Plasticity of laccase generated by homeologous recombination in yeast.
Laccase-encoding sequences sharing 65-71% identity were shuffledin vivo by homeologous recombination. Yeast efficiently repaired linearized plasmids containing clac1, clac2 or clac5 Trametes sp. C30 cDNAs using a clac3 PCR fragment. From transformants secreting active variants, three chimeric laccases (LAC131, LAC232 and LAC535), each resulting from double crossovers, were purified, and their apparent kinetic parameters were determined using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and syringaldazine (SGZ) as substrates. At acidic pH, the apparent kinetic parameters of the chimera were not distinguishable from each other or from those obtained for the LAC3 enzyme used as reference. On the other hand, the pH tolerance of the variants was visibly extended towards alkaline pH values. Compared to the parental LAC3, a 31-fold increase in apparent k(cat) was observed for LAC131 at pH 8. This factor is one of the highest ever observed for laccase in a single mutagenesis step.
Cusano
Angela M
AM
Laboratoire Biosciences, Institut des Sciences Moléculaires de Marseille, Université Aix-Marseille, ISM2 CNRS UMR 6263, Marseille Cedex 20, France.
Mekmouche
Yasmina
Y
Meglecz
Emese
E
Tron
Thierry
T
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019020:Directed Molecular Evolution; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D055454:Trametes / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D042845:Laccase