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The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O 2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency ( k cat / K m ) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-β-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.

Objective: The dye-decolorizing peroxidase from Bacillus amyloliquefaciens, BaDyP, was identified to be an efficient catalyst for the degradation of phenolic β-ether lignin model dimer guaiacylglycerol-β-guaiacyl ether (GGE) and dyes., Results: Efeb gene encoding BaDyP from B. amyloliquefaciens MN-13 consisted of 1257 bp and the open reading frame encoded 418 amino acids. The efeb gene was expressed in Escherichia coli BL21 and a recombinant BaDyP of 50 kDa was achieved. The BaDyP exhibited activity in oxidizing GGE and decolorizing azo and triphenylmethane dyes. At pH 4.5 and 30 °C the BaDyP not only completely degraded GGE by the cleavage of β-O-4 ether bond and Cα-C β bond, and Cα oxidation without any oxidative mediator, but also decolorized four synthetic dyes, including congo red, bromine cresol green, eriochrome black T and crystal violet. This was achieved with decolorization rates of 65.7%, 70.62%, 80.06% and 62.09%, respectively, after 72 h of incubation., Conclusions: BaDyP was identified as a bacteria peroxidase with great potential for the degradation of lignin and bioremediation of dye-contamination.

Two 8-O-4'-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC 50  = 45.56 μM) and 2b (IC 50  = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC 50  = 82.66 μM) and 2a (IC 50  = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: MyoNovin is a novel skeletal muscle-regenerating compound developed through synthesis of two nitro groups onto a guaifenesin backbone to deliver nitric oxide to skeletal muscle with a potential to treat muscle atrophy. The purpose of this study was to utilize in silico, in vitro, and in vivo approaches to characterize MyoNovin and examine its safety, biodistribution, and feasibility for drug delivery., Methods: In silico software packages were used to predict the physicochemical and biopharmaceutical properties of MyoNovin. In vitro cardiotoxicity was assessed using human cardiomyocytes (RL-14) while effects on CYP3A4 metabolic enzyme and antioxidant activity were examined using commercial kits. A novel HPLC assay was developed to measure MyoNovin concentration in serum, and delineate initial pharmacokinetic and acute toxicity after intravenous administration (20 mg/kg) to male Sprague-Dawley rats., Results: MyoNovin showed relatively high lipophilicity with a LogP value of 3.49, a 20-fold higher skin permeability (19.89 cm/s*107) compared to guaifenesin (0.66 cm/s*107), and ~10-fold higher effective jejunal permeability (2.24 cm/s*104) compared to guaifenesin (0.26 cm/s*104). In vitro, MyoNovinwas not cytotoxic to cardiomyocytes at concentrations below 8 μM and did not inhibit CYP3A4 or show antioxidant activity. In vivo, MyoNovin had a short half-life (t1/2) of 0.16 h, and a volume of distribution Vss of 0.62 L/kg. Biomarkers of MyoNovincardiac and renal toxicity did not differ significantly from baseline control levels., Conclusions: The predicted high lipophilicity and skin permeability of MyoNovin render it a potential candidate for transdermal administration while its favourable intestinal permeation suggests it may be suitable for oral administration. Pharmacokinetics following IV administration of MyoNovin were delineated for the first time in a rat model. Preliminary single 20 mg/kg dose assessment of MyoNovin suggest no influenceon cardiac troponin or β-N-Acetylglucosaminidase. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-β-guaiacyl ether (GGE), which contains a key β-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities., (Copyright © 2015, Palamuru et al.)

Lignin, an aromatic polymer of phenylpropane units joined predominantly by β-O-4 linkages, is the second most abundant biomass component on Earth. Despite the continuous discharge of terrestrially produced lignin into marine environments, few studies have examined lignin degradation by marine microorganisms. Here, we screened marine isolates for β-O-4 cleavage activity and determined the genes responsible for this enzymatic activity in one positive isolate. Novosphingobium sp. strain MBES04 converted all four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGGE), a structural mimic of lignin, to guaiacylhydroxypropanone as an end metabolite in three steps involving six enzymes, including a newly identified Nu-class glutathione-S-transferase (GST). In silico searches of the strain MBES04 genome revealed that four GGGE-metabolizing GST genes were arranged in a cluster. Transcriptome analysis demonstrated that the lignin model compounds GGGE and (2-methoxyphenoxy)hydroxypropiovanillone (MPHPV) enhanced the expression of genes in involved in energy metabolism, including aromatic-monomer assimilation, and evoked defense responses typically expressed upon exposure to toxic compounds. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of lignin-containing materials in marine environments.

This paper reported the modulation of enzyme activity by organic small molecule. The esterification activity of Penicillin G acylase (PGA) was improved more than 70-fold by the addition of 10% N-methylimidazole. Some control experiments have been designed to demonstrate the catalytic specificity of PGA. The structure and the amount of additive were optimized to improve the product yield. The influence of N-methylimidazole on the PGA conformation was investigated by FTIR and autodock simulation. Seven substrates were used to evaluate the effect of structure on the PGA-catalyzed transesterification. A series of products were successfully synthesized with the yield ranged from 56% to 84% and PGA showed specific recognition on the substrate with phenyl group in the presence of 10% N-methylimidazole., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors + P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD.

Mixtures of equal amounts of the erythro and threo forms of the phenolic arylglycerol beta-aryl ether 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol were oxidized (i) with laccases from Trametes versicolor, Agaricus bisporus, Myceliophthora thermophila and Rhus vernicifera, (ii) with laccase-mediator systems consisting of T. versicolor laccase and ABTS or HBT, and (iii) with various model oxidants including cerium(IV) ammonium nitrate (CAN), lignin peroxidase, Fenton's reagent, and lead(IV) tetraacetate (LTA). All the laccases exhibited a similar preferential degradation of the threo form. The mediator ABTS counteracted the threo preference of laccase, but the mediator HBT did not affect it. The outer-sphere model oxidants CAN and lignin peroxidase showed a preferential degradation of the threo form. LTA and Fenton's reagent did not exhibit any stereo-preference. The results suggest that laccases of different origin, primary structure, and redox potential behave as typical outer-sphere oxidants in their interaction with the diastereomers of the arylglycerol beta-aryl ether.

Cleavage of the arylglycerol beta-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial beta-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the beta-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The beta-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The beta-aryl ether cleavage enzyme converts the guaiacylglycerol beta-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the C alpha alcohol structure and p-hydroxyl group and specifically attacks the beta-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the C alpha and C beta positions.

The catalytic cycle of lignin peroxidase (LiP, ligninase) isozyme L3 from the white-rot fungus Phlebia radiata was investigated using stopped-flow techniques. Veratryl (3,4-dimethoxybenzyl) alcohol and a lignin model compound, non-phenolic beta-O-4 dimer 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol, were used as electron donors. This is the first report on the detailed kinetic analysis of a LiP-catalysed C alpha-C beta bond cleavage of the dimer, representing the major depolymerisation reaction in the lignin polymer. The native enzyme showed a typical heme peroxidase absorbance spectrum with a Soret maximum at 407 nm. Following the reaction with H2O2, the Soret band decreased in absorbance, shifted to 403 nm and then to 421 nm, demonstrating the formation of compound I followed by the formation of compound II, respectively. Similar results have been reported for the LiP from Phanerochaete chrysosporium upon reaction with H2O2. However, compound I of L3 was more stable in the absence of additional electron donors. The second-order rate constant of compound I formation by H2O2 was determined to be 6 x 10(5) M-1 s-1 and was the same at pH 3.0 and 6.0. Compound I was rapidly reduced to compound II and further to native enzyme when either veratryl alcohol or the beta-O-4 dimer was supplied as electron donor and in both cases veratraldehyde appeared as the major product. At pH 6.0, the second-order rate constant for compound II formation was similar with either veratryl alcohol or the beta-O-4 dimer (6.7 x 10(3) and 6.5 x 10(3) M-1 s-1, respectively). At pH 3.0 formation of compound II with either reductant proceeded so rapidly that determination of the respective rate constants was not possible. The results point to identical catalytic cycles of L3 with veratryl alcohol or the beta-O-4 dimer involving both compounds I and II as intermediates and participation of the same veratryl alcohol radical as the most appropriate reductant for compound II. Chemical evidence of such a radical, formed after the initial LiP-catalysed one-electron oxidation of beta-O-4 dimeric lignin models, is presented in a separate article [Lundell, T., Schoemaker, H., Hatakka, A. & Brunow, G. (1993) Holzforschung, in the press]. The catalytic redox-cycle and the oxidation mechanism presented here reconcile seemingly contradictory results obtained in previous studies on LiP kinetics during the last decade.

The effect of manganese on lignin peroxidase activity was studied. The enzyme was produced with a new process using an air-lift-type reactor. The experiments were performed with veratryl alcohol and a dimeric lignin model compound. It was shown that when Mn(II).lactate complex was present the amount of veratraldehyde formed and the uptake of oxygen were significantly enhanced during the aerobic oxidation of veratryl alcohol. A similar effect can be obtained with superoxide dismutase. These results strongly suggest that the superoxide anion can occur during the reaction; its scavenging by Mn(II) or superoxide dismutase generates H2O2. In contrast, no evidence for the formation of superoxide anion was found during oxidation of the lignin model, compound veratrylglycerol-beta-guaiacyl ether.

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.