scholarly journals THE EVIDENCE OF NON N-GLYCAN LINKED MANNOSE IN EXOCHITINASE (42 kDa) FROM Trichoderma harzianum BIO10671 GLYCOSYLATION

2017 ◽  
Vol 25 (2) ◽  
pp. 295-302
Author(s):  
Muskhazli Mustafa ◽  
Faridah Qamaruz Zaman ◽  
Salfarina Ramli ◽  
Nor Farizan Tohfah ◽  
Nalisha Ithnin ◽  
...  
Keyword(s):  
Trichoderma Harzianum ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Strong Response ◽  
Negative Results ◽  
Cell Wall Lysis ◽  
Lectin Staining ◽  
Sds Page ◽  
Series Of Experiments ◽  
Terminal Mannose

Chitinase 42 kDa produced by Trichoderma harzianum has been proven as a prime compound to be excreted onto the hyphae of the pathogen causing localised cell wall lysis at the point of interaction. This finally initiate the process of the host cell becomes empty of cytoplasm, disintegrates and shows a rapid collapse. This study investigates the existence of N-glycan linked mannose in chitinase 42 kDa produced by the Malaysian T. harzianum strain BIO10671.  The chitinase 42 kDa from T. harzianum BIO10671 was initially purified using anion exchange chromatography prior to a series of experiments such as immunoblotting against the chitinase 42 kDa antibody, lectin staining for detecting any terminal linked mannose, and galactofuranose detection to determine the presence of galatofuranose components in glycoproteins. The enzyme purification harvested about 12-fold of chitinase 42 kDa from T. harzianum BIO10671 with strong indication of the presence chitinase 42 kDa presence on SDS-Page. This was confirmed by immunoblotting with a strong response around 42 kDa after overnight incubation in chitinase 42 kDa antibody suggesting that the gene for chitinase 42 kDa was greatly expressed in this strain. There are no intervation of galatofuranose on any of the terminal mannose in chitinase 42 kDa as shown by negative results on samples treated with or without endoglycosidase-H and lectin staining. Therefore, it can be concludeed that glycosylation occurred in the chitinase 42 kDa from T. harzianum 42 kDa was not in the form of N-glycan linked mannose as expected.    

scholarly journals Coal Depolymerising Activity and Haloperoxidase Activity of Mn Peroxidase fromFomes durissimusMTCC-1173

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Sunil Kumar Singh ◽  
Meera Yadav ◽  
Sudha Yadava ◽  
Kapil Deo Singh Yadav
Keyword(s):  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Sodium Lactate ◽  
Low Ph ◽  
Exchange Chromatography ◽  
Fungal Strain ◽  
Mn Peroxidase ◽  
Sds Page ◽  
Catalytic Rate ◽  
Temperature Optima

Mn peroxidase has been purified to homogeneity from the culture filtrate of a new fungal strainFomes durissimusMTCC-1173 using concentration by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The molecular mass of the purified enzyme has been found to be 42.0 kDa using SDS-PAGE analysis. The values using MnSO4and H2O2as the variable substrates in 50 mM lactic acid-sodium lactate buffer pH 4.5 at were 59 μM and 32 μM, respectively. The catalytic rate constants using MnSO4and H2O2were 22.4 s−1and 14.0 s−1, respectively, giving the values of 0.38 μM−1s−1and 0.44 μM−1s−1, respectively. The pH and temperature optima of the Mn peroxidase were 4 and , respectively. The purified MnP depolymerises humic acid in presence of H2O2. The purified Mn peroxidase exhibits haloperoxidase activity at low pH.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Antithrombin Milano, Single Amino Acid Substitution at the Reactive Site, Arg393 to Cys

1988 ◽  
Vol 60 (03) ◽  
pp. 471-475 ◽  
Author(s):  
H Erdjument ◽  
D A Lane ◽  
H Ireland ◽  
V Di Marzo ◽  
M Panico ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Fast Atom Bombardment ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Reducing Conditions ◽  
Sds Page ◽  
Two Peaks

SummaryAntithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; anti thrombin Milano peak 1 of Mr ∼60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr ∼120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the ∼120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr ∼60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the ∼120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxy-methylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, North-wick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Adding value in barley malt rootlets as a source of 5’-phosphodiesterase

2020 ◽  
Vol 15 (1-2) ◽  
pp. 27-37
Author(s):  
Sunčica Beluhan ◽  
Ivana Karmelić ◽  
Mirela Ivančić Šantek
Keyword(s):  
Half Life ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Barley Malt ◽  
First Order Kinetics ◽  
Unit Fractions ◽  
Sds Page ◽  
Phosphodiesterase 5

A thermostable 5’-phosphodiesterase (5’-PDE, EC 3.1.4.1) was extracted from barley (Hordeum distichum var. Rex) malt rootlets. The purification procedure comprised acetone precipitation, S-Sepharose cation-exchange and DEAE-Sepharose anion-exchange chromatography. The enzyme was purified 101-fold with a recovery of 22% and a specific activity of 81.9 U mg-1 protein, Optimum enzyme activity was obtained at 70 °C, and pH 8.9. The SDS-PAGE profiling of the purified protein exhibited molecular weight of 116 kDa and revealed three sub-unit fractions of 26, 43, and 56 kDa making up its active configuration. The kinetic constants Km and Vmax were determined as 0.25 mM and 0.816 mmol min-1, respectively. Thermodynamic studies showed that the thermal inactivation of purified barley malt rootlets 5’-PDE followed the first-order kinetics, indicating inactivation energy (Ed) of 134 kJ mol-1. The half-life (t1/2) at 70 °C was estimated as 169 min. Thermodynamic parameters ΔH*, ΔS* and ΔG* were determined as a function of temperature and were 131.15 kJ mol-1, 37.01 kJ mol-1 K-1 and 118.4 kJ mol-1, respectively. The purified enzyme has long half-life with 11 days at 0 °C, 37 hours at 4 °C and 11 hours at room temperature. These results provide useful information about the factors that affects the activity of barley malt rootlets 5’-PDE and suggests a good indication for application of this enzyme in pharmaceutical and food industry.

scholarly journals Isolation of H-protein loaded with methylamine as a transient species in glycine decarboxylase reactions

1991 ◽  
Vol 278 (3) ◽  
pp. 765-769 ◽  
Author(s):  
M Neuburger ◽  
A Jourdain ◽  
R Douce
Keyword(s):  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Transient Species ◽  
Protein Isolation ◽  
Nitrobenzoic Acid ◽  
P Protein ◽  
Sds Page ◽  
Rapid Fixation ◽  
High Concentrations ◽  
H Protein

A three-step protocol was devised to purify H-protein, which can be readily released as a soluble protein from pea mitochondria. After the final step of purification (anion-exchange chromatography) the native enzyme was eluted as two distinct peaks at 250 and 350 mM-KCl if the lysis buffer contained glycine. Each from exhibited an identical Mr of 15000 on SDS/PAGE and they were not distinguishable by PAGE under non-denaturating conditions. Both forms catalysed the rapid fixation of [14C]bicarbonate to the carboxy group atom of glycine during the exchange reaction, whereas the reversible exchange of electrons between NADH and lipoamide bound to the H-protein in the presence of 5,5′-dithiobis-(2-nitrobenzoic acid) was seen only with the form eluted at 350 mM-KCl. During the early steps of H-protein isolation, when P- and H-protein react together in the presence of glycine, the methylamine intermediate bound to the lipoamide of the H-protein accumulates in the medium at the expense of oxidized H-protein. Under these conditions the methylamine intermediate, which is a rather stable structure, was easily separated from the oxidized H-protein on ion-exchange chromatography. The methylamine bound to the lipoamide of the H-protein prevented the reversible exchange of electrons between NADH and lipoamide. High concentrations of glycine were required for the loading of H-protein with methylamine catalysed by a large excess of P-protein.

scholarly journals Isolation of Plasma Proteins from Bovine Blood by Cold Ethanol Precipitation and Anion Exchange Chromatography

1970 ◽  
Vol 26 ◽  
pp. 2-12
Author(s):  
Patricia Adamma Ekwumemgbo ◽  
James Adagadzu Kagbu ◽  
Andrew Jonathan Nok ◽  
Israel Kehinde Omoniyi ◽  
Paul Ocheme Ameh ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Plasma Proteins ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Bovine Blood ◽  
Protein Assay ◽  
Molecular Weights ◽  
Standard Curve ◽  
Ethanol Precipitation ◽  
Sds Page

Plasma (100.00 ml) obtained from bovine blood by centrifugation was fractionatedinto seven precipitates by cold ethanol precipitation. The yield (amount) of proteins in theprecipitates calculated from the standard curve of BSA was 1160.83 mg, 806.57 mg,1149.94 mg, 8.79 mg, 19.88 mg, 21.98 mg, and 13.97 mg respectively, while the totalamount of protein obtained was 3180.00 mg. The precipitates were fractionated into 25fractions each by Anion Exchange Chromatography (AEC) and the amount of protein ineach fraction was obtained by Bradford protein assay. SDS-PAGE analysis was performedon the fractions with proteins and their estimated molecular weights were obtained with theaid of the molecular weight marker. The result indicated that cold ethanol precipitation andanion exchange chromatographic techniques are valuable tools for the purification ofbovine blood to obtain high grade α, β and γ-fibrinogen, IgM (μ-globulin), IgG (γ -globulin),alpha (α –globulin), β-globulin (E-globulin) and albumin.Keywords: Plasma; Chromatography; Electrophoresis; Albumin; Fibrinogen; GlobulinDOI: 10.3126/jncs.v26i0.3624Journal of Nepal Chemical SocietyVol. 26, 2010Page: 2-12

scholarly journals Biotransformation of Aromatic Methyl Groups to Aldehyde Groups Using Laccase of Gloephyllum Stratum MTCC-1117

2020 ◽  
Author(s):  
RAM SAHAY
Keyword(s):  
White Rot Fungi ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
White Rot ◽  
Show Absorption Band ◽  
Yellow Colour ◽  
Sds Page ◽  
Coir Dust ◽  
Laccase Enzyme ◽  
Aldehyde Groups

Abstract Laccases has been produced by white rot fungi are involved in lignin containing natural substrates wheat-straw, bagasse, saw-dust, corn cob and coir dust particle on the production of laccase enzyme in the aqueous cultivation medium of Gloephyllum stratum MTCC1117. The approach involved concentration of aqueous filtrate by ultrafiltration and anion exchange chromatography on DEAE (diethyl aminoethyl cellulose). From SDS-PAGE analysis the molecular mass of the purified enzyme is 57 kDa. The Km and kcat values of the laccase are found to be 18 μM and 0.34 s-1 using 2,6-dimethoxyphenol as the substrate, giving a kcat/Km value is 1.70 x 103 M-1 s-1. The pH and temperature optimum were 4.5 and 40 °C respectively. The purified enzyme has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover the conversion of methylbenzene to benzaldehyde in the lack of mediator molecules, property exhibited by yellow laccases.

scholarly journals Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

2012 ◽  
Vol 3 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Abd El Aziem Farouk ◽  
Ralf Greiner ◽  
Anis Shobirin Meor Hussin
Keyword(s):  
Relative Rate ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enterobacter Sakazakii ◽  
Degrading Enzyme ◽  
Purification And Properties ◽  
Sds Page ◽  
Rate Of Hydrolysis

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM).

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