Philippe Demarche

Combined cross-linked enzyme aggregates from versatile peroxidase and glucose oxidase: Production, partial characterization and application for the elimination of endocrine disruptors

Versatile peroxidase (VP) from Bjerkandera adusta was insolubilized in the form of cross-linked enzyme aggregates (CLEA®s). Of the initially applied activity 67% was recovered as CLEA®s. Co-aggregation of VP with glucose oxidase from Aspergillus niger led to an increased activity recovery of 89%. The combined CLEA®s showed higher stability against H(2)O(2) and exerted VP activity upon glucose addition. The elimination of the endocrine disrupting chemicals bisphenol A, nonylphenol, triclosan, 17α-ethinylestradiol and the hormone 17β-estradiol (10 mg L(-1) each) and the removal of their estrogenic activity by combined CLEA®s were tested in batch experiments. Within 10 min, the combined CLEA®s were able to remove all the endocrine disruptors except triclosan (residual concentration 74%). The removal of the estrogenic activity was higher than 55% for all compounds, except triclosan. A membrane reactor continuously operated with combined CLEA®s could almost completely remove bisphenol A (10 mg L(-1)) for 43 h.

Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater

Over the past decades, water pollution by trace organic compounds (ng L(-1)) has become one of the key environmental issues for developed countries. To date there is no effective and sustainable remediation strategy available. Laccases from white rot fungi were found particularly attractive for the removal of some micropollutants such as the plasticizer bisphenol A (BPA), the anti-inflammatory drug diclofenac (DF) and the steroidal hormone 17-α-ethinylestradiol (EE2). Laccase immobilization is a prerequisite for their use in continuous water treatment processes. In this study, laccase from Coriolopsis gallica was immobilized on mesoporous silica spheres in a two-step adsorption-crosslinking process. The initial laccase activity, crosslinker (glutaraldehyde) concentration and extra protein (albumin) concentration were varied following a central composite experimental design and optimized with respect to the immobilization yield, activity and thermal stability of the biocatalysts. After a multi-objective optimization of the biocatalyst formulation, a maximum biocatalyst activity of 383 Ug(-1), determined with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) at pH 4.5, was obtained. Biocatalyst particles were physically characterized by means of scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses revealing few modifications of the surface area and structure during/after the immobilization procedure. The biocatalyst showed a significantly higher thermostability than the free enzyme with a half-life of 31.5 hours and 3.9 hours compared to 6.1 hours and 0.6 hours at 55°C and 75°C respectively. The biocatalyst was able to eliminate in a continuously stirred membrane reactor more than 95% of BPA 10 μM and EE2 10 μM and 70% of DF 10 μM when treated individually and more than 90% when treated as a mixture in aqueous buffered solution (pH 5) for more than 60 reactor volumes. In real wastewater conditions (pH 7.8) the biocatalyst could degrade more than 85% of BPA and EE2 along with 30% of DF when tested in mixture for more than 80 hours, which illustrates the potential of this biocatalyst for the treatment of aquatic micropollutants.

Design-of-experiment strategy for the formulation of laccase biocatalysts and their application to degrade bisphenol A

Immobilizing enzymes can expand their applicability to continuous process operations and facilitates process intensification. An optimized formulation of immobilized biocatalysts is therefore of strategic interest in the field of industrial biotechnology. Nevertheless, biocatalyst formulation still largely relies on empirical approaches which lack effectiveness in the identification of optimum immobilization conditions. In the present study, design of experiments, multiple linear regressions and modeling were used to screen, interpret and finally optimize crucial immobilization parameters. A laccase preparation from Coriolopsis polyzona MUCL38443 was immobilized via a sequential adsorption-crosslinking process on mesoporous silica particles. As a target variable, biocatalyst activity was doubled (∼280 U g(-1)) while dramatically reducing processing time (two hours instead of 26 hours) and reagent inputs (80 mm instead of 1m glutaraldehyde (GLU)). Immobilization yield (∼50%) and thermostability (∼60% residual activity after 24 hours at 45°C) could be maintained under the optimized conditions. As an example of its application in environmental biotechnology, the optimized biocatalyst was implemented in a continuous stirred-tank membrane reactor (CSTMR) to continuously degrade the endocrine disruptor bisphenol A (BPA) in wastewater. A 90% removal of 50 μm BPA was achieved over 30 reactor volumes (hydraulic residence time (HRT) of 1.85 hours, 50 mL working volume).

Stimulation of laccases from Trametes pubescens: Use in dye decolorization and cotton bleaching

ABSTRACT The production of laccases from Trametes pubescens was investigated along with the role of nutrients and elicitors. Copper proved to be a fundamental inducer, although productivity yields were consistently enhanced only in the presence of additional compounds (textile dyes). Using a central composite design, the optimal culture condition was examined, by taking into consideration the three distinct variables and their combinatorial effect. The 290u2009Uu2009ml−1 of laccases were produced after setting nitrogen, copper, and reactive blue 19 concentration; in a bioreactor, activity recovery was lower (90u2009Uu2009ml−1) and pellet morphology was different. The activity of the laccase crude extract was maximal at 60°C and stable for 14u2009h at 50°C and for 2 months at pH 6 and room temperature. The biotechnological potential was assessed, confirming the capacity to decolorize single or mixed solutions of textile dyes and to enhance the whitening yield of raw cotton fibers, working in synergism with the conventional H2O2-based method.

Dynamic measurement of oxidase activity based on oxygen consumption in open systems

Oxidases catalyze the oxidation of a variety of substrates with the concomitant reduction of molecular oxygen as a final electron acceptor. UV‐visible spectrophotometry is a simple and high‐throughput method commonly used to measure oxidase activities. However, drawbacks such as light scattering exist especially concerning the activity assessment of enzymes immobilized on supports. Monitoring of the universal cosubstrate O2 circumvents these drawbacks. This study aimed at developing a methodology that allows activity measurement of many types of oxidases based on O2 consumption applicable to various open systems. Dissolved oxygen in the reaction medium was monitored by an O2 sensor and the reaction rate was deduced from the O2 mass balance equation correcting for atmospheric diffusion. Common activity units (μMproduct min−1 or U/L) could be subsequently derived using calibration curves. The sensitivity of the method toward temperature, atmospheric pressure, and ionic strength variations was evaluated, and made it possible to define operating windows for the simplification of the proposed methodology.