Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching.
Pycnoporus cinnabarinus laccase was fused to the C-terminal linker and carbohydrate binding module (CBM) of Aspergillus niger cellobiohydrolase B (CBHB). The chimeric enzyme of molecular mass 100 kDa was successfully produced in A. niger. Laccase-CBM was further purified to determine its main biochemical properties. The Michaelis-Menten constant and pH activity profile were not modified, but the chimeric enzyme was less thermostable than either the P. cinnabarinus laccase or the recombinant laccase produced in the same strain. Laccase-CBM was able to bind to a cellulosic substrate and, to a greater extent, to softwood kraft pulp. Binding to the pulp was shown to be mainly time and temperature-dependent. Laccase-CBM was further investigated for its softwood kraft pulp biobleaching potential and compared with the P. cinnabarinus laccase. Addition of a CBM was shown to greatly improve the delignification capabilities of the laccase in the presence of 1-hydroxybenzotriazole (HBT). In addition, ClO(2) reduction using 5 U of chimeric enzyme per gram of pulp was almost double than that observed using 20 U of P. cinnabarinus laccase per gram of pulp. We demonstrated that conferring a carbohydrate binding capability to the laccase could significantly enhance its biobleaching properties.
INRA, UMR1163, Biotechnologie des Champignons Filamenteux, Case 932, 163 avenue de Luminy, F-13000 Marseille, France. holy.ravalason@esil.univmed.fr
Holy
H
Ravalason
Isabelle
I
Herpoël-Gimbert
Eric
E
Record
Frédérique
F
Bertaud
Sacha
S
Grisel
Sandra
S
de Weert
Cees A M J J
CA
van den Hondel
Marcel
M
Asther
Michel
M
Petit-Conil
Jean-Claude
JC
Sigoillot
D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D017606:Chlorine Compounds / Q000737:chemistry; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010087:Oxides / Q000737:chemistry; D010209:Paper; D055494:Pycnoporus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014934:Wood / Q000737:chemistry*
D002241:Carbohydrates; D017606:Chlorine Compounds; D005656:Fungal Proteins; D010087:Oxides; D011993:Recombinant Fusion Proteins; C025109:chlorine dioxide; D042845:Laccase