RU2032743C1 - Method for producing of yeast alcohol oxidase - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves cultivation of yeast as producer of alcohol oxidase (AO). Collected cells of cultivated biomass are suspended. Thus prepared suspension is disintegrated, cellular homogenate is centrifuged, supernatant liquid which contains AO and catalase being purified. Said purification is carried out by sorption of cellular-free centrifugate, 0.01-0.04 M tric-HCl buffer together with 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithioteitole being used. Said process takes place at pH 7.6-8.2, it is followed by eluation of protein with the help of 0.1-0.5 M NaCl, mentioned above buffer being used. The latter process is carried out at pH7.6-8.2 in the presence of 0.1 mM EDTA and 0.01 mM dithioteitole. Fractions by 4 ml are collected at rate 8 ml/h. Thus ferment preparation of AO having no catalase impurities is prepared. Desired product yield is 43.4 % , its specific activity is 12.5 units. EFFECT: improves efficiency of the method. 6 dwg, 3 tbl

Description

 The invention relates to methods for producing enzyme preparations, namely alcohol oxidase (AO), and can be used in biotechnology to obtain AO used in the enzymatic determination of the concentration of alcohol in the blood and other biological fluids of humans and animals.

A known method for producing alcohol oxidase by culturing methylotrophic catalase-deficient yeast in formate-glucose or formaldehyde glucose mixtures in a nutrient medium, wherein glucose serves as a carbon source, and formate and formaldehyde are energy substrates. The methylotrophic yeast with a catalase-deficient strain used to produce AOs is not able to synthesize catalase; therefore, the AOs contained in them can be used for the quantitative and qualitative determination of ethanol [1]
However, the use of a mutant catalase-deficient strain leads to an increase in the cost of the target product. In addition, the known method is characterized by reduced synthesis of the enzyme AO, which makes the method economically disadvantageous.

The closest in technical essence and the achieved positive effect (prototype) is a method for producing AO from Hansenula polymorpha methylotrophic yeast, including growing yeast biomass on a mineral medium, isolating cells from the culture medium by centrifugation, triple cell destruction in a high-pressure press, protein precipitation in region 30 -60% saturation with ammonium sulfate followed by dialysis (to remove catalase) of the protein solution against a 20-fold buffer containing 10 mM catalase-3- inhibitor amino-1,2,4 triazole and 0.05% H ₂ O ₂ chemical modification method [2]
The isolated by a known preparation method (100 g of yeast, 6 g protein) had a specific activity of 10-12 U / mg protein and was stored without loss of activity for years at -40 ° ^C. This was the amount of enzyme sufficient to 12000 colorimetric assays in the determination of ethanol.

The known method made it possible to remove a significant amount (about 90%) of catalase, however, to complete the process, i.e. it was not possible to obtain an AO preparation completely devoid of catalase impurities in a known manner, since continued dialysis will lead to the fact that the remaining small amount of catalase (about 10%) does not have a protective effect on AO from H ₂ O ₂ and the AO is partially or completely inactivated. The presence of a catalase impurity in the AO preparation makes it necessary to take a large amount of the enzyme 500 μg per sample in the incubation mixture for colorimetric determination of primary alcohols. Therefore, given the high prices of enzyme preparations, the use of AO obtained by a known method for determining the concentration of alcohol in the blood and other biological fluids of humans and animals makes this analysis quite expensive.

 The aim of the invention is to increase the purity of the enzyme preparation.

 The invention consists in the fact that protein purification is carried out on a DEAE-Toyoperl 650 M ion exchanger balanced with 0.01-0.04 M Tris-HCl buffer in the presence of 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol with pH 7.6-8.2.

 In FIG. 1 for example 1 shows the chromatography of a protein solution on DEAE Toyoperl 650 M; figure 2 for example 2; figure 3 for example 3; in FIG. 4,5, respectively, examples 4 and 5; in Fig.6 the dependence of the reaction rate on time, where the ethanol content: 1-10 mg / l; 2-20 mg / l, 3-40 mg / l.

 The method is as follows.

 A boidinii yeast culture is grown on a mineral medium. The amount of biomass grown is determined spectrophotometrically, and the purity of the culture is monitored by microscopy. Cells are harvested in a logarithmic growth phase. The collected cells are suspended, the suspension is disintegrated in a homogenizer in the presence of silica sand. Cell homogenate is centrifuged, followed by the removal of intact cells. The supernatant, including AO and catalase, is used for subsequent purification.

 The obtained cell-free centrifugate is sorbed on DEAE-Toyoperl 650M followed by protein elution, obtaining at this stage of purification the enzymatic preparation AO completely free of catalase impurities. Then, by the method of sulfoammonium fractionation, stabilization of AO is carried out.

 In the proposed method for producing AO at the stage of working out its optimal parameters, it was found that the best effect was recorded when the extracted proteins were separated on a DEAE-Toyoperl 650M ion exchanger balanced with 0.01-0.04 M Tris-HCl buffer in the presence of 0.05 M NaCl, 0 , 1 mM EDTA and 0.1 mM dithiothreitol at 7.6-8.2. A decrease in the molarity of Tris-HCl buffer below 0.01 M leads to a loss of stability of the AO preparation due to its dissociation into subunits, and an increase in molarity above 0.04 M leads to a decrease in the degree of sorption of the protein preparation. Lowering the pH of the buffer below 7.6 destabilizes the structure of AO, while using a pH above 8.2 impairs the separation of catalase and AO. An increase in the concentration of EDTA and dithiothreitol, which stabilize the structure of AO, does not lead to an increase in the effect compared to the applied concentrations. The used molarity of NaCl 0.05 M was selected experimentally and is optimal for achieving the initial strong binding of the protein to the DEAE-Toyoperl carrier of 650 M. When choosing an ion exchanger, it was found that neither DEAE-Sephadex nor DEAE-cellulose provided complete separation of alcohol oxidase and catalase. It has been experimentally confirmed that only DEAE-Toyoperl 650 M has the necessary resolving power, allowing to separate these two enzymes.

PRI me R 1. The cultivation of C. boidinii yeast is carried out at 29 ^about With on a mineral medium of the following composition (g / l of water): (NH ₄ ) ₂ SO ₄ 5; KH ₂ PO ₄ 1; MgSO ₄ 0.5; NaCl 0.1. As a carbon source, methanol is added at a concentration of 0.5% by total volume. The amount of biomass grown is found spectrophotometrically at 623 nm. The purity of the culture is controlled by microscopy. Cells were harvested in the logarithmic growth phase by centrifugation at 5000g for 10 min followed by washing with 0.02 M Na-phosphate buffer, pH 7.6 at 6 ° ^C. The culture is maintained on an agar slant of the following composition (grams / liter of water): glucose 20.0; peptone 20.0; yeast autolysate 10.0; agar 20.0. The cells were stored at -15 ^{° C} throughout the year.

10 g of harvested cells (thawed) are taken and suspended in 30 ml of 0.02 M Tris-HCl, buffer, pH 8.0 containing 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol. The resulting suspension is disintegrated in a homogenizer in the presence of 18 g of silica sand for 15 minutes at 1000 g. Cell homogenate is centrifuged (30 min, 20,000 g), followed by removal of cell debris. The supernatant, including a mixture of alcohol oxidase and catalase, is used for subsequent purification. To do this, take 27 ml (810 mg) of the obtained acellular centrifuge and apply to a column (2x20 cm) with DEAE-Toyoperl 650 M. The gel is equilibrated with 0.02 M Tris-HCl buffer, pH 8.0 in the presence of 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol. After adsorption of the protein preparation, the column is washed with 300 ml of 0.02 M Tris-HCl buffer, pH 8.0 containing 0.08 M NaCl, 0.1 mm EDTA and 0.1 mm dithiothreitol, followed by elution of the protein with a linear NaCl gradient of 0.1 -0.5 M in 0.02 M Tris-HCl buffer, pH 8.0 with 0.1 mM EDTA and 0.1 mM dithiothreitol at a rate of 8 ml / h, collecting 4 ml fractions. The volume of the tank and mixer is 150 ml in each vessel. Catalysis elutes in the region of 0.22-0.27 M NaCl, and AO somewhat later, in the region of 0.30-0.34 M (see figure 1). As a result, at this stage of purification, an AO enzyme preparation was obtained with a maximum specific activity of 15 U (average value of 12.5 U), completely free of catalase impurity (Fig. 1). AO stabilization is carried out by the method of sulfonammonium fractionation. For this purpose, the most active fractions were pooled and AO enzymatic eluate to a protein concentration of 6.1 mg / ml was added under stirring finely divided ammonium sulfate to a final saturation of 70% after 2-hour incubation at ⁴ ° C cmec centrifuged 20 min at 20000g. The precipitate AO are stored in the form of paste at -20 ^C. The final yield of protein of 43.4% A significant loss in activity was observed over a year. All operations on the isolation and purification of AO is carried out at 6 ° ^C. The results are summarized in Table 1.

 PRI me R 2. The cultivation of the yeast C. boidinii and obtaining cell homogenate is carried out in a manner identical to the method described in example 1.

 27 ml (810 mg) of a cell-free centrifugate obtained after destruction of the cells in a homogenizer is applied to a column (2x20 cm) with DEAE-Toyoperl 650 M. The gel is equilibrated with 0.01 M Tris-HCl with a pH of 7.6 containing 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol. After protein binding to the ion exchanger, the column is washed with 300 ml of 0.01 M Tris-HCl buffer, pH 7.6, containing 0.08 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol, followed by elution of the protein with a linear NaCl gradient of 0.1 -0.5 M in 0.01 M Tris-HCl buffer, pH 7.6 with 0.1 mM EDTA and 0.1 mM dithiothreitol, at a rate of 8 ml / h, collecting 4 ml fractions. The volume of the tank and mixer is 150 ml in each vessel. Catalase elutes in the region of 0.21-0.26 M NaCl, and AO somewhat later, in the region of 0.28-0.33 M (see Fig. 2). As a result, at this stage of purification, an AO enzyme preparation was obtained with a maximum specific activity of 12 E (average value of 9E), completely free of catalase impurity (Fig. 2). AO stabilization was carried out in a manner identical to the method described in example 1. The final protein yield was 40.4%. There was no significant loss of activity during the year. The results are summarized in table 1.

 PRI me R 3. The cultivation of yeast C. boidinii and obtaining cell homogenate is carried out in a manner identical to the method described in example 1.

 27 ml (810 mg) of cell-free centrifugate obtained after the destruction of cells in a homogenizer is applied to a column (2x20 cm) with DEAE-Toyoperl 650M. The gel was equilibrated with 0.04 M Tris-HCl buffer, pH 8.2 containing 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol. After protein binding to the ion exchanger, the column is washed with 300 ml of 0.04 mM Tris-HCl buffer containing 0.08 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol, followed by elution of the protein with a linear NaCl gradient of 0.1-0.5 M in 0.04 M Tris-HCl buffer, pH 8.2 with 0.1 mM EDTA and 0.1 mM dithiothreitol at a rate of 8 ml / h, collecting 4 ml fractions. The volume of the tank and mixer is 150 ml in each vessel. Catalase elutes in the region of 0.24-0.29 M NaCl, and AO somewhat later, in the region of 0.33-0.37 M (see Fig. 3). As a result, at this stage of purification, an AO enzyme preparation was obtained with a maximum specific activity of 12.5 U (average value of 10.5 U), completely free of catalase impurity (Fig. 3). AO stabilization was carried out in a manner identical to the method described in example 1. The final protein yield was 41.2%. No significant loss of activity was observed during the year. The results are summarized in table 1.

PRI me R 4. The cultivation of the yeast Hansenula polymorpha is carried out at 29 ^about C on a mineral medium of the following composition (g / l): (NH ₄ ) ₂ SO ₄ 5; KH ₂ PO ₄ 1; MgSO ₄ 0.5; NaCl 0.1. The mineral medium is designed for 1 liter of water. As a carbon source, methanol is added at a concentration of 0.5% by total volume. The amount of biomass grown is found spectrophotometrically at 623 nm. The purity of the culture is controlled by microscopy. Cells were harvested in the logarithmic growth phase by centrifugation at 5000g for 10 min followed by washing with 0.02 M Na-phosphate buffer, pH 7.6 at 6 ° ^C. The culture was maintained on solid medium of the following composition (g / l): glucose 20, 0; peptone 20.0; yeast autolysate 10.0; agar 20.0. Cells are stored at -15 ^o C.

10 g of harvested cells (thawed) are taken and suspended in 30 ml of 0.02 M Tris-HCl buffer, pH 8.0, containing 0.05 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol. The resulting suspension is disintegrated in a homogenizer in the presence of 18 g of silica sand for 15 minutes at 1000 g. Cell homogenate is centrifuged (30 min, 20,000 g), followed by removal of cell debris. The supernatant, including a mixture of alcohol oxidase and catalase, is used for subsequent purification. To do this, take 26 ml (594 mg) of the obtained acellular centrifuge and apply to a column (2x20 cm) with DEAE-Toyoperl 650 M. The gel is equilibrated with 0.02 M Tris-HCl buffer, pH 8.0, in the presence of 0.05 M NaCl 0.1 mM EDTA; 0.01 mM dithiothreitol. After sorption of the protein preparation, the column is washed with 300 ml of 0.02 M Tris-HCl buffer, pH 8.0 containing 0.08 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol, followed by elution of the protein with a linear NaCl gradient of 0.1 -0.5 M in 0.02 M Tris-HCl buffer, pH 8.0 with 0.1 mM EDTA and 0.1 mM dithiothreitol at a rate of 8 ml / h, collecting 4 ml fractions. The volume of the tank and mixer is 150 ml in each vessel. Catalase elutes in the region of 0.29-0.34 M NaCl, and AO somewhat later in the region of 0.4-0.44 M (see Fig. 1). As a result, at this stage of purification, an AO enzyme preparation was obtained with a maximum specific activity of 17 E (average value of 14E), completely free of catalase impurity (Fig. 4). AO stabilization is carried out by the method of sulfonammonium fractionation. For this purpose, the most active fractions were pooled and AO enzymatic eluate to a protein concentration of 4.5 mg / ml was added under stirring finely divided ammonium sulfate to a final saturation of 70% after 2-hour incubation at ⁴ ° C the mixture was centrifuged 20 min at 20000g. The precipitate AO are stored in the form of paste at -20 ^C. The final yield of protein was 58.5% All operations of isolation and purification AO conducted at 6 ° ^C. The results are summarized in Table 2.

 PRI me R 5. The cultivation of the yeast Pichia pastoris and obtaining cell homogenate is carried out in a manner identical to the method described in example 4.

 25 ml (524 mg) of a cell-free centrifugate obtained after destruction of the cells in a homogenizer is applied to a column (2x20 cm) with DEAE-Toyoperl 650 M. The gel is equilibrated with 0.02 M Tris-HCl buffer, pH 8.0, containing 0.05 M NaCl, 0.1 mm EDTA and 0.1 mm dithiothreitol. After protein binding to the ion exchanger, the column is washed with 300 ml of 0.02 M Tris-HCl buffer, pH 8.0, containing 0.08 M NaCl, 0.1 mM EDTA and 0.1 mM dithiothreitol, followed by elution of the protein with a linear gradient of NaCl 0 , 1-0.5 M in 0.02 M Tris-HCl buffer, pH 8.0, with 0.1 mM EDTA and 0.1 mM dithiothreitol at a rate of 8 ml / h, collecting 4 ml fractions. The volume of the tank and mixer is 150 ml in each vessel. Catalase elutes in the region of 0.25-0.31 M NaCl, and AO somewhat later in the region of 0.37-0.41 M (see Fig. 5). As a result, at this stage of purification, an AO enzyme preparation was obtained with a maximum specific activity of 13.5 U (average value of 12 U), completely free of catalase impurity (Fig. 5). AO stabilization was carried out in a manner identical to the method described in example 4. The final protein yield was 48.0%. The results are summarized in table 3.

 Thus, as can be seen from the graphs of FIGS. 4 and 5, the proposed method makes it possible to achieve the goal if a yeast culture of Hansenula polymorpha (prototype), as well as other types of methylotrophic yeast, for example, Pichia pastoris, is used as an alcohol oxidase source.

The method involves the use of cultures stored in the catalog of cultures of the All-Union collection of non-pathogenic microorganisms. The yeast strains that are the producers of the target product (alcohol oxidase, AO) have the following numbers in the official collection of microorganisms: Candida boidinii 34 Hansenula polymorpha 1397 Pichia polymorpha 1392 Pichia pastoric 1379 Pichia angusta 2599 Pichia pinus 1616
The enzyme preparation isolated by the method, in which 62 mg of protein was obtained from 10 g of yeast cells, has an average specific activity of 12.5 U (Example 1). This amount of AO (62 mg) is enough for 20,000 colorimetric assays to determine ethanol concentrations, i.e. for one analysis, 3 μg of the enzyme is used with an analysis duration of 3 minutes (see Fig. 6), which reflects the dependence of the rate of alcohol oxidase reaction on time with an ethanol content of 10 mg / L 1; 20 mg / l 2; 40 mg / l 3. The incubation medium in 1 ml contained: 0.06 ml of 0.07% 2,6-dichlorophenolindophenol, 30 μg in 0.03 ml of horseradish peroxidase, 3 μg of AO and 0.9 ml of 0.02 M phosphate buffer pH 7.6.

 Therefore, given the high cost of enzyme preparations, the determination of ethanol concentration using AO obtained by this method that does not contain catalase impurities significantly accelerates the analysis time and reduces its final cost compared to the prototype.

Claims (1)

 METHOD FOR PRODUCING YEAST ALCOHOLOXIDASE, which involves culturing a producing microorganism, isolating cell biomass, destroying cells to obtain a cell-free extract, purification from catalase, characterized in that, in order to increase the purity of the enzyme preparation, the cell-free extract is purified by ion exchange chromatography using T, 0.01 0.04 M Tris-HCl buffer, in the presence of 0.05 M sodium chloride, 0.1 mmol EDTA and 0.1 mmol dithiothreitol at pH 7.6 8.2.

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