Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution.

This study was carried out in order to assess several modifications of carbon nanotube-based nanomaterials for their applications in laccase electrodes and model biofuel cells. The modified MWCNTs served as adapters for the immobilization of laccase from Catenuloplanes japonicus VKM Ac-875 on the surface of electrodes made of graphite rods and graphite paste. The electrochemical properties of the electrodes were tested in linear and cyclic voltammetrical measurements for the determination of the redox potential of the enzyme and achievable current densities. The redox potential of the enzyme was above 500 mV versus NHE, while the highest current densities reached hundreds of µA/cm². Model biofuel cells on the base of the laccase cathodes had maximal power values from 0.4 to 2 µW. The possibility of practical application of such BFCs was discussed.

Three-dimensional structures of six closely related hydrogenases from purple bacteria were modeled by combining the template-based and ab initio modeling approach. The results led to the conclusion that there should be a 4Fe3S cluster in the structure of these enzymes. Thus, these hydrogenases could draw interest for exploring their oxygen tolerance and practical applicability in hydrogen fuel cells. Analysis of the 4Fe3S cluster’s microenvironment showed intragroup heterogeneity. A possible function of the C-terminal part of the small subunit in membrane binding is discussed. Comparison of the built models with existing hydrogenases of the same subgroup (membrane-bound oxygen-tolerant hydrogenases) was carried out. Analysis of intramolecular interactions in the large subunits showed statistically reliable differences in the number of hydrophobic interactions and ionic interactions. Molecular tunnels were mapped in the models and compared with structures from the PDB. Protein–protein docking showed that these enzymes could exchange electrons in an oligomeric state, which is important for oxygen-tolerant hydrogenases. Molecular docking with model electrode compounds showed mostly the same results as with hydrogenases from E. coli, H. marinus, R. eutropha, and S. enterica; some interesting results were shown in case of HupSL from Rba. sphaeroides and Rvi. gelatinosus.

(Ca²⁺)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6β6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9–10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (K_M = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002–0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.