Improved recovery of active recombinant laccase from maize seed.
Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.
Bailey
M R
MR
ProdiGene, 101 Gateway Blvd. Suite 100, College Station, TX 77845, USA.
Woodard
S L
SL
Callaway
E
E
Beifuss
K
K
Magallanes-Lundback
M
M
Lane
J R
JR
Horn
M E
ME
Mallubhotla
H
H
Delaney
D D
DD
Ward
M
M
Van Gastel
F
F
Howard
J A
JA
Hood
E E
EE
D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D042845:Laccase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D008031:Lignin / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D001051:Apoenzymes; D002712:Chlorides; D005656:Fungal Proteins; D011994:Recombinant Proteins; D003300:Copper; D008031:Lignin; D042845:Laccase