RU2207370C1 - Strain of bacterium pseudoalteromonas issachenkonii kmm 3549t as producer of fucoidane hydrolase and nutrient medium for its culturing - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry. SUBSTANCE: the strain Pseudoalteromonas issachenkonii KMM 3549^T is isolated from brown algae thallome Fucus evanescens as result of search of producers of glycoside hydrolases and designated for production of hydrolytic enzymes. Nutrient medium for it culturing comprises sources of nitrogen, carbon, energy and growth substances. Medium comprises peptone as source of nitrogen, xylose as source of carbon and energy and sea water as source of growth substances. Nutrient medium comprises the following ratio of components, g/l: peptone, 2.5-5.0; xylose, 1.0- 5.0; sea water, up to 1 l. Invention provides elevation of biosynthesis of fucidane hydrolase and reduction of activity of concomitant glycosidases-hydrolases, enhanced yield of fucoidane hydrolase in medium of simple composition and reduced cost of product. EFFECT: valuable properties of strain. 2 cl, 7 tbl, 4 ex

Description

 The invention relates to biotechnology and is intended for use in the production of hydrolytic enzymes used in the preparation of biologically active water-soluble fucoooligosaccharides.

 Sulfated water-soluble polysaccharides of brown algae - fucoidans, as well as their low molecular weight fragments (fuco-oligosaccharides) have a wide range of biological effects and can be used in the treatment of certain human diseases [1-3]. The structure of fucoidans and their corresponding biological activity vary from source to source. In general, brown algae fucoidans are a family of homo- and heteropolysaccharides, consisting mainly of sulfated fucose residues sometimes in the third [4], more often in the second and / or fourth positions, which are connected in the main chain α-1,3-, or α -1,2-, or, possibly, α-1,4-O-glycosidic bonds [5, 6]. In addition to fucose, fucoidans may contain minor monosaccharides: mannose, galactose, glucose, xylose, rhamnose, uronic acids [1].

 It is known that fucoidan hydrolases that specifically catalyze the hydrolytic cleavage of fucoidans are synthesized by both macro- and microorganisms - inhabitants of the marine environment. No fucoidan hydrolases were found in terrestrial organisms.

 Microorganisms as sources of production of enzymes with unique specificity are attracting increasing attention, since they have a huge reproduction rate and allow synthesis to be carried out under controlled conditions.

 Known marine bacteria (strain 2-40), which degrades complex polysaccharides, including fucoidan [7]. However, the fucoidan-degrading properties of this bacterium have not been studied.

 A known strain of the marine halophilic bacterium Flavobacierium sp., From which fucoidan-degrading enzymes were isolated [8]. Using these enzymes, the structures of the fucoidan fragments of one of the brown algae were established; the type of O-glycosidic bond between the residues of mannose, galactose, and glucuronic acid was determined. However, this strain is not available to us.

 Closest to the proposed invention is a strain of marine bacteria Vibrio sp. N 5 - inhabitants of sea sand. Three endo-fucoidan hydrolases were isolated from the biomass of this bacterium, which hydrolyze fucoidan from the brown alga Kjelmaniella crassifolia to sulfated fucoooligosaccharides [9]. The level of specific fucoidan-hydrolase activity in the extract according to published data does not exceed 0.14 u / mg protein. In this case, a nutrient medium is used to induce the enzyme, which contains, along with nitric acid ammonium, magnesium sulfate, calcium chloride, phosphoric acid disubstituted sodium, sodium chloride and a yeast extract, an expensive inducer is fucoidan (5 g - $ 120).

 The objective of the invention is the identification of a new strain of marine bacteria producing fucoidan hydrolase, capable of growing on simple nutrient media, and optimization of the nutrient medium for its cultivation, which allows to increase the yield of fucoidan hydrolase and reduce the level of activity of concomitant glycoside hydrolases, as well as the cost of the product.

The problem was solved by the fact that under laboratory conditions the degradation of the thallus of the brown alga Fucus evanescens by the microbial community was caused (the alga was collected in Craternaya Bay). A strain of bacteria of the genus Pseudoalteromonas was isolated from this microbial community, which was degraded by the thallus of the brown alga Fucus evanescens. It is selected for its ability to synthesize fucoidan hydrolase. The strain was assigned collection number KMM 3549 ^T in the Collection of Marine Microorganisms of the TIBOh FEB RAS (official KMM acronym and number 644 in the World Federation of Cultural Collections - WFCC). The strain is deposited in the Belgian collection of microorganisms under the number LMG 19697.

The strain was isolated by direct seeding from an accumulation culture on an agar medium of the following composition (g / l): peptone - 5.0; yeast extract - 2.0; glucose - 1.0; K ₂ HPO ₄ 0.2; casein hydrolyzate - 2.0; MgSO ₄ - 0.05; agar-agar - 20.0; distilled water - 500 ml, sea water - 500 ml, pH of 7.8, after five days of incubation at 28 ^o C.

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 and in a 30% glycerol solution at a temperature of 80 ^o C.

The strain has the following characteristics:
Cultural and morphological features
Strain KMM 3549 ^T forms round (5-8 mm) convex mucous transparent colonies on smooth agar media with smooth edges. Cells are mobile small (0.5-0.9 • 1.0-1.5 microns) Gram-negative bacilli, having an aerobic type of metabolism, capable of growing at temperatures from 4 to 37 ^o C.

Physiological and biochemical characteristics
Strain KMM 3549 ^{T is} oxidase- and catalase-positive, needs sodium ions for growth, is characterized by the ability to hydrolyze DNA, gelatin, chitin, alginate, casein, tween-85 and the lack of the ability to hydrolyze starch, agar, carrageenan. Does not produce hydrogen sulfide, indole, acetone. Assimilates D-galactose, D-fructose, acetate, pyruvate, fumarate, citrate, maltose, sucrose, melibiosis, lactose, succinate, mannitol. Does not absorb mannose, gluconate, glycerin, xylose, trehalose and fucose.

Chemotaxonomic signs
The main fatty acids are represented by 15: 1 (n-8) (5.0%), 16: 1 (n-7) (45.6%), 16: 0 (15.1%), 18: l (n- 7) (6.6%), 17: l (n-8) (8.2%).

Based on physiological, biochemical, and chemotaxonomic characters, strain KMM 3549 ^{T is} assigned to bacteria of the genus Pseudoalteromonas.

Molecular genetic traits
The composition of G + C bases in DNA is 43.3 mol%.

 Phylogenetic analysis of the nucleotide sequences of the 16S rRNA gene revealed 98.6-99.8% homology with the 16S rRNA genes of Pseudoalteromonas tetraodonis, Pseudoalteromonas espejiana, Pseudoalteromonas atlantica. Pseudoalteromonas carrageenovora.

DNA-DNA experiments on hybridization of strain KMM 3549 ^T with phylogenetically similar type strains showed 50.0% homology with P. tetraodonis IAM 14160 ^T , 38% with P. espejiana IAM 13640 ^T , 54% with P. atlantica IAM 12927 ^T and 48% with P. carrageenova IAM 12662 ^T , which allowed the identification of strain KMM 3549 as a new species [10] - Pseudoalteromonas issachenkonii.

The initial cultivation of the strain KMM 3549 ^{T is} carried out on a standard medium of the following composition (g / l): peptone - 5.0; yeast extract - 2.0; casein hydrolyzate - 2.0; MgSO ₄ - 0.05; agar-agar - 20.0; fucoidan - 1.0; sea water - 1000 ml, pH 7.8.

The seed Pseudoalteromonas issachenkonii KMM 3549 ^{T is} grown in 100 ml flasks containing 50 ml of fermentation medium for 20-24 hours at a temperature of 20-25 ^o C on a shaker with a rotation speed of 150 rpm, until 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 20 hours at a temperature of 20-25 ^o C on a rocking chair with a rotation speed of 120 rpm

 Preparation of bacterial extracts for testing glycoside hydrolase activities.

After cultivation in 500 ml of a liquid medium with fucoidan for 24 hours at 25 ^° C, cells (1 g of fresh weight) are collected by centrifugation for 30 minutes at 5000 rpm at 4 ^° C, suspended in 10 ml of 0.01 M phosphate buffer, pH 7.2 and destroy by ultrasound (20 kHz). The resulting homogenate is centrifuged at 15,000 rpm for 20 minutes. The supernatant is used as a crude cellular extract of the enzyme.

 The glycanase activity of the crude extract was tested using brown seaweed fucoidan Fucus evanescens [11], brown alga Laminaria cichorioides [12], pustulan from Umbellicaria russica lichen [13]. Alginic acid and agar (Serva, USA) are used as substrates to determine the activity of alginase and agarase. The action of the enzyme is recorded by the Nelson calorimetric method [14] by the appearance of reducing sugars, the hydrolysis products of the above polysaccharides. The amount of protein is measured by the Lowry method using bovine serum albumin as a standard [15]. The specific activity of glycanases is expressed as the amount of enzyme releasing 1 μmol of reducing sugars in 1 hour per 1 mg of protein.

Glycosidase activities are tested using p-nitrophenyl derivatives of the corresponding monosaccharides (Sigma, USA) in 0.01 M phosphate buffer, pH 7.2 at 20 ^° C. The reaction mixture consists of 0.05 ml of crude extract and 0.2 ml of p-nitrophenyl glycoside (1 mg / ml in 0.01 M phosphate buffer, pH 7.2). The reaction is stopped by the addition of 1.25 ml of 1M Na ₂ CO ₃ . The specific activity of glycosidases is defined as the amount of enzyme under the action of which 1 μmol of p-nitrophenol per min per 1 mg of protein is formed.

The activity of glycanases and glycosidases from the cell extract of Pseudoalteromonas issachenkonii KMM 3549 ^{T is} shown in table. 1. The biomass extract of these bacteria contains highly active laminarinase, alginase, less active pustulanase and fucoidan hydrolase. Carrageenase and agarase were not detected. In addition, glycosidases that catalyze the hydrolysis of mainly β-O-glycosidic bonds (β-galactosidase, β-glucosidase, β-xylosidase and N-acetyl-β-D-glucosaminidase) were found in the cell extract of Pseudoalteromonas issachenkonii KMM 3549. Glycosidases that catalyze the hydrolysis of α-O-glycosidic bonds (α-galactosidase, N-acetyl-α-D-galactosaminidase, α-mannosidase, α-fucosidase) were not detected.

To identify conditions for optimal culture growth and the accumulation of glycoside-hydrolase activities in the medium, the cultivation of P. issachenkonii KMM 3549 ^T strain is carried out on nutrient media of various compositions. The experiments are carried out in flasks on a thermostatically controlled shaker (170 rpm) at a temperature of 28 ^o C for 20 hours. Each flask contains 100 ml of culture medium and an equal amount (5%) of seed. All experiments are carried out under the same conditions in two replicates.

 Culture media contain peptone as a nitrogen source, yeast extract as a component containing the necessary growth factors, poly- or monosaccharides as sources of carbon and energy and sea water.

Initially, the optimization of the composition of the nutrient medium for the strain P. issachenkonii KMM 3549 ^T for the main components is carried out. As can be seen from the table. 2, of the six variants, only two modifications of the medium, including peptone and glucose or only peptone, have a positive effect on the synthesis of fucoidan hydrolases. The presence in the nutrient medium of one yeast extract leads to a relatively weak growth of bacteria and the absence of fucoidan-hydrolase activity. On the medium containing peptone and yeast extract, and on the medium containing peptone, yeast extract and glucose, intensive bacterial growth is observed, which is 3 times higher than that on the medium with peptone, but there is no fucoidan-hydrolase activity. An analysis of the results of the first series of experiments suggests that only peptone is significant for the synthesis of fucoidan hydrolase, the presence of yeast extract and glucose is not significant.

 The effect of various peptone concentrations on the synthesis of fucoidan hydrolase, laminarinase, alginase, and N-acetyl-β-D-glucosaminidase is further investigated (Table 3). According to the results obtained, a peptone concentration of 2.5 g / L is significant for the synthesis of fucoidan hydrolase. The maximum specific activity of alginase is observed at a peptone concentration of 1 g / L. The effect of various peptone concentrations on the synthesis of N-acetyl-β-D-glucosaminidase is practically absent. An increase in peptone concentration to 10 g / l leads to an intensive growth of bacteria, but not to an increase in the level of glycoside-hydrolase activities.

 The results of the next series of experiments contain data on the effect of fucose, xylose, ramnose and galactose as monosaccharide fragments of fucoidans on the synthesis of glycoside hydrolases (Table 4). Higher fucoidan hydrolase activity is achieved using xylose and galactose. However, in the presence of galactose, the activity of concomitant glycoside hydrolases is also markedly increased.

 Varying the xylose concentration in the fourth series of experiments (Table 5) shows that the specific activity of fucoidan hydrolase becomes maximum at a xylose concentration of 5 g / L. A high concentration of xylose (10 g / l) in the culture medium inhibits bacterial growth.

 Nutrient optimization experiments using polysaccharides — fucoidan, laminaran, alginate, agarose, and carrageenan — show that agarose and carrageenan inhibit fucoidan hydrolase synthesis (Table 6). Fucoidan weakly induces the synthesis of fucoidan hydrolase, but is a positive factor for the synthesis of N-acetyl-β-D-glucosaminidase. A positive factor for the production of fucoidan hydrolase is the presence of both laminaran and alginate, but the addition of both laminaran and alginate to the medium increases the specific activity of laminarinase, a high content of which is not desirable during the purification procedure of fucoidan hydrolase. The only enzyme, the synthesis of which is not affected by the presence of polysaccharide inducers, was alginase.

 It is important to note that experiments on optimizing the nutrient medium using polysaccharides show that it is inappropriate to include polysaccharides as a carbon source in the composition of nutrient media, since the activity of fucoidan hydrolases in this case, as well as other glycoside hydrolases, is very low.

 The invention is illustrated by the following examples.

Example 1. The cultivation of the strain Pseudoalteromonas issachenkonii KMM 3549 ^{T is} carried out on a medium of the following composition (g / l): peptone - 2.5; xylose - 1.0; sea water - up to 1 l, pH 7.8. The cultivation of bacteria, obtaining cell extracts and determination of glycosidase activities in this and subsequent examples are performed as described above (table. 7).

Example 2. The cultivation of the strain Pseudoalteromonas issachenkonii KMM 3549 ^{T is} carried out on a medium of the following composition (g / l): peptone - 2.5; xylose - 5.0; sea water - up to 1 l, pH 7.8.

Example 3. The cultivation of the strain Pseudoalteromonas issachenkonii KMM 3549 ^{T is} carried out on a medium of the following composition (g / l): peptone - 5.0; xylose - 1.0; sea water - up to 1 l, pH 7.8.

Example 4. The cultivation of the strain Pseudoalteromonas issachenkonii KMM 3549 ^{T is} carried out on a medium of the following composition (g / l): peptone - 5.0; xylose - 5.0; sea water - up to 1 l, pH 7.8.

Thus, by varying the composition of the nutrient medium, the synthesis of fucoidan hydrolase, laminarinase, and N-acetyl-β-D-glucosaminidase can be regulated. The use of the proposed strain Pseudoalteromonas issachenkonii KMM 3549 ^T and the medium for its cultivation can significantly simplify the process of obtaining fucoidan hydrolases and reduce the cost of the resulting products, and also allows to increase the specific activity of fucoidan hydrolases by 3 times.

Claims (2)

1. The strain Pseudoalteromonas issachenkonii KMM 3549 ^T is a producer of fucoidan hydrolases. 2. A nutrient medium for the cultivation of a producer of fucoidan hydrolase, containing sources of nitrogen, carbon and energy, a source of growth substances, characterized in that it contains peptone as a source of nitrogen, xylose as a carbon and energy source, and sea water in the following ratio of components, g / l:
Peptone - 2.5-5.0
Xylose - 1.0-5.0
Sea water - Up to 1 hp

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