title: Characterization of C-terminally engineered laccases.
abstract: Extremities of proteins are potent sites for functionalization. Carboxy terminus variants of the Trametes sp. strain C30 LAC3 laccase were generated and produced in Saccharomyces cerevisiae. A variant deleted of the last 13 residues (CΔ) and its 6 His tagged counterpart (CΔ6H) were found active enzymes. The production of CΔ6H resulted in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of CΔ6H noted CΔ6Hh. Properties of CΔ, CΔ6H and CΔ6Hh were compared. Globally, LAC3 catalytic efficiency was moderately affected by terminal modifications except in CΔ for which the kcat/KM ratio decreased 4 fold (with syringaldazine as substrate) and 10 fold (with ABTS as substrate) respectively. The catalytic parameters kcat and KM of CΔ6H and CΔ6Hh were found to be strictly comparable revealing that over glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduced its activity. So, despite a complex glycosylated pattern observed for some of the variant enzymes, terminal sequences of laccases appear to be appropriate sites for the functionalization/immobilization of laccase.
authors.affiliation: Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), Beijing 100048, China; Aix Marseille Université, CNRS, iSm2 UMR 7313, 13397 Marseille, France. Electronic address: liuyingli@th.btbu.edu.cn.
authors.forename: Yingli
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Italian Institute of Technology, Center For Advanced Biomaterials For Health Care, Largo Barsanti e Matteucci, 53, 80125 Napoli, Italy.
authors.forename: Angela Maria
authors.initials: AM
authors.lastname: Cusano
authors.affiliation: Illumina, 25861 Industrial Blvd., Hayward, CA 94545, USA.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Wallace
authors.affiliation: Aix Marseille Université, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Mekmouche
authors.affiliation: Aix Marseille Université, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Sana
authors.initials: S
authors.lastname: Ullah
authors.affiliation: Aix Marseille Université, CNRS, iSm2 UMR 7313, 13397 Marseille, France.
authors.forename: Viviane
authors.initials: V
authors.lastname: Robert
authors.affiliation: Aix Marseille Université, CNRS, iSm2 UMR 7313, 13397 Marseille, France. Electronic address: thierry.tron@univ-amu.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Tron
mesh_terms: D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D055454:Trametes / Q000201:enzymology / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D006023:Glycoproteins; D042845:Laccase
keywords: Glycosylation; Multi-copper enzyme; Tagged protein