RU2077577C1 - Strain of bacterium deleya marina - a producer of alkaline phosphatase and a method of alkaline phosphatase preparing - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymology. SUBSTANCE: strain of bacterium Deleya marina BKM B-2021D is isolated from coelomic liquid of Far Eastern mollusk Crenomytilus grayanus. Strain shows high level of the parent specific activity of alkaline phosphatase (40-45 U/mg protein). Method of alkaline phosphatase preparing involves the growing of the strain-producer on nutrient medium, separation of bacterial biomass from cultural fluid followed by isolation of the end product by an aqueous extraction of microbe cells and three-stage chromatography purification of the crude enzyme preparation by methods of anion-exchange, hydrophobic chromatography and gel-filtration. Specific activity of the purified alkaline phosphatase is 14000-15000 U/mg protein. The end product is used in medicine, genetic engineering, molecular biology. EFFECT: improved method of enzyme preparing. 2 cl, 2 tbl

Description

 The invention relates to biotechnology, in particular to the production of enzymes, and can be used to obtain highly active alkaline phosphatase used in medicine, genetic engineering, molecular biology.

Marine microorganisms are promising sources of new alkaline phosphatases. In the literature, there is some information about the ability of marine microorganisms to produce alkaline phosphatases. Several alkaline phosphatases isolated from bacterial strains of marine origin are described. It was shown that these enzymes differ from the already known alkaline phosphatases in some physicochemical properties. So, from the marine bacteria Pseudomonas sp. extracellular alkaline phosphatase was isolated, the activity of which is affected by an increase in pressure, as well as the presence of the main ions contained in sea water. The enzyme has a specific activity of 192 u / mg protein [1] From the Antarctic marine bacteria NK-47, thermolabile alkaline phosphatase is isolated, which is used to cleave terminal 5'-phosphate groups in nucleic acid molecules, and after the reaction is completed, it is easily inactivated by heating at 60 ^o C The specific activity of the enzyme is 906 units / mg protein [3]
In recent years, strains of symbiotic marine bacteria, mainly bivalve mollusks (Crenomytilus grayanus), have been found that are distinguished by the ability to synthesize highly active alkaline phosphatases. A known strain of marine bacteria, originally identified as Acinetobacter sp. later reclassified as Alteromonas macleodii KMM 162 (VKPM B-3905, 40 MS), producing alkaline phosphatase with high specific activity (6000 6700 u / mg protein).

Alkaline phosphatase of strain KMM 162 has a molecular weight of 90 kDa, exhibits maximum activity at a pH of 9.5 to 9.8 in the presence of 1 mM MgCl ₂ , and EDTA is inhibited. The enzyme is thermostable, but in the presence of 2-mercaptoethanol or dithiothreitol is completely inactivated at 55 - 60 ^o C, which allows it to be used for labeling nucleic acids and removed from the reaction mixture by heat treatment [2] Despite the high specific activity, this alkaline phosphatase was not used in an enzyme-linked immunosorbent assay to obtain conjugates, since the enzyme is unstable to chemical modifications and loses its activity during appropriate processing and storage.

 The objective of the invention is the identification of a new strain producing alkaline phosphatase with a higher, in comparison with analogues, specific activity.

 The problem was solved in that a strain of the bacterium Deleya marina was selected from the coelomic fluid of the Far Eastern bivalve mollusk Crenomytilus grayanus, selected according to the high initial specific phosphatase activity.

 The strain of bacteria Deleya marina, the authors assigned the number KMM 296 (4 MS 37), it is deposited in the All-Russian collection of microorganisms under the number VKM B-2021 D.

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

 Characterization of the strain.

Cultural and morphological signs:
a) the bacteria form round with smooth edges, convex colonies up to 5 mm in diameter, the surface of the colonies is smooth, the mucous consistency. The pigment does not accumulate;
b) strain KMM 296 (4 MS 37) is a small gram-negative bacilli, mobile using flagella. In bacteria, 1 2 polar flagella and 1 2 lateral flagella are formed. Chemorganotrophs with an oxidative type of metabolism. Oxidase-negative, 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 KMM 296 (4 MS 37) gelatin, starch, tween-80 does not hydrolyze. Arginine dehydrolase is absent, not a denitrifier. Indole and hydrogen sulfide does not form. Lactose, melibiosis, maltose, sucrose, glycerin does not utilize. The molar percentage of G + C bases in DNA is 65.5.

 Strains of the genus Deleya are neither pathogenic nor toxic to warm-blooded animals.

 The strain Deleya marina KMM 296 (4 MS 37) differs from the well-known strain of the prototype Alteromonas macleodii KMM 162 (40 MS) in cultural and morphological characteristics: it forms whitish opaque colonies, from 1 to 4 polar flagella, including lateral ones, are formed in cells; according to physiological and biochemical characteristics: oxidase-negative, practically does not utilize carbohydrates, and also has a high molar percentage of G + C bases in DNA.

 The objective of the invention is the development of a method for producing alkaline phosphatase from strain KMM 296, which allows to obtain an enzyme with higher, in comparison with analogues, specific activity and higher resistance to chemical modifications.

 The problem is solved in that the proposed method allows to obtain alkaline phosphatase with a specific activity of 14,000 to 15,000 units / mg of protein, which also has high stability, which makes it possible to use the enzyme in an enzyme immunoassay.

 The proposed method for producing alkaline phosphatase involves the cultivation of a producer strain on a nutrient medium, the separation of biomass from the culture fluid, the destruction of microbial cells, followed by isolation of the target product by aqueous extraction, as well as chromatographic purification of the crude enzyme preparation. The bacterium strain Deleya marina KMM 296 (4 MS 37; VKM B-2021 D) is used as a producer. Purification of the enzyme is carried out first by anion exchange chromatography on DEAE cellulose, then by hydrophobic chromatography on butyl toyoperl and gel filtration on Sephadex G-100.

 The method is illustrated by the following examples.

EXAMPLE 1. A freshly seeded culture is washed from a jamb with nutrient medium into a 1000 ml flask containing 500 ml of nutrient medium of the following composition (g / l): peptone 5.0; 2.5 yeast extract; glucose 1; K ₂ HPO ₄ 0.2; MgSO ₄ 0.05; 750 ml of natural sea water (a replacement of 25 g / l NaCl is acceptable); 250 ml of distilled water; The pH of the medium is 7.5 to 7.8 and grown 24 hours in an incubator at 28 ^o C. Thus prepared inoculum inoculate flasks with a capacity of 1000 ml (with 500 ml of culture medium). Grown for 24 28 hours on a rocking chair (120 rpm) at 24 ^o C.

 To obtain the enzyme, bacterial cells are precipitated in a flow centrifuge at 5000 rpm. The output of bacterial biomass 5 g with 500 ml of culture fluid. Protein productivity 95 mg / l. The specific activity of alkaline phosphatase in the aqueous extract is 45 u / mg protein. The total phosphatase activity of the aqueous extract is 4275 units. with 1 liter of culture fluid.

 A comparison of the data obtained for strain KMM 296 with the characteristics of strain KMM 162 shows that under similar conditions this strain gives a higher yield of total phosphatase activity from 1 liter of culture medium, as well as alkaline phosphatase with a higher specific activity (Table 1).

 EXAMPLE 2

 The strain is grown as described in example 1.

To 200 g of crude biomass obtained from 20 l of the liquid nutrient medium described in example 1, add 1000 ml of 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% NaN ₃ (buffer A) and cell suspension disintegrate in portions of 80 ml for 5x30 sec, cooling in ice. Then centrifuged at 3000 rpm for 30 minutes The supernatant was collected and the resulting aqueous extract was mixed with 700 ml of equilibrated buffer A DEAE cellulose in a cold room for 1 hour. Then DEAE cellulose was washed on a Schott filter with 0.2 M NaCl in buffer A. The enzyme was eluted with 0.4 M NaCl in buffer A. Dry ammonium sulfate salt was added to the eluate to a final concentration of 1.5 M; after dissolving the salt, the protein solution is applied to a column (V 32 ml) with butyl-tooper, balanced with 1.5 M ammonium sulfate in buffer A, and elution is carried out with a gradient of ammonium sulfate from 1.5 M to 0.5 M (250 ml each solution), at a rate of 40 ml / h, taking fractions of 6 ml. The enzyme leaves at a concentration of ammonium sulfate 1.1 M 0.9 M. Active fractions are collected, concentrated by ultrafiltration on a PM-30 membrane (Amersham) and applied to a column (V 140 ml) with Sephadex G-100, equilibrated with buffer: 0.01 M Tris-HCl, 0.005 M MgCl ₂ , 0.05 M NaCl, 0.01% NaN ₃ , pH 8.0. The enzyme is eluted with the same buffer at a rate of 12 ml / h, taking 4 ml fractions. Active fractions are collected and concentrated on a PM-30 membrane. Table 2 summarizes the product characteristics during the cleaning process. This isolation method provides high stability of the alkaline phosphatase preparation.

Alkaline phosphatase activity is determined by p-NFF cleavage. A standard incubation mixture in a volume of 500 μl contains 15 mm paranitrophenyl phosphate (p-NFF), 1 M diethanolamine (DEA), pH 10.3 and the enzyme. After 30 minutes of incubation at 37 ^° C., the reaction was stopped by the addition of 2 ml of 0.5 M NaOH. The amount of p-NFF formed during the enzymatic reaction is determined spectrophotometrically at 400 nm. The unit of alkaline phosphatase activity is the amount of an enzyme that catalyzes the release of 1 μM p-NFF (E ₄₀₀ nm 18600) during 1 min of incubation. Specific activity is expressed in units of enzyme activity per 1 mg of protein. The protein concentration in the solution is determined by the Bradford method.

The study of the properties of alkaline phosphatase produced by strain KMM 296 and isolated according to the invention showed that the enzyme has a molecular weight of 55 kDa, is stable in the range of pH 6.0 to 11.0, and exhibits maximum activity in 1 M DEA buffer at pH 10.3 in the presence of divalent metal cations. This alkaline phosphatase is thermostable and does not lose its initial activity after 15 minutes of heating in the temperature range of 20-50 ^o C. A similar preincubation at 60 ^o C leads to the loss of 80% of the original activity. The enzyme does not require the presence of divalent metal cations to exhibit its maximum activity, and the addition of 5 mM EDTA to the incubation mixture does not lead to a noticeable decrease in alkaline phosphatase activity. The enzyme is cleaved at the same rate with p-NFP 5'-AMP, 5'-CMP and 5'-dCMP. Alkaline phosphatase of strain KMM 296 was successfully used in the first stage of enzymatic labeling of lambda phage DNA, as well as for the preparation of enzyme conjugates.

 All of the above testifies in favor of the widespread use of the isolated enzyme in the practice of laboratory research, as well as in immunodiagnostics.

Claims (1)

1 1. The strain of the bacterium Deleya marina VKM B-2021 D is an alkaline phosphatase producer. 2 2. A method of producing alkaline phosphatase, which involves growing a producer strain on a nutrient medium to produce biomass, separating biomass from the culture fluid, followed by isolation of the target product by aqueous extraction of microbial cells and chromatographic purification of the crude enzyme preparation, characterized in that the strain is used as a producer bacteria Deleya marina VKM B-2021 D, and purification is carried out first by anion exchange chromatography on DEAE cellulose, then by hydrophobic chromatography on butyl toyoper and gel filtration on Sephadex G-100.

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