by employing S-hexG-Ag to remove the bulk of GST before chromatography on GSH-Ag. The purified GST eluted from GSH-Ag was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities (19·2 and 1·67 U/mg respectively). The bovine enzyme also showed high GST activity towards 4-hydroxynonenal (5·09 U/mg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the bovine GST contains two distinct polypeptides, one with an Mr of 25 900 and the other with an Mr of 26 500. An abundant α-class GST was also purified from human adrenal cortex that possessed properties which were similar to the bovine α-class GST described above; however, unlike the bovine enzyme, the corresponding human α-class GST bound to S-hexG-Ag. As with the bovine enzyme, the purified human GST displayed marked peroxidase and isomerase activities (27 and 4·02 U/mg respectively). Further analysis on SDS-PAGE (Mr 25 800) and reverse-phase highperformance liquid chromatography established that this abundant α-class GST in human adrenal cortex is equivalent to the human liver GST B1B1 enzyme. As both human and bovine adrenal cortex contain high levels of α-class GST with similar catalytic properties, we discuss the possible functions of these enzymes in this tissue. Journal of Endocrinology (1992) 132, 83–92

scholarly journals Purification and characterization of β-N-acetylglucos-aminidase from Grifola frondosa

BioResources ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. 7234-7248
Author(s):  
Yi-Cheng Wang ◽  
Te-Sheng Lien ◽  
Nan-Yin Chen ◽  
Tai-Hao Hsu
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolysis Product ◽  
Chitinase Activity ◽  
Grifola Frondosa ◽  
Purification And Characterization ◽  
Carbohydrate Utilization ◽  
Highly Active

Using commercial API-ZYM screening kits, highly active α-glucosidase, β-glucosidase, and β-N-acetylglucosaminidase were found in Grifola frondosa, having potential for carbohydrate utilization. Of these, β-N-acetylglucosaminidase, which converts chitin to N-acetylglucosamine, was purified and characterized. The recovery was 24.5%, and the purified enzyme had a specific activity 0.67 U/mg protein. Chitinase activity was confirmed by zymogram analysis. The enzyme was also shown to be β-N-acetylglucosaminidase, as N-acetylglucosamine was the main hydrolysis product from colloidal chitin. Thus, the molecule was named NAG38, to indicate β-N-acetylglucosaminidase activity and a molecular weight of 38 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity was optimal at pH 7.0 and 50 °C, with Km and Vmax values of 0.112 mM and 0.570 μmol/min/mg protein against p-nitrophenyl N-acetyl-β-D-glucosaminide. The bioactivity was inhibited by Hg2+, Ag+, Mg2+, Zn2+, Ca2+, and Mn2+, with residual enzyme bioactivity only 11.1% after incubation in Hg2+, but was not substantially inhibited by Ba2+, K+, and Na+.

scholarly journals Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Preeti Anand ◽  
Jay Prakash Pandey ◽  
Dev Mani Pandey
Keyword(s):  
San Francisco ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Evolutionary Genetics ◽  
Imaging Analysis ◽  
Silk Cocoon ◽  
Natural Color ◽  
Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

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