RU2817282C1 - Photocolorimetric method for assessing dehydrogenase activity of protein extracts - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a method for evaluating dehydrogenase activity of protein extracts obtained from microorganisms, which consists in the fact that in a measuring temperature-controlled cell, which includes a signal recording system, working solution containing a protein extract, a substrate and potassium ferricyanide are introduced, and dehydrogenase activity is determined from the rate of change in the concentration of potassium ferricyanide in the linear phase of the enzymatic reaction.

EFFECT: invention enables rapid assessment of dehydrogenase activity.

3 cl, 1 dwg, 1 tbl

Description

The invention relates to biotechnology and can be used to evaluate the dehydrogenase activity of protein extracts obtained from microorganisms using potassium ferricyanide. Protein extracts are a new type of bioelectrocatalyst for biofuel cells. The protein extract contains a cascade of enzymes and associated coenzyme systems that carry out oxidative and reductive transformations. Technological production of protein extract requires methods for assessing dehydrogenase activity to control product quality.

Currently, there are a number of photocolorimetric methods for determining the dehydrogenase activity of biological objects.

Photocolorimetric methods using tetrazolium salts are based on the ability of dehydrogenases to abstract hydrogen from various substrates (organic acids, alcohols, carbohydrates, etc.) and reduce tetrazolium salts to colored formazan (patent RU 2 491137 C1 “Method for monitoring the effectiveness of reclamation of disturbed tundra soils of various types granulometric composition by analyzing dehydrogenase activity", patent RU 2 387 996 C1 "Method for monitoring the purification of soils contaminated with hydrocarbons and neutralizing hydrocarbon sludge by analyzing dehydrogenase activity", patent RU 2 199 211 C2 "Method for determining the viability of earthworm cocoons", methodological instructions on sanitary microbiological study of soil (approved by the chief state sanitary doctor of the USSR 02/19/81 No. 2293-81 “Determination of dehydrogenase activity of microorganisms”, Zhang, Xinying, et al. “Responses of Scirpus triqueter, soil enzymes and microbial community during phytoremediation of pyrene contaminated soil in simulated wetland." Journal of Hazardous Materials 193 (2011): 45-51).

These methods based on tetrazolium salts are characterized by the following disadvantages:

1. Duration of analysis;

2. The need to work with a volatile solvent for formazan;

3. The inability to determine the dehydrogenase activity of biological material containing copper due to the negative effect of copper on the absorption of the reduction product of the hydrogen acceptor tetrazolium salts.

Photocolorimetric methods using methylene blue as a hydrogen acceptor (patent RU 2 476 598 C2 “Method for quantitative determination of dehydrogenase activity of microorganisms”, Chekalov V.P. Determination of sorption capacity and dehydrogenase activity of bottom sediments using methylene blue // Sea Ecology. - 2006 . - T. 72. - P. 103-109., Varakin E. A. et al. Determination of dehydrogenase activity of activated sludge microorganisms in the process of biological wastewater treatment // Biotechnology in the chemical forest complex - 2014. - P. 99. -104.) are characterized by the following disadvantage - the need to create anaerobic conditions for carrying out measurements due to the oxidation of the reduced form of the colored reagent by atmospheric oxygen.

The problem to be solved by the present invention is the development of a method that allows for rapid assessment of dehydrogenase activity and is devoid of the above-mentioned disadvantages.

This is achieved by the fact that when quantitatively determining the dehydrogenase activity of protein extracts, which involves their contact with a substrate and a hydrogen acceptor, potassium ferricyanide is used as a hydrogen acceptor, and the activity is determined by the rate of change in the concentration of potassium ferricyanide in the linear phase of the enzymatic reaction.

In the proposed method, dehydrogenase activity is assessed by reducing the concentration of potassium ferricyanide, which is continuously recorded optically. The test sample of protein extracts is introduced into a thermostated measuring cell, including a signal recording system (LED, photosensor), a substrate mixture is added to the extract, which is prepared by mixing 15 mM potassium ferricyanide in a buffer solution and 1 M glucose in a buffer solution in a 1:1 ratio. The duration of the experiment is 10-15 minutes.

An illustrative example of the implementation of the proposed method includes the following steps: a working solution is prepared by mixing 100 μl of the protein extract being determined and 200 μl of the substrate mixture, which is prepared by mixing 15 mM potassium ferricyanide in a buffer solution and 1 M glucose in a buffer solution in a 1:1 ratio. Then the working solution is transferred to the measuring tablet and the spectrophotometer is turned on, preheated to 37°C. The spectrophotometer begins to record a decrease in the concentration of potassium ferricyanide during the enzymatic reaction by the optical density of the working solution. Under conditions of excess substrate and hydrogen acceptor, the reaction progresses linearly for approximately 300 seconds, after which the measurement process is stopped. The obtained values of the optical density of the solution were converted to ferricyanide concentration using the formula C(FC)=OD/0.4824, where

C(FC) - potassium ferricyanide concentration,

OD - obtained optical density values.

Based on the calculated values, a graph of C(FC) versus t is plotted. Photosensor readings are recorded once every 2 seconds and approximated by a linear dependence on time (Fig. 1). For the resulting graph, the initial linear section is determined, for which a tangent is constructed, and the tangent of the angle of inclination of this tangent is found (Fig. 1, curve 2). The formula C=v*t allows you to find v as the angle of inclination of the constructed tangent, thus finding the reaction rate v, which characterizes dehydrogenase activity as a kinetic parameter. Using the example of figure 1, the dehydrogenase activity estimated using the described method was 0.00159 mmol/s.

Instruments for measuring optical density are known to one skilled in the art. In particular, a spectrophotometer can be used that has a computer interface, has a thermostated cell and provides mixing of the solution. A calibration must first be made between the optical density and the concentration of potassium ferricyanide in solution (which is obvious to one skilled in the art).

Table 1 presents a comparison of DHA extracts obtained from E. Coli, evaluated using ferricyanide and TTX

As can be seen, the activity measured by both methods decreases from sample 1 to sample 4. Thus, the dehydrogenase activity of extracts obtained from E. Coli, assessed by the photocolorimetric method using ferricyanide, correlates with the data obtained using the photocolorimetric method using TTX.

Claims (3)

1. A method for assessing the dehydrogenase activity of protein extracts obtained from microorganisms, which consists in introducing a working solution containing a protein extract, substrate and potassium ferricyanide into a measuring thermostated cell, including a signal recording system, and the activity of dehydrogenases is determined by the rate of change in the ferricyanide concentration potassium in the linear phase of the enzymatic reaction. 2. The method according to claim 1, characterized in that a working solution consisting of 100-200 μl of protein extract and 200-300 μl of a substrate mixture, which is prepared by mixing 15 mM potassium ferricyanide in a buffer solution and 1 M glucose in a buffer solution in a 1:1 ratio. 3. The method according to claim 1, characterized in that an LED or photosensor is used as a signal recording system.

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