scholarly journals Isolation and Biochemical Characterization of Cellulase Produced by Bacterial Isolates from Sugarcane Waste Soil

2021 ◽  
pp. 28-35
Author(s):  
A. U. Hassan ◽  
R. Jafaru ◽  
I. B. Mato ◽  
E. Kereakede ◽  
A. H. Galadanci ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cellulase Production ◽  
Enzyme Concentration ◽  
Microbiological Analysis ◽  
Cellulase Enzyme ◽  
Sds Page ◽  
Important Enzyme

Cellulase is one of the most economically important enzyme, which aids in catalyzing cellulolysis, the decomposition of cellulose and other related polysaccharides. So the demand/importance of this enzyme in both domestic and commercial sectors cannot be over emphasized. In this research cellulase-producing bacteria were isolated from soil around sugarcane waste dumping area, which was identified to be P. fulorescens after numerous biochemical and microbiological analysis. The bacteria were then grown and used to ferment certain biomass, with the aim of using the organisms to produce the cellulase enzyme. The total protein/cellulase enzyme activity of the medium was ascertained. Optimization/characterization for maximum cellulase activity was done by varying the temperature, pH, enzyme concentration and substrate concentration, in which the optimum condition for cellulase production was ascertain to be at a temperature and pH of 40˚C and pH 7 respectively. SDS-PAGE electrophoresis was carried out to determine and reconfirm the presence and molecular weight of the isolated enzyme. The estimated extrapolated molecular weight of the enzyme was found to be 13.5KDa.

scholarly journals Characterization of cellulase from E. coli BPPTCC-EGRK2

2018 ◽  
Vol 52 ◽  
pp. 00024
Author(s):  
Mas Gunawan Haryanto ◽  
Siswa Setyahadi ◽  
Muhammad Sahlan ◽  
Masafumi Yohda ◽  
Yosuke Fukutani ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Animal Feed ◽  
Maximum Velocity ◽  
Cellulase Production ◽  
Luria Bertani ◽  
E Coli ◽  
Sds Page ◽  
Weight Analysis

Cellulase enzymes are widely used in various industries such as detergent industry, bioethanol, animal feed, textile and paper. This research focused on characterization of cellulase produced from Eschericia coli BPPT-CC EgRK2, which is a recombinant that can produce protein enzymes endo- β-1,4-glucanase. Eschericia coli BPPT-CC EgRK2 was cultured in 1 litre liquid medium Luria Bertani. Because the bacteria is intracellular, sonication is needed for cell disruption to get the cellulase enzyme. The enzyme activity was then analyzed by CMC substrate at different concentration. The protein content analysis was carried out using Bradford method; the molecular weight analysis was done using SDS-PAGE; while the enzyme kinetics was plotted using Michaelis-Menten model. Our results showed the highest enzyme activity was 2.403 U/ml and the protein concentration was 5.352 mg/ml. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for CMC substrate hydrolysis were 0.07 μmol/ml and 2.49 μmol/ml/sec, respectively. The cellulase molecular weight was 58 kDa using SDS-PAGE with 7.5% stacking gel. The results indicated that Eschericia coli BPPT-CC EgRK2 is a promising renewable source for cellulase production for industrial application.

scholarly journals Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen.

1986 ◽  
Vol 34 (2) ◽  
pp. 209-214 ◽  
Author(s):  
J U Alles ◽  
K Bosslet
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Specific Antigen ◽  
Similar Molecular Weight ◽  
Immunochemical Characterization ◽  
Sds Page

A new monoclonal antibody (moab BW 200) of IgG3 kappa-isotype was generated which recognizes an epitope located on an antigen molecule restricted to human neoplastic and non-neoplastic endothelial cells. The molecular weight of the antigen was determined using immunoprecipitation analysis followed by SDS-PAGE. Despite its similar molecular weight to FVIII-RAG, the antigen detected by moab BW 200 was shown to be different from FVIII-RAG.

Isolation and biochemical characterization of septate junctions: differences between the proteins in smooth and pleated varieties

1989 ◽  
Vol 93 (1) ◽  
pp. 123-131
Author(s):  
NANCY J. LANE ◽  
STEPHEN M. DILWORTH
Keyword(s):  
Periplaneta Americana ◽  
Biochemical Characterization ◽  
Septate Junction ◽  
Septate Junctions ◽  
Molecular Weights ◽  
Thin Sectioning ◽  
Sds Page ◽  
High Concentrations ◽  
Membrane Components

Septate junctions are found only in invertebrate tissues, and are almost ubiquitous within them. In arthropods, the two major types are the ‘pleated’ and the ‘smooth’ varieties. Using tissues from different species, including the cockroach Periplaneta americana, procedures have been established for obtaining membrane fractions selectively enriched in septate junctions. The junctions have been identified in pellets of these fractions by both thin sectioning and freeze-fracturing. SDS-PAGE of these membrane fractions reveals two major polypeptide species with apparent molecular weights of 22000–24000 and 17000–18000. Consistent differences in these apparent molecular weights are observed between the pleated and smooth varieties of septate junction. These polypeptides are probably integral membrane components, as they remain associated after treatment with high concentrations of urea. Evidence suggests a plane of weakness in the mid-line of the extracellular septal ribbons.

Characterization Of The ADP-Induced Fibrinogen Btnd1Ng Sites On Human Platelets Using Photoaffinity Techniques: Some Preliminary Findings

1981 ◽  
Author(s):  
Ellinor I Peerschke ◽  
Mariorie B Zucker ◽  
Avner Rotman
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Fragment ◽  
Fibrinogen Receptor ◽  
Congenital Deficiency ◽  
Sds Page ◽  
Platelet Membrane Receptor

The interaction of fibrinogen with its, platelet membrane receptor was investigated using 125-labeled fibrinogen which was photoaffinity labeled with a light-sensitive azide. This photoreactive material (125I-NPA-fibr) was indistinguishable from unlabeled fibrinogen as well as from iodinated fibrinogen on SDS-PAGE. It bound specifically to platelets stimulated with ADP and was crosslinked to the platelet membrane after exposure to light ( λ >300 nm) for 4 min. No crosslinking was observed in the presence of EDTA or with platelets that failed to aggregate with ADP either due to the congenital deficiency thrombasthenia or following incubation with EDTA for 8 min at 37° , pH 7.8 and recalcification. SDS-PAGE of platelets bearing crosslinked 125I-NPA-fibr revealed a radiolabeled band of about 450,000 daltons in addition to the 340,000 dalton radioactive band of fibrinogen, suggesting that fibrinogen had been covalently bound to a platelet membrane component with an intact apparent molecular weight of approximately 110,000 daltons. Following reduction, an extra radioactive band was noted at 80,000 daltons. As the A∝-chains of fibrinogen were too weakly labeled to be detected by autoradiography, this indicated that either the Bβ or γchain of fibrinogen was attached to a 25,000-35,000 molecular weight platelet membrane fragment. We conclude that the additional radioactive bands observed after electrophoresis of platelets bearing specifically bound-photoaffinity labeled 125I-fibrinogen most likely represent the binding of the B β or γ chains of fibrinogen to the platelet fibrinogen receptor which may be GPIIb.

Cloning and Characterization of a Novel N-acetylglucosaminidase (AtlD) from Enterococcus faecalis

2018 ◽  
Vol 28 (1) ◽  
pp. 14-27 ◽  
Author(s):  
Carlos Eduardo Serrano-Maldonado ◽  
Israel García-Cano ◽  
Augusto González-Canto ◽  
Eliel Ruiz-May ◽  
Jose Miguel Elizalde-Contreras ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Heterologous Protein ◽  
Biochemical Characterization ◽  
Ph Values ◽  
Food Borne ◽  
Sds Page ◽  
Exclusion Chromatography

The <i>atlD</i> gene from<i></i> an <i>Enterococcus faecalis</i> strain isolated from a Mexican artisanal cheese was cloned, sequenced and expressed in <i>Escherichia coli</i> in order to perform a biochemical characterization<i>.</i> A partial amino acid sequence of the heterologous protein was obtained by LC-MS/MS, and it corresponded to a novel peptidoglycan hydrolase designated AtlD. Its molecular mass was 62–75 kDa, as determined by SDS-PAGE, zymography, Western blot, and exclusion chromatography. Electrofocusing rendered an isoelectric point (pI) of 4.8. It exhibited N-acetylglucosaminidase activity, with an optimal pH and temperature between 6–7 and 50°C, respectively. It retained 85% activity with NaCl at 1,000 mM, but it was susceptible to divalent ions, particularly Zn<sup>2+</sup>. It showed antibacterial activity against <i>Listeria monocytogenes</i>,<i> Staphylococcus aureus</i>, and enterococcal<i></i> strains of clinical origin. Due to the fact that it showed activity versus pathogenic bacteria, and because of its capabilities under ionic strength, temperature, and pH values present in food matrices, it could be applied as an additive in the food industry. This study will aid in the design of new antibacterial agents of natural origin to combat food-borne diseases, and it could be used as an industrial or hospital hygiene agent as well.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

1996 ◽  
Vol 38 (6) ◽  
pp. 401-406 ◽  
Author(s):  
Yano Tomomasa ◽  
Cleide Ferreira Catani ◽  
Michiko Arita ◽  
Takeshi Honda ◽  
Toshio Miwatani
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Hela Cells ◽  
Immunofluorescence Test ◽  
Native Form ◽  
Bacterial Surface ◽  
E Coli ◽  
Sds Page ◽  
Adhesive Factor

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals Biochemical Characterization of a Thiol-Activated, Oxidation Stable Keratinase from Bacillus pumilus KS12

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Rinky Rajput ◽  
Richa Sharma ◽  
Rani Gupta
Keyword(s):  
Ion Exchange ◽  
Bacillus Pumilus ◽  
Biochemical Characterization ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amidolytic Activity ◽  
Alkaline Serine Protease ◽  
Sds Page ◽  
Temperature Optima

An extracellular keratinase from Bacillus pumilus KS12 was purified by DEAE ion exchange chromatography. It was a 45 kDa monomer as determined by SDS PAGE analysis. It was found to be an alkaline, serine protease with pH and temperature optima of 10 and 60C, respectively. It was thiol activated with two- and eight-fold enhancement in presence of 10 mM DTT and β-mercaptoethanol, respectively. In addition, its activity was stimulated in the presence of various surfactants, detergents, and oxidizing agents where a nearly 2- to 3-fold enhancement was observed in presence of H2O2 and NaHClO3. It hydrolyzed broad range of complex substrates including feather keratin, haemoglobin, fibrin, casein,and α-keratin. Analysis of amidolytic activity revealed that it efficiently cleaved phenylalanine → leucine → alanine- p-nitroanilides. It also cleaved insulin B chain between Val2- Asn3, Leu6-Cys7 and His10-Leu11 residues.

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