George Songulashvili

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants.

The white-rot fungus Cerrena unicolor C-139 produced 450 000 U l(-1) of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K(m) value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.

Production of a high level of laccase by submerged fermentation at 120-L scale of Cerrena unicolor C-139 grown on wheat bran

Submerged fermentation in a stirred bioreactor of the white rot fungus Cerrena unicolor C-139 was done at a 120-L scale in the presence of wheat bran as a cheap lignocellulosic substrate for fungus growth and laccase production. Enzyme monitoring showed that laccase production started after 2 days of cultivation, attaining a maximum activity of 416.4 U·mL(-1) at day 12 of fermentation. After treatment of culture liquid by successive micro- and ultrafiltration (5kDa), a liquid concentrate containing 22203176 units of laccase was obtained. Obtaining large amount of laccase is essential for various industrial applications, including detoxification of industrial effluents, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated area, stabilization of beverages, production of cosmetics, manufacture of anti-cancer drugs, and nanobiotechnology. The cultivation method and the fungal strain used here provided a substantial amount of enzyme produced at a price lower than 0.01 € cent/unit enzyme.

The potential of Cerrena unicolor laccase immobilized on mesoporous silica beads for removal of organic micropollutants in wastewaters

AbstractMicropollutants (MPs) can be defined as inorganic and organic substances present at low concentrations (pg/L–ng/L) in the environment, however, having adverse consequences for living organisms even at these low concentrations. To date, an effective and sustainable global strategy against this insidious contamination of aquatic environments barely exists. Source controls and technical systems, such as wastewater treatment plants, function only as partial barriers or not at all. The enzyme laccase was identified as active in the degradation of different MPs. We present data showing that laccase from Cerrena unicolor immobilized on mesoporous silica beads controlled porosity carrier (CPC) in a continuous column reactor was suitable for elimination of MPs including bisphenol A(BPA), 4-nonylphenol (NP), and triclosan (TCS). A system equipped with a 3.5 × 0.7 cm column packed with immobilized laccase CPC beads (43 units/g of enzyme activity), and eluted at 1 mL/min with a solution containing BPA, NP, an...

Using Laccases for Food Preservation

Abstract Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) have a number of applications in the field of the food industry. These oxidative enzymes are mainly produced by white-rot fungi in copious amounts and are characterized by high substrate versatility. In addition to lignin, their natural substrate, laccases may catalyze oxidative transformation of several substrates including phenols, substituted phenols, aromatic acids, aromatic and nonaromatic alcohols, aromatic aldehydes, aromatic amines, anilines, dyes, lignin derivatives, polyaromatic hydrocarbons (PAHs), quinones, and thiols residues. Many of these components are present in various foods and beverages and their modification may result in quality upgrading: wine and beer stabilization, decreased interaction between proteins and polyphenols during fruit juice processing, increased machinability of dough, and so forth. Laccases could also be used to eliminate O2 from packaging or dissolved O2 to control odors and enhance taste of food. Relatively well documented in patent literature, laccases are described as potential actors for a large range of applications in industry. Yet current implementations in food industry remain limited due to high laccase production cost and poor control of the enzymatic reaction byproducts. However, new developments, as immobilization of laccases in nanostructured materials, might provide renewed perspectives for food industry.

Assessment of methods of detection of water estrogenicity for their use as monitoring tools in a process of estrogenicity removal

ABSTRACT Methods of monitoring of estrogenicity in water were gathered, compared, and tested within the context of their practical use as measurement and design tools, in the development of a process of degradation of estrogenic endocrine disruptors. In this work, the focus was put on in vitro assays, with the use of analytical techniques as additional analysis when possible. Practically, from a literature review, four methods that seemed most suitable to practical use required in a process development were tested: the Yeast Estrogen Screen assay, the Lyticase-assisted Yeast Estrogen Screen assay (LYES), the MMV-LUC assay and the HPLC-UV analytical method. Dose–response curves in response to estrogenic standard 17β-estradiol were compared. Bisphenol A estrogenicity was measured by the methods as well. The model for the calculation of estradiol equivalents as measurements units was adapted. The methods were assessed in terms of ranges of detection, time of experiment, cost, ease of the experiment, reproducibility, etc. Based on that assessment, the LYES assay was selected and successfully applied to the monitoring of estrogenicity removal from 17β-estradiol and bisphenol A. More precisely, the bioassay allowed the acquisition of kinetic curves for a laboratory-scaled process of estrogenicity removal by immobilized enzymes in a continuous packed-bed reactor. The LYES assay was found to have a real methodological potential for scale-up and design of a treatment process. The HPLC-UV method showed good complementarity with the LYES assay for the monitoring of bisphenol A concentrations in parallel with estrogenicity, reporting no significant estrogenicity from degradation byproducts, among others.