RU2113479C1 - Strain of bacterium pseudoalteromonas species - a producer of alpha-galactosidase - Google Patents

Abstract

FIELD: biochemistry, enzymes. SUBSTANCE: strain of bacterium Pseudoalteromonas sp. VKM B-2135 D is used as a producer of galactosidase that is capable to remove α-1,3-bound galactose residues from glycoproteins of B-blood substances and to convert the latter to substances of group H (0). Strain is isolated from sea water by method of direct plating. Strain is cultured on medium of the following composition, g/l: bactopeptone 5.0; glucose 1.0; K₂HPO₄ 0.2; yeast extract (Difco) 2.0; distilled water 500 ml, and sea water 500 ml at pH 7.8, at 25 C for 24 h. Culture produces α-galactosidase. Preparation of α-galactosidase is isolated from biomass by its releasing from cells by ultrasonic treatment followed by enzyme purification by ion-exchange chromatography on DEAE-Toyoperl 650-M, gel-filtration on Sepharose CL-4B and rechromatography on DEAE-Toyoperl 650-M. The yield of α-galactosidase preparation at specific activity 10 U/mg protein is 30% of its content in crude extract. Enzyme shows optimum pH at 6.7-7.7, molecular mass 200 kDa. Enzyme inactivates the group specificity of human blood-B erythrocytes. Invention can be used in microbiology, biotechnology and medicine. EFFECT: strain of bacterium indicated above, improved method of enzyme preparing, increased yield of enzyme. 3 tbl

Description

 The invention relates to biotechnology, in particular to the production of enzymes that are important in solving the problem of universal donation, and can be used to obtain highly active α -galactosidase used in medicine, genetic engineering, molecular biology.

 The development of an effective way to obtain red blood cells that are universal in their group properties for emergency transfusiological care for victims continues to be very relevant in our country.

 Of great practical interest are α -galactosidases capable of removing α-1,3-linked galactose residues from glycoproteins of group substances in the blood with the conversion of the latter into substances of group 0 (H). Such α-galactosidases are quite rare. From the literature data, only six α-galactosidases are known that can catalyze the transformation of erythrocytes from human blood group B to blood group 0. These include α-galactosidases of actinomycete Streptomyces sp. (99178) [1], zygomycete Mortierella vinacea [2], protozoa Trichomonas foetus [3], coffee beans 14.5], sweet almonds [6] and sweet potatoes Colocacia esculenta [7].

 In the literature, information was provided on successful transfusion of transformed red blood cells (α-galactosidase from coffee beans) to humans (volunteers) [8-11]. However, for the wide practical application of such red blood cells, highly purified specific enzymes are necessary, which can be obtained from accessible and easily reproducible raw materials.

 The most technologically advanced sources of enzymes are microorganisms, since they have an enormous reproduction rate and synthesize biologically active substances under human-controlled conditions.

 Among known bacteria of terrestrial origin, an enzyme producer with the required specificity was not found.

 In addition to narrow substrate specificity, the pH optimum of the enzyme is of great importance. So, all the enzymes that are effective on red blood cells described above have an optimum in the acidic pH region, which is extremely undesirable for red blood cells.

 The analysis of numerous literature data showed that it is bacteria that contain enzymes, one of the properties of which (optimum action at pH 7.0) satisfies the technological requirements for the modification of red blood cells in physiological conditions.

 Marine bacteria as sources of enzymes with unique specificity are attracting increasing attention of researchers. The Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences is the owner of an extensive collection of marine bacteria collected in various regions of the World Ocean during the expeditions of the academic vessel Akademik Oparin. Screening among almost two thousand strains of marine bacteria of various genera and habitats [12] revealed several promising strains producing α -galactosidases that inactivate the group specificity of human erythrocytes of blood group B.

 The objective of the invention is the identification of a new strain producing α -galactosidase with the above properties (narrow substrate specificity, optimum action at pH 7.0 - 7.5), necessary for biotechnology.

 The problem is solved in that a bacterial strain Pseudoalteromonas sp. Was selected from seawater, selected according to the high initial α-galactosidase activity for p-nitro-phenyl-α - D-galactopyranoside.

 The bacterial strain Pseudoalteromonas sp. assigned number KMM 701, it is deposited in the All-Russian collection of microorganisms under the number VKM V-2135 D.

The strain was isolated by direct seeding on an agar medium of the following composition (g / l): peptone - 5, yeast extract -2.5, glucose - 1, MgSO ₄ - 0.5, K ₂ HPO ₄ - 0.2; seawater - 750 ml, distilled water - 250 ml, pH - 7.5.

The culture is stored in test tubes under a layer of mineral oil in a semi-liquid medium of the above composition, at a temperature of 10 ^o C.

 The strain has the following characteristics.

 Cultural and morphological characters.

 a) Bacteria form round with smooth edges, convex colonies up to 4 mm in diameter, the surface of the colonies is smooth, the mucous consistency. The pigment does not accumulate.

b) The strain VKM B-2135 D is a small gram-negative rods, mobile using a single polar flagellum. Chemorganotrophs with an oxidative type of metabolism. Oxidase- and catalase-positive, do not grow in the medium without NaCl, and do not accumulate pigment. Luminescence and fluorescence are absent. Grow at 4 ^o C, at 41 ^o C no growth.

 Physiological and biochemical characteristics.

 Strain VKM V-2135 D does not hydrolyze gelatin, starch, tween-80. Arginine dehydrolase is absent, not a denitrifier. Indole and hydrogen sulfide does not form. Melibiosis, maltose, sucrose, glycerin does not utilize. Strains of the genus Pseudoalteromonas are neither pathogenic nor toxic to warm-blooded animals.

 An example of using a strain of marine bacteria Pseudoalteromonas sp. VKM B-2135 D for the production of the α-galactosidase enzyme.

Fermentation is carried out on a modified nutrient medium YK of the following composition, g / l:
Bactopeptone (Difco) - 5.0
Glucose - 1.0
K ₂ HPO ₄ - 0.2
Yeast Extract (Difco) - 2.0
Distilled Water - 500 ml
Sea water - 500 ml
pH - 7.8
Seed Pseudoalteromonas sp. VKM B-2135 D is grown in 250 ml flasks containing 50 ml of fermentation medium for 18-20 hours at 25 ^° C on a shaker at a rotation speed of 150 rpm to obtain a density of 10 ⁹ cells / ml.

1000 ml flasks containing 500 ml of a fermentation medium of the same composition are seeded with the resulting seed. Fermentation is carried out for 24 hours at 25 ^o C on a rocking chair with a rotation speed of 120 rpm

 The sequence of procedures for the isolation and purification of α -galactosidase from the biomass of bacteria and its purification is traditional.

 α-galactosidase activity is concentrated in the biomass of bacteria, and not in the culture medium. A portion of the raw biomass is suspended in 0.01 M phosphate buffer, pH 6.8-7.0. The ratio of biomass and buffer does not exceed 0.5 mg per 1 ml. The suspension is voiced by ultrasound with a frequency of 22 kHz (at a current of 0.4 A) 4-5 times for 40 s with a 10-20-second break on the ultrasonic disperser. The homogenate is centrifuged for 30-35 minutes in a centrifuge at 15,000 rpm.

 The supernatant is subjected to ion exchange chromatography on a DEAE-Toyoperl 650 M column from Toya Soda, Japan (15.5 x 2), equilibrated with 0.01 M phosphate buffer, pH 6.8-7.0. Elution is carried out with a linear gradient of NaCl (0 - 0.5 M) 300/300 ml at a rate of 22 ml / h.

 Fractions containing α -galactosidase activity are combined, concentrated by ultrafiltration on a PM-ZO membrane (Amicon, England) and applied to a Pharmacia CL-4B Sepharose column (Sweden) (62x2.5 cm), equilibrated with the same buffer. Elute at a rate of 28 ml / h.

 After gel filtration, fractions containing α -galactosidase activity are rechromatographed on a DEAE-Toyoperl 650 M under the conditions indicated above.

 The total α -galactosidase activity of the biomass extract prepared as described above is completely adsorbed on the anion exchanger, and 65% of it is eluted with a linear NaCl gradient at 0.14 M with one symmetric narrow peak. As a result, the enzyme is purified 5.5 times (table. 1). Subsequent concentration by ultrafiltration on a PM-ZO membrane and further gel filtration on sepharose of CL-6B fractions containing α -galactosidase activity leads to a 65-fold purification of the enzyme preparation with almost no loss of activity. Rechromatography on a DEAE-Toyoperl 650 M helps separate a number of protein contaminants. For further studies, fractions of the enzyme preparation are taken that correspond to the central part of the peak of activity at 0.19 M NaCl. The yield of α -galactosidase activity is 30% of the activity of the extract, the specific activity increases from 0.1 to 10 units / mg (table. 1).

The standard hydrolytic activity of α -galactosidase (U) is determined as follows: 450 μl of a solution of p-nitrophenyl-α-D-α-galactopyranoside manufactured by Sigma, USA (1 mg / ml) is added to 50 μl of the enzyme solution 1 M phosphate buffer, pH 7.0, incubated at a temperature of 20 ^o C. The reaction is stopped after 5-20 minutes, depending on the speed of development of the yellow color, adding 2.5 ml of 1 M Na ₂ CO ₃ and measure the optical density of the solution at 412 nm on SF-26 (USSR). The amount of p-nitrophenol released is determined from the calculation: 23 μg / ml corresponds to 0.5 units of the optical density of the solution. The unit of activity is the amount of enzyme that catalyzes the formation of 1 μmol of p-nitrophenol in 1 min under the conditions of determination.

 The specific activity of α -galactosidase is considered as the ratio of h standard units per mg protein per unit volume (U / mg protein). Protein is determined by the Lowry method.

 As a result of the purification scheme used, an enzyme preparation is obtained that is 98 times purified and does not contain other glycanases and glycosidases.

 The enzyme has an optimum effect at a pH of 6.7-7.7, required for the reaction of the transformation of human blood group B (III) red blood cells into H (O) under sparing red blood cells. This property gives a significant advantage of the obtained enzyme over the already known α-galactosidases from other sources indicated above, having optimum action at pH <6.

The enzyme does not withstand freezing, but remains unlimited at 4 ^° C and during the week at 20 ^° C in the presence of 0.1% Na azide, although at 30 ^° C it loses 50% activity in 10 minutes.

 The molecular weight of the enzyme is 200 kDa according to gel filtration on Sepharose CL-6B company "Pharmacia" (Sweden).

 α-galactosidase obtained from the bacterial strain Pseudoalteromonas VKM B-2135 D was tested at the Hematology Research Center of the Russian Academy of Medical Sciences (Moscow) for the ability to transform human blood group B (III) red blood cells into H (O) red blood cells.

 Native red blood cells, blood, and standard group sera were obtained from the blood transfusion station of the Hematology Research Center of the Russian Academy of Medical Sciences. Monoclonal antibodies (anti-A, anti-B, anti-H, anti-M, anti-N) - from the company "Hematologist" RAMS.

 In long-term transformation experiments, when hemolysis is likely, erythrocytes fixed with glutaraldehyde were used.

The effectiveness of the enzyme when acting on red blood cells is established by the following procedures. Well-washed fixed red blood cells are mixed with the enzyme solution and, after 24 hours of incubation with stirring at 26 ^° C, they are washed 3 times with buffered saline pH 7.3 and then mixed with the corresponding sera. After 1 h of incubation at room temperature with gentle stirring, the agglutinating ability of such absorbed sera is determined. Agglutination tests are carried out on 96-well plates in a series of double dilutions of normal and / or absorbed ("depleted") serum (antibody solutions) using native red blood cells pre-collected from several samples of donated blood.

 Red blood cells fixed with glutaraldehyde completely retain the structure of ABO-group antigens and, accordingly, are capable of specifically adsorbing antibodies [13]. Therefore, if the enzyme effectively inactivates the B antigen, that is, converts it to H (0), the antigen that characterizes the first (zero) blood group, then anti-B antibodies do not adsorb (the serum does not "deplete"), and the agglutination titer native red blood cells of the corresponding blood group will not differ from the titer of fresh (not "depleted") serum. Accordingly, it is possible to evaluate the different degree of inactivation of antigens by changing the titer of "depleted" sera. In the table. 2 shows titers of "depleted" serum after treatment of B (III) fixed red blood cells with α -galactosidase from Pseudoalteromonas sp. VKM B-2135 D, which testify to the effectiveness of the action of this enzyme preparation on the transformation of human blood erythrocytes.

 In the table. Figure 3 shows the results of an immunological study of native uncommitted erythrocytes treated with α -galactosidase, which has been shown to be effective on fixed erythrocytes (Table 2). It can be seen that B (III) antigens are transformed into O (I) antigen, since there is no agglutination with anti-B serum. The titer of agglutination with anti-H serum is naturally slightly increased due to the appearance of new H antigens (0 (I) antigens) instead of the previously B antigens. This picture also indicates the absence of fucosidase in the samples, otherwise the H antigen would be inactivated. Enzymatic treatment also does not change the titer of agglutination with anti-M or anti-N serum, which may indicate the absence of sialidases in the studied enzyme samples. This is very important from the point of view of engraftment of transformed red blood cells with the introduction of their g into the bloodstream. α-galactosidase, past the purification procedure shown in table. 1 also does not cause non-specific aggregation of red blood cells and their hemolysis, which indicates a fairly high purity of the enzyme preparation.

 The problem is solved in that an enzyme preparation is obtained that is able to meet technological requirements: the optimum action is pH 7.0, it effectively acts on red blood cells of blood group B (III).

Sources of information
l. Oishi K., Aida K. // Agric. Biol. Chem. 1972. Vol. 35. P. 578.

 2. Ulezlo I. V., Zaprometova O. M. // Applied biochem. microbiol. 1982, vol. 18, p. 5.

 3. Yates A.D., Morgan W.T.J., Watkins W.M. // FEBS Letts. 1975. Vol. 60.P. 281.

 4. Zamitz M. L., Kabat L. A. // J. Am. Chem. Soc. 1960. Vol. 82. P. 3953.

 5. Dey P. M., Pridham J. B. // Adv. Enzymol. 1972. Vol. 36 P. 91.

 6. Malhotra O. P., Dey P.M. // Biochem. J. 1967. Vol. 103. P. 508.

 7. Chien S.-F., Lin-Chu M. // Carbohydr. Res. 1991. Vol. 217. P. 508.

 8. Goldstein J., Lenny L. a.a. // Science. 1982. Vol. 215. P.164-170.

 9. Goldstein J., in The Red Cell. 1984. P. 130-157.

 10. Lenny L., Goldstein J. a.a. // Blood. 1991. Vol. 77. P.1383-1388.

 11. Lenny L., Goldstein J. a.a.// Blood. 1994. Vol. 84. P.469.

12. Bakunina I.Yu., Ivanova EP, Nedashkovskaya O.M., Gorshkova N.M., Mikhailov V.V., Elyakova L.A. // Biology of the sea. 1997,
13. Kovner V. Ya., Kulman R.A. and others // Hematology and transfusiology. 1986.V.31. S. 21-25.

Claims (1)

 The bacterial strain Pseudoalteromonas VKM B - 21315-D - producer of alpha galactosidase.

Images