Modified nanodiamonds (MNDs) produced by detonation synthesis can be used as an effective adsorbent to immobilize extracellular peroxidases of the luminous basidiomycete Neonothopanus nambi. The enzymes are firmly immobilized on MND particles and exhibit catalytic activity. The indicator system (the MND–enzyme complex) reused many times retains its ability to catalyze reaction of co-oxidation of phenol and 4-aminoantipirine in the presence of hydrogen peroxide and remains functionally active during long-term storage (for 1 month or longer) in aqueous suspensions at 4 °C. MNDs and enzymes of higher fungi can be effectively used to construct new reusable indicator systems for analytical applications such as monitoring contamination of aquatic environments by phenolic compounds.

In vitro experiments proved the usefulness of electron paramagnetic resonance (EPR) spectrometry for detecting detonation nanodiamonds (NDs) in samples of biomaterials (blood and homogenates of organs of mice). A characteristic EPR signal ( g = 2.003, Δ H ≃ 10 G) was detected in biomaterials containing NDs, and its intensity linearly increased at nanoparticle concentrations of between 1.6 and 200 µg/ml. In vivo experiments demonstrated that EPR spectrometry was effective for monitoring the inter‐organ distribution of NDs intravenously injected to mice. In 2.5 h after the injection of NDs, the nanoparticles mainly accumulated in the lungs and liver of the animals – about 25 and 20%, respectively, of the initially injected NDs. The amounts of NDs accumulated in the heart and kidneys were considerably lower. Also, EPR spectrometry did not detect NDs in the blood, spleen, brain, and femoral muscles of mice. Ten days after injection, EPR spectrometry detected redistribution of NDs in mice. The amounts of nanoparticles decreased approximately by a factor of 3.5 in the lungs and increased almost by a factor of 3 in the liver; NDs were detected in the spleen. This study suggests ways to use EPR spectrometry to study the distribution, accumulation, and elimination of detonation NDs injected into laboratory animals.

A stimulator of light emission of the fungus was found in an aqueous extract from mycelium of the luminous basidiomycete Neonothopanus nambi after its treatment with β-glucosidase. The addition of the extract to the luminous mycelium increases the level of light emission from several times to 1.5 orders of magnitude or more. The luminescence stimulator is a low-molecular-weight thermostable compound: it is detected in the permeate after filtering the extract through a 10-kDa cutoff membrane and it retains the stimulating effect after heat treatment at 100°C for 5 min. In the absorption spectrum of the aqueous sample of the stimulator, two main peaks are observed in the shortwave region (205 and 260 nm) and a shoulder in the range of 350–370 nm can be seen. The luminescence stimulator exhibits blue fluorescence with an emission maximum at 440 nm when excited at 360 nm. It was established that the luminescence-stimulating component is not a substrate (or its precursor) of the luminescent system of the N. nambi fungus.

In vivo studies have revealed that the addition of caffeic acid and a low-molecular-weight compound, a bioluminescence stimulator, discovered in our research, to the mycelium of the luminous fungus Neonothopanus nambi leads to a rapid and significant (by an order of magnitude or more) increase in the intensity of its light emission. It has been suggested that the observed effect of activation of fungal bioluminescence may be mediated by the oxidation of added substances by enzymes of the ligninolytic complex of basidiomycetes (in particular, peroxidases) with the emission of visible light quanta. Parallel in vivo experiments showed that additions of hispidin (a luciferin precursor in the luminous higher fungi) have no effect on the light emission intensity of the mycelium. At the same time, in vitro studies have reported that caffeic acid and the detected lowmolecular luminescence stimulator do not affect the level of light emission of the enzyme luminescent system isolated from the N. nambi mycelium in the presence of NADPH and significantly suppress the emission reaction of the system activated by NADPH and hispidin. A set of data collected demonstrate that different biochemical pathways present in the luminous higher fungi and different enzymes (or enzyme systems) and different substrates are involved in generation of visible light quanta.

The paper presents data that testify in favor of the participation of the cytochrome P450 system in the light emission of higher fungi. Extracts from mycelia of different species of luminous basidiomycetes containing fungal luminescent systems that provide luminescence in vitro were obtained. Applied conditions for the isolation of luminescent systems (sonication, centrifugation at 40000g) indicate the presence of membrane structures in the extracts, in particular, microsomes formed as a result of ultrasonic disintegration of the endoplasmic reticulum (ER). Differential spectral analysis of the extracts revealed the presence of two absorption peaks at 410 nm and 450 nm, which indicates the presence of cytochromes b5 and P450. The luminescence of the extracts is stimulated by reduced pyridine nucleotides, however, the addition of NADPH causes a higher level of luminescence compared with NADH. The addition of hydrogen peroxide significantly (from several times to 1-2 orders of magnitude) increases the luminescence intensity of extracts activated by NAD(P)H. The addition of fluconazole significantly inhibits the light emission of extracts. The data obtained indicates that the cytochrome P450 system associated with ER membranes may participate in the mechanism of light emission of higher fungi with the involvement in the process of electron transport enzyme systems: NADPH-dependent reductase of cytochrome P450 - cytochrome P450 and NADH-dependent reductase of cytochrome b5 - cytochrome b5 - cytochrome P450. In this case, cytochrome P450 may hydroxylate hispidin (precursor of the luminescent reaction substrate) to form luciferin and catalyze its oxidation in the presence of ROS with light emission.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND–oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND–oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND–oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.