The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Ming Tien - One of the best experts on this subject based on the ideXlab platform.
-
oxidation of a tetrameric nonphenolic Lignin model compound by Lignin Peroxidase
Journal of Biological Chemistry, 2001Co-Authors: Tunde Mester, Katia Ambertbalay, Simone Ciofibaffoni, Lucia Banci, Daniel A Jones, Ming TienAbstract:Abstract The present study maps the active site of Lignin Peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant Lignin Peroxidases. A nonphenolic tetrameric Lignin model was synthesized that contains β-O-4 linkages. The fungal and recombinant wild type Lignin Peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that Lignin Peroxidase is able to attack any of the Cα-Cβ linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant Lignin Peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with Lignin Peroxidase having an exposed active site capable of directly interacting with the Lignin polymer without the advent of low molecular weight mediators.
-
Oxidation of 1,2,4,5-tetramethoxybenzene by Lignin Peroxidase of Phanerochaete chrysosporium.
Archives of Biochemistry and Biophysics, 1996Co-Authors: Rao S. Koduri, Steven D. Aust, Ross E. Whitwam, David P. Barr, Ming TienAbstract:Abstract We have reinvestigated the Lignin Peroxidase-catalyzed oxidation of 1,2,4,5-tetramethoxybenzene (TMB) by using presteady-state and steady-state kinetic methods. Our presteady-state kinetic results show that the reaction of compound I with TMB obeyed second order kinetics with a rate constant of 1.1 × 10 7 M −1 s −1 . The reaction of compound II with TMB exhibits a hyperbolic concentration dependence with a K d of 16 μ M and k = 24 s −1 . The stoichiometry of TMB oxidation during steady state is two TMB cation radicals formed per H 2 O 2 consumed. These results clearly show that TMB is a good substrate for both compounds I and II of Lignin Peroxidase.
-
Oxidation of Guaiacol by Lignin Peroxidase ROLE OF VERATRYL ALCOHOL
Journal of Biological Chemistry, 1995Co-Authors: Rao S. Koduri, Ming TienAbstract:Abstract We have investigated the Lignin Peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of Lignin Peroxidase. The rate constant for the reaction with compound I is 1.2 × 10M s. The reaction of guaiacol with compound II exhibits a K of 64 μM and a first-order rate constant of 17 s. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a K of 160 μM and a k of 7.7 s. Veratryl alcohol, a secondary metabolite of Ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the Lignin Peroxidase reaction.
-
Oxidation of Guaiacol by Lignin Peroxidase
1995Co-Authors: Rao S. Koduri, Ming TienAbstract:We have investigated the Lignin Peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of Lignin Peroxidase. The rate constant for the reaction with compound I is 1.2 3 10 6 M 21 s 21 . The reaction of guaiacol with compound II exhibits a Kd of 64 mM and a first-order rate constant of 17 s 21 . Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with aKmof 160 mMand akcatof 7.7 s 21 . Veratryl alcohol, a secondary metabolite of Ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the Lignin Peroxidase reaction. This study investigates the ability of 3,4-dimethoxybenzyl (veratryl) alcohol to mediate the Lignin Peroxidase-catalyzed oxidation of guaiacol. Lignin Peroxidases are hemeproteins secreted by the white rot fungus Phanerochaete chrysosporium
-
kinetic analysis of Lignin Peroxidase explanation for the mediation phenomenon by veratryl alcohol
Biochemistry, 1994Co-Authors: Rao S. Koduri, Ming TienAbstract:We investigated the role of veratryl alcohol in Lignin Peroxidase-catalyzed oxidation of anisyl alcohol with pre-steady-state and steady-state kinetic methods. Veratryl alcohol has been proposed to act as a redox mediator for substrates that are not directly oxidized by the enzyme. Alternatively, its mediation activity has also been attributed to its ability to protect the enzyme from H 2 O 2 -dependent inactivation. As previously reported, veratryl alcohol was able to stimulate the oxidation of anisyl alcohol. However, this stimulation is not due to mediation or protection of the enzyme. The stimulation can be attributed to the relative reactivity of anisyl alcohol with compounds I and II of Lignin Peroxidase. We found that anisyl alcohol reacts with compound I, but not with compound II
Steven D. Aust - One of the best experts on this subject based on the ideXlab platform.
-
Expression of the Lignin Peroxidase H2 Gene fromPhanerochaete chrysosporiuminEscherichia coli
Biochemical and Biophysical Research Communications, 1998Co-Authors: N. Scott Reading, Steven D. AustAbstract:The DNA sequence for the extracellular Lignin Peroxidase isozyme H2 fromPhanerochaete chrysosporium,obtained from cDNA clone λML-6, was synthesized by PCR and successfully expressed inEscherichia coliunder control of the T7 promoter. The portion of the cDNA encoding the signal peptide, not found in the mature native enzyme, was not included. Recombinated Lignin Peroxidase H2 (rLiPH2) was produced in inclusion bodies in an inactive form. Active enzyme was obtained by refolding with glutathione-mediated oxidation in a medium containing urea, Ca2+, and hemin. The recombinant enzyme had spectral characteristics and kinetic properties identical to that of native enzyme isolated fromP. chrysosporium.Surprisingly, rLiPH2, like the native enzyme, also exhibited some manganese Peroxidase activity.
-
Effect of Calcium on the Reversible Thermal Inactivation of Lignin Peroxidase
Archives of Biochemistry and Biophysics, 1997Co-Authors: Steven D. AustAbstract:Abstract This study investigated the effects of calcium on the thermal inactivation of Lignin Peroxidase from Phanerochaete chrysosporium. The monophasic loss of veratryl alcohol oxidase activity corresponded to the loss of calcium when the enzyme was thermally inactivated. Addition of calcium slowed and oxalate and EGTA increased the apparent inactivation rate. The thermally inactivated Lignin Peroxidase could be readily reactivated by addition of Ca 2+ . The amount of activity recovered was dependent on temperature, Ca 2+ concentration, and incubation conditions. Enzyme activity could be recovered up to 95% of its original value by addition of Ca 2+ when Lignin Peroxidase was depleted of Ca 2+ by incubation with EGTA. Although heme absorbance decreased when the enzyme was thermally inactivated, the amount of iron in the enzyme did not change. Changes in the heme environment of the inactivated enzyme were suggested by changes in the electronic absorption in which the Soret band shifted from 408 to 410 nm, the absorption at 502 nm shifted to 532 nm, and the absorption at 634 nm disappeared upon inactivation. Upon the addition of Ca 2+ , the bands returned to the original wavelength. Therefore, it is proposed that the inactivation mechanism of Lignin Peroxidase is that the loss of calcium causes heme environmental changes resulting in the loss of enzyme activity.
-
Oxidation of 1,2,4,5-tetramethoxybenzene by Lignin Peroxidase of Phanerochaete chrysosporium.
Archives of Biochemistry and Biophysics, 1996Co-Authors: Rao S. Koduri, Steven D. Aust, Ross E. Whitwam, David P. Barr, Ming TienAbstract:Abstract We have reinvestigated the Lignin Peroxidase-catalyzed oxidation of 1,2,4,5-tetramethoxybenzene (TMB) by using presteady-state and steady-state kinetic methods. Our presteady-state kinetic results show that the reaction of compound I with TMB obeyed second order kinetics with a rate constant of 1.1 × 10 7 M −1 s −1 . The reaction of compound II with TMB exhibits a hyperbolic concentration dependence with a K d of 16 μ M and k = 24 s −1 . The stoichiometry of TMB oxidation during steady state is two TMB cation radicals formed per H 2 O 2 consumed. These results clearly show that TMB is a good substrate for both compounds I and II of Lignin Peroxidase.
-
Reductive Dehalogenation of Aliphatic Halocarbons by Lignin Peroxidase of Phanerochaete chrysosporium.
Environmental Science & Technology, 1995Co-Authors: Aditya Khindaria, Thomas A. Grover, Steven D. AustAbstract:Contamination of soils and aquifers by aliphatic halocarbons is a serious environmental pollution problem. We report here the novel observation that the halocarbons trichloroethylene (TCE) and CCl 4 were mineralized by Phanerochaete chrysosporium under aerobic conditions. Ligninolytic cultures of this white rot fungus mineralized 20.3% of 10 ppm TCE and 18.8% of 10 ppm CCl 4 in 9 days. These chemicals were not mineralized by nonLigninolytic cultures of P. chrysosporium, indicating that Lignin Peroxidases play an important role in the mineralization of these chemicals. In a previous study, we reported Lignin Peroxidase-catalyzed reductive dehalogenation of CCl 4 with the resultant formation oftrichloromethyl radical. We have extended this study and report here reductive dehalogenation of CHCl 3 , CH 2 Cl 2 , TCE, and 1,1,1-trichloroethane. Dehalogenation was catalyzed by a reductive reaction system containing Lignin Peroxidase, veratryl alcohol, EDTA or oxalate, H 2 O 2 , and the halocarbon with phenyl N-tert-butylnitrone as a spin trap for electron spin resonance detection of the resulting radicals. Since all the components of the reductive system with oxalate as an electron donor are excreted by P. chrysosporium, we propose that this mechanism may be involved in the degradation of these halocarbons by the fungus.
-
Conversion of Lignin Peroxidase compound III to active enzyme by cation radicals.
Archives of Biochemistry and Biophysics, 1994Co-Authors: David P. Barr, Steven D. AustAbstract:Abstract It has been previously reported that the catalytically inactive compound III form of Lignin Peroxidase is formed during the oxidation of certain chemicals such as phenols (P. J. Harvey and J. M. Palmer, 1990, J. Biotechnol. 13, 169-179). Here we provide evidence that the cation radicals of methoxybenzenes such as 1,2,4,5-tetramethoxybenzene (TMB) and veratryl alcohol promote the oxidative conversion of compound III back to ferric enzyme. Two kinetic phases were observed during the oxidation of TMB by Lignin Peroxidase. In the first phase the formation of TMB cation radical and compound III were observed simultaneously. The second phase involved a rapid disappearance of compound III and the TMB cation radical. Ferric enzyme appeared concomitantly with the disappearance of compound III. The TMB cation radical, generated electrochemically, was able to convert compound III to ferric enzyme. Comparative studies using veratryl alcohol were performed and supported the idea that the cation radical of these chemicals are capable of reactivating compound III. The significance of these reactions with respect to Lignin Peroxidase catalysis are discussed.
Paramjit Kaur Gill - One of the best experts on this subject based on the ideXlab platform.
-
involvement of Lignin Peroxidase manganese Peroxidase and laccase in degradation and selective Ligninolysis of wheat straw
International Biodeterioration & Biodegradation, 2002Co-Authors: Daljit Singh Arora, Mukesh Chander, Paramjit Kaur GillAbstract:Some white-rot fungi have been studied to evaluate their potential to degrade wheat straw with specific reference to their Ligninolytic ability and associated enzymes, viz. Lignin Peroxidase, manganese Peroxidase, and laccase. The data have been compared with the much studied Phanerochaete chrysosporium. In the present study, Daedalea flavida and two of the Phlebia spp. were found to be capable of degrading Lignin selectively and hence hold better prospects in various biotechnological applications than P. chrysosporium. Phlebia radiata and P. floridensis were the best producers of manganese Peroxidase and laccase, respectively, whereas P. chrysosporium was best for Lignin Peroxidase.
-
comparison of two assay procedures for Lignin Peroxidase
Enzyme and Microbial Technology, 2001Co-Authors: Daljit Singh Arora, Paramjit Kaur GillAbstract:The most widely accepted assay for detecting Lignin Peroxidase, based on the oxidation of veratryl alcohol to veratraldehyde, suffers from some drawbacks. At 310 nm, the wavelength at which the assay is performed, some other materials like Lignins, quinonic compounds and aromatics also exhibit strong absorbance thus interfering with the estimation when present in the media. The present study reports the Lignin Peroxidase production by some white rot fungi under different nutritional conditions. The veratryl alcohol oxidation assay procedure for Lignin Peroxidase has been compared with another method based on the oxidation of the dye azure B involving absorbance measurements in the visible range. The latter method proved to be much more advantageous over the veratryl alcohol oxidation method, in media supplemented with malt extract, Lignin preparations and agricultural residues. The enzyme production by veratryl alcohol assay could be detected only in mineral salts broth. By the azure B assay the enzyme activity was detected in all the media tested. The supplements gave varied response in different media. Veratryl alcohol enhanced the enzyme production in malt extract broth and mineral salts malt extract broth. Among the Lignin preparations Indulin AT increased the Lignin Peroxidase titres from 2 to 20 fold in different fungi. Similarly, wheat straw supplemented in mineral salts broth and malt extract broth, separately, strongly stimulated the Lignin Peroxidase production. The above studies revealed that azure B assay may act as a substitute or equivalent method.
Mark A Moen - One of the best experts on this subject based on the ideXlab platform.
-
depolymerization of a synthetic Lignin in vitro by Lignin Peroxidase
Enzyme and Microbial Technology, 1991Co-Authors: Kenneth E Hammel, Mark A MoenAbstract:Abstract Lignin Peroxidases, although believed to catalyze the first step of fungal Ligninolysis in vivo , have never been demonstrated to depolymerize unmodified Lignin in vitro . It is shown here that crude Phanerochaete chrysosporium Lignin Peroxidase, in the presence of H 2 O 2 and veratryl alcohol, will catalyze the partial fragmentation of a 14 C β -labeled synthetic hardwood Lignin in Na acetate (pH 4.5)/ N , N -dimethylformamide, 9:1. Gel permeation chromatography of the treated Lignin demonstrated that fragments with molecular weights as low as ca. 170 were products of this reaction. Determinations of total 14 C showed that 25–30% of the radiolabel originally present was converted to volatile products during enzymatic oxidation. No evidence for depolymerization was found in control reactions from which Lignin Peroxidase or H 2 O 2 had been omitted, and little change in the Lignin was discernible in reactions that lacked veratryl alcohol.
David A Ramirez - One of the best experts on this subject based on the ideXlab platform.
-
effects of different wavelengths of light on Lignin Peroxidase production by the white rot fungi phanerochaete chrysosporium grown in submerged cultures
Bioresource Technology, 2010Co-Authors: David A Ramirez, Sandra V Munoz, Lucia Atehortua, Frederick C MichelAbstract:In this study, the effects of different wavelengths of light (UV, blue, green, yellow, red) and white light on Lignin Peroxidase (LiP), protein, biomass and exo-polysaccharide production and glucose uptake by Phanerochaete chrysosporium BKM-F-1767 were determined. The experiments were conducted under aerated (CS) and oxygenated (RS) culture conditions. The results showed that only green light significantly increased maximum LiP production (by 20% and 27% in CS and RS cultures respectively). Green light also increased biomass production in oxygenated cultures (RS). Blue and UV light both significantly reduced maximum LiP activity. Yellow, red and white lights had mixed effects on culture properties. This is the first time that the effects of different wavelengths of light on Lignin Peroxidase production and other culture properties have been investigated. The novel findings may be important in improving the yield of Lignin modifying enzymes for biomass conversion processes and understanding their regulation.