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 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 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 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.

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.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Comparative study on amylosucrases derived from Deinococcus species and catalytic characterization and use of amylosucrase derived from Deinococcus wulumuqiensis

Amylase ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 19-31 ◽  
Author(s):  
Ki-Tae Kim ◽  
Chan-Su Rha ◽  
Young Sung Jung ◽  
Ye-Jin Kim ◽  
Dong-Hyun Jung ◽  
...  
Keyword(s):  
Reaction Products ◽  
Sucrose Hydrolysis ◽  
Transglycosylation Activity ◽  
Acceptor Specificity ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Optimal Reaction Temperature ◽  
Optimal Reaction ◽  
Catalytic Characterization ◽  
Hydrolysis Activity

Abstract Amylosucrase (ASase; EC 2.4.1.4), a versatile enzyme, exhibits three characteristic activities: hydrolysis, isomerization, and transglycosylation. In this study, a novel ASase derived from Deinococcus wulumuquiensis (DWAS) was identified and expressed in Escherichia coli. The optimal reaction temperature and pH for the sucrose hydrolysis activity of DWAS were determined to be 45 °C and 9.0, respectively. DWAS displays relatively high thermostability compared with other ASases, as demonstrated by half-life of 96.7 and 4.7 min at 50 °C and 55 °C, respectively. DWAS fused with 6×His was successfully purified to apparent homogeneity with a molecular mass of approximately 72 kDa by Ni-NTA affinity chromatography and confirmed by SDS-PAGE. DWAS transglycosylation activity can be used to modify isovitexin, a representative flavone C-glucoside contained in buckwheat sprouts to increase its limited bioavailability, which is due to its low absorption rate and unstable structure in the human body. Using isovitexin as a substrate, the major transglycosylation product of DWAS was found to be isovitexin monoglucoside. The comparison of transglycosylation reaction products of DWAS with those of other ASases derived from Deinococcus species revealed that the low sequence homology of loop 8 in ASases may affect the acceptor specificity of ASases and result in a distinctive acceptor specificity of DWAS.

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