Natural and recombinant fungal laccases for paper pulp bleaching

Abstract

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l(-1). Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

Heterologous laccase production and its role in industrial applications

“…161 Laccases have also found interesting applications in biopulping and biobleaching of lignocellulosic materials for paper manufacturing. 141,148 Sigoillot and co-workers investigated the pulp bleaching efficiency of P. cinnabarinus laccase expressed in two distinct Aspergilli hosts, in comparison with the native enzyme. The results obtained, together with the observed differences in redox potentials of the recombinant laccases, have been ascribed to the host-specific processing.…”

“…The results obtained, together with the observed differences in redox potentials of the recombinant laccases, have been ascribed to the host-specific processing. 141 In order to improve laccase treatments of pulp, Ravalson and co-workers synthesized a chimeric laccase by fusing P. cinnabarinus laccase lac1 to the carbohydrate binding module (CBM) of A. niger cellobiohydrolase B. 148 The chimeric protein was investigated for its softwood kraft pulp biobleaching potential in comparison with the native counterpart.…”

Laccases and Their Applications: A Patent Review

“…In order to improve laccase production, fungal laccases have been expressed heterologously in Saccharomyces cerevisiae [48,49,[60][61][62][63], Trichoderma reesei [48,54,64], Aspergillus oryzae [65][66][67][68][69][70][71], Pichia pastoris [51,[72][73][74][75][76], Yarrowia lipolitica [77], Aspergillus sojae [78], Aspergillus niger [52,79], Aspergillus nidulans [69,79,80], Aspergillus ficuum [81], tobacco [82][83][84][85] and maize [86][87][88]. Bacterial laccases from B.…”

“…Moreover, laccase can also offer a simple and convenient alternative to using peroxidases with H 2 O 2 , because laccases are available on an economically feasible scale. Otterbein et al [73] Record et al [52] Sigoillot et al [71] Alves et al [96,97] * The reported production levels have been obtained in shake flask cultivation, except in the case of P. radiata and M. albomyces laccases which were produced in a laboratory fermentor. …”

Imobilização da lacase em micropartículas de quitosana obtidas por spray drying e usadas na construção de biossensores

“…[15][16][17] As lacases são usadas em diversas aplicações industriais, incluindo biorremediação, clarificação de vinho, análise de drogas, produção de etanol e construção de biossensores. 18,19 Vários biossensores contendo lacase imobilizada têm sido desenvolvidos para a determinação de compostos fenólicos. [20][21][22][23][24] A rutina, Figura 1, é um derivado de flavona e como resultado da atividade farmacológica é empregada em fármacos de efeito antiinflamatório, antitumor, antialérgico, antiviral, entre outras propriedades.…”