Vaidyanathan Vinoth Kumar

Magnetic cross-linked laccase aggregates — Bioremediation tool for decolorization of distinct classes of recalcitrant dyes

The increasing use of laccase in waste water industries is useful to explore the high benefit/cost ratio of insolubilization technologies like cross linked enzyme aggregates (CLEAs) for the decolorization and detoxification of distinctive classes of recalcitrant dyes. Amino-functionalized magnetic nanoparticles bonded to CLEAs increased the potential of laccase-based CLEAs and are applicable for commercial implementation of this technology in environmental applications. The activity recovery obtained from the stable rigid structure of magnetic CLEAs was around 32%. High volumetric activity, increased in thermal and operational stability of laccase and its resistance to extreme conditions were the properties provided by these magnetic CLEAs. Kinetic studies show that the catalytic efficiency of the enzyme, based on the kcat/km value, changed significantly upon CLEAs and magnetic CLEA formations. When 0.2U/mL of magnetic CLEAs was used, the biocatalyst rapidly decolorized 61-96% of remazol brilliant blue R, malachite green and reactive black 5 initially at 50mgL(-1) at 20°C and pH7.0. Investigation of dye degradation using both active and heat denatured CLEAs revealed a slight adsorption of dyes on inactivated biocatalysts. A laboratory scale perfusion basket reactor (BR) was used to study the continuous decolorization of dyes. The efficient decolorization (>90%) of remazol brilliant blue R and slight decrease in CLEA activity were measured over a 10h period of continuous operation, which illustrates the potential of CLEAs for the wastewater treatment. The present findings will advance the understanding of dye decolorization mechanism by CLEA laccase, which could provide useful references for developing industrial wastewater treatment.

Preparation and characterization of porous cross linked laccase aggregates for the decolorization of triphenyl methane and reactive dyes.

The production of porous cross-linked enzyme aggregates (p-CLEAs) is a simple and effective methodology for laccase immobilization. A three-phase partitioning technique was applied to co-precipitate laccase and starch, followed by cross-linking with glutaraldehyde and removal of starch by α-amylase to create pores in the CLEAs. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. The half-life of free laccase at 55°C was calculated to be 1.3h, while p-CLEAs did not lose any activity even after 14 h. p-CLEAs also exhibited improved storage stability, catalytic efficiency and could be recycled 15 times with 60% loss of activity. The catalysts decolorized triphenylmethane and reactive dyes by 60-70% at initial dye concentrations of 2 and 0.5 g L(-1), respectively, without any mediators. These results suggest the potential of CLEA technology in waste-water treatment.

Chemometric formulation of bacterial consortium-AVS for improved decolorization of resonance-stabilized and heteropolyaromatic dyes.

A bacterial consortium-AVS, consisting of Pseudomonas desmolyticum NCIM 2112, Kocuria rosea MTCC 1532 and Micrococcus glutamicus NCIM 2168 was formulated chemometrically, using the mixture design matrix based on the design of experiments methodology. The formulated consortium-AVS decolorized acid blue 15 and methylene blue with a higher average decolorization rate, which is more rapid than that of the pure cultures. The UV-vis spectrophotometric, Fourier transform infra red spectrophotometric and high performance liquid chromatographic analysis confirm that the decolorization was due to biodegradation by oxido-reductive enzymes, produced by the consortium-AVS. The toxicological assessment of plant growth parameters and the chlorophyll pigment concentrations of Phaseolus mungo and Triticum aestivum seedlings revealed the reduced toxic nature of the biodegraded products.

Towards high potential magnetic biocatalysts for on-demand elimination of pharmaceuticals

The present study investigated the applicability of a laccase based bioprocess for the treatment of a mixture containing 13 selected pharmaceuticals. To do so, laccase was immobilized as cross-linked enzyme aggregates (MAC-CLEAs) on amine functionalized magnetic nanoparticles using chitosan/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) as the cross-linking system. The activity recovery of laccase reached 61.4% under the optimal conditions of MAC-CLEAs formation. The latter exhibited enhanced storage stability over one year at 4°C and showed better temperature resistance compared to its soluble counterpart. The biocatalysts were properly recycled and the catalytic activity recovery was good even after a hundred and fifty batch reactions. Complete removal of pharmaceuticals like acetaminophen, diclofenac, mefenamic acid, atenolol and epoxy carbamazepine and partial removal of fenofibrate, diazepam, trimethoprim, and ketoprofen by laccase was achieved within 12h of incubation, whereas efficient removal of indometacin required the presence of mediator.

Application of Response Surface Methodology to Optimize Three Phase Partitioning for Purification of Laccase from Pleurotus ostreatus

We used a novel approach to purify Pleurotus ostreatus laccase using the three phase partitioning (TPP) methodology. The aim of our research was to study the effect of TPP parameters on laccase purity and yield. The response surface methodology (RSM) has been applied to design the experiments to evaluate the interactive effects of the three most important operating variables: ammonium sulphate saturation (w/v) (20–80%), ratio of crude extract to t-butanol (v/v) (1:1 to 1:3), and temperature (20–60°C). Using this methodology, the optimum values for the critical components were obtained as follows: ammonium sulphate saturation (w/v), 50–60%; ratio of crude extract to t-butanol (v/v) 1.0:1.8; temperature 42–45°C, respectively. Under optimal conditions, the experimental laccase yield and purity was 184% and 7.22-fold, respectively. SDS-PAGE and RP-HPLC revealed that the laccase was purified by TPP. The determination coefficients (R2) were 0.9891 and 0.9728 for laccase purity and yield, respectively, indicating an adequate degree of reliability in the model. To our knowledge, the present work demonstrates for the first time the successful application of RSM to TPP.

Recent developments in the use of tyrosinase and laccase in environmental applications

Abstract Our current global environmental challenges include the reduction of harmful chemicals and their derivatives. Bioremediation has been a key strategy to control the massive presence of chemicals in the environment. Enzymes including the phenoloxidases, laccases and tyrosinases, are increasingly being investigated as “green products” in the removal of many chemical contaminants in waters and soils. Both phenoloxidases are widespread in nature and attractive biocatalysts due to their ability to use readily available molecular oxygen as sole cofactor for their catalytic elimination of a large number of chemicals. Taking advantage of their catalytic potentials, remarkable advances have been made in the engineering of laccases to produce suitable biocatalysts in environmental applications. Studies about novel strategies of laccase immobilization and insolubilization for the treatment of chemical contaminants were provided. Likewise, tyrosinases are gaining increasing interest in environmental applications due to their catalytic similarities with laccases although they remain far less investigated to date. This disparity was addressed in this review along with the molecular features and catalytic mechanism of tyrosinases relevant in environmental applications. A perspective on the future use of laccases and tyrosinases in bioremediation was discussed.

Kinetics, mechanism, isotherm and thermodynamic analysis of adsorption of cadmium ions by surface-modified Strychnos potatorum seeds

The surface-modified Strychnos potatorum seeds (SMSP) were used as an effective low-cost adsorbent for the removal of cadmium ions from aqueous solution. SMSP was characterized by Fourier transform infrared spectroscopy and scanning electron microscopic analyses. The effect of operating variables such as solution pH, adsorbent dose, contact time, initial cadmium ions concentration and temperature on the removal of cadmium ions were studied in a batch mode adsorption operation. The optimum conditions for the adsorption of cadmium ions onto the SMSP were found to be: pH of 5.0, SMSP dose of 2 g/L, contact time of 30min, temperature of 30 °C for an initial cadmium ions concentration of 100 mg/L. Kinetic data were analyzed using the pseudo-first order and pseudo-second order kinetic equations, intraparticle diffusion model, Boyd kinetic model and shrinking core model. The characteristic parameters for each model have been estimated. Adsorption of cadmium ions onto the SMSP follows the pseudo-second order kinetic model. The rate-limiting steps in the adsorption process were found to be external and internal diffusion. Equilibrium data were well described by the Langmuir isotherm model than the Freundlich isotherm model, which yields a maximum monolayer adsorption capacity of 200 mg/g. Thermodynamic parameters such as standard free energy change, enthalpy change and entropy change were also estimated. The results show that the removal of cadmium ions by the SMSP was found to be spontaneous and exothermic.

Aspergillus niger exo‐inulinase purification by three phase partitioning

Inulinase (2, 1‐β‐D‐fructan fructanohydrolase, EC 3.2.1.7) hydrolyses inulin into nearly pure fructose, which is an excellent alternative for the production of fructose syrup. Growing inulinase utilization in different industries encourages the search for high benefit/cost ratio purification techniques for such enzymes. Here, we adapted the three‐phase partitioning (TPP) technique for the downstream process of inulinase obtained from Aspergillus niger. TPP is a simple non‐chromatographic process used for purification and concentration of protein. The various conditions required for attaining efficient purification of inulinase were optimized. The optimum conditions for TPP were found to be 30% w/v ammonium sulfate saturation with 1.0 : 0.5 v/v ratio of t‐butanol to crude extract at pH 4.0 and temperature 25°C. The enzyme was purified by 10.2‐fold using two‐step TPP with an overall recovery of 88%. The enzymes molecular mass was found to be 63.8 kDa by SDS‐PAGE analysis. Terminal hydrolysis fructose units from the inulin show that enzymes are exo‐inulinase. The recovery of purified exo‐inulinase achieved in this work shows the technical viability of enzyme purification by TPP.

Xenobiotic Compounds Degradation by Heterologous Expression of a Trametes sanguineus Laccase in Trichoderma atroviride

Fungal laccases are enzymes that have been studied because of their ability to decolorize and detoxify effluents; they are also used in paper bleaching, synthesis of polymers, bioremediation, etc. In this work we were able to express a laccase from Trametes (Pycnoporus) sanguineus in the filamentous fungus Trichoderma atroviride. For this purpose, a transformation vector was designed to integrate the gene of interest in an intergenic locus near the blu17 terminator region. Although monosporic selection was still necessary, stable integration at the desired locus was achieved. The native signal peptide from T. sanguineus laccase was successful to secrete the recombinant protein into the culture medium. The purified, heterologously expressed laccase maintained similar properties to those observed in the native enzyme (Km and kcat and kcat/km values for ABTS, thermostability, substrate range, pH optimum, etc). To determine the bioremediation potential of this modified strain, the laccase-overexpressing Trichoderma strain was used to remove xenobiotic compounds. Phenolic compounds present in industrial wastewater and bisphenol A (an endocrine disruptor) from the culture medium were more efficiently removed by this modified strain than with the wild type. In addition, the heterologously expressed laccase was able to decolorize different dyes as well as remove benzo[α]pyrene and phenanthrene in vitro, showing its potential for xenobiotic compound degradation.

Indigenously acclimatized bacterial consortium for anthracene biotransformation

ABSTRACT A bacterial consortium was screened from the soil exposed to frequent petroleum spills and was employed to degrade and detoxify anthracene at normal operating parameters under static condition. The anthracene degradation was facilitated by the bacterial cells mediated metabolism and biodegradation was confirmed by the different spectral and chromatographic techniques. The distinct products of biotransformation were due to the enzymatic activity of the bacterial consortium.