tailieunhanh - Báo cáo khoa học: 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 plas-mids containingclac1, clac2 orclac5 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. . | ỊFEBS Journal Plasticity of laccase generated by homeologous recombination in yeast Angela M. Cusano f Yasmina Mekmouche Emese MegleczJ and Thierry Tron Laboratoire Biosciences Institut des Sciences Moleculaires de Marseille Université Aix-Marseille ISM2 CNRS UMR 6263 Marseille Cedex 20 France Keywords cupredoxin domains functional hybrids heterologous expression multicopper enzyme recombination Correspondence T. Tron Laboratoire Biosciences Institut des Sciences Moleculaires de Marseille Universite Aix-Marseille ISM2 CNRS UMR 6263 Avenue Escadrille Normandie Niemen case 342 F-13397 Marseille Cedex 20 France Fax 33 491 288440 Tel 33 491 289196 E-mail These authors contributed equally to this work Laccase-encoding sequences sharing 65-71 identity were shuffled in vivo by homeologous recombination. Yeast efficiently repaired linearized plasmids containing clacl 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 kcat was observed for LAC131 at pH 8. This factor is one of the highest ever observed for laccase in a single mutagenesis step. Present addresses Ị-INRA NANCY UMR 1136 Interactions Arbres-Micro-organismes Equipe de Pathologie Forestiere Route d amance 54280 Champenoux France ỊIMEP Case 36 Universite de Provence 3 Place Victor Hugo 13331 Marseille Cedex 3 France Database The sequences of the laccase hybrid .