2021  1,540
2020  1,374
2019  1,023
2018  0,932
2017  0,977
2016  0,799
2015  0,662
2014  0,740
2013  0,739
2012  0,637
2011  0,658
2010  0,654
2009  0,570
2008  0,849
2007  0,805
2006  0,330
2005  0,435
2004  0,623
2003  0,567
2002  0,641
2001  0,490
2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 56(2022) N 4 p. 600-609; DOI 10.1134/S0026893322040021 Full Text

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 Laccase

1Centro 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