Method for separate determination of activity of three-domain, two-domain laccases and abiotic oxidative activity in forest litter and soil

Abstract

FIELD: materials research.SUBSTANCE: invention can be used to study biochemical factors that control the processes of decomposition and formation of soil organic matter. The method provides for separate determination of the activity of three- and two-domain laccases and the abiotic oxidizing capacity of soil or forest litter in relation to 2.2' -azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid in the presence of sodium azide inhibitor NaN3. At the first stage, selective inhibition of three-domain laccases is carried out with sodium azide at a concentration of 0.1 mM±5% while maintaining the activity of two-domain laccases. At the second stage, the biotic component of oxidative activity is separated from the abiotic one by simultaneously inactivating three-domain and two-domain laccases while maintaining the oxidative activity of abiotic soil components, by completely inhibiting laccase activity with sodium azide at a concentration of 200 mM±5%. Based on the obtained data, the activity values of two- and three-domain laccases and abiotic activity maintained at a concentration of 200 mM±5% are calculated.EFFECT: accuracy and time reduction in the separate determination of the activity of three-domain and two-domain laccases, as well as abiotic oxidative activity in soil and forest litter.1 cl, 4 dwg, 2 tbl, 4 ex

Description

[0001] Field of technology to which the invention relates

[0002] The invention relates to the field of materials research and can be used to study biochemical factors that control the processes of decomposition and formation of soil organic matter.

[0003] Technology Level

[0004] Catalytic oxidation activity in forest litters and soils is crucial for the accumulation and stabilization of soil organic matter. Laccases are widespread enzymes that catalyze the oxidation of phenolic compounds and aromatic amines with molecular oxygen [Janusz, G., Pawlik, A., Świderska-Burek, U., Polak, J., Sulej, J., Jarosz-Wilkołazka, A., Paszczyński, A., 2020. Laccase properties, physiological functions, and evolution. Int J Molecular Sci 21, 966. https: / / doi.org / 10.3390 / ijms21030966]. As a result, phenoxyl radicals and quinones are formed, which undergo spontaneous condensation. Laccases are three- or two-domain proteins. Three-domain laccases are synthesized by bacteria and fungi, oxidize phenolic substrates under acidic conditions (pH 4-6), and participate in both the synthesis and degradation of organic matter [Baldrian, P., 2006. Fungal laccases—occurrence and properties.FEMS Microbiology Reviews 30, 215-242. https: / / doi.org / 10.1111 / j.1574-4976.2005.00010.x]. Two-domain laccases are produced only by bacteria, oxidize phenolic substrates under neutral and alkaline conditions (pH 7-9) and catalyze polymerization reactions, thus stabilizing organic carbon [Janusz, G., Pawlik, A., Świderska-Burek, U., Polak, J., Sulej, J., Jarosz-Wilkołazka, A., Paszczyński, A., 2020. Laccase properties, physiological functions, and evolution. Int J Molecular Sci 21, 966. https: / / doi.org / 10.3390 / ijms21030966]. Soil organic and mineral colloids, which are abiotic catalysts, also participate in the processes of oxidative transformation of organic matter in soils [Huang, PM, 2000. Abiotic catalysis. In: Handbook of Soil Science: Properties and Processes, pp. 303-334]. To study the abiotic component of the oxidative activity of soils, sterilization by autoclaving is usually carried out.However, this technique changes the soil particle size distribution, reducing the content of silt and fine dust [Li, H., Liu, L., Li, C. Liu, X., Ziadi, N., Shi, Y., 2023. Efficiency of different soil sterilization approaches and their effects on soil particle size distribution. J Soil Sci Plant Nutr 23, 3979–3990. https: / / doi.org / 10.1007 / s42729-023-01315-2], and partially destroys organic matter [Berns, AE, Philipp, H., Narres, H.-D., Burauel, P., Vereecken, H., Tappe, W., 2008. Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV, and fluorescence spectroscopy. Europ J Soil Sci 59, 540-550. https: / / doi.org / 10.1111 / j.1365-2389.2008.01016.x], which may lead to an underestimation of the oxidative activity of soil colloids in sterilized soils.

[0005] Methods for determining laccase activity in soil based on the catalytic oxidation of ABTS are known [CN 105200118 B, published 13.04.2018, CN 104020122 B, published 24.02.2016, CN 111235219 B, published 28.02.2023, CN 106248604 B, published 21.12.2016]. However, existing methods do not allow for separate determination of the activity of two- and three-domain laccases. Another drawback of the known methods is the inability to separate the abiotic component, while it can be significant in mineral horizons of soils with high content of manganese and iron [Huang, PM, 2000. Abiotic catalysis. In: Handbook of Soil Science: Properties and Processes, pp. 303-334]. There are no universal methods for separate determination of the activity of three- and two-domain laccases, as well as abiotic oxidative activity in soils and forest litter.Separate determination of two- and three-domain laccases, as well as the abiotic oxidative activity of forest litter and soils, will allow for a more detailed assessment and control of the factors responsible for the processes of decomposition and formation of soil organic matter.

[0006] The method for measuring laccase activity of soils according to CN 105200118 B, published 13.04.2018, consists of treating a fresh soil sample with a sterile 0.9% NaCl solution, followed by microtitering the extract with a solution of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid sodium salt (hereinafter referred to as ABTS) at a concentration of 20-30 mM in a 96-well plate for 1-5 hours by measuring the optical density at 420 nm every 5-30 min. The disadvantages of this method are: the stage of extraction of laccases from soils, which leads to underdetermination of laccase activity, since a significant part of the enzyme remains bound to the mineral component; long time of analysis; high consumption of reagents and the impossibility of separate determination of two- and three-domain laccases and abiotic oxidizing activity.

[0007] The method for determining laccase activity in soil according to CN 104020122 B, published on 24.02.2016, consists of extracting the enzyme with a phosphate buffer at pH 7.1 using ultrasound for 10-20 min, keeping it in a water bath for 20-30 min to restore the enzymatic activity and monitoring the change in the optical density of ABTS at a wavelength of 420 nm for 5 min. The disadvantages of the method are: the need for protein extraction and restoration of enzymatic activity in the samples; the short lifetime of the ABTS cation radical at neutral pH values ​​and the impossibility of separately determining two- and three-domain laccases and abiotic oxidizing activity.

[0008] The method for determining laccase activity in litter CN 111235219 B, published 02 / 28 / 2023, consists of extracting laccase with a universal buffer with pH 4.5, separating the extract from the litter material by centrifugation, incubating in a universal buffer at pH 4.5 for 60 min at 4°C, incubating an aliquot of the extract in the presence of ABTS for 30 min at 22°C, stopping the reaction by placing it in a freezer and measuring the optical density at a wavelength of 420 nm. The disadvantages of the method are: the extraction step, which leads to possible incomplete extraction and dilution of the enzyme solution, which reduces the sensitivity of the method, as well as the impossibility of separate determination of different types of laccases and abiotic oxidizing activity.

[0009] The method for determining laccase activity in soil according to CN 106248604 B, published December 21, 2016, involves preparing a system of laccase reverse micelles using isobutyltrimethylmethane to maintain enzyme stability during the isolation process, extracting laccases from the reverse micelles with acetate buffer, and measuring enzymatic activity using a colorimetric method using ABTS. A disadvantage of this solution is the use of toxic isobutyltrimethylmethane and the inability to separately determine laccases and abiotic oxidizing activity.

[0010] The closest analogue of the invention is a solution that allows for the separate determination of three-domain and two-domain laccases in soils, which consists in determining the oxidizing activity of soils in relation to 2 mM ABTS without using an inhibitor and using an inhibitor of sodium azide at a concentration of 0.1 mM and 50 mM, with subsequent calculation of the activity of three-domain laccases as the difference between the total oxidizing activity measured without an inhibitor and the activity in the presence of 0.1 mM NaN3, and the activity of two-domain laccases - by the difference between the oxidizing activity in the presence of 0.1 mM NaN3 and the activity in the presence of 50 mM NaN3 [Zavarzina, AG, Kulikova, NA, Trubitsina, LI, Belova, OV, Pyatova, MI, Danilin, IV, Pogozhev, PE, Kuzyakov, Y., Lisov, A.V., 2025. Disentangling two and three domain laccases in soils: contribution of fungi, bacteria and abiotic processes to oxidative activities. Soil Biol Biochem 208, 109861. https: / / doi.org / 10.1016 / j.soilbio.2025.[No. 109861, publication date: May 25, 2025, hereinafter [D1]]. A disadvantage of this solution is the high residual oxidative activity (from 5 to 80% of the total), which is partially or completely suppressed during autoclaving, suggesting its mixed nature, caused by both biotic and abiotic components in unknown ratios. Consequently, the technical solution does not allow for the separation of soil oxidative activity caused by the presence of biotic and abiotic components. The present invention utilizes a sodium azide concentration of 200 mM, which solves the problem of separating the activity of two-domain and three-domain laccases from the abiotic component of soil and litter oxidative activity without sterilization.

[0011] Thus, the technical problem solved by the claimed invention is the development of a method for separately determining the enzymatic activity caused by two- and three-domain laccases, as well as the abiotic component of oxidative activity in soils and forest litter. These indicators are important characteristics of the catalytic activity of soils, influencing the formation and stabilization of organic matter.

[0012] Disclosure of the essence of the invention

[0013] The technical result of the invention is the accuracy and reduction of time in the separate determination of the activity of three-domain and two-domain laccases, as well as abiotic oxidative activity in soil and forest litter.

[0014] The result is achieved due to the use of sodium azide (NaN3) inhibitor in certain concentrations, which allows for selective inactivation of three-domain laccases while maintaining the activity of two-domain laccases (NaN3 concentration of 0.1 mM with an acceptable deviation from the specified value of up to 5%) or simultaneous inactivation of three-domain and two-domain laccases while maintaining the oxidative activity of abiotic soil components (NaN3 concentration of 200 mM with an acceptable deviation from the specified value of up to 5%).

[0015] The technical result is achieved by a method for separately determining the laccase and abiotic components of oxidative activity in soil or forest litter, which method provides for separate determination of the activity of three- and two-domain laccases and the abiotic oxidative capacity of soil or forest litter in relation to 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid in the presence of a sodium azide inhibitor NaN3, in which, according to the invention, at the first stage, selective inhibition of three-domain laccases is carried out with sodium azide at a concentration of 0.1 mM while maintaining the activity of two-domain laccases. At the second stage, the biotic component of oxidative activity is separated from the abiotic by simultaneous inactivation of three-domain and two-domain laccases while maintaining the oxidative activity of abiotic soil components, achieving complete inhibition of laccase activity with sodium azide at a concentration of 200 mM.Based on the obtained data, the activity values ​​of two- and three-domain laccases and abiotic activity maintained at a concentration of 200 mM are calculated.

[0016] Using this method for determining the oxidative activity of litter and soil is convenient for determining biotic and abiotic components and eliminates sterilization, reducing analysis time. This method is useful for unifying and standardizing methods for measuring laccase activity in soils.

[0017] The novelty of the invention is due to the use of sodium azide in various concentrations for the selective inhibition of three-domain laccases or the inhibition of both types of laccases in the soil while maintaining the oxidative activity of abiotic components of the soil.

[0018] Brief description of drawings

[0019] Fig. 1 shows a graph of the dependence of the activity of three-domain laccases of L. tigrinus and C. comatus fungi immobilized on kaolinite-Al(OH)x on the concentration of NaN3.

[0020] Fig. 2 shows a graph of the dependence of the activity of two-domain laccases of actinobacteria S. carpinensis, S. viridochromogenes and Cat. japonicus immobilized on soil on the concentration of NaN3.

[0021] Figure 3 shows a graph of the dependence of the oxidative activity of coniferous and broad-leaved forest litter on the concentration of NaN3. The initial activity was 1.30 ± 0.10 U / g in coniferous litter and 1.32 ± 0.10 U / g in broad-leaved litter.

[0022] Fig. 4 shows a graph of the dependence of the oxidative activity of humus horizons of soils on the concentration of NaN3.

[0023] Implementation of the invention

[0024] The method is carried out as follows.

[0025] To determine the total oxidative activity, a litter sample of natural moisture content is ground in a mill, and soil of natural moisture content is passed through a sieve with a mesh diameter of 2 mm. Next, 50 mM Na-acetate buffer (pH 4.5) is added to a weighed portion of the litter or soil at a ratio of 1:20 (w / v). The sample is equilibrated with the buffer for 10 min while stirring (shaking manually or on a thermoshaker at a temperature of 25°C), the mixture is centrifuged, and then the supernatant (supernatant solution) is replaced with a solution of 2 mM ABTS in 50 mM Na-acetate buffer (pH 4.5), in a ratio of 1:30. A sample to which 50 mM Na-acetate buffer (pH 4.5) was added instead of the ABTS solution was used as a control. The mixture is then shaken for 2-4 min at 25°C, centrifuged for 1 min at 18,000 g, an aliquot of the supernatant is taken, diluted in 50 mM Na-acetate buffer (pH 4.5) in a ratio of 1:10 and the optical density is determined at a wavelength of 420 nm.At low optical density (D. 420 <0.2) the supernatant is not diluted. The soil moisture content (W) is determined in a separate sample. Based on the data obtained, the total oxidative activity (TOA) is calculated. общ ) soils using formula (1):

[0026] (1)

[0027] where A420experiment is the optical density of the ABTS solution at 420 nm; A420control is the optical density of the control sample at 420 nm; V is the volume of the ABTS solution added to the soil, ml; k = (100+W) / 100 is the conversion factor for an absolutely dry sample; 1000 is the conversion to micromoles; t is the total time of interaction of ABTS with the soil (shaking and centrifugation), min; 36000 is the extinction coefficient of ABTS at a wavelength of 420 nm, M -1 cm -1 [Heinfling et al., 1998], l is the optical path length, cm; m is the mass of soil at the initial moisture content, g.

[0028] For separate determination of oxidative activity of soils caused by two-domain laccases (OA 2D), three-domain laccases (OA 3D ), as well as abiotic oxidative activity (OA аб ) in separate weighed portions of soil or forest litter, the oxidizing activity is determined as described above, but using 50 mM Na-acetate buffer (pH 4.5) and a 2 mM ABTS solution containing 0.1 mM NaN3 or 200 mM NaN3 for equilibration. When preparing solutions containing 200 mM NaN 3, After adding sodium azide to the buffer, the pH of the solutions should be adjusted to 4.5 with glacial acetic acid, as sodium azide has an alkalizing effect. Next, using formula (1), determine the oxidizing activity in the presence of 0.1 mM or 200 mM NaN3. Then, calculate the activity of three-domain and two-domain laccases using formulas (2) and (3), respectively:

[0029] (2)

[0030] (3)

[0031] Abiotic oxidative activity of OA аб corresponds to the oxidative activity at 200 mM NaN3.

[0032] Examples of the invention implementation

[0033] Example 1. Determination of the activity of immobilized three-domain laccases in the presence of sodium azide

[0034] Enzyme preparations secreted by white rot fungi Lentinus tigrinus VKM F-160 and Coprinus comatus VKM 2940 (All-Russian Collection of Microorganisms, http: / / www.vkm.ru / index.htm) were used. To induce laccase, submerged cultivation of fungi was carried out in the presence of 0.1 mM Cu 2+Isolation and purification to an electrophoretically homogeneous state were carried out as described in [Zavarzina, AG, Demin, VV, Belova, OV, Leontievsky, AA, Lisov, AV, 2022. Heterophase synthesis of humic substances at low substrate concentrations and flow-through conditions. Euras Soil Sci 55, 911–925. https: / / doi.org / 10.1134 / S1064229322070146]. Immobilization of laccase on kaolinite containing aluminum hydroxide deposited on its surface (kaolinite-Al(OH)x) was carried out according to D1. The laccase activity was determined by the oxidation rate of 2 mM ABTS in 50 mM Na-acetate buffer (pH 4.5) in the presence of NaN3 at various concentrations: the initial activity of immobilized laccases in the absence of sodium azide, corresponding to 100%, was (0.198±0.030) conventional units / g and (0.833±0.008) conventional units / g for L. tigrinus and C. comatus, respectively. The change in the optical density of the ABTS solution in 1 min was taken as the conventional units of activity. The results are shown in Fig. 1.

[0035] As can be seen from Fig. 1, a significant decrease in laccase activity occurs already at a NaN concentration of 30.001 mM, and at a NaN concentration of 30.1 mM and higher, the activities of three-domain laccases are 0. Thus, sodium azide at a concentration of 0.1 mM completely inhibits the activity of immobilized three-domain laccases. Similar results can be obtained for other three-domain laccases in a free state or when immobilized on other mineral and organomineral supports.

[0036] Example 2. Determination of the activity of immobilized two-domain laccases in the presence of sodium azide

[0037] Recombinant enzymes of actinobacteria Streptomyces carpinensis VKM Ac-1300, Streptomyces viridochromogenes VKM Ac-629 and Сatenuloplanes japonicus VKM Ac-825 (All-Russian Collection of Microorganisms, http: / / www.vkm.ru / index.htm) were used. Expression, purification and characterization of laccase were carried out according to [Trubitsina, LI, Tishchenko, SV, Gabdulkhakov, AG, Lisov, AV, Zakharova, MV, Leontievsky, AA, 2015. Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes. Biochimie 112, 151-159. https: / / doi.org / 10.1134 / 2015.10.1015. org / 10.1016 / j.biochi.2015.03.005; Trubitsina, LI, Abdullatypov, AV, Larionova, AP, Trubitsin, IV, Alferov, SV, Ponamoreva, ON, Leontievsky, AA, 2021. Expression of thermophilic two-domain laccase from Catenuloplanes japonicus in Escherichia coli and its activity against triarylmethane and azo dyes. PeerJ 9, e11646. https: / / doi.org / 10.7717 / peerj.11646; Trubitsina, LI, Trubitsin, IV, Lisov, AV, Gabdulkhakov, AG, Zavarzina, AG, Belova, OV, Larionova, AP, Tishchenko, SV, Leontievsky, AA, 2023. A novel two-domain laccase with middle redox potential: physicochemical and structural properties. Biokhimiia 88, 1658-1667. https: / / doi.org / 10.1134 / S0006297923100188]. Immobilization of laccases on the humus horizon of sod-podzolic soil was carried out according to D1. Laccase activity was determined by the oxidation rate of 2 mM ABTS in 50 mM Na-acetate buffer (pH 4.5) in the presence of NaN3 in different concentrations: the initial activity corresponding to 100% was (0.240±0.003) conv. units / g, (0.024±0.001) conventional units / g and (0.060±0.001) conventional units / g for S. carpinensis, S. viridochromogenes and Cat. japonicus, respectively. The change in the optical density of the ABTS solution in 1 min was taken as the conventional units of activity. The results are shown in Fig. 2.

[0038] As can be seen from Fig. 2, two-domain laccases, unlike three-domain laccases (Fig. 1), are more resistant to the action of sodium azide. At a sodium azide concentration of 0.1 mM, the activity of two-domain laccases remains at a level close to 100%, and complete inhibition is observed only at a NaN3 concentration of 200 mM and higher. At a sodium azide concentration of 50 mM, the laccase of S. viridochromogenes immobilized on soil retains approximately 20% of the initial activity, while that of S. carpinensis laccase retains approximately 10%. This indicates that the sodium azide concentration of 50 mM used in D1 may be insufficient for complete inhibition of two-domain laccases. The results demonstrate that the different sensitivities of three- and two-domain laccases to the inhibitory effect of NaN3 makes it possible to separately determine them in natural samples. Similar results can be obtained for other two-domain laccases in a free state or when immobilized on other mineral and organomineral supports.

[0039] Example 3. Determination of oxidative activity of laccases in forest litter

[0040] The materials used were coniferous litter collected under a wood sorrel spruce forest (Moscow Region) and deciduous litter collected in a broad-leaved forest (oak and maple litter, Tula Region). The organic carbon content in the coniferous and broad-leaved litters was 321.9 g / kg and 184.8 g / kg, respectively. The oxidative activity of the litters was determined by the oxidation rate of 2 mM ABTS in 50 mM Na-acetate buffer (pH 4.5) in the presence of NaN3 at concentrations from 0.1 to 200 mM. Additionally, the oxidative activity was determined in litter samples sterilized twice in an autoclave (120°C, 1 atm., 30 min). The experiments were repeated five times. The results are shown in Fig. 3 and Table 1.

[0041] As can be seen from Fig. 3, with an increase in the sodium azide concentration, the oxidative activity of the litter in relation to ABTS decreases, which is consistent with the trend of a decrease in laccase activity in the presence of sodium azide (Figs. 1, 2) and indicates the contribution of the laccase component to the oxidative activity. At a sodium azide concentration of 200 mM, complete inhibition of activity is achieved in both coniferous and broadleaf litter. At a concentration of 50 mM NaN3, residual enzymatic activity remained at a level of 26% and 16% of the initial, respectively, indicating incomplete inhibition of laccase activity, since forest litter is characterized by a low content of mineral particles that determine abiotic oxidative activity. The obtained results show that the use of sodium azide at a concentration of 200 mM allows for the complete inhibition of the biotic component of oxidative activity caused by the presence of laccases.

[0042] Based on the obtained data, the activities of two- and three-domain laccases, as well as the abiotic oxidizing activity of forest litter, were calculated (Table 1).

[0043] Table 1. Activities of two- and three-domain laccases, as well as abiotic oxidizing activity of forest litter, U / g (mean ± standard deviation, fivefold replication)

[0044] Total oxidizing activity (TOAtotal) Activity of three-domain laccases (OA3D) Activity of two-domain laccases (OA2D) Abiotic oxidizing activity (AOA) Oxidizing activity of autoclaved sample Coniferous litter 1,30 ± 0,06 0,17 ± 0,07 1,07 ± 0,04 0,06 ± 0,04 0,00 ± 0,00 Broadleaf litter 1,32 ± 0,09 0,18 ± 0,08 1,14 ± 0,08 0,00 ± 0,00 0,00 ± 0,00

[0045] As can be seen from Table 1, higher activity rates of two-domain laccases are observed in the litter, while the abiotic component of oxidative activity is absent.

[0046] Example 4. Determination of the oxidative activity of laccases in humus horizons of soils

[0047] Samples from the 0-5 cm depth of the humus horizons of sod-podzolic (southern taiga, Moscow region), gray forest, dark gray forest soils (broad-leaved forests, Tula region) and chernozem (forest-steppe, Lipetsk region) were used. The organic carbon content in the samples was 35.3 g / kg, 37.8 g / kg, 69.1 g / kg and 69.3 g / kg, respectively. The Mn content was 2.3 mmol / kg, 19.7 mmol / kg, 6.9 mmol / kg and 6.2 mmol / kg, respectively. Laccase activity was determined by the oxidation rate of 2 mM ABTS in 50 mM Na-acetate buffer (pH 4.5) in the presence of NaN3 at concentrations from 0.1 to 200 mM. Oxidative activity was additionally measured in soil samples sterilized twice in an autoclave (120°C, 1 atm, 30 min). The results are shown in Fig. 4 and Table 2.

[0048] As can be seen from Fig. 4, the inhibitory effect of sodium azide in soil samples is weaker than in litter, especially in samples with a high Mn content (gray forest soil). This is due to the oxidative contribution of the mineral component, i.e., abiotic oxidation. The residual oxidative activity at 200 mM NaN3 was 17-29% of the activity without sodium azide in sod-podzolic, dark gray forest, and chernozem soil samples, and 68% in the gray forest soil sample. Since other phenoloxidases resistant to high concentrations of sodium azide, except for two-domain bacterial laccases, are unknown, the residual oxidative activity determined in soils in the presence of 200 mM NaN3 is due to the abiotic component (soil colloids).

[0049] The activities of two- and three-domain laccases, calculated on the basis of the obtained data, as well as the abiotic oxidizing activity of soil organic horizons are presented in Table 2.

[0050] Table 2. Activities of two- and three-domain laccases, as well as abiotic oxidizing activity of organic horizons of soils of different soil-geographical zones, U / g (mean ± standard deviation, fivefold replication)

[0051] Total oxidizing activity (TOAtotal) Activity of three-domain laccases (OA3D) Activity of two-domain laccases (OA2D) Abiotic oxidizing activity (AOA) Oxidizing activity of autoclaved sample Sod-podzolic soil (Moscow region) 1,19 ± 0,10 0,00 ± 0,13 0,98 ± 0,05 0,21 ± 0,02 0,00 ± 0,00 Gray forest soil (Tula region) 1,06 ± 0,07 0,00 ± 0,10 0,35 ± 0,05 0,72 ± 0,02 0,01 ± 0,00 Dark gray forest soil (Tula region) 0,87 ± 0,07 0,06 ± 0,11 0,56 ± 0,05 0,25 ± 0,01 0,00 ± 0,00 Black soil (Lipetsk region) 0,72 ± 0,03 0,08 ± 0,04 0,46 ± 0,02 0,18 ± 0,01 0,00 ± 0,00

[0052] In the humus horizons of soils, the total oxidative activity is comparable (sod-podzolic and gray forest soil) or slightly lower than in the litter (Table 1); the activity of three-domain laccases is low or absent. The activity of two-domain laccases accounts for a significant proportion (30-80%) of the total oxidative activity. Residual activity stable to 200 mM NaN3 accounts for 17-68% of the total. The absence of activity in autoclaved samples indicates a disruption of the native state of soil colloids during sterilization.

[0053] Conclusion: the given examples confirm the implementation of the purpose - the possibility of separate determination of the activity of three-domain, two-domain laccases and the abiotic component of the oxidative activity of soils and litters.

[0054] Based on the analysis of the oxidative activity of soils and litter at sodium azide concentrations from 0.1 to 200 mM, an inhibitor concentration (200 mM) was established that allows separating the biogenic and abiotic components of oxidative activity without autoclaving the samples.

[0055] By analyzing the oxidative activity of soils and litter at sodium azide concentrations ranging from 0.1 to 200 mM, it was demonstrated that a concentration of 200 mM completely suppresses oxidative activity in litter (where the mineral component of soils, which determines the abiotic component of oxidative activity, is absent), which clearly indicates the predominantly biogenic nature of the residual activity, caused by two-domain laccases stable in the presence of 50 mM sodium azide (the high stability of some 2D laccases to an azide concentration of 50 mM is also shown in Fig. 2). It was established that a concentration of 200 mM is sufficient to suppress the activity of two-domain laccases. The activity retained at 200 mM is abiotic. This activity is higher than the activity of autoclaved samples, indicating that sterilization by autoclaving disrupts the native state of soil colloids and leads to underdetermination of the abiotic component of soil oxidative activity.Thus, in D1, using a 50 mM sodium azide concentration leads to underestimation of 2D laccase activity, while autoclaving leads to underestimation of the abiotic component of oxidative activity. Using 200 mM sodium azide eliminates both problems of the previous solution and represents not just a selection of optimal conditions, but a fundamentally new and previously unused approach to separating the biogenic and abiotic components of oxidative activity in soils and forest litter.

Claims

A method for the separate determination of the laccase and abiotic components of oxidative activity in soil or forest litter, which provides for the separate determination of the activity of three- and two-domain laccases and the abiotic oxidative capacity of soil or forest litter in relation to 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid in the presence of a sodium azide inhibitor NaN3, characterized in that - at the first stage, selective inhibition of three-domain laccases is carried out with sodium azide at a concentration of 0.1 mM ± 5% while maintaining the activity of two-domain laccases, - at the second stage, the biotic component of oxidative activity is separated from the abiotic component by simultaneously inactivating three-domain and two-domain laccases while maintaining the oxidative activity of abiotic soil components, by completely inhibiting laccase activity with sodium azide at a concentration of 200 mM ± 5%, - based on the obtained data, the activity values ​​of two- and three-domain laccases and abiotic activity maintained at a concentration of 200 mM ± 5% are calculated.

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