scholarly journals One-step purification and characterization of two novel Thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278

2020 ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography

Abstract A newly screened cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) from fermentation solution were recovered by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable under 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values ​​of 2.76 mg/mL, 20.6 U/mg for pNPG. Thin-layer chromatography and high-performance liquid chromatography analysis showed that cellobiose BG FH1and BG FH2 had hydrolysis activity and can hydrolyze cellobiose into glucose. In addition, both enzymes also exhibited transglycoside activity, which can use low molecular weight monosaccharides to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 can produce heat-resistant β-glucosidase with both hydrolytic activity and transglycosidic activity, and has potential application value in bioethanol and papermaking industries.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

2020 ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

2020 ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Identification and Quantification of an Adulterant in a Dietary Supplement Marketed for Sexual Enhancement

2018 ◽  
Vol 1 (4) ◽  
pp. 25-33 ◽  
Author(s):  
Flavia Redko ◽  
Sabrina Flor ◽  
Silvia Lucangioli ◽  
Jerónimo Ulloa ◽  
Rafael Ricco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Performance ◽  
Spectroscopic Techniques ◽  
Easy Method ◽  
Isolation And Purification ◽  
Chromatography Analysis ◽  
Pharmaceutical Ingredients ◽  
Emerging Risk ◽  
The Mean ◽  
Layer Chromatography

In recent years, the consumption of dietary supplements (DS) has increased worldwide. In Argentina, approximately 14 million DS units were sold between 2015 and 2017. The adulteration of DS with active pharmaceutical ingredients or their analogues has been reported. This represents an alarming emerging risk to public health. The aim of this work was to detect the possible adulteration of a DS marketed in Argentina for the treatment of erectile dysfunction. Initially, thin layer chromatography analysis of the DS capsules content suggested the presence of a major compound. For the isolation and purification of this compound, an easy method consisted of a liquid-liquid extraction (water/CH2Cl2) followed by re-crystallisation from ethanol, is reported. Spectroscopic techniques such as mono- and bidimensional nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry allowed its identification as tadalafil. A rapid and reliable method was developed for the quantification of tadalafil in this DS by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The mean content of tadalafil per capsule was 21.2 mg which represents a slightly higher value than that found in approved products in Argentina (5 or 20 mg per tablet). In addition, an undeclared alga was identified in the DS by microscopic techniques.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14

1993 ◽  
Vol 39 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Mohamed Blaghen ◽  
Dominique J. M. Vidon ◽  
Mohamed Said El Kebbaj
Keyword(s):  
Molecular Weight ◽  
Yersinia Enterocolitica ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Mercury Resistance ◽  
Thiol Compounds ◽  
Mercuric Reductase ◽  
Purification And Properties ◽  
Layer Chromatography

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70 000. The isolated enzyme had a molecular weight of about 200 000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140 000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105 000 composed of two identical subunits of 52 000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.Key words: Yersinia enterocolitica, mercury resistance, mercuric reductase.

TUMOR-ASSOCIATED FIBRINOLYSIS IN OVARIAN CARCINOMA - HPLC AND N-TERMINAL AMINO ACID ANALYSIS REVEAL THE PATHWAY OF DEGRADATION OF CROSSLINKED FIBRIN

1987 ◽  
Author(s):  
O Wilhelm ◽  
A Henschen ◽  
R Hafter ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
Ovarian Carcinoma ◽  
Ascitic Fluid ◽  
High Performance ◽  
Degradation Products ◽  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Reversed Phase ◽  
Fibrin Degradation Products ◽  
Sds Page

Crosslinked fibrin has been demonstrated by immunohistochemi-cal tests to occur around tumor plugs, on the surface and in the stroma of the tumor in ovarian cancer. High levels of D-Dimer (200-800μg/ml), the characteristic terminal degradation product of crosslinked fibrin, are found in ascitic fluid of patients with advanced ovarian cancer. These findings suggest that fibrin polymerisation and degradation are related to and even may influence tumor growth. The kind of proteases which are responsible for degradation of crosslinked fibrin is, however, unknown.lt was the aim of this study to evaluate whether plasmin and/or other proteases are involved in tumor-associated fibrinolysis. Therefore the total high-molecular-weight fibrin degradation products in ascitic fluid were purified by protamine sulfate precipitation, gel filtration, immunoadsorption and compared with the components of plasmin-degraded crosslinked fibrin, i.e. DD,DY,YX,DXD and DXY, by direct SDS-PAGE in the absence of mercaptoethanol and after excision of the bands, mercaptolysis and re-electrophoresis. Pronounced similarity between the two sets of fragments was observed. For further information the fragments from the two sources were mercaptolysed and their polypeptide chain components separated by reversed-phase high-performance liquid chromatography, the components being identified by N-terminal sequence analysis and SDS-PAGE. Highly similar patterns were obtained and components corresponding to γ-γ ,γ-γ1, β, β2 and α1 could be recognized. The findings provide strong evidence for plasmin being the primary protease involved in ovarian carcinoma-related fibrinolysis, (supported by Deutsche Forschungsgemeinschaft.SFB 207, A2).

Processing of cell-bound insulin by capillary and macrovascular endothelial cells in culture

1985 ◽  
Vol 248 (2) ◽  
pp. E244-E251 ◽  
Author(s):  
K. D. Dernovsek ◽  
R. S. Bar
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Potential Role ◽  
Umbilical Vein ◽  
Gel Filtration ◽  
Storage Site ◽  
Surface Receptors ◽  
Chromatography Analysis ◽  
Degradation Rates ◽  
Cellular Processing

The processing of cell-bound insulin was determined in endothelial cells cultured from three large blood vessels (human umbilical vein, bovine pulmonary artery, and bovine aorta) and one microvascular source (bovine fat capillaries). Cells were exposed to monoiodinated TyrA14-insulin, the rates of dissociation of cell-bound TyrA14-insulin determined, and cell alteration of insulin assessed by gel filtration and high-performance liquid chromatography analysis. We found that 1) overall degradation rates of insulin are low for all cultured endothelial cells, 2) cell-bound insulin is rapidly processed to a nonreceptor compartment and then rapidly dissociated from all cells, primarily as biologically intact insulin, and 3) degradation of cell-bound insulin, although relatively low, does occur in endothelial cells with the least degradation by capillary cells. The presence of specific surface receptors for insulin on endothelial cells coupled with rapid cellular processing of intact insulin is consistent with a potential role for endothelial cells in either the transport of intact insulin out of the bloodstream or as a regional storage site for intact hormone.

Partial Primary Structure Of Human α2-Antiplasmin

1981 ◽  
Author(s):  
H R Lijnen ◽  
B Wiman ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Single Chain ◽  
Tryptic Digest

α2-Antiplasmin (α2AP), the main physiological inhibitor of plasmin in human plasma, is a single–chain glycoprotein with a molecular weight of 67,000 consisting of about 510 amino acids and containing 13 percent carbohydrate.A tryptic digest on 400 mg of reduced, carboxymethylated and citraconylated purified α2AP was performed. Peptides were separated by combinations of ion exchange chromatography, gel filtration and high performance liquid chromatography, and sequenced using the manual Edman degradation. Some peptides were further digested in order to establish overlaps. At the time of submission of this abstract we have sequenced 7 out of the approximately 21 arginyl peptides completely (each between 3 and 21 residues) and are working on the others. At present we have about 200 residues of sequence. Here we only report the stretches of 10 amino acids or more, which may be useful to compare the structure of α2AP with that of other serine protease inhibitors.

Antibacterial Activities of Antibacterial Proteins/Peptides Isolated from Organs and Mucus of Clarias Gariepinus Reared at High Stocking Density

2012 ◽  
Vol 455-456 ◽  
pp. 455-460 ◽  
Author(s):  
Xiao Mei Wang ◽  
Wei Dai ◽  
Ke Zhing Xing ◽  
Tian Jun Li ◽  
Xin Wang
Keyword(s):  
Molecular Weight ◽  
Antibacterial Activity ◽  
Crude Protein ◽  
Clarias Gariepinus ◽  
Gel Filtration ◽  
Stocking Density ◽  
Antibacterial Activities ◽  
Edwardsiella Tarda ◽  
Bacterial Strains ◽  
Sds Page

. Antibacterial proteins/peptides are important parts of the innate immune system in Clarias gariepinus. To examine potential antibacterial proteins/peptides in organs and mucus of C. gariepinus, crude protein/peptide extracts were isolated with ammonium sulfate precipitation from mucus, skin, gill, suprabranchial organ and intestine. Following further extraction using Sephadex G-50 gel filtration chromatography, the proteins/peptides associated with two absorption peaks (AP1 and AP2) were pooled, respectively, and assayed for their antibacterial activities against Escherichia coli, Aeromonas hydrophila and Edwardsiella tarda. The results showed that AP1 and AP2 from all the sampled tissues and mucus at concentration of 100 mg mL-1 exhibited antibacterial activity against the tested bacterial strains. Differences in antibacterial activity were observed among sample extracts. The protein profiles of AP1 obtained by Tricine-SDS-PAGE gel showed a broad range of peptides/proteins, and molecular weight of the mutual abundant peptide obtained was about 27 kDa. Antibacterial activity of AP2 extracted from intestine was due to peptide with molecular weight of 5.5 kDa.

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