Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta.

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy - AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family.

Properties of two laccases from the Trametes hirsuta 072 multigene family: Twins with different faces.

Utilization of laccases in biotechnology and bioremediation has created a strong demand for the characterization of new enzymes and an increase in production of known laccases. Thus, additional research into these enzymes is critically needed. In this study, we report a comparative study of the biochemical and transcriptional properties of two different laccase isozymes from Trametes hirsuta 072 - the constitutive and inducible forms. A recombinant LacC enzyme was expressed in Penicillium canescens to characterize its properties. LacC is single-purpose enzyme, unlike LacA, which can operate efficiently under a wide range of temperatures and pHs (55-70 °C and pH 3-5, respectively). LacC has a lower RedOx potential than LacA and does not oxidize substrates containing amine groups. Expression of the lacC gene was selective compared to that of the lacA gene and increased significantly in the presence of complex synthetic compounds such as dyes and xenobiotics. This study shows that laccases from the multigene families of basidiomycetes differ significantly in their properties, thus providing a complementary effect during lignin degradation.

Immunoassays of fungal laccases for screening of natural enzymes and control of recombinant enzyme production.

Because of the wide application of laccases in different biotechnological processes and intense studies of the enzymes from different sources, the development of efficient techniques for monitoring laccase level is a task of significant importance. Enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques were developed to control total content and isoform composition of laccases, including their recombinant preparations. Because glycosylated and nonglycosylated forms have different structures and sets of epitopes, two kinds of polyclonal antibodies were obtained and applied. The first antibody recognized the native (glycosylated) laccase purified from Trametes hirsuta and the second one reacted with recombinant (nonglycosylated) laccase expressed in Escherichia coli. Titers of the antibodies were analyzed by indirect ELISA with laccases isolated from several strains of basidiomycetes. The obtained cross-reactivity data for both antibodies demonstrated a correspondence with sequence homology of the laccases. The antibodies raised against recombinant (nonglycosylated) laccase had higher titers and thus were preferable for screening of recombinant laccase in cultural media. Thus, optimal antibody preparations were selected for screening of laccase-producing strains, and the control of recombinant enzymes and the efficiency of their use in immunochemical control of laccase levels were confirmed.