Purification and Characterization of Laccase fromChaetomium thermophilium and Its Role in Humification

1998 ◽  
Vol 64 (9) ◽  
pp. 3175-3179 ◽  
Author(s):  
Benny Chefetz ◽  
Yona Chen ◽  
Yitzhak Hadar
Keyword(s):  
Municipal Solid Waste ◽  
Solid Waste ◽  
Laccase Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Type I ◽  
Light Spectrum ◽  
Solid Media ◽  
Wide Range

ABSTRACT Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process.C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

scholarly journals Isolation, Structural Characterization, and Valorization of Pectic Substances from Algerian Argan Tree Leaves (Argania spinosa(L.) Skeels)

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Kadda Hachem ◽  
Abderrahmane Labani ◽  
Meriem Kaid-Harche
Keyword(s):  
Antioxidant Activities ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Resonance Spectroscopy ◽  
Type I ◽  
Tree Leaves ◽  
Pectic Polysaccharides ◽  
Pectic Substances ◽  
Argan Tree

Pectic polysaccharides were solubilized from Algerian argan tree leaves by sequential extraction with water at 100°C (water-soluble pectin; AL-WSP) and EDTA solution at 80°C (chelating-soluble pectin; AL-CSP). Both AL-WSP and AL-CSP were rich in arabinose (28% and 74.5%, resp.) and had a high content of uronic acid (38.5% and 21.5%, resp.). Pectic substances were deesterified and fractionated by anion exchange chromatography, giving five fractions for each extract. Most of the fractions were characterized by methylation analysis and then analyzed by13C nuclear magnetic resonance spectroscopy. The results showed that AL-WSP consisted of rhamnogalacturonan type I, with arabinan and galactan branching at the O-4 position of the main rhamnose chain, while AL-CSP consisted of rhamnogalacturonan type I and a block of homogalacturonan. Antioxidant activities of AL-WSP and AL-CSP were evaluated by electronic spin resonance. The results showed that the antioxidant potential of AL-WSP (8.1%) and AL-CSP (−1.2%) was significantly lower than that of vitamin E.

Comparison of different anion-exchange chromatography resins for the purification of cyanobacterial microcystins

2015 ◽  
Vol 16 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Theerasak Somdee ◽  
Anchana Somdee
Keyword(s):  
Liquid Chromatography ◽  
Anion Exchange ◽  
High Performance ◽  
Total Yield ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ionization Mass ◽  
Freeze Dried ◽  
Wide Range

For the first time, different types of diethylaminoethyl (DEAE) anion-exchange resins, widely used in previous studies, were investigated to determine the most effective resin for the purification of microcystins (MCs). MCs were extracted from freeze-dried Microcystis aeruginosa cells that had been harvested from the Bueng Nong Khot reservoir, Khon Kaen, Thailand. The toxins were precipitated with ammonium sulfate and then fractionated using five different anion-exchange chromatography resins, followed by chromatography with a C18 cartridge. The toxins were further identified via liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) analysis, and the yields and purity were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection. DEAE Sephadex A-25 exhibited the best overall performance for MC purification regarding both yield and purity, followed by DEAE cellulose, DEAE Sephacel, DEAE Sepharose Fast Flow and Toyopearl DEAE. Four MC variants, MC-RR, MC-FR, [Dha7]MC-LR and MC-WR, were obtained, and [Dha7]MC-LR was the major variant, with a total yield of 53.08 mg and a purity of 95% using the Sephadex resin. This study indicates that protein precipitation and single-column chromatography using DEAE Sephadex A-25 constitute an effective method for the purification of a wide range of MC variants.

Analysis of the substituent distribution along the chain of water-soluble methyl cellulose by combination of enzymatic and chemical methods

Holzforschung ◽  
2004 ◽  
Vol 58 (1) ◽  
pp. 97-104 ◽  
Author(s):  
B. Saake ◽  
S. Lebioda ◽  
J. Puls
Keyword(s):  
Anion Exchange ◽  
Size Exclusion Chromatography ◽  
Methyl Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Degree Of Substitution ◽  
Water Soluble ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
C Nmr

Abstract Four methyl cellulose samples in the degree of substitution range from 0.5 to 2.0 were characterised by combination of different analytical methods. Samples were analysed regarding their partial degree of substitution by hydrolysis and anion exchange chromatography with pulsed amperometric detection. For calibration of the chromatographic system, standard substances were isolated by preparative HPLC and their structure was confirmed by 13C-NMR spectroscopy. For two methyl cellulose samples per-acetylation and 13C-NMR with inverse gated decoupling was carried out for comparison with the chromatographic analysis. Endoglucanase fragmentation of methyl celluloses was performed and water-soluble and insoluble fractions were analysed separately. A preparative size exclusion chromatography system for enzymatic-degraded water-soluble methyl cellulose was developed and the molar masses of the individual fractions were examined by analytical size exclusion chromatography. By combination of endoglucanase fragmentation, preparative chromatography, hydrolysis and anion exchange chromatography an approach for the analysis of the substitutent distribution along the polymeric chain of water-soluble methyl cellulose could be established.

The state of municipal solid waste management in Israel

2018 ◽  
Vol 36 (6) ◽  
pp. 527-534 ◽  
Author(s):  
Shira Daskal ◽  
Ofira Ayalon ◽  
Mordechai Shechter
Keyword(s):  
Waste Management ◽  
Municipal Solid Waste ◽  
Solid Waste ◽  
Financial Incentives ◽  
Solid Waste Management ◽  
Management Strategies ◽  
The State ◽  
Municipal Solid Waste Management ◽  
Msw Management ◽  
Wide Range

Regulation is a key tool for implementing municipal solid waste (MSW) management strategies and plans. While local authorities in Israel are responsible for the storage, collection, and disposal of MSW, Israel’s Ministry of Environmental Protection (MoEP) is responsible for the formulation and implementation of waste management policies and legislation. For the past 12 years, about 80% of the MSW in Israel has been landfilled and recycling rates have not increased, despite regulations. This paper presents the state of MSW management in Israel in light of the MoEP’s strategic goal of landfilling reduction, the regulations and legislation designed and implemented for achieving this goal, and the ensuing results. Among other things, the results indicate the importance of monitoring and assessing policy and regulations to examine whether regulation is in fact effective and whether it keeps track of its own targets and goals or not. It is also concluded that even when there is an extensive regulation that includes a wide range of laws, economic penalties and financial incentives (such as landfill levy and financing of MSW separation at source arrangements), this does not guarantee proper treatment or even an improvement in waste management. The key to success is first and foremost a suitable infrastructure that will enable achievement of the desired results.

scholarly journals Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation

1987 ◽  
Vol 247 (1) ◽  
pp. 165-174 ◽  
Author(s):  
M Whitman ◽  
D Kaplan ◽  
T Roberts ◽  
L Cantley
Keyword(s):  
Kinase Activity ◽  
Energy Charge ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Type I ◽  
Type Ii ◽  
Ionic Detergents ◽  
Rous Sarcoma ◽  
3T3 Fibroblasts ◽  
Specific Association

Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.

Fungal Polygalacturonases Exhibit Different Substrate Degradation Patterns and Differ in Their Susceptibilities to Polygalacturonase-Inhibiting Proteins

1999 ◽  
Vol 12 (8) ◽  
pp. 703-711 ◽  
Author(s):  
B. J. Cook ◽  
R. P. Clay ◽  
C. W. Bergmann ◽  
P. Albersheim ◽  
A. G. Darvill
Keyword(s):  
Anion Exchange ◽  
Plant Species ◽  
High Performance ◽  
Hydrolytic Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polygalacturonic Acid ◽  
Substrate Degradation ◽  
Wide Range ◽  
Substrate Cleavage

Polygalacturonic acid (PGA) was hydrolyzed by polygalacturonases (PGs) purified from six fungi. The oligogalacturonide products were analyzed by HPAEC-PAD (high performance anion exchange chromatography-pulsed amperimetric detection) to assess their relative amounts and degrees of polymerization. The abilities of the fungal PGs to reduce the viscosity of a solution of PGA were also determined. The potential abilities of four polygalacturonase-inhibiting proteins (PGIPs) from three plant species to inhibit or to modify the hydrolytic activity of the fungal PGs were determined by colorimetric and HPAEC-PAD analyses, respectively. Normalized activities of the different PGs acting upon the same substrate resulted in one of two distinct oligogalacturonide profiles. Viscometric analysis of the effect of PGs on the same substrate also supports two distinct patterns of cleavage. A wide range of susceptibility of the various PGs to inhibition by PGIPs was observed. The four PGs that were inhibited by all PGIPs tested exhibited an endo/exo mode of substrate cleavage, while the three PGs that were resistant to inhibition by one or more of the PGIPs proceed by a classic endo pattern of cleavage.

scholarly journals Structural Characterization and Antioxidative Activity of Low-Molecular-Weights Beta-1,3-Glucan from the Residue of ExtractedGanoderma lucidumFruiting Bodies

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Pai-Feng Kao ◽  
Shwu-Huey Wang ◽  
Wei-Ting Hung ◽  
Yu-Han Liao ◽  
Chun-Mao Lin ◽  
...  
Keyword(s):  
Cell Death ◽  
High Performance ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Dependent Manner ◽  
Ros Production ◽  
Fruiting Bodies ◽  
Concentration Dependent Manner

The major cell wall constituent ofGanoderma lucidum(G. lucidum) isβ-1,3-glucan. This study examined the polysaccharide from the residues of alkaline-extracted fruiting bodies using high-performance anion-exchange chromatography (HPAEC), and it employed nuclear magnetic resonance (NMR) and mass spectrometry (MS) to confirm the structures. We have successfully isolated low-molecular-weightβ-1,3-glucan (LMG), in high yields, from the waste residue of extracted fruiting bodies ofG. lucidum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay evaluated the capability of LMG to suppress H2O2-induced cell death in RAW264.7 cells, identifying that LMG protected cells from H2O2-induced damage. LMG treatment decreased H2O2-induced intracellular reactive oxygen species (ROS) production. LMG also influenced sphingomyelinase (SMase) activity, stimulated by cell death to induce ceramide formation, and then increase cell ROS production. Estimation of the activities of neutral and acid SMasesin vitroshowed that LMG suppressed the activities of both neutral and acid SMases in a concentration-dependent manner. These results suggest that LMG, a water-solubleβ-1,3-glucan recycled from extracted residue ofG. lucidum, possesses antioxidant capability against H2O2-induced cell death by attenuating intracellular ROS and inhibiting SMase activity.

scholarly journals The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes

1986 ◽  
Vol 238 (2) ◽  
pp. 491-499 ◽  
Author(s):  
S Palmer ◽  
P T Hawkins ◽  
R H Michell ◽  
C J Kirk
Keyword(s):  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Ionophore A23187 ◽  
Inositol Tetrakisphosphate ◽  
Inositol Phosphate Accumulation ◽  
Phosphatidylinositol 4 ◽  
Phosphate Groups

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.

scholarly journals MULTIVARIATE ANALYSIS OF THE RELATIONSHIP BETWEEN VARIOUS SOCIAL ASPECTS AND THE PROCESS OF UTILIZATION OF MUNICIPAL SOLID WASTE ACROSS EUROPEAN COUNTRIES

2021 ◽  
Vol 23 (5) ◽  
pp. 47-52
Author(s):  
E. Gubar ◽  
◽  
N. Gladyshev ◽  
A. Bogomolov ◽  
◽  
...  
Keyword(s):  
Sustainable Development ◽  
Multivariate Analysis ◽  
Municipal Solid Waste ◽  
Solid Waste ◽  
Integrated System ◽  
Social Aspects ◽  
Important Place ◽  
Msw Management ◽  
Wide Range ◽  
The Relationship

The development of the municipal sector leads to the emergence of a wide range of waste. In such conditions, an important place is occupied by the problems of environmental safety and sustainable development. The article contains a multivariate analysis of the relationship between various social aspects and the process of utilization of municipal solid waste (MSW). The influence of economic, cultural, legislative and environmental factors on these relations has been presented. Methods of utilization of MSW in the countries of the European Union (EU) have been studied. As a result, a significant amount of information on solving the MSW problem in the EU countries has been revealed. Also groups of indicators have been identified and presented, which provide a comprehensive assessment of the state of the MSW management sector. It is concluded that the main hierarchy of MSW management methods is determined by the principles of sustainable development. The waste management system implies an integrated system of various aspects, namely social, economic, regulatory, managerial and technical. A promising area is the use of chemometric analysis for the development and interpretation of selected development scenarios for the MSW management sector.

scholarly journals Expression, purification, and bioactivity of a soluble recombinant ovine interferon-tau in Escherichia coli

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Hai-Yang Yu ◽  
Dong-Mei Gao ◽  
Wei Zhou ◽  
Bing-Bing Xia ◽  
Zhi-Yuan He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Restriction Enzymes ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Type I ◽  
Interferon Tau ◽  
E Coli ◽  
Nickel Affinity Chromatography

Abstract Introduction Ovine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein. Materials and Methods First, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography. Results The protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays. Conclusion Purified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

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