Enhanced large-scale production of laccases from Coriolopsis polyzona for use in dye bioremediation
Creators: name: Cui, Tingting
Publisher: name: University of Westminster
Year: 2009

description: Pollution from synthetic dyes, released by textile and paper pulping plants, draws major concern. Textile effluents have negative impact both on the environment and human health because they are toxic and some are carcinogenic. Apart from the textile industry, dyes are also widely used in manufacturing industries for leather products, cosmetics, pharmaceuticals, foods and beverages. It is known that white rot fungi can decolourise and detoxify various industrial effluents through the production of extracellular lignin modifying enzymes, a major class of which are laccases (EC 1.10.3.2). Considering the above, three strains of white rot fungi, Coriolopsis polyzona (MUCL 38443), Pleurotus ostreatus (ATCC no. MYA-2306) and Pycnoporus sanguineus (MUCL 41582) were studied for their ability to produce laccases in liquid media. The effects of mannan oligosaccharides (MO) as elicitor and ferulic acid as inducer were studied using central composite experimental design in liquid cultures of the three strains. The results showed that MO, either added alone or combined with ferulic acid, enhanced laccase activity in the three different cultures and the enhancement was species specific. The highest increase was in liquid cultures of P. sanguineus (88-fold) followed by P. ostreatus (3-fold) and C. polyzona (2-fold), among which C. polyzona resulted in the highest laccase activity. The combined addition of 150 mg/l of MO and 1 mM ferulic acid resulted in the optimal laccase activity by C. polyzona, whereas additions of 75 mg/l MO to the cultures of P. sanguineus and P. ostreatus led to the optimal activity. Extracellular laccase activity was considerably increased when C. polyzona was grown in glucose-bactopetone based culture medium induced by ferulic acid. The effect of inoculum conditions on laccase production was studied at reactor scales. Laccase activity achieved with conidia inoculation was higher compared with mycelium inoculation at the early stage of fermentation. However, the laccase levels were similar after 23 days of fermentation (110 U/ml and 100 U/ml for the conidia and mycelia pre-culture respectively). The conidia inoculation is preferred in scale-up when time-cost is considered. The maximal laccase activity with conidia inoculation in a 2 litre stirred tank reactor was 27% higher compared to that in shaken flasks. This showed that C. polyzona cultures have the potential to be scaled-up for increased laccase activity by applying conidia inoculum. The fermentation of C. polyzona was scaled-up to 20 litre and 150 litre stirred tank reactors applying fed-bath strategy. This resulted in 100 % enhancement of laccase activity. Addition of oak wood powder in the culture medium increased total laccase activity indicating the potential of lignocellulosic wastes as alternative substrates for enhanced laccase production with reducing cost. In order to investigate the application of laccases in dye decolourisation, two major laccase isozymes (Lac I &amp; II) from C. polyzona were purified to apparent eletrophoretic homogenetity using hydrophobic interaction chromatography and ionexchange chromatography. Both enzymes were found to be monomeric proteins with the same molecular mass of 63 kDa, and isoelectronic point of 4.3. Their catalytic activities were studied under various substrates, pHs and temperatures. The highest enzyme affinity and efficiency were obtained with 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Compared with other fungal laccases, the laccases from C. polyzona have very low Km values with ABTS as a substrate. The optimum pHs were 2.8, 3.0 and 5.0 on ABTS, 2, 6-dimethoxyphenol (DMP) and syringaldazine, respectively. Both isozymes had acidic optimal pH values. However, they were more stable in netural pH rather than at acidic pH. Moreover, mass spectrometry (MS) analysis of tryptic digestion products of the two isozymes was performed, which showed further similarity of these two isozymes. As common physical or chemical methods for dye removal are expensive, have low efficiency and sometimes generate other pollutants, the decolourisation of industrial effluents containing single and mixed dyes was investigated using purified laccase (Lac I) from C. polyzona as well as whole cell culture. The method appeared to be an attractive alternative for dye removal. Anthraquinone dyes were found to be more easily decolourised by Lac I compared to azo dyes. The addition of redox mediator ABTS and violuric acid (VA) improved considerably the catalytic efficiencies of azo dyes. Decolourisation, 40-50 %, was achieved for the reactive and the direct dye baths. Response surface technology (RSM) was applied to optimise the decolourisation of the diazo dye reactive black 5 (RB 5) by Lac I. Box-Behnken experimental design with three variables including laccase activity (100, 200, 300 U/l), pH (5, 7, 9) and VA concentration (0, 1.25, 2.5 mM) was studied to identify a significant correlation between the effect of these variables on decolourisation of RB5. The experimental values were in good agreement with the predicted values with the correlation coefficient of 97.4%.