The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Kiyohisa Mizumoto - One of the best experts on this subject based on the ideXlab platform.
-
mrna capping Enzyme Isolation and characterization of the gene encoding mrna guanylyltransferase subunit from saccharomyces cerevisiae
Journal of Biological Chemistry, 1992Co-Authors: Kiyohisa Mizumoto, Yoshio Shibagaki, Naoto Itoh, H Yamada, Shigekazu NagataAbstract:Abstract The highly purified yeast mRNA capping Enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping Enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping Enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.
-
mRNA capping Enzyme. Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae.
The Journal of biological chemistry, 1992Co-Authors: Yoshio Shibagaki, Naoto Itoh, H Yamada, Shigekazu Nagata, Kiyohisa MizumotoAbstract:The highly purified yeast mRNA capping Enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, 1989-1995). The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping Enzyme. The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase. The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues. From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping Enzyme are encoded by two separate genes, not as a fused protein. CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis. Gene disruption experiment indicated that CEG1 is essential for the growth of yeast. We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.
László Poppe - One of the best experts on this subject based on the ideXlab platform.
-
Production and Application of Novel Sterol Esterases from Aspergillus Strains by Solid State Fermentation
Journal of the American Oil Chemists' Society, 2007Co-Authors: Enikő R. Tőke, Viviána Nagy, Katalin Recseg, György Szakács, László PoppeAbstract:Among numerous mesophilic fungi screened for sterol esterase activity followed by the esterification reaction between plant β-sitosterol and lauric acid in organic solvent, six Aspergillus strains were selected as the most active producers. These fungi had not been studied previously for sterol esterase production. The fungi were cultivated under solid state fermentation (SSF) conditions. The gently dried SSF cultures as such were tested in the esterification reactions, without any special Enzyme Isolation and purification (downstream) processes. All the six Aspergillus SSF preparations were able to synthesize sterol esters. Sterol esterase activity of these GRAS cleared Aspergillus strains was inducible by sterol ester supplementation to the SSF medium and showed remarkably different moisture optimum during growth as compared to the production of lipase (determined by pNP-palmitate). Genome analysis revealed that sterol esterase production might be a common feature of many Aspergillus species. The synthetic usefulness of the best SSF preparations of A. oryzae NRRL 6270 and A. sojae NRRL 6271 was demonstrated by synthesis of esters of plant sterols with lauric acid resulting in 45–63% conversions (GC) and 27–38% isolated yields of steryl laurates. The isomer preference of A. oryzae NRRL 6270 towards the 10 E ,12 Z isomer of conjugated linoleic acid (CLA) in the esterification reaction with plant sterols was also determined.
-
Kinetic resolutions with novel, highly enantioselective fungal lipases produced by solid state fermentation
Journal of Molecular Catalysis B-enzymatic, 2006Co-Authors: Viviána Nagy, Enikő R. Tőke, György Szakács, Lee Chee Keong, Gábor Szatzker, Darah Ibrahim, Ibrahim Che Omar, László PoppeAbstract:Abstract Thirty-eight filamentous fungi cultivated under solid state fermentation (SSF) conditions were screened for lipase activity and enantioselectivity in kinetic resolutions of racemic secondary alcohols ( rac - 1a – c ) by acetylation with vinyl acetate performed in organic solvents. Many of the target fungi have not been studied previously for lipase/esterase activity and enantioselectivity. Without special Enzyme Isolation processes, the room temperature (25 °C) dried SSF cultures as such were tested in the enantiomer selective biotransformations. The majority of these SSF preparations proved to be effective as enantiomer selective biocatalysts exhibiting high but usual enantioselectivities according to the Kazlauskas rule. However, the SSF preparation of Mucor hiemalis origin acted as a selective anti-Kazlauskas catalyst. The best SSF products were successfully applied in preparative scale resolutions.
Lo Gorton - One of the best experts on this subject based on the ideXlab platform.
-
Comparative spectroelectrochemical studies of lyophilized and nonlyophilized laccases from Cerrena unicolor basidiomycete
Electroanalysis, 2007Co-Authors: Sergey Shleev, Jerzy Rogalski, Maciej Klis, Yan Wang, Renata Bilewicz, Lo GortonAbstract:The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen Enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET-based biocatalytic reduction of O-2 by C unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady-state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3-D structures of laccase molecules, conformational changes, and partial denaturation of the Enzyme, which appeared after Enzyme Isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high Km-value towards O-2, and the unusual electrochemical behavior of C unicolor laccase used in the present study. In spite of the comparable specific activity and long-term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of Enzyme than its lyophilized counterpart. (Less)
-
Comparative spectroelectrochemical studies of lyophilized and nonlyophilized laccases from Cerrena unicolor basidiomycete
'Wiley', 2007Co-Authors: Shleev Sergey, Rogalski Jerzy, Bilewicz Renata, Klis Maciej, Wang Yan, Lo GortonAbstract:The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen Enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET-based biocatalytic reduction of O-2 by C unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady-state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3-D structures of laccase molecules, conformational changes, and partial denaturation of the Enzyme, which appeared after Enzyme Isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high Km-value towards O-2, and the unusual electrochemical behavior of C unicolor laccase used in the present study. In spite of the comparable specific activity and long-term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of Enzyme than its lyophilized counterpart
William E Broderick - One of the best experts on this subject based on the ideXlab platform.
-
pyruvate formate lyase activating Enzyme strictly anaerobic Isolation yields active Enzyme containing a 3fe 4s cluster
Biochemical and Biophysical Research Communications, 2000Co-Authors: Joan B Broderick, Timothy F Henshaw, Jennifer Cheek, Kristi Wojtuszewski, Matthew R Trojan, Ryan M Mcghan, Amy Kopf, Megan Kibbey, Sheila Smith, William E BroderickAbstract:Abstract Pyruvate formate-lyase-activating Enzyme (PFL-AE) from Escherichia coli ( E. coli ) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S -adenosyl- l -methionine (AdoMet). Although PFL-AE is an iron-dependent Enzyme, Isolation of the Enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first Isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV–visible, EPR, and resonance Raman data support the presence of a [3Fe–4S] + cluster in the purified Enzyme. The isolated native Enzyme, but not apo-Enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.
-
Pyruvate Formate-Lyase-Activating Enzyme: Strictly Anaerobic Isolation Yields Active Enzyme Containing a [3Fe–4S]+ Cluster
Biochemical and Biophysical Research Communications, 2000Co-Authors: Joan B Broderick, Timothy F Henshaw, Jennifer Cheek, Kristi Wojtuszewski, Matthew R Trojan, Ryan M Mcghan, Amy Kopf, Megan Kibbey, Sheila R. Smith, William E BroderickAbstract:Abstract Pyruvate formate-lyase-activating Enzyme (PFL-AE) from Escherichia coli ( E. coli ) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S -adenosyl- l -methionine (AdoMet). Although PFL-AE is an iron-dependent Enzyme, Isolation of the Enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first Isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV–visible, EPR, and resonance Raman data support the presence of a [3Fe–4S] + cluster in the purified Enzyme. The isolated native Enzyme, but not apo-Enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.
-
Pyruvate formate-lyase-activating Enzyme: strictly anaerobic Isolation yields active Enzyme containing a [3Fe-4S](+) cluster.
Biochemical and biophysical research communications, 2000Co-Authors: Joan B Broderick, Timothy F Henshaw, Jennifer Cheek, Kristi Wojtuszewski, Matthew R Trojan, Ryan M Mcghan, Amy Kopf, Megan Kibbey, Sheila R. Smith, William E BroderickAbstract:Abstract Pyruvate formate-lyase-activating Enzyme (PFL-AE) from Escherichia coli ( E. coli ) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S -adenosyl- l -methionine (AdoMet). Although PFL-AE is an iron-dependent Enzyme, Isolation of the Enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first Isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV–visible, EPR, and resonance Raman data support the presence of a [3Fe–4S] + cluster in the purified Enzyme. The isolated native Enzyme, but not apo-Enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity.
Kin Yip Tam - One of the best experts on this subject based on the ideXlab platform.
-
silica encapsulated nanomagnetic particle as a new recoverable biocatalyst carrier
Journal of Physical Chemistry B, 2006Co-Authors: Shik Chi Tsang, Xin Gao, Kin Yip TamAbstract:Enzymes are versatile biocatalysts with major advantages of ultrahigh reaction selectivity and specificity under mild conditions, which currently find increasing applications. However, their applications are often hampered by difficulties in recovery and recycling. As a result, we carried out detailed investigations on the synthesis and characterization of silica-encapsulated iron oxide magnetic nanoparticles of controlled dimension as an Enzyme carrier. It is shown that the relatively smaller sized silica-coated magnetic nanoparticle prepared by the microemlusion technique can a carry bulky Enzyme, β-lactamase, via chemical linkages on the silica overlayer without severely blocking the enzymatic active center (which is commonly encountered in conventional solid supports). An activity study by Michalis−Menten kinetics reflects that this new type of immobilization allows Enzyme Isolation with accessibility as good as free Enzyme. The recovery and reusability of the nanoparticle-supported Enzyme upon applic...
-
Colloidal stable silica encapsulated nano-magnetic composite as a novel bio-catalyst carrier
Chemical communications (Cambridge England), 2003Co-Authors: Xin Gao, Kin Yip Tam, Shik Chi TsangAbstract:A colloidal stable silica-encapsulated magnetic nano-composite of a controlled dimension is, for the first time, employed to carry beta-lactamase via chemical linkage on the silica overlayer: activity study reflects that this new type of immobilisation allows site (Enzyme) Isolation, accessibility as good as free Enzyme and recovery & reusability upon application of magnetic separation.