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.

Representatives of the colorless sulfur bacteria of the genus Beggiatoa use reduced sulfur compounds in the processes of lithotrophic growth, which is accompanied by the storage of intracellular sulfur. However, it is still unknown how the transformation of intracellular sulfur occurs in Beggiatoa representatives. Annotation of the genome of Beggiatoa leptomitoformis D-402 did not identify any genes for the oxidation or reduction of elemental sulfur. By searching BLASTP, two putative persulfide dioxygenase (PDO) homologs were found in the genome of B. leptomitoformis. In some heterotrophic prokaryotes, PDO is involved in the oxidation of sulfane sulfur. According to HPLC-MS/MS, the revealed protein was reliably detected in a culture sample grown only in the presence of endogenous sulfur and CO₂. The recombinant protein from B. leptomitoformis was active in the presence of glutathione persulfide. The crystal structure of recombinant PDO exhibited consistency with known structures of type I PDO. Thus, it was shown that B. leptomitoformis uses PDO to oxidize endogenous sulfur. Additionally, on the basis of HPLC-MS/MS, RT-qPCR, and the study of PDO reaction products, we predicted the interrelation of PDO and Sox-system function in the oxidation of endogenous sulfur in B. leptomitoformis and the connection of this process with energy metabolism.

The green unicellular algae Chlamydomonas reinhardtii contains 12–13 carbonic anhydrases (CAs). For a long time, the two closely related α-CAs of the periplasmic membrane CAH1 and CAH2 were considered to be the CAs with the highest CO₂ hydration activity. The recombinant protein α-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. Long-term sustainability of the enzyme was observed at alkaline pH (>8), with maintenance of half of its activity at 4 °C for up to 50 days. Thermostability of rCAH3 indicated the retention of the activity at 20 °C for one hour at pH 9–10 with its ~50% decrease at pH 6–7. However, the residual activity of rCAH3 after incubation at an extremely high temperature (75 °C) for 15 min led to the formation of the double-hump graph with maxima at pH 6 and 9. The enzyme demonstrated high sensitivity to ethoxyzolamide and acetazolamide at nM concentrations, to Zn²⁺ and Cu²⁺ cations at 1 mM concentrations, and L-cysteine was able to completely inhibit CA activity of rCAH3 through reduction of sulfhydryl groups. Esterase activity of rCAH3 was well detected with values comparable to those of bovine CAII, but with a maximum at pH 8 instead of pH 9, which is usual for bovine CAII. The results indicated that CAH3 may be the most active CA of C. reinhardtii and that its role in the photosynthetic apparatus function could have been underestimated in previous works.

The green unicellular algae Chlamydomonas reinhardtii contains 12–13 carbonic anhydrases (CAs). For a long time, the two closely related α-CAs of the periplasmic membrane CAH1 and CAH2 were considered to be the CAs with the highest CO2 hydration activity. The recombinant protein α-CA CAH3 (rCAH3) from the thylakoid lumen obtained in the present study showed more than three times higher activity compared to CAH1 and more than 11 times higher compared to previous studies with rCAH3. Long-term sustainability of the enzyme was observed at alkaline pH (>8), with maintenance of half of its activity at 4 °C up to 50 days. Thermostability of rCAH3 indicated the retention of the activity at 20 °C for one hour at pH 9–10 with its ~50% decrease at pH 6–7. However, the residual activity of rCAH3 after incubation at extremely high temperature (75 °C) for 15 min led to formation of the double-hump graph with maxima at pH 6 and 9. The enzyme demonstrated high sensitivity to ethoxyzolamide and acetazolamide at nM concentrations, to Zn2+ and Cu2+ cations at 1 mM concentrations, and L-cysteine was able to completely inhibit CA activity of rCAH3 through reduction of sulfhydryl groups. Esterase activity of rCAH3 was well detected with values comparable to those of bovine CAII, but with a maximum at pH 8 instead of pH 9, which is usual for bovine CAII. The results indicated that CAH3 may be the most active CA of C. reinhardtii and that its role in the photosynthetic apparatus function could have been underestimated in previous works.

A novel two-domain small laccasefrom Streptomyces ochraceiscleroticus (SoSL) was produced through recombination in Escherichia coli and purified by affinity chromatography. The properties (thermal optimum and thermostability, pH optima and pH-stability), kinetic characteristics, substrate specificity and dye decolorization ability were estimated. Laccase SoSL was able to oxidize 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS)and 2,6-dimethoxyphenol (2,6-DMP) with at a maximal rate at pH 3.5 and 9.0, respectively, and was stable at pH 9.0 (retained 75% activity after incubation at room temperature for 120 h). High enzyme affinity to ABTS is caused by an expanded area occupied by aromatic amino acid residues on its surface. Substrate-directed immobilization of the enzyme was performed using naphthylated multiwalled carbon nanotubes (MWCNTs), and a high oxygen reduction reaction potential (+0.62 V vs. normal hydrogen electrode (NHE)) was observed. The above-mentioned features make this enzyme a promising one for further studies in bioremediation and biological fuel cell technologies.