SU1620483A1 - Method of producing enzymic preparation for quantitative analysis of thiamine diphosphate in biological objects - Google Patents

Abstract

The invention relates to methods for producing enzyme preparations from microbiological raw materials and can be used in the enzyme industry, and the resulting complex is for quantitative determination of the level of thiamine diphosphate (TDF). The purpose of the invention is to expand the technological capabilities of the method, accelerate the process and reduce the cost of the target product. The method involves the use of a fresh pasty mass of brewer's yeast 7-9 regenerations. A suspension is prepared to extract the protein preparation, which is sonicated at a frequency of 20-22 kHz for 18-25 minutes, followed by two-stage fractionation with acetone and ammonium sulfate. At the first stage of acetone fractionation, 55 ml of acetone is added to every 1UU ml of yeast extract, and 10 ml is added to the second. Final purification is performed by adsorption chromatography on axiapatite by elution of a complex of 0.12 M phosphate buffer to a ratio of pyruvate decarboxylase and alcoholdehydrogenase activities in the enzyme complex 1: (0.15-0.5) with pyruvate decarboxylase activity of at least 14 units. act./mg protein. After final purification of the protein preparation, apopiruvated decarboxylase is obtained in the composition of the complex with its subsequent stabilization. 3 il. 3 tab. and SS О5 ГС О Ј 00 with

Description

The invention relates to biotechnology for producing enzyme preparations from microbiological raw materials and can be used in the enzyme industry, and the resulting complex is used for quantitative determination of the level of thiamine diphosphate (TDF) in

blood or other biological fluids for the purpose of diagnosing early 1 Bi-deficiency during the examination of 1 different layers of the population at stationary clinics or under normal conditions, as well as in animal husbandry when growing young on livestock complexes and in animal husbandry.

Determination of total TDF in biological objects is carried out by an enzymatic method, which is based on the ability of a diphosphoric ester as a coenzyme to recombine in an apopyruvate decarboxylase. The resulting active protein choloform oxidizes pyruvate to acetic aldehyde, which in the conjugate system with alcohol dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) is converted into ethyl alcohol. The reaction rate is judged by the decrease in NADH absorption at 340 nm. The quantitative content of TDF, which is proportional to the act of introducing the formed holopiruvate decarboxylase and a decrease in the reduced NAD, is determined using a calibration curve with known concentrations of the coenzyme. The main components of the system, which ensure the recombination of thiamine diphosphate and the subsequent oxidation of pyruvate, and then NADH, are alcohol dehydrogenogenase and apopiruvate decarboxylase.

The purpose of the invention is to expand the technological capabilities of the method, speeding up the process and cheapening the target product.

The method is carried out as follows.

A fresh pasty mass of Saccharomyces carls-bergensis 7-9 yeast beer yeast is taken for regeneration and a suspension is prepared to extract the protein preparation. The prepared suspension is treated with ultrasound at a frequency of 20-22 kHz for 18-25 minutes and two-stage fractionation with acetone and ammonium sulfate is carried out. At the pseudo stage of acetone fractionation, 55 ml of acetone is added to each 100 ml of yeast extract, and 10 ml of acetone is added to the second. Then, adsorption chromatography on hydroxyapatite is performed by elution of a complex of 0.12 M phosphate buffer to a ratio of pyruvate decarboxylase and alcohol dehydrogenase activity in the resulting enzyme complex 1: (0.15-0.5), and the pyruvate decarboc strength should be at least 14 2d by acto. / mg protein.

After the final purification of the protein preparation on hydroxyapatite, apopiruvate decarboxylase is obtained in

the composition of the complex with its subsequent stabilization, as the enzymatic method for the determination of TDF in biological objects using

The enzyme complex is based on the ability of a diphosphoric ester as a coenzyme to recombine with an apopiru-BL decarboxylase.

The best effect from use. of the method is observed when the acetone fractionation is carried out in the first stage by adding 55 ml of acetone for every 100 ml of yeast extract

and final purification of the target product by elution of a complex of 0.12 M phosphate buffer to the ratio of pyruvate decarboxylase and alcohol dehydrogenase activities in the enzyme

0 complex 1: (0,15-0,5), with pyruvate decarboxylase activity of at least 14.0 units of act. / Mg protein.

Increasing the amount of acetone added over 55 ml for every 100 ml

5 of the yeast extract in the first stage of acetone fractionation leads to the removal of one of the two components from the enzyme complex - alcohol-dehydrogenase. With an increase in the initial volume of acetone from 58 to 60 ml, added for every 100 ml of yeast extract, the enzyme complex contains residual concentrations (traces) of alcohol dehydrogenase, and an increase in the amount of acetone over 60 ml does not reveal alcohol dehydrogenase in the complex. Reducing the volume of acetone below 55 ml added to each yeast extract will drastically reduce the pyruvate decarboxylase content in the enzyme complex. When adding 52-53 ml of acetone for every 100 ml of yeast extract, the enzyme complex contains traces of pyruvate decarboxylase; when 54 ml of acetone is added, the activity of pyruvate decarboxylase is reduced.

An increase in the phosphate buffer morbidity above 0.12 M leads to a decrease in the activity of the enzyme comtex due to the elution of impurity proteins, and a decrease in the molarity of the same buffer below 0.12 M promotes the separation of alcohol dehydrogenase and pyruvate decarboxylase (alcohol dehydrogenase is found on the column with adsorbent) . To quantify thiamine diphosphate in biological objects using the enzymatic method,

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so that the ratio of pyruvate decarboxylase and alcohol dehydrogenase activities in the enzyme complex was 1: (0.15-0.5) with pyruvate decarboxylase activity of at least 14.0 units of act. / mg of protein.

Exceeding these upper limits does not lead to a significant increase in the sensitivity of the enzymatic method for the determination of TDF using an enzyme complex in biological objects compared to

with an applied ratio of pyruvate-decarboxylase and alcohol dehydrogenase activities, and a decrease in these limits contributes to a sharp decrease in sensitivity. In addition, a decrease in pyruvate decarboxyl activity lower than 14.0 units of act./mg protein also leads to a significant weakening of the sensitivity of the enzymatic method for determining TDF using this complex in biological objects.

The invention is illustrated by examples, i

Example

1. Preparation of the starting material, isolation and purification of the complex of pyruvate decarboxylase and alcohol dehydrogenase. Take 5 kg of fresh beer yeast Saccharomyces carlsbergensis 7-9 regeneration. Fresh yeast cells are washed several times with cold water and centrifuged for 15 minutes at 5000 g. The resulting pasty mass is used to prepare a suspension and extract the protein preparation. To do this, 400 g of yeast cells are mixed with 1750 ml of an aqueous solution containing 67 ml of glycerol, 0.668 g of EDTA, 13.2 g and 2.8 g (. The prepared suspension is vigorously stirred for 10 minutes. The mixture is cooled to -2 ° C and sonicate at a frequency of 22 kHz for 20 minutes, until complete destruction of the cells is achieved, contributing to the maximum yield of the final product. The obtained yeast extract is centrifuged for 30 minutes at 4000 g and then 1 hour at 105000 g. The subcellular fractions that have settled after centrifugation are removed and for every 100 ml nadosad 55 ml of acetone, cooled to -20 ° C, are added with continuous stirring to prevent the solution from heating above 0 ° C.

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After 15 minutes at this temperature, the suspension is centrifuged for 5 minutes at 4000 g and the precipitate is discarded. For every 100 ml of supernatant, 10 ml of acetone is re-added, reducing the temperature of the solution to -2 ° C. After 15 minutes, the extract is centrifuged, and the final residue is quickly suspended in 400 ml of 0.02 M Tris-HCl buffer, pH 7.3, with 1 M EDTA and 5 mM cysteine hydrochloride. After 30 minutes of stirring at 0 ° C, homogeneous protein growth is

5 The thief is subjected to ammonium sulfate fractionation by adding 108 g of dry ground (K1C), 504. After 1 h, the mixture is centrifuged, the precipitate is discarded, and for every 100 ml of supernatant, 7 g of ammonium sulfate are re-introduced within 20 minutes with stirring. Salted protein is collected by centrifugation.

Final purification is carried out by the method of adsorption chromatography on hydroxyapatite. The pH and ionic strengths of the equilibrium buffer were experimentally selected, in which the enzyme complex does not bind with the adsorbent and is found in the very first fractions of the eluting solution, while the associated impurities are still effectively retained on the carrier. To this end, protein (3 g) of sulfo-ammonium paste is dialyzed for 4 hours against a 20-fold volume of 0.12 M sodium phosphate buffer, pH 6.8, with 2 mM Mg2, 1 mM EDTA, 0.5 mM TDF and 5 mM cysteine

hydrochloride, and the precipitated precipitate is removed by centrifugation. The supernatant was diluted with the original 0.12 M sodium phosphate buffer to a protein concentration of 10 mg / ml, mixed with 3 times the volume of hydroxyapatite compacted sediment, equilibrated with the same buffer, and centrifuged at 10 ° C for 10 min. 10 min at 12000g. The supernatant contains a mixture of alcohol-dehydrogenase and pyruvstedecarboxylase. For completeness of extraction, the adsorbent is rewashed, the protein of the supernatant is combined, precipitated (NH4) SO (salting out) and collected by centrifugation. At this stage of purification (Table 1), the specific activity of pyruvate decarboxylase increases 2.9 times (from 4.8 units of act. / Mg

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, 0 units of act. / Mg), and alcoholdehyde of rogenase - 1.75 times (from 1, 2 units of act. / To 2, units of act. / Mg) and is acceptable for enzymatic determination of the content of TDF . The ratio of pera vatdecarboxylase (14 units of act. / Mg of protein) and alcohol dehydrogenase (2.1 units of act ,, / mg of protein) activity in the enzyme complex is 1: 0.15 (Table 1).

The excretion process takes 12 hours.

Example 2: Preparation of apopiru vatdecarboxylase in the complex and its stabilization.

The preparation of apopiruvate decarboxylase is based on the ability of a holoform farm to easily dissociate in an alkaline medium with the release of TDF and ions.

l ji

Mg. To remove the coenzyme, the enzyme complex (680 mg) precipitated after fractionation on hydroxy patite is dissolved in 5 ml of 0.01 M Tris-HCl buffer, pH 950, with 1 mM EDTA and 5 mM cysteine hydrochloride, is removed for complete dissociation at pH for 15 min and applied to a column (5.4 x 70 cm) with dextran gel ED-1.0 (fraction up to 50 µM), equilibrated with the initial buffer. By

as protein yield solution is collected in a volume of 30 ml. TDF elutes later starting with 40 ml. In this state without coenzyme, the apoenzyme is sensitive to the presence of lyophhydryl reagents, metal ions, the effect of pH, temperature and ionic strength of the buffer solution, therefore the flow rate of the liquid through the column should be quite high (36-48 mp / h), and the eluted protein after release is immediately stabilized (the capacity for recombination of yeast apopiruvate decarboxylase is retained in 0.01 M Tris-HCl buffer, pH 9.0 for an hour), All operations for the isolation of the apoenzyme and subsequent protection against inactivation and is carried out at.

The stabilization of the apopiruvate decarboxylase and alcohol dehydrogenase complex is carried out with glycerol or ammonium sulfate in the presence of Mg2-4 ions. In the first case, the pH of the apoferment solution eluted from the column is reduced by 0.05 M sodium phosphate buffer to 6.8, and then glycerol and 0.5 M MgSO $ are added to the final concentration of components 30 and 0.02 M, respectively. Wto

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 (

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rum - an equal volume of 5.4 M ammonium sulfate in 0.1 M sodium phosphate buffer, 8, with 0.04 M MgSO4 is poured into the protein eluate with continuous stirring, after which the protein is lyophilized using a freeze-drying unit at a temperature of

and pressure b .. At 30%

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glycerol in the presence of Mg, the reactivation of apopiruvate decarboxylase with low concentrations of TDF and alcohol-dehydrogenase activity remains high within 1.0-1.5 weeks, allowing serial studies of the level of TDF in biological objects without the daily practice of selecting the required apoenzyme protein concentration, observed in work with sulfo-ammonium preparations.

Lyophilized from 2.7 M (0.02 M MgSOq. Complex is stable throughout the year (Table 2).

Example 3. Model system for quantification of TDF in biological objects: in animal tissues, blood.

In order to diagnose B-deficiency with the use of an enzyme complex of animals, 12 hours before slaughter, they are deprived of food. After decapitation of the animal, the studied tissues are quickly removed, cleaned from the membranes: they are forced through a press with a pore diameter of 1 mm and homogenized in a homogenizer with a Teflon pestle (7–9 movements) with 5 volumes of 17% trichloroxy xenoic acid cooled to 0–2 ° C. The homogenate is centrifuged for 10 minutes at

3000 rpm, and the precipitate is re-mixed with 5 volumes of 7% trichloroxy acid at 0-2 C, followed by centrifugation. The supernatant obtained from two centrifugations is treated twice with 3 volumes of a saturated aqueous solution of ether to remove the trichloroxy acid. The final dilution for the liver, kidney, heart tissues is 1:20, and the brain and muscles 1:10 is achieved by adding to the protein-free extract of 0.05 M sodium phosphate buffer pH 6.8, after which 0.1 ml aliquots are taken for recombination of TDF contained in tissues with aprivate decarboxylase of the furancy complex. Recombination is carried out at room temperature for 30 minutes.

To do this, to the analyzed aliquots, add 0.1 ml of 0.02 M MgSO1 and 0.1 ml of a solution of the purified enzyme complex of apopiruvatecarcinyl oxidase with alcohol dehydrogenase (200-600 µg of protein) diluted to the required concentration of 0.05 M sodium phosphate buffer, pH 6.8. Overall

The abscissa axis is the concentration of TDF used, and the ordinate is the corresponding extrusion value minus the control fixing the degree of spontaneous oxidation. NADH in the absence (see Fig. 1-3). B during the enzymatic detection of total TDF in the analyzed blood or tissue samples

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the volume was adjusted with 0.05 M sodium-JQ baseline, recorded in the experiment with phosphate buffer, to 0.8 ml. After the time of incubation, 2.0 ml of 0.15 M sodium citrate buffer, pH 5.9 and 0.1 ml of 0.2 M sodium pyruvic acid are introduced into each sample. The reaction is started by adding 0.1 ml of 0.5 mM NAD N, measuring the change in the optical density of the solution for the first 3 minutes at 340 nm. From the total value of the spectrophotometrically recorded optical density (OD), they find the corresponding value of a point on the calibration curve. Its parameters, multiplied by the original tissue dilution, correspond to the desired value of the concentration of total TDF.

Blood is taken from the bezim of the finger of the examined patients who have not taken food for 8-10 hours prior to the analysis. To determine the level of TDF, 0.2 ml of capillary blood is mixed with 0.4 ml of 0.05 M sodium phosphate buffer, pH 6.8, containing 2% heparin cooled to 4 ° C. After a 15-minute exposure at 4 ° C, the mixture is boiled for 2 minutes, centrifuged for 10 minutes at 3000 rpm, and 0.2 ml aliquots are taken from here to recombine the TDF of the test samples with apopiruvate decarboxylase. The conditions for recombination are identical to those for animal tissue.

The desired points for the calibration graph are in conditions similar to recombination of the blood or tissue samples under test, instead of using 0.1 ml of tissue

extract or 0.2 ml of blood, known concentrations (0.005-0.2 μg) of the chromatographically pure preparation TDF. According to the resulting extrinsic differences of reduced and oxidized NAD, recorded the first 3 minutes of the conjugate reactive reaction of a reactively slapped decarboxylase and the alcohol dehydrogenase of yeast at 340 nm, a calibration curve is constructed, putting aside

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the values are extrapolated to a calibration curve, and then to the absolute axis with known concentrations of TDF.

Example 4. The interval of concentrations of TDF in which the linearity of the conjugate process is maintained with the use of the complex.

N # FIG. Figures 1-3 show the dependences of the total rate of the conjugate enzymatic process on the concentration of TDF at various polar-to-center concentrations of the proposed complex in the absence (Fig. 1-2 a) and when additionally applied (Fig. 1 and 2 b) to the dimensional alcohol dehydrogenase. K can be seen from the graphs, in the test and in the interval of coenzyme concentrations, n protein content of 0.18-0.60 mg, satisfactory linearity of the total process rate is maintained up to 0.3 µg TDF. At a lower than 0.18 mg content of the complex in the strength of the insufficient extinction difference between the control and the experimental samples, in the case of low concentrations of TDF, the accuracy of the enzymatic determination decreases (see Table 3). A concentration of 0.6 mg of the complex practically provides protein saturation and its increase contributes to the non-productive costs of the complex. The addition of commercial alcohol dehydrogenase (50 µg per sample) to the complex does not lead to an increase in the rate (see Fig. 1-2) of the conjugate process, thereby indicating the sufficiency of the enzymatic activity of the yeast alcohol dehydrogenase in the complex.

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Example 5. Sensitivity accuracy.

According to the table. 3, the enzymatic method of quantitative determination of total TDP using the complex allows one to fix the concentration of coenzyme in a dose of 0.005 microns, and the measurement accuracy averages 1.96%.

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Q her recorded in the experiment

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the values are extrapolated to the calibration curve, and then to the abscissa axis with known concentrations of TDF.

Example 4. A concentration range of TDF, in which the linearity of the conjugate process using the complex is maintained.

N # FIG. 1-3 show the dependences of the total rate of the conjugated enzymatic process on the concentration of TDF at various polar concentrations of the proposed complex in the absence (Fig. 1-2 a) and with additional addition (Fig. 1 and 2) of commercial alcohol dehydrogenase. As can be seen from the graphs, in the studied concentration range of coenzyme, with a protein content of 0.18-0.60 mg, satisfactory linearity of the total process rate is maintained up to 0.3 µg TDF. With a lower content of the complex than 0.18 mg, due to the insufficient extinction difference between the control and experimental samples in the case of low concentrations of TDF, the accuracy of the enzymatic determination decreases (see Table 3). A concentration of 0.6 mg of the complex practically provides protein saturation and its increase contributes to the overhead of the complex. The addition of commercial alcohol dehydrogenase (50 µg per sample) to the complex does not lead to an increase in the rate (see Fig. 1-2) of the conjugate process, thereby indicating that the enzymatic activity of the yeast alcohol dehydrogenase in the complex is sufficient.

and

Example 5. Sensitivity accuracy.

According to the table. 3, an enzymatic method for quantifying total TDP using a complex allows the concentration of coenzyme to be recorded at a dose of 0.005 µg, and the measurement accuracy is on average 1.96%.

Claims (1)

The invention The method of obtaining an enzyme preparation for the quantitative determination of thiamine diphosphate. in biological objects, providing for the isolation of pyruvate decarboxylase from brewer's yeast extract by fractionation with acetone, fractionation with ammonium sulfate in the concentration range of 44-53% saturation and chromatographic purification, characterized in product together with pyruvate decarboxylase, alcohol dehydrogenase is separated, the fractionation of the extract with acetone is carried out in a concentration range of 35.5-41.1% and chromatographic purification is carried out by the method of adsorption chromatography on hydroxyapatite in a 0.12 M phosphate buffer to the ratio of pyruvate decar – oxylase and alcoholidehydride - rogenase activities in the enzyme preparation 1: (0.15-0.5) and up to a value of pyruvate decarboxylase activity of not less than 14.0 units / mg of protein, and pyruvat decarboxylase in the preparation is obtained as an apoenzyme. Table t Continuation of table 2