scholarly journals Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1→4)-β-d-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli

1993 ◽  
Vol 296 (1) ◽  
pp. 235-243 ◽  
Author(s):  
V Garcia-Campayo ◽  
S I McCrae ◽  
J X Zhang ◽  
H J Flint ◽  
T M Wood
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Physicochemical Characterization ◽  
Silver Ions ◽  
Anion Exchange Chromatography ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Ruminococcus Flavefaciens ◽  
Oat Spelt

Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli. The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing. XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa. The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum. XYLA-A1 showed a higher sensitivity to pH than XYLA-C2. The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions. Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased. Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis. With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues. Neither domain was able to release arabinose from oat spelt xylan.

scholarly journals Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakienseGlucose Oxidase Expressed in Escherichia coli

1998 ◽  
Vol 64 (4) ◽  
pp. 1405-1411 ◽  
Author(s):  
Susanne Witt ◽  
Mahavir Singh ◽  
Henryk M. Kalisz
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Pcr Amplification ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Peptide Fragments ◽  
Thermal Stabilities ◽  
E Coli

ABSTRACT The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC1.1.3.4 ]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. RecombinantEscherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml−1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg−1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX inE. coli.

scholarly journals A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-d-myo-inositol 3,4,5-trisphosphate

1991 ◽  
Vol 280 (2) ◽  
pp. 533-539 ◽  
Author(s):  
G Reiser ◽  
R Schäfer ◽  
F Donié ◽  
E Hülser ◽  
M Nehls-Sahabandu ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Anion Exchange ◽  
Protein Band ◽  
Anion Exchange Chromatography ◽  
Bovine Brain ◽  
Exchange Chromatography ◽  
Receptor Protein ◽  
Radioactive Label ◽  
Binding Reaction ◽  
High Affinity

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.

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).

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 Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association

1998 ◽  
Vol 334 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Ravi MEHROTRA ◽  
David J. THORNTON ◽  
John K. SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Peptide Mapping ◽  
Minor Component ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Average Molecular Mass ◽  
Self Association ◽  
A Minor

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30–120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

Saccharide and fructooligosaccharide accumulation across leaf-bases during growth and bulb development of onion ( Allium cepa L.)

2008 ◽  
Vol 56 (1) ◽  
pp. 21-31 ◽  
Author(s):  
N. Shiomi ◽  
N. Benkeblia ◽  
S. Onodera ◽  
N. Takahashi ◽  
M. Fujishima ◽  
...  
Keyword(s):  
Allium Cepa ◽  
High Performance ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Glucose Content ◽  
Bulb Formation ◽  
Total Carbohydrates ◽  
June July

The accumulation of saccharides and fructooligosaccharides (FOS) in the individual leaf-bases of onion ( Allium cepa L.) was investigated during growth and bulb development. Saccharides and FOS were analysed by means of high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD). The glucose content was the highest, while the content of saccharides (glucose, fructose and sucrose) increased during June, July and August and decreased slightly during September. The trisaccharides all accumulated to a similar extent, although the neokestose [ 3b , 6 G - β -D fructofuranosylsucrose] content was higher than that of 1-kestose [ 3a , 1 F - β -D-fructofuranosylsucrose]. Tetra-, penta- and high-DP (degree of polymerization) FOS also showed a similar pattern, though the contents of 4b [6 G (1- β -D-fructofuranosyl) 2 sucrose] and 5b [6 G (1- β -D-fructofuranosyl) 3 sucrose] were higher compared with that of other tetra-[ 4a , 1 F (1- β -D-fructofuranosyl) 2 sucrose and 4c , 1 F , 6 G -di- β -D-fructofuranosyl sucrose] and penta-saccharides [ 5a , 1 F (1- β -D-fructofuranosyl) 3 sucrose]. Total FOS accumulated to a greater extent in the inner (youngest) leaf-bases than in the outer (oldest) leaf-bases, and their content was high during August. The total carbohydrates content was 6.71, 7.25, 8.10 and 6.30 g 100 g −1 FW during June, July, August and September, respectively. During bulb formation, a balance was observed between the glucose, fructose, sucrose and FOS contents, with an average ratio of 20:10:10:60 of total carbohydrates, respectively.

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