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Improvement of Catalytic Efficiency, Thermo-stability and Dye Decolorization Capability of Pleurotus ostreatus IBL-02 laccase by Hydrophobic Sol Gel Entrapment
Asgher, Muhammad
Kamal, Shagufta
Iqbal, Hafiz Muhammad Nasir
Chemistry Central Journal
In serious consideration of the worldwide environmental issues associated with the extensive use of the textile dyes and effluents generated thereof, the scientists across the world are in search for potential treatment technologies for their treatment. In such scenario the ligninolytic enzymes provide a potential alternative because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents.
Laccase produced from Pleurotus ostreatus IBL-02 during decolorization of the reactive textile dye Drimarene brilliant red K-4BL (DBR K-4BL) was purified and immobilized by hydrophobic gel entrapment. The crude laccase was 4.2-fold purified with specific activity of 573.52 U/mg after passing through the DEAE-Sepharose ion exchange and Sephadex-G-100 chromatography columns. P. ostreatus IBL-02 laccase was found to be a homogenous monomeric protein as evident by single band corresponding to 67 kDa on native and sodium dodesylsulfate polyacrylamide gel electrophoresis (PAGE). The laccase was immobilized by entrapment in Sol–gel matrix of trimethoxysilane (T) and proplytetramethoxysilane (P) prepared using different T:P molar ratios. The free and immobilized laccases were compared to investigate the effect of immobilization on catalytic efficiency and thermo-stability features. Laccase immobilized in the Sol–gel of 1:5 T:P ratio was optimally active and thermo-stable fraction at pH 5, 60°C with half-life of 3 h and 50 min. Laccases immobilized in 1:2 and 1:5 T:P ratio gels had significantly higher Km (83 and100mM) and Vmax (1000 and 1111 mM/mg) values as compared to free laccase. After 5 h reaction time varying decolorization percentages with a maximum of 100% were achieved for different dyes and effluents.
In summary, P. ostreatus IBL-02 laccase was immobilized by entrapping in a Sol–gel matrix with an objective to enhance its catalytic and stability properties. Sol–gel entrapped laccase presented potential efficiency as a biocatalyst when applied for decolorization of different dyes and effluents. The main benefits of the Sol–gel matrix immobilization processes are the eco-friendly approach, chemical free and energy saving reaction conditions.
P. ostreatus IBL-02; Laccase; PAGE; Sol–gel immobilization; Kinetics; Textile dye; Waste water effluent; Decolorization
Related Articles
A Novel Halophilic Lipase, LipBL, Showing High Efficiency in the Production of Eicosapentaenoic Acid (EPA)
Pérez, Dolores
Martín, Sara
Fernández-Lorente, Gloria
Filice, Marco
Guisán, José Manuel
Ventosa, Antonio
García, María Teresa
Mellado, Encarnación
PLoS ONE
Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.
Methods and Findings
A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.
In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.
Related Articles
Enhanced decolorization of Solar brilliant red 80 textile dye by an indigenous white rot fungus Schizophyllum commune IBL-06
Asgher, Muhammad
Yasmeen, Qamar
Iqbal, Hafiz Muhammad Nasir
Saudi Journal of Biological Sciences
An indigenously isolated white rot fungus, Schizophyllum commune IBL-06 was used to decolorize Solar brilliant red 80 direct dye in Kirk's basal salts medium. In initial screening study, the maximum decolorization (84.8%) of Solar brilliant red 80 was achieved in 7 days shaking incubation period at pH 4.5 and 30 °C. Different physical and nutritional factors including pH, temperature and fungal inoculum density were statistically optimized through Completely Randomized Design (CRD), to enhance the efficiency of S. commune IBL-06 for maximum decolorization of Solar brilliant red 80 dye. The effects of inexpensive carbon and nitrogen sources were also investigated. Percent dye decolorization was determined by a reduction in optical density at the wavelength of maximum absorbance (λmax, 590 nm). Under optimum conditions, the S. commune IBL-06 completely decolorized (100%) the Solar brilliant red 80 dye using maltose and ammonium sulfate as inexpensive carbon and nitrogen sources, respectively in 3 days. S. commune IBL-06 produced the three major ligninolytic enzymes lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase (Lac) during the decolorization of Solar brilliant red 80. LiP was the major enzyme (944 U/mL) secreted by S. commune IBL-06 along with comparatively lower activities of MnP and Laccase.
S. commune IBL-06; Direct dye; Solar brilliant red 80; Bio-remediation; Ligninolytic enzymes
Related Articles
The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases
Chow, Jennifer
Kovacic, Filip
Dall Antonia, Yuliya
Krauss, Ulrich
Fersini, Francesco
Schmeisser, Christel
Lauinger, Benjamin
Bongen, Patrick
Pietruszka, Joerg
Schmidt, Marlen
Menyes, Ina
Bornscheuer, Uwe T.
Eckstein, Marrit
Thum, Oliver
Liese, Andreas
Mueller-Dieckmann, Jochen
Jaeger, Karl-Erich
Streit, Wolfgang R.
PLoS ONE
Triacylglycerol lipases (EC 188.8.131.52) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.
Related Articles
Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface
Jiang, Zhengbing
Gao, Bei
Ren, Ren
Tao, Xingyi
Ma, Yushu
Wei, Dongzhi
BMC Biotechnology
For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.
Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the α-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40°C at pH8.0, they retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.
The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.
Related Articles
A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization
Cheng, Yuan-Yuan
Qian, Yun-Kai
Li, Zhi-Feng
Wu, Zhi-Hong
Liu, Hong
Li, Yue-Zhong
International Journal of Molecular Sciences
Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C10), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.
Sorangium cellulosum; cold-adapted lipase; detergent tolerant; organic synthesis
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Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica▿
Salameh, Moh'd A.
Wiegel, Juergen
Applied and Environmental Microbiology
Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca2+ and one Mn2+ ion per monomeric enzyme unit. The addition of 1 mM MnCl2 to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.
Related Articles
First co-expression of a lipase and its specific foldase obtained by metagenomics
Martini, Viviane Paula
Glogauer, Arnaldo
Müller-Santos, Marcelo
Iulek, Jorge
de Souza, Emanuel Maltempi
Mitchell, David Alexander
Pedrosa, Fabio Oliveira
Krieger, Nadia
Microbial Cell Factories
Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil.
Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni2+ column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg−1, being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg−1, respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser103, Asp250, His272), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/β hydrolase folding type I.
This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.
Lipases; Metagenomics; Biocatalysis; Lipase-foldase co-expression
Related Articles
Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum
Muhammad Nasir Iqbal, Hafiz
Asgher, Muhammad
BMC Biotechnology
An eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents.
A large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the permissible limits.
In conclusion, G. lucidum MnP was immobilized by sol–gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.
Bio-catalysis; G. lucidum; MnP; PAGE; Sol–gel; Immobilization; Textile effluents; Decolorization; Toxicity reduction
Related Articles
Global Analysis of Outer Membrane Proteins from Leptospira interrogans Serovar Lai
Cullen, Paul A.
Cordwell, Stuart J.
Bulach, Dieter M.
Haake, David A.
Adler, Ben
Infection and Immunity
Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from Leptospira interrogans serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of Leptospira.
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In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E. coli
Wu, Xiangping
You, Pengyong
Su, Erzheng
Xu, Jingjing
Gao, Bei
Wei, Dongzhi
BMC Biotechnology
Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process.
In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively.
The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.
Pseudomonas aeruginosa; Lipase; Chaperone; Dual expression plasmid; Esterification
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Silica-Antibiotic Hybrid Nanoparticles for Targeting Intracellular Pathogens ▿
Seleem, Mohamed N.
Munusamy, Prabhakaran
Ranjan, Ashish
Alqublan, Hamzeh
Pickrell, Gary
Sriranganathan, Nammalwar
Antimicrobial Agents and Chemotherapy
We investigated the capability of biodegradable silica xerogel as a novel carrier of antibiotic and the efficacy of treatment compared to that with the same dose of free drug against murine salmonellosis. The drug molecules (31%) entrapped in the sol-gel matrix remained in biologically active form, and the bactericidal effect was retained upon drug release. The in vitro drug release profiles of the gentamicin from the xerogel and that from the xerogel-polyethylene glycol (PEG) were distinctly different at pH 7.4. A delayed release of gentamicin was observed from the silica xerogel network (57% in 33 h), and with the addition of 2% PEG, the release rate reached 90% in 33 h. Administration of two doses of the silica xerogel significantly reduced the Salmonella enterica serovar Typhimurium load in the spleens and livers of infected AJ 646 mice. The silica xerogel and xerogel-PEG achieved a 0.45-log and a 0.41-log reduction in the spleens, respectively, while for the free drug there was no reduction. On the other hand, silica xerogel and xerogel-PEG achieved statistically significant 1.13-log and 1.15-log reductions in the livers, respectively, while for the free drug the reduction was a nonsignificant value of 0.07 log. This new approach, which utilizes a room-temperature synthetic route for incorporating therapeutic drugs into the silica matrix, should improve the capability for targeting intracellular pathogens.
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Halophilic Bacteria as a Source of Novel Hydrolytic Enzymes
de Lourdes Moreno, María
Pérez, Dolores
García, María Teresa
Mellado, Encarnación
Life : Open Access Journal
Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity.
halophiles; extremophiles; hydrolases; saline environments
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Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium
Ollikka, Pauli
Alhonmäki, Kirsi
Leppänen, Veli-Matti
Glumoff, Tuomo
Raijola, Timo
Suominen, Ilari
Applied and Environmental Microbiology
The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.
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LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis
Shen, Guomiao
Singh, Krishna
Chandra, Dinesh
Serveau-Avesque, Carole
Maurin, Damien
Canaan, Stéphane
Singla, Rupak
Behera, Digambar
Laal, Suman
Infection and Immunity
We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif "GXSXG" characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV−) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines.
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Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.
Katagiri, N
Tsutsumi, Y
Nishida, T
Applied and Environmental Microbiology
Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.
Related Articles
The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor
Pereira, Eridan Orlando
Tsang, Adrian
McAllister, Tim A
Menassa, Rima
Biotechnology for Biofuels
Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries.
We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively.
Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.
Lipase; Acetylxylan esterase Acremonium alcalophilum; Nicotiana benthamiana; Heterologous expression
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Fast and economic immobilization methods described for non-commercial Pseudomonas lipases
Cesarini, Silvia
Infanzón, Belén
Pastor, F I Javier
Diaz, Pilar
BMC Biotechnology
There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications.
To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000.
The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.
Pseudomonas; Lipase; Immobilization; Accurel; Celite; FAMEs
Related Articles
Whole-cell fungal transformation of precursors into dyes
Polak, Jolanta
Jarosz-Wilkołazka, Anna
Microbial Cell Factories
Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes.
An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water.
This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation.
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Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY▿
Mishra, Kanhu C.
de Chastellier, Chantal
Narayana, Yeddula
Bifani, Pablo
Brown, Alistair K.
Besra, Gurdyal S.
Katoch, Vishwa M.
Joshi, Beenu
Balaji, Kithiganahalli N.
Kremer, Laurent
Infection and Immunity
PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(ΔPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipYmar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipYmar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(ΔPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(ΔPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.
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CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production
Wang, Jianjun
Zhu, Junge
Min, Cong
Wu, Sheng
BMC Biotechnology
γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened.
We prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The Vmax and kcat value of Avicel immobilized Gla-linker-Cbd were 381 U mg-1 and 4.7 × 105 s-1 respectively. And the values of the free Gla-linker-Cbd were 151 U mg-1 and 1.8 × 105 s-1 respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value.
Cellulose is the most abundant and renewable material on the Earth. The absorption between Cbd domain and cellulose is a bio-green process. The cellulose immobilized fusion Gla exhibited good catalytic characters, therefore we think the cellulose immobilized Gla is a promising catalyst for the industrial preparation of (-) - γ-lactam.
Avicel; Cellulose binding domain; γ-lactamase; (-) γ-lactam
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Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability
Fernández-Fueyo, Elena
Ruiz-Dueñas, Francisco J
Martínez, María Jesús
Romero, Antonio
Hammel, Kenneth E
Medrano, Francisco Javier
Martínez, Angel T
Biotechnology for Biofuels
The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation.
Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences.
The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing enzymes of biotechnological interest.
Genome; Pleurotus ostreatus; Ligninolytic peroxidase genes; Heterologous expression; Crystal structure; Catalytic properties; Thermal stability; pH stability; Gene duplication; Peroxidase evolution
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Immobilization of the Antarctic Bacillus sp. LX-1 α-Galactosidase on Eudragit L-100 for the Production of a Functional Feed Additive
Lee, Jaekoo
Park, Inkyung
Cho, Jaiesoon
Asian-Australasian Journal of Animal Sciences
Partially purified α-galactosidase from Bacillus sp. LX-1 was non-covalently immobilized on a reversibly soluble-insoluble polymer, Eudragit L-100, and an immobilization efficiency of 0.93 was obtained. The optimum pH of the free and immobilized enzyme was 6.5 to 7.0 and 7.0, respectively, while there was no change in optimum temperature between the free and immobilized α-galactosidase. The immobilized α-galactosidase was reutilized six times without significant loss in activity. The immobilized enzyme showed good storage stability at 37°C, retaining about 50% of its initial activity even after 18 d at this temperature, while the free enzyme was completely inactivated. The immobilization of α-galactosidase from Bacillus sp. LX-1 on Eudragit L-100 may be a promising strategy for removal of α-galacto-oligosaccharides such as raffinose and stachyose from soybean meal and other legume in feed industry.
α-Galactosidase; Bacillus; Eudragit L-100; Immobilization; α-Galacto-oligosaccharides; Feed Industry
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Freeze-Dried Targeted Mannosylated Selenium-Loaded Nanoliposomes: Development and Evaluation
Youngren, Susanne R.
Mulik, Rohit
Jun, Byoung
Hoffmann, Peter R.
Morris, Kenneth R.
Chougule, Mahavir B.
AAPS PharmSciTech
The aim of this investigation was to develop and evaluate freeze-dried mannosylated liposomes for the targeted delivery of selenium. Dipalmitoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol were dissolved in a chloroform and methanol mixture and allowed to form a thin film within a rotatory evaporator. This thin film was hydrated with a sodium selenite (5.8 μM) solution to form multilamellar vesicles and homogenized under high pressure to yield unilamellar nanoliposomes. Se-loaded nanoliposomes were mannosylated by 0.1% w/v mannosamine (Man-Lip-Se) prior to being lyophilized. Mannosamine concentration was optimized with cellular uptake studies in M receptor expressing cells. Non-lyophilized and lyophilized Man-Lip-Se were characterized for size, zeta potential, and entrapment efficiency. The influence of liposomal composition on the characteristics of Man-Lip-Se were evaluated using acidic and basic medium for 24 h. Thermal analysis and powder X-ray diffraction were used to determine the interaction of components within the Man-Lip-Se. The size, zeta potential and entrapment efficiency of the optimum Man-Lip-Se were observed to be 158 ± 28.9 nm, 33.21 ± 0.89 mV, and 77.27 ± 2.34%, respectively. An in vitro Se release of 70–75% up to 24 h in PBS pH 6.8 and <8% Se release in acidic media (0.1 N HCl) in 1 h was observed. The Man-Lip-Se were found to withstand gastric-like environments and showed sustained release. Stable freeze-dried Man-Lip-Se were successfully formulated with a size of <200 nm, ∼75% entrapment, and achieved controlled release of Se with stability under acidic media, which may be of importance in the targeted delivery of Se to the immune system.
Electronic supplementary material
The online version of this article (doi:10.1208/s12249-013-9988-3) contains supplementary material, which is available to authorized users.
mannosylation; nanoliposome; selenium; thermal properties
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Functionalized Graphene Sheets As Immobilization Matrix for Fenugreek β-Amylase: Enzyme Kinetics and Stability Studies
Srivastava, Garima
Singh, Kritika
Talat, Mahe
Srivastava, Onkar Nath
Kayastha, Arvind M.
PLoS ONE
β-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek β-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries.
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American Cancer Society Estimates Nearly 19 Million People Will Survive Cancer by 2024
Updated: 06/02/2014 - People with cancer are living longer.
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Jude Deveraux brilliantly entwines passion, memory, and a touch of intrigue in this spellbinding "New York Times" bestseller.
At age twelve, Cassandra Madden fell in love with Jefferson Ames, a young man she met at one of her mother's business conferences. For years, Cassandra held on to this unrequited love in order to cope with her loneliness and the pain of her mother's coldness; even after meeting a man she thought she might marry, her heart still yearned for Jeff.
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About the Author
Jude Deveraux is the author of historical and contemporary women's fiction featuring women of strong character and gorgeous, exciting men. Jude has had more than thirty books on the New York Times bestsellers list, 60 million copies in print, and has been translated into 18 languages. When she's not writing, she enjoys reading murder mysteries, working in her garden, and in boxing class she likes to show much younger males that she can throw a mean right cross.
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I need some emotional support here.
It would be ruled justifiable homicide.
and who'd support a liar anyway?
•Mar 12, 2013 7:48 PM jacktrade175 posts
My wife will kill me if she know how deep i am in sapx. -------------- Hey guess what jack, your wife would be doing us all a favor if she silences you permanently.
You just made my night, Thank You!
You need to be the idiot I've come to know. Continue swapping in & out of your aliases posting garbage. Now get busy.
Will you be mad at me if you find out how deep i am in your wife?
On another thread this evening you try to play the sensitive injured party role. As if you had done something to me personally. That's not going to work. What you've stated to anyone/everyone that reads this board is that you went in big and kept that secret from your wife. YOU posted that. If you are to be taken for being truthful, then you have chosen to keep something important from someone close to you. Concealment is dishonest. If what you've claimed isn't true then you are a liar. YOU deserve disdain because of who you've made your self out to be. Not sure whether you grasp the inarguable point I've made. You are not trustworthy.
The sooner she does the better.
That said, if she kills you, do not include your shares going to her in your final will and testament. Glad I could help out.
You've come to the right place. That said, if she kills you, do not include your shares going to her in your final will and testament. Glad I could help out.


text:
A negative personal and family history for venous thrombotic events is not sufficient to exclude thrombophilia in patients with cerebral venous thrombosis
Version of Record online: 23 JUL 2004
DOI: 10.1111/j.1468-1331.2004.00862.x
European Journal of Neurology
Volume 11, Issue 8, pages 555–558, August 2004
Additional Information
How to Cite
Amberger, N., Masuhr, F., Valdueza, J. M., Vetter, B. and Weih, M. (2004), A negative personal and family history for venous thrombotic events is not sufficient to exclude thrombophilia in patients with cerebral venous thrombosis. European Journal of Neurology, 11: 555–558. doi: 10.1111/j.1468-1331.2004.00862.x
Publication History
- Issue online: 23 JUL 2004
- Version of Record online: 23 JUL 2004
- Received 25 October 2003 Accepted 4 February 2004
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Logo Design for Graphic Design Firm Compney
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text:
We all know about spaghetti, macaroni and lasagna….. but the Italian kitchen is so much more and widely varied. Depending on where you are and when you are there, you will find many dishes that are totally awesome and you've never heard of it. That, and sweet stuff of course ! One of the reasons…(Read More)
Here's another great place to stay when in Umbria. A little bit of paradise in Italy's Green Heart, re-built and managed by two people from Holland. Wonderful video ! Find more on their website HERE Polmone Turismo Verde from Polmone on Vimeo…(Read More)
Enrico from Hear the Road in Italy posted this great video on Youtube. You'll find the link to his company in the right-hand column of this page.  …(Read More)
Hi everybody….. these past two years have been difficult and EuropeRides has been left at the side for a while. But we're still here (and now in Italy) and even if we ourselves cannot take you to ride, we are here at least help you find others who can ! The latest such contact is…(Read More)
At long last we've managed to get ourselves in the saddle again. Not without some problems, mind you. After my rides 3 weeks ago, I found the GS's NEW battery empty again. Should have tried it before mounting up, but no, we got all dressed up and ready at 95 F, pushed the…(Read More)
This is the first winter that I haven't started the BMW('s). Shame on me ! And we did not even HAVE a winter in the sense of cold, ice and snow. Haven't seen any of it. But since we returned from France last September we have been in some (partly self-inflicted) turmoil…(Read More)
Le Tour 2013
We've been riding our 1200GS a lot these past few weeks, visiting many of our favorite alpine passes again, but first let me share our yesterday's ride to see Stage 19 of the Tour de France 2013. For now I'll provide you the link to my Facebook Album, but I'll add…(Read More)
With our customers safely on a plane heading back to Dallas Forth Worth Airport, we can look back on some fun guiding days this month. Nina and I helped our guests tour some of the nice places in the North of our country, including the Windmill Museum in the Schermer, visiting the 400 year old…(Read More)
As of today you will spot the image of, and link to, the Alpentourer Magazin in the side bar. Stephan Fennel produces this German language motor touring mag and does a great job at it. Even if you don't read/speak German it stil lis a great source of photos and routes for 'anything…(Read More)
Some lucky fellow found the new, but camouflaged, RT's on Teneriffe of all places. Dunno if 2014 will be 2013 even? With all the silly stickers on them, the new form is hard to make out – which is why they do it – but nice to watch nonetheless. We are getting ready for the…(Read More)
The good news: WINTER IS OVER ! You may think "well, duh!" But really, this winter has been the longest for many decades and although I have been riding our GS in France a few times along snow lined mountain roads, and the RT here in Holland in between frost periods, last week was the…(Read More)
Being one of the first users of Paper.li, the Swiss attempt at making automated, curated on-line newspapers, we have the 'EuropeRides MotorMag' appear twice daily. The way it works is that it crawls through many thousands Twitter and Facebook pages and accounts that WE have liked, and picks up the articles on topics…(Read More)
To all of our friends, motoring or not…(Read More)
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On the main connection between France and Italy in the eastern Alps, connecting the French Tarantaise region with the Italian Aosta Valley, lies this wonderfully equipped, motor-friendly hotel ! As you may remember, Nina and I checked them out earlier this year to get them their Motorrad-Tourer certificate of quality. And since we check…(Read More)
…….. *cough*…….. …. but I could NEVER remember the track…(Read More)
Via one of our valued contacts we got THIS LINK outlining 'what's different' in France. Well, regarding motoring laws and such…… we could fill 3 entire websites if we'd that that too literally … *grin*. Especially about the breathalysers, ethylotests, alcohol testers or whatever else you may want to call them, be aware that…(Read More)
Voor mijn Nederlandse lezers die misschien nog niet van de Motorrrijders Aktie Groep hebben gehoord: op onze site staat een goed overzicht voor motorrijders die naar het buitenland gaan: http://www.mag-nl.org/cms/index.php?page=vakantietips-internationaal Een leuk feit bij de debiele Franse wetgeving aangaande de verplichting om TWEE alcoholtesters bij…(Read More)
Of course you all being smart smartphone users, and I nothing but a rookie, you've all found this long time ago! But for me it was a brain wave! As Nina and I also hike the mountain trails in our are, besides riding the BMW, I always pack a lot of 'ordnance maps'. I…(Read More)
Het is weer vakantietijd en net als wij gaat u straks weer op stap met de (liefst) motor, auto of anders. En u checkt natuurlijk nog even uw verzekeringen. Of niet? Hierbij een stukje van André Janssen over dat onderwerp. Zijn al die verzekeringen wel echt nodig? door André Janssen Verzekerd op reis gaan…(Read More)