Design and Engineering of an Efficient Peroxidase Using Myoglobin for Dye Decolorization and Lignin Bioconversion

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 O2 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 (kcat/Km) 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.

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Fe3O4@SiO2-L-Cu(II): An efficient and easily recoverable catalyst for reducing/degrading environmental pollutants in aqueous medium

10.21203/rs.3.rs-611020/v1 ◽

2021 ◽

Author(s):

Jaber Dadashi ◽

Mohammad Khaleghian ◽

Babak Mir Tamizdoust

Keyword(s):

High Efficiency ◽

Fourier Transforms ◽

Organic Dyes ◽

Aqueous Media ◽

Environmental Pollutants ◽

Catalytic Efficiency ◽

Significant Loss ◽

Homogeneous Catalyst ◽

X Ray ◽

Recoverable Catalyst

Abstract In the present study, a magnetically recoverable catalyst is synthesized through immobilizing copper (II) over the Fe3O4@SiO2 nanoparticles (NPs) surface [Fe3O4@SiO2-L-Cu (II)] (L = pyridine-4-carbaldehyde thiosemicarbazide). Accordingly, synthesized catalysts were determined and characterized by energy dispersive X-ray spectrometry (EDS), Fourier transforms infrared spectroscopy (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM), and thermogravimetric-differential thermal analysis (TG-DTA) procedures. Using [Fe3O4@SiO2-L-Cu (II)], 4-Nitrophenol (4-NP), Cr(VI), and organic dyes, e.g., Methylene blue (MB) and Congo Red (CR) were reduced in aqueous media. As shown by results of Catalytic performance investigations, [Fe3O4@SiO2-L-Cu (II)] performance was outstanding concerning reduction reactions in mild conditions. Remarkable attributes of this method are high efficiency, removal of a homogeneous catalyst, easy recovery from the reaction mix, and uncomplicated route. The amount of activity in this catalytic system was almost constant after several stages of recovery and reuse. The results show that the catalyst was efficient and easily recoverable in the successive run without a significant loss of catalytic efficiency.

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pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

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The Sustainable Production of a Novel Laccase from Wheat Bran by Bordetella sp. JWO16: Toward a Total Environment

Catalysts ◽

10.3390/catal11060677 ◽

2021 ◽

Vol 11 (6) ◽

pp. 677

Author(s):

John Onolame Unuofin

Keyword(s):

Wheat Bran ◽

Contaminated Soils ◽

Residual Activity ◽

Dye Decolorization ◽

Fermentation Medium ◽

Biochemical Properties ◽

Synthetic Dyes ◽

High Concentrations ◽

Capital Intensive ◽

Agroindustrial Residue

Laccase is increasingly adopted in diverse industrial and environmental applications, due to its readily accessible requirements for efficient catalytic synthesis and biotransformation of chemicals. However, it is perceived that its industrial production might incur some unfavorable overhead, which leads to expensive market products, and the corresponding negative environmental feedback, due to the use of capital-intensive and precarious chemicals. To this end, this study was designed to evaluate the performance indicators of the valorization of wheat bran by a novel Jb1b laccase and its subsequent application in waste minimization and water management, on a laboratory scale. Optimal Jb1b laccase was produced in submerged fermentation medium containing wheat bran, an agroindustrial residue, through response surface methodology (RSM) algorithm, and was applied in dye decolorization and denim bioscouring, respectively. Results showed that the resultant enzyme manifested unique biochemical properties, such as enhanced tolerance at certain physicochemical conditions, with a residual activity of at least ca. 76%. Furthermore, phenomenally high concentrations of synthetic dyes (0.2% w v−1) were decolorized over 56 h, and a 6 h mediator-supported simultaneous denim bleaching and decolorization of wash effluent was observed. The sustainability of the production and application processes were inferred from the reusability of the fermentation sludge as a potential biofertilizer, with subsequent prospects for the biostimulation and bioaugmentation of contaminated soils, whereas the decolorized water could be adopted for other uses, amongst which horticulture and forestry are typical examples. These phenomena therefore authenticate the favorable environmental feedbacks and overhead realized in this present study.

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Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

Bioresources and Bioprocessing ◽

10.1186/s40643-019-0267-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Hui-Hui Su ◽

Fei Peng ◽

Pei Xu ◽

Xiao-Ling Wu ◽

Min-Hua Zong ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Rational Design ◽

Glucuronic Acid ◽

Specific Activity ◽

Value Added ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glucaric Acid ◽

Triple Mutant ◽

Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

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Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

Scientific Reports ◽

10.1038/s41598-021-96524-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nur Suhanawati Ashaari ◽

Mohd Hairul Ab. Rahim ◽

Suriana Sabri ◽

Kok Song Lai ◽

Adelene Ai-Lian Song ◽

...

Keyword(s):

Active Site ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Homology Model ◽

Biochemical Properties ◽

Terpene Synthase ◽

Monoterpene Synthase ◽

Terminal Domain ◽

Catalytic Active Site ◽

Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

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α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

Biochemical Journal ◽

10.1042/bj3600727 ◽

2001 ◽

Vol 360 (3) ◽

pp. 727-736 ◽

Cited By ~ 9

Author(s):

Bernd NIDETZKY ◽

Christian EIS

Keyword(s):

Steady State ◽

Transition State ◽

Schizophyllum Commune ◽

Catalytic Efficiency ◽

Kinetic Studies ◽

Competitive Inhibitor ◽

Chemical Mechanism ◽

Steady State Kinetic ◽

State Kinetic ◽

Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

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Synthesis of amine-epoxy based polymers and their potential application in the remediation of selected organic dyes from synthetic effluents

10.51415/10321/2655 ◽

2017 ◽

Author(s):

◽

Sharista Raghunath

Keyword(s):

Electron Microscopy ◽

Catalytic Activity ◽

Ir Spectroscopy ◽

1H Nmr Spectroscopy ◽

Organic Dyes ◽

Synthetic Dyes ◽

X Ray ◽

Dye Reduction ◽

Ft Ir ◽

Ft Ir Spectroscopy

The presence of dyes in effluent poses various environmental as well as health hazards for many organisms. Although various remediation strategies have been implemented to reduce their effect, dyes still manage to infiltrate into the environment and hence new strategies are required to address some of the problems. This study investigated the innovation of two cationic water-soluble polymers viz., Proline-Epichlorohydrin-Ethylenediamine Polymer (PEP) and Thiazolidine-Epichlorohydrin-Ethylenediamine Polymer (TEP) that were used to remediate selected synthetic dyes from synthetic effluent by adsorption and dye reduction. Both polymers were synthesized using monomers of a secondary amine, epichlorohydrin and ethylenediamine and were subsequently characterized and modified and their remediation potential studied. In the first study, PEP was synthesized and characterized by 1H-NMR Spectroscopy, FT-IR Spectroscopy, dynamic light scattering, and thermogravimetric analysis (TGA). Thereafter PEP was modified with bentonite clay, by simple mixing of the reactants, to form a Proline-Epichlorohydrin-Ethylenediamine Polymer-bentonite composite (PRO-BEN); it was characterized by FT-IR Spectroscopy, scanning electron microscopy (SEM)/ energy dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Adsorption studies were then undertaken with a synthetic effluent containing three textile dyes, viz., Reactive Blue 222 (RB 222), Reactive Red 195 (RR 195) and Reactive Yellow (RY 145). Various conditions were investigated including pH of the solution, temperature, sodium chloride concentration, initial dye concentration and the dosage of adsorbent used. The experimental data for all dyes followed a Langmuir isotherm. The adsorption process was found to be pseudo-second order. According to the thermodynamic parameters, the adsorption of the dyes was classified as physisorption and the reaction was spontaneous and exothermic. The data were also compared using studies with alumina as an adsorbent. Results showed that PRO-BEN exhibited better absorptivity and desorption than alumina making its use a better recyclable remediation strategy for the removal of organic dyes in wastewater treatment plants. In the second study, TEP was synthesized and then characterized by FT-IR Spectroscopy, 1H-NMR Spectroscopy, TGA and DLS. Thereafter, TEP was used to prepare TEP capped gold nanoparticles (TEP-AuNPs). Herein, two methods were investigated: the Turkevich method and an adaptation of the Turkevich method using bagasse extract. The TEP-AuNPs was characterized by FT-IR Spectroscopy, SEM, EDX, DLS and TEM. Thereafter the reduction of each of Allura Red, Congo Red and Methylene Blue was investigated with the TEP-AuNPs for its catalytic activity toward dye reduction. This study showed that the batch of AuNPs prepared by the Turkevich method had higher rates of dye reduction compared with AuNPs prepared using bagasse extract. Also the quantity of TEP used as capping agent greatly influenced the size, shape and surface charge of the nanoparticles as well as their catalytic performance: the Vroman effect explained this behavior of the TEP-AuNPs. It was finally concluded that whilst PRO-BEN, in the first study, showed excellent dye remediation properties, the second study on TEP-AuNPs showed good catalytic activity for the reduction of selected dyes, however, it was more effective at lower polymer concentration. Finally, both materials displayed good potential for the clean-up of selected synthetic dyes from synthetic effluents.

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Organic Dyes Adsorption on the Almond Shell (Prunus dulcis) as Agricultural Solid Waste from Aqueous Solution in Single and Binary Mixture Systems

Biointerface Research in Applied Chemistry ◽

10.33263/briac122.20222040 ◽

2021 ◽

Vol 12 (2) ◽

pp. 2022-2040

Keyword(s):

Aqueous Solution ◽

Binary Mixture ◽

Contact Time ◽

Binary Systems ◽

Organic Dyes ◽

Low Cost ◽

Kinetic Studies ◽

Prunus Dulcis ◽

Order Kinetic ◽

Almond Shell

Almond shell (AS) is a low-cost adsorbent used in this study for the removal of methylene blue (MB), crystal violet (CV), and Congo red (CR) from an aqueous solution in single and mixture binary systems. The low-cost adsorbent was characterized by FTIR and SEM analysis. The effects of AS dose, contact time, initial dye concentration, pH, and temperature on MB, CV, and CR adsorption were studied in a single system. In a binary system, the MB, CV, and CR were removed from the mixture of MB+CR, CV+MB, and CV+CR with a percentage in volume ranging from 0 to 100 % in MB and CV, and CR. Kinetic studies showed rapid sorption following a second-order kinetic model with of contact time of 10 min. The modulation of adsorption isotherms showed that retention follows the Langmuir model. The thermodynamic parameters proved that the MB, CV, and CR adsorption process was feasible, spontaneous, and exothermic. The synergy adsorption between dyes in a binary mixture of MB+CR and CV+CR, while the competition adsorption between dyes in a binary mixture of MB+ CV.

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Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications

10.21203/rs.3.rs-19704/v1 ◽

2020 ◽

Author(s):

Wissal BEN ALI ◽

Delphine Chaduli ◽

David Navarro ◽

Christian Lechat ◽

Annick Turbé-Doan ◽

...

Keyword(s):

Culture Supernatant ◽

Cost Effective ◽

Optimal Growth ◽

Dye Decolorization ◽

Growth Conditions ◽

Active Cell ◽

Oxidative Enzymes ◽

Synthetic Dyes ◽

Biotechnological Applications ◽

Fungal Strains

Abstract Background : Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost–effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization.Results : Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum , Stemphylium lucomagnoense and Aspergillus nidulans . Among these five isolates, one T. asperellum strain ( T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator.Conclusions : The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO 4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

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Service sector (services) key features of Russia and Azerbaijan

RUDN Journal of Economics ◽

10.22363/2313-2329-2019-27-1-18-34 ◽

2019 ◽

Vol 27 (1) ◽

pp. 18-34

Author(s):

Mikhail E. Savlov

Keyword(s):

Service Sector ◽

Value Added ◽

Developed Countries ◽

Structural Features ◽

Heterogeneous Structure ◽

General Description ◽

Material Production ◽

Brics Countries ◽

Macroeconomic Indicators ◽

Per Capita

The presented article gives a general description of the sphere of non-material production of Russia and Azerbaijan, which is based on the key macroeconomic indicators. The internal heterogeneous structure of the sector is also illustrated, structural features of the service sector and its individual segments in both countries are revealed. The objectivity and adequacy of the sphere characteristics of non-material production of Russia and Azerbaijan is based on intercountry comparisons. Macroeconomic indicators of main developed countries (the USA, Japan, Germany, France, the United Kingdom), the BRICS countries and the former Soviet republics serve as a background for the illustration of the service sectors of Russia and Azerbaijan. It is too early to refer Russia and Azerbaijan to post-industrial countries, even considering only one economic parameter - the structure of the economy. Considering the gross value added (GVA) of the service sector per capita in current prices and the GVA of the service sector per capita in constant 2010 prices, Russia and Azerbaijan lag behind the leading economies of the world, some BRICS countries and even some former republics of USSR. In this regard, the study of the sphere of intangible production is not so popular in Russia and Azerbaijan, as the economic background for the actualization of these studies has not been yet created.

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