Vol 56(2022) N 4 p. 600-609; DOI 10.1134/S0026893322040021
A.A. Arteaga-Castrejón1, M.R. Trejo-Hernández1, Y. Mekmouche2, A. Amouric2, P. Rousselot-Pailley2, V. Robert2, T. Tron2*, F. Martínez-Morales2**
Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase1Centro de Investigación en Biotecnología, Morelos, 62209 México
2Aix Marseille Université, Centrale Marseille, CNRS, Marseille, 13397 France
Received - 2021-09-05; Revised - 2021-12-02; Accepted - 2021-12-10
Fungal laccases are oxidoreductases with low-specificity for substrates. The characterization of laccase's surface is a prerequisite used to obtain hybrid catalysts with new properties. Surface-exposed lysine residues are targets in immobilization reactions. In this work, LAC3-K0, an enzyme devoid of lysine, was used as a platform to detect potential surface-exposed sites suitable for replacement with a lysine residue. Seven sites were selected from a LAC3-K0 3-D model, and single lysine mutants (UNIKn, n = residue number) were obtained by site-directed mutagenesis. All mutants were expressed in Saccharomyces cerevisiae W303-1A and detected as functional secreted proteins by their ability to oxidize guaiacol or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) on agar plates. All variants were active at acidic pH but presented no activity at neutral pH, as expected. Likewise, variants were stable a temperature between 15-55°C, and were completely inactivated at 70°C. Oxidation assays revealed that the replacement of one or two surface residues with lysine greatly affected enzyme activity and substrate specificity. The catalytic; parameters (KMapp and kcatapp) determined with ABTS were found to be different among the variants; Vmaxapp was 1.5-2 fold higher in UNIK269 and triple mutant, with a KMapp of 0.27 and 0.30, respectively; kcatapp was 30.25 in UNIK238 and 32.34 in the triple mutant. The role of hydrophobic patches detected on the surface of LAC3-K0 was determined to be a favorable factor to be considered in the interaction of hybrid materials. All variants with uniquely surface located lysine created in this work can be in demand for obtaining laccases with a certain substrate specificity in the design of hybrid materials.
laccase, oriented functionalization, site-directed mutagenesis, UNIK