Natural and recombinant fungal laccases for paper pulp bleaching.
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).
UMR 1163 INRA/Université de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, C.P. 925, 13288 Marseille 09, France. cecile.sigoillot@esil.univ-mrs.fr
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Sigoillot
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Record
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Belle
J L
JL
Robert
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Levasseur
P J
PJ
Punt
C A M J J
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van den Hondel
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Fournel
J C
JC
Sigoillot
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Asther
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D007218:Industrial Microbiology; D042845:Laccase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008031:Lignin / Q000378:metabolism; D010084:Oxidation-Reduction; D010209:Paper; D011124:Polyporaceae / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008031:Lignin; D042845:Laccase