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Bioelectrochemistry (Amsterdam,... Feb 2015Two modified electrodes with immobilized glucose oxidase were developed. Modification with poly(3,4-ethylenedioxythiophene) (PEDOT) and polyacrylic acid (PAA) doped with...
Two modified electrodes with immobilized glucose oxidase were developed. Modification with poly(3,4-ethylenedioxythiophene) (PEDOT) and polyacrylic acid (PAA) doped with poly(4-lithium styrenesulfonic acid) (PSSLi) in a newly elaborated procedure was used in the first electrode. The second one presents innovative solution and consists of two sublayers; one of them was PEDOT doped with PSSLi and the other was composed of PEDOT and anthranilic acid (AA) doped with poly(4-styrenesulfonic acid) (PSSH). Glucose oxidase was covalently bonded with the carboxyl groups of the polymer through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (WSC). The activity of immobilized enzyme was confirmed by spectrophotometry using the reaction of the produced hydrogen peroxide with o-dianisidine. The procedure for immobilization was optimized. It was found that the choice of an appropriate doping agent and its concentration were significant and 0.1M PSSLi proved to be the best doping agent. The most efficient immobilization was established for WSC and GOD concentration at the level of 4mg/ml and 5mg/ml respectively. In both cases, it was found that a small deviation from the concentrations determined to cause a sharp decrease in the activity of the enzyme, which was proven by spectrophotometric measurements. Prepared electrodes were active over a month with repeatable measurement results.
Topics: Acrylic Resins; Bridged Bicyclo Compounds, Heterocyclic; Carbodiimides; Electrochemical Techniques; Enzymes, Immobilized; Glucose Oxidase; Methylamines; Polymers; Styrenes; Sulfonic Acids; ortho-Aminobenzoates
PubMed: 25023029
DOI: 10.1016/j.bioelechem.2014.06.009 -
Analytical Biochemistry Oct 2014D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino...
D-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent KM and kcat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in kcat/KM relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.
Topics: Amino Acid Substitution; Amino Acids; Colorimetry; D-Amino-Acid Oxidase; Dianisidine; Horseradish Peroxidase; Hydrogen Peroxide; Kinetics; Substrate Specificity; Transaminases
PubMed: 24949900
DOI: 10.1016/j.ab.2014.06.006 -
Applied Biochemistry and Biotechnology Aug 2014Vast applications of peroxidases create an increasing demand to characterize peroxidases from new sources with more applicability potential. The aim of the present study...
Vast applications of peroxidases create an increasing demand to characterize peroxidases from new sources with more applicability potential. The aim of the present study was to check the presence of peroxidase activity from Caralluma umbellata. This is the first report on the C. umbellata peroxidase (CUP). The presence of peroxidase was revealed by the histochemical analysis of the stem sections, zymographic studies, and in vitro peroxidase activity assay using various reducing substrates viz., 2, 2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), guaiacol, o-dianisidine, and ferulic acid. The band pattern in zymogram confirms that CUP has a molecular weight less than that of horseradish peroxidase (44 kDa). Comparative evaluation of peroxidase activity of CUP with respect to horseradish peroxidase (HRP) indicates that CUP catalyzes ABTS and ferulic acid in a similar pattern as HRP but with guaiacol, the extent of catalysis shown by CUP over HRP is high. The standard inhibitors sodium azide and sodium meta bisulphite inhibited CUP activity in a dose dependent manner.
Topics: Apocynaceae; Biocatalysis; Coumaric Acids; Guaiacol; Kinetics; Peroxidases; Plant Proteins; Substrate Specificity
PubMed: 24943097
DOI: 10.1007/s12010-014-1013-0 -
Pakistan Journal of Biological Sciences... Jan 2014Pleurotus fossulatus (Cooke) Sace is member of oyster mushroom can produced extracellular laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) in submerged...
Pleurotus fossulatus (Cooke) Sace is member of oyster mushroom can produced extracellular laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) in submerged fermentation. To analyze the optimum production for laccase P. fossulatus was cultured both in stationary and shaking condition in different media. Partial purification of laccase was done after 0-80% ammonium sulphate precipitation, followed by DEAE (Diethylaminoethyl) Sephadex (A-50) anion exchange chromatography. Potato-sucrose peptone (PSP) medium and Potato-dextrose (PD) medium showed highest laccase production in shaking and stationary conditions, respectively. Though the time required for optimum laccase production in stationary condition was much more than the shaking condition but the amount of laccase was about 2.75t greater in former condition. The laccase produced in stationary condition was more stable than the enzyme produced in shaking condition. The partially purified enzyme showed highest affinity towards o-dianisidine than guaiacol and ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as evidenced by their K(m). The physico-chemical properties of the laccase suggested the significance of this enzyme in industrial applications.
Topics: Chemical Phenomena; Culture Media; Laccase; Pleurotus
PubMed: 24783799
DOI: 10.3923/pjbs.2014.173.181 -
Research in Veterinary Science Jun 2014The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix...
The aim of the present work was to evaluate whether concentrations of the carboxy-terminal cross-linked fragment of type II collagen (CTX-II), the activities of matrix metalloproteinase-2 and -9 (MMP-2/-9) and Myeloperoxidase (MPO) in canine synovial fluids (SF) can reflect structural alterations of articular cartilage in dogs with fragmented medial coronoid process (FMCP). Elbow joints with FMCP underwent radiographic and arthroscopic examination. Commercially available assays were used to analyze SF for CTX-II concentration and MMP-2/-9 activity. MPO activity was measured by o-dianisidine-assay. The MMPs were further evaluated by zymography. CTX-II concentration and MMP-2 activity showed age-dependent trends in controls. Increased enzyme activities of MPO and MMP-2/-9 were found in diseased dogs. MMP-9activity seems suitable to underline the subjective assessment of the degree of cartilage damage. These initial data of the study suggest that MPO and MMP-2/9 may be used as objective biomarkers in the diagnosis of canine osteoarthritis due to FMCP.
Topics: Animals; Biomarkers; Cartilage, Articular; Collagen Type II; Dog Diseases; Dogs; Female; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Osteoarthritis; Peroxidase; Radiography; Statistics, Nonparametric
PubMed: 24684895
DOI: 10.1016/j.rvsc.2014.02.012 -
Journal of Neural Transmission (Vienna,... Oct 2014Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the...
Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.
Topics: Automation, Laboratory; Blood Chemical Analysis; Ceruloplasmin; Dianisidine; End Stage Liver Disease; Fasting; Hepatic Encephalopathy; Hepatolenticular Degeneration; Humans
PubMed: 24663495
DOI: 10.1007/s00702-014-1196-0 -
The International Journal of... Apr 2014Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including...
Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hbαe1, hbβe1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3'-UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1.
Topics: 3' Untranslated Regions; Animals; Animals, Genetically Modified; Base Sequence; Blood Vessels; Blotting, Western; Embryo, Nonmammalian; GATA1 Transcription Factor; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; HCT116 Cells; Hematopoiesis; Homeodomain Proteins; Humans; In Situ Hybridization; MicroRNAs; Myeloid Ecotropic Viral Integration Site 1 Protein; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Nucleic Acid; Trans-Activators; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 24448023
DOI: 10.1016/j.biocel.2014.01.005 -
3 Biotech Feb 2014A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was...
A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200-700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest K value being for ortho-dianisidine and highest K and K/K for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.
PubMed: 28324461
DOI: 10.1007/s13205-013-0129-1 -
International Journal of Analytical... 2013One titrimetric and two spectrophotometric methods are described for the determination of ketotifen fumarate (KTF) in bulk drug and in tablets using cerium(IV) as the...
One titrimetric and two spectrophotometric methods are described for the determination of ketotifen fumarate (KTF) in bulk drug and in tablets using cerium(IV) as the oxidimetric agent. In titrimetry (method A), the drug was treated with a measured excess of cerium(IV) in H2SO4 medium and after a standing time of 10 min, the surplus oxidant was determined by back titration with iron(II). The spectrophotometric procedures involve addition of a known excess of cerium(IV) to KTF in acid medium followed by the determination of unreacted oxidant by reacting with either p-dimethyl amino benzaldehyde and measuring the resulting colour at 460 nm (method B) or o-dianisidine and subsequent measurement of the absorbance of coloured product at 470 nm (method C). Titrimetric assay is based on a 1 : 2 reaction stoichiometry between KTF and cerium(IV) and the method is applicable over 2-18 mg range. In spectrophotometry, regression analysis of Beer's law plots showed a good correlation in 0.4-8.0 and 0.4-10.0 g mL(-1) KTF ranges for method B and method C, respectively, and the corresponding molar absorptivity coefficients are calculated to be 4.0 × 10(4) and 3.7 × 10(4) L mol(-1) cm(-1).
PubMed: 24324496
DOI: 10.1155/2013/697651 -
Journal of Ethnopharmacology Nov 2013Various parts of the plant pineapple (Ananas comosus) are used in traditional medicine worldwide for treatment of a number of diseases and disorders. In folk medicine,...
ETHNOPHARMACOLOGICAL RELEVANCE
Various parts of the plant pineapple (Ananas comosus) are used in traditional medicine worldwide for treatment of a number of diseases and disorders. In folk medicine, pineapple leaf extract was used as an antimicrobial, vermicide, purgative, emmenagoogue, abortifacient, anti-oedema and anti-inflammatory agent. Compared to the fruit and stem extracts of pineapple, information about its leaf extract is limited. The potential of pineapple crown leaf extract as an ethno-medicine has been evaluated in terms of its enzymatic activities related to wound healing, antimicrobial property and toxicity.
MATERIALS AND METHODS
Major protein components of the extract were revealed by 2-D gel electrophoresis followed by MS/MS analysis. Zymography, DQ-gelatin assay were performed to demonstrate proteolytic, fibrinolytic, gelatinase and collagenase activities. DNase and RNase activities were revealed from agarose gel electrophoresis. Antimicrobial activity was evaluated spectrophotometrically from growth inhibition. Sprague-Dawley rat model was used to measure acute and sub-acute toxicity of the extract by analyzing blood markers.
RESULT
The extract contains several proteins that were clustered under native condition. Proteomic studies indicated presence of fruit bromelain as major protein constituent of the extract. It showed nonspecific protease activity, gelatinolytic, collagenase, fibrinolytic, acid and alkaline phosphatase, peroxidase, DNase and RNase activities along with considerable anti-microbial property. The leaf extract did not induce any toxicity in rats after oral administration of acute and sub-acute doses.
CONCLUSION
Pineapple leaf extract is nontoxic, contains enzymes related to damage tissue repairing, wound healing and possibly prevents secondary infections from microbial organisms.
Topics: Amylases; Ananas; Animals; Anti-Bacterial Agents; Bacteria; Caseins; Deoxyribonucleases; Esterases; Female; Peptide Hydrolases; Peroxidases; Phosphoric Monoester Hydrolases; Plant Extracts; Plant Leaves; Plant Proteins; Proteomics; Rats; Rats, Sprague-Dawley; Ribonucleases; Wound Healing
PubMed: 24076462
DOI: 10.1016/j.jep.2013.08.024