K. V. Purtov

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Mechanism and color modulation of fungal bioluminescence

Study of fungal bioluminescence mechanisms generates development of a multicolor enzymatic chemiluminescence system. Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

Isolation of luminescence system from the luminescent fungus Neonothopanus nambi

56 This study is devoted to the problem of isolation of the lighttemitting system able to luminesce in vitro from the fungus Neonothopanus nimbi. The study was performed with the mycelium of the luminous higher fungus N. nambi, inhabiting the tropp ical forests of South Vietnam. [1] The fungus culture was kindly provided for experiments by Vietnamese researcher Dao Thi Van (private collection of strains To obtain fungal biomass, the mycelium was cultured in Petri plates in a liquid nutrient medium by the techh nology described earlier [2]. The grown mycelium was taken from the Petri dish and washed with deionized (DI) water (MilliiQ system, Millipore, United States) to remove the residual components of the nutrient medium and exometaboliltes. After washing, the remaining water was removed from the mycelial biom ass with filter paper. The isolation of the luminescent system of N. nambi mycelium included the following steps, which were carried out at 0–4°C. Mycelium washed with DI water was ground in the cold with scissors and transferred to a beaker placed in an ice bath. The bioo mass was poured with cold 0.1 M phosphate buffer (pH 7.0) supplemented with 0.1–1.0% BSA (Serva, Germany) in the ratio 1 : 5 (wet biomass weight : buffer volume). The biomass was destroyed with a Volna ultrasonic disintegrator (Russia). Sonication was perr formed at a power of 200 W three times for 5 s at 11min intervals. The homogenate was transferred into chilled tubes and centrifuged at 48 000 g for 30–60 min in an Avanti ® JE centrifuge (BeckmannCoulter, United States). The pellet was discarded, and the supernatant was either used immediately for study (in this case, it was stored at 4°C) or immediately frozen at –20°C and stored at this temperature. The luminescence of the supernatants was meaa sured using a BLM 8801 luminometer (Nauka Special Engineering and Design Department, Krasnoyarsk, Russia) calibrated using the Hastings–Weber radioacc tive standard [3] (one luminescent unit was 10 8 phoo tons per 1 s). The signals were recorded using an LKB 2210 recorder (LKB, Sweden). It was found that supernatants isolated from the mycelium of the luminous fungus N. nambi by the method described above emitted long luminescence (Fig. 1). This fact allowed us to conclude that a selff sufficient luminescent system that ensures luminess cence in vitro was isolated from this fungal species. After filtering the supernatant through a membrane with an …

Why does the bioluminescent fungus Armillaria mellea have luminous mycelium but nonluminous fruiting body

By determining the components involved in the bioluminescence process in luminous and nonluminous organs of the honey fungus Armillaria mellea, we have established causes of partial luminescence of this fungus. The complete set of enzymes and substrates required for bioluminescence is formed only in the mycelium and only under the conditions of free oxygen access. Since the synthesis of luciferin precursor (hispidin) and 3-hydroxyhispidin hydroxylase in the fruiting bodies is blocked, the formation of luciferin—the key component of fungal bioluminescent system—was not observed. That is why the fruiting body of Armillaria mellea is nonluminous despite the presence of luciferase, the enzyme that catalyzes the oxidation of luciferin with a photon emission.

Model drug delivery system based on nanodiamonds

This study investigates the possibility of using a detonation nanodiamond as a targeted drug-delivery vehicle. Supramolecular complexes composed of nanodiamond particles and address protein molecules (nanodiamond-IgGI125 and RAM-nanodiamond-BSAI125) have been obtained. In vitro experiments have revealed that these complexes are stable and exhibit high colloidal stability in blood serum, as well as passivity to the cellular components of blood. It has been shown that, after the simultaneous covalent immobilization of two proteins (rabbit anti-mouse antibody (RAM) and bovine serum albumin (BSAI125)) on the nanoparticles, the resulting complex is able to specifically bind to the target antigen (mouse IgG).

Isolation and Purification of Fungal Luciferase from Neonothopanus nimbi

This is the first study to obtain a high-purity luciferase from the fungus Neonothopanus nambi biomass that is suitable for subsequent sequencing.

Structure of fungal oxyluciferin, the product of the bioluminescence reaction

The structure of fungal oxyluciferin was determined, the enzymatic bioluminescence reaction under substrate saturation conditions with discrete monitoring of formed products was conducted, and the structures of the end products of the reaction were established. On the basis of these studies, the scheme of oxyluciferin degradation to the end products was developed. The structure of fungal oxyluciferin was confirmed by counter synthesis.

Components of the luminescent system of the luminous fungus Neonothopanus nambi

65 Earlier, we reported that a luminescent system with continuous luminescence in vitro was isolated from the mycelium of the luminous fungus Neonothopanus nambi [1]. The results of this study led us to the conn clusion that the isolated luminescent system is a fairly stable complex that includes protein and nonprotein components required for the light emission reaction. The aim of this work was to separate the protein and nonprotein components of the lighttemitting syss tem of the fungus Neonothopanus nambi and to study some of their properties. The study was performed with the mycelium of the higher luminous fungus N. nambi, inhabiting tropical forests of Southern Vietnam [2]. The culture of fungus for experiments was kindly provided by Dao Thi Van (BIOOLUMI Co., Ltd., Vietnam). The film mycelium biomass was obtained by culturing the fungus in Petri dishes on a liquid nutrient medium by the technology developed by us earlier [3]. Grown mycelium was taken from the Petri dishes and incubated in distilled water to remove the residual components of the nutrii ent medium and exometabolites. To obtain the extracts containing the luminescent reaction substrate, the fungal biomass was incubated in water for 8 h. To obtain the extracts containing the protein components of the luminescent system (primarily the enzymes involved in the light emission reaction), the mycelial biomass was incubated in water for 16 h. The luminescent reaction substrate was extracted from the mycelium biomass as follows. Mycelium incubated in distilled water was placed in a heattresiss tant glass, in which distilled water was added in a 1 : 1 ratio (wet biomass weight : water volume). Thus pree pared sample was placed in a microwave oven and heated to water boiling. Thereafter, the sample was rapidly cooled in an ice bath. The liquid portion of the sample was collected, transferred into chilled tubes, and centrifuged at 30000 g for 20 min at 4°C in an Avanti® JJE centrifuge (BeckmannCoulter, United States). The pellet was discarded, and the supernatant was collected and frozen at –20°C and stored at this temperature until use. The protein components (including the enzymes involved in the light emission reaction) were extracted from the N. nambi mycelium at 0–4°C. Fungal biom ass incubated in distilled water frozen at –80°C (ILS kelvinator, Nuaire Inc., Korea), after which the biomass was thawed and repeatedly washed with diss tilled water. The washed mycelium was ground and transferred …