Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases.
Laccases have numerous biotechnological applications, among them food processing. The widespread use of laccases has increased the demand for an inexpensive and safe source of recombinant enzyme. We explored the use of a rice-based system for the production of two fungal laccases derived from the ascomycete Melanocarpus albomyces and the basidiomycete Pycnoporus cinnabarinus. High-expression levels of active recombinant laccases were achieved by targeting expression to the endosperm of rice seeds. The laccase cDNAs were fused to a plant-derived signal sequence for targeting to the secretory pathway, and placed under the control of a constitutive seed-specific promoter fused to an intron for enhanced expression. This construct enabled the recovery of on average 0.1-1% of soluble laccase in total soluble proteins (TSP). The highest yields of recombinant laccases obtained in rice seeds were 13 and 39 ppm for riceMaL and ricePycL, respectively. The rice-produced laccases were purified and characterized. The wild-type and the recombinant proteins showed similar biochemical features in terms of molecular mass, pI, temperature and optimal pH and the N-terminus was correctly processed. Although presenting lower kinetic parameters, the rice-produced laccases were also suitable for the oxidative cross-linking of a food model substrate [maize-bran feruloylated arabinoxylans (AX)].
CropDesign NV, a BASF Plant Science Company, Technologiepark 3, Zwijnaarde-Gent, Belgium.
Chris
C
de Wilde
Eva
E
Uzan
Zhongyi
Z
Zhou
Kristiina
K
Kruus
Martina
M
Andberg
Johanna
J
Buchert
Eric
E
Record
Marcel
M
Asther
Anne
A
Lomascolo
D001203:Ascomycota / Q000201:enzymology*; D015153:Blotting, Western; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012275:Oryza / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010084:Oxidation-Reduction; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids; D011124:Polyporaceae / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D013696:Temperature; D014990:Xylans / Q000737:chemistry / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry / Q000378:metabolism
D003373:Coumaric Acids; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014990:Xylans; C004999:ferulic acid; D042845:Laccase