RU2223322C2 - Method for preparing roquefortin and strain for its realization - Google Patents

Abstract

FIELD: biotechnology, microbiology. SUBSTANCE: invention proposes the novel strain Penicillium roquefortii f39 that was obtained by ultraviolet mutagenesis method of culture Penicillium roquefortii VKM F- 2389. The obtained strain shows high level of roquefortin synthesis and its parts in alkaloids sum. Also, invention proposes method for preparing roquefortin involving culturing the strain Penicillium roquerfortii f39 under aeration condition and stirring in medium containing mannitol, succinic acid, urea, MgSO₄×7H₂O, KH₂PO₄ and mixture of trace elements. Proposed method for preparing roquefortin using the novel strain Penicillium roquefortii f39 provides reducing culturing time significantly and to enhance the yield of the end product. Proposed inventions can be used in veterinary science for preparing alkaloid roquefortin used as biochemical reagent. EFFECT: improved preparing method. 2 cl, 5 ex

Description

 The invention relates to the field of biotechnology and microbiology, and can be used to produce rockfortin alkaloid, suitable for use as a biochemical reagent, as well as in veterinary medicine.

Roquefortin-10β- (1.1-dimethyl-2-propenyl) -3- (imidazol-4-ylmethylene) -5α, 10β, 11.11α-tetrahydro-2H-pyrazino [1 ^' , 2 ^' : 1,5] pyrrolo [2 , 3-b] indole -] - 1,4- (3Н, 6Н) - dione is a secondary metabolite (alkaloid, mycotoxin) of many fungi of the Penicillium genus, for which pronounced neurotoxic, antibiotic and antiprotozoal properties are determined.

 Its producers are widespread in nature and often affect food and feed, the consumption of which can lead to poisoning. This problem is especially acute in veterinary medicine, given the high sensitivity of some farm and domestic animals to rockfortin (Schell M. M. // Veterinary Medicine. 2000. V.95. N.4. P.283-286). This makes it necessary to control the content of this mycotoxin in feed. Rockfortin is also used as a chemotaxonomic marker in the determination of certain species of fungi of the genus Penicillium (Frisvad J.C., Filtenborg O. // Appl. Environment Microbiol. 1983. V.46. N.6. P. 1301-1310).

 Known methods for microbiological production of rockfortin alkaloid by culturing various fungi of the genus Penicillium.

 The production of rockfortin by culturing Penicillium commune AUA 827 micromycete is described (Wagener R.E., Davis N. D., Diener U. I. // Appl. Environment. Microbiol. 1980. V.39. N.4. P.882-887). The culture is grown for 7-10 days superficially on a medium containing sucrose (50 g / l) and yeast extract (20 g / l) as a source of nitrogen. The content of rockfortin reached 100 mg / L.

 Quantitative assessment was carried out by thin-layer chromatography by comparing the size of the spots with spots containing a known amount of substance, the error rate of the method can reach 20%. Rockfortin is isolated from mycelium homogenate by extraction with chloroform.

 In this method, in addition to rockfortin, the mushroom producer synthesized a significant amount of another metabolite, penitrem A. The presence of impurities of this biologically highly active compound in rockfortin preparations is extremely undesirable when using the latter as a biochemical reagent. In addition, it is known that the fungus P.commune is capable of causing allergic diseases in humans. The intracellular localization of the alkaloid complicates the procedure for its isolation by the need to destroy the mycelium.

There is also a method of producing rockfortin with the fungus Penicillium farinosum (= Penicillium crustosum) VKM F-1746 (Dudina L.P., Reshetilova T.A., Kozlovsky A.G., Eroshin V.K. // Prikl. Biochemistry and microbiology . 1993. V.29. Issue 5. S.700-705). The producer is grown in a deep way with stirring and aeration on an Abe medium containing (g / l): mannitol - 50.0; succinic acid - 5.4; KH ₂ PO ₄ - 10 MgSO ₄ • 7H ₂ O - 0.3, ammonia solution - up to pH 5.3. The cultivation duration is 5-8 days. The authors proposed procedure for fractional subtraction of the pH of the medium in the range of 6-8 during the cultivation process allows to increase the content of extracellular rockfortin to 100 mg / l.

 The main disadvantage of this method is the use of pathogenic fungus as a producer of rockfortin. It is known that P.crustosum belongs to microorganisms of the III group of pathogenicity and causes penicilliosis (SP 1.2.011-94, approved by the State Sanitary and Epidemiological Supervision of the Russian Federation).

 In addition, the biosynthesis of the target product during the cultivation of P.crustosum on the Abe medium is complex with several maxima and minima. At the same time, a high rate of change in the concentration of roquefortin complicates the choice of the optimal moment of alkaloid release and is an important reason for the low reproducibility of the results by fermentation.

 Closest to the proposed is a method of producing rockfortin, including growing a surface culture of Penicillium roquefortii NRB 111275 on a liquid medium containing sucrose (150 g / l) and yeast extract (20 g / l) (Scott R.M., Kennedy B.P. C., Harwig J., Blanchfield, B.J. // Appl. Environment. Microbiol. 1977. V. 33. N 2. P.249-253). In this case, intracellular accumulation of the alkaloid to a concentration of 100 mg / L occurs.

 However, the low growth rate of the producer under the proposed conditions led to a longer cultivation time. The highest alkaloid content was observed on days 16-20 of fermentation.

 In the case of isolation of roquefortin, its intracellular localization complicates this procedure: mechanical destruction of mycelium and multistage extraction of roquefortin using two organic solvents, chloroform and acetone, are necessary.

 Known producer of rockfortin - a strain of the fungus Penicillium roquefortii VKM F-2389. (A. G. Kozlovsky, T. A. Reshetilova. Microbiology. 1984. V. 53, issue 1, pp. 81-84). However, the level of synthesis of rockfortin is low, the maximum accumulation requires about 10 days of cultivation.

 The problem to which the invention is directed, is to create a method of microbiological production of rockfortin alkaloid using a new producer strain.

 The technical result that can be obtained by carrying out the invention is that the cultivation period is significantly reduced.

The essence of the proposed method lies in the fact that they use the strain of the fungus Penicillium roquefortii f 39, cultivate it under aeration and stirring on a medium containing mannitol, succinic acid, urea, MgSO ₄ • 7H ₂ O, KH ₂ PO ₄ and a mixture of trace elements in the following quantities (g / l):
Mannitol - 60.0-75.0
Succinic acid - 2.9-9.8
Urea - 2.0-5.0
MgSO ₄ • 7H ₂ O - 0.1-0.5
KN ₂ RO ₄ - 0.5-1.5
A mixture of trace elements - 1-5 ml / l of medium
Water - Up to 1 L
pH (adjusts with NaOH solution) - 3.8-5.5
Isolation of rockfortin is carried out by known methods.

 The strain of the fungus Penicillium roquefortii f39 was obtained by ultraviolet mutagenesis of a culture of Penicillium roquefortii VKM F-2389. The strain of the fungus Penicillium roquefortii f39 is stored in the collection of mushrooms IBPM them. G.K. Scriabin RAS.

 The obtained mutant strain was characterized by a high level of synthesis of rockfortin and its share in the total alkaloids (55-60% compared to 15% in P. roquefortii VKM F-2389). Morphological properties. The strain of the fungus Penicillium roquefortii f39 (wort agar, 24C, 7 days) forms compact colonies 20-25 mm in diameter with scalloped edge, flat, salad-colored, the growing edge is white, 1.0-2.0 mm wide. The reverse side of the colony is flesh-colored. The culture has a pleasant mushroom smell. Exudate is absent. Conidial structures differentiate relatively slowly. The tassels are typically asymmetric, with long chains of conidia. Conidiophores 200.0-300.0 • 4.5-5.0 microns. Twigs 12.0-18.0 • 3.5-4.0 microns. 3-4 formulas per whorl, 10.0-12.0 • 3.5 microns. Sterigma 6.0-8.02.2-2.5 microns. Conidia are smooth, spherical and hemispherical, 3.5-4.0 microns in diameter.

Features of the development of the strain of the fungus Penicillium roquefortii f39 in comparison with the original culture of P. roquefortii VKM F-2389 are shown in the table
In addition, 1) a reduced content of biomass, 2) a large content of extracellular rockfortin per unit of biomass - up to 3.5 mg / g (about 2.4 in the wild type).

 The species P.roquefortii is not pathogenic, as indicated by the U.S. Environmental Protection Agency (URL: http://www.era.gov/opptintr/biotech/fra/fd008.htm).

 The proposed strain is characterized by high activity of formation of rockfortin, despite the fact that the release of the main amount of metabolite occurs on Wednesday.

 Rockfortin biosynthesis by P. roquefortii f 39 culture is stimulated by the medium composition described above.

 Mannitol in combination with succinic acid provides the possibility of deep fermentation and accumulation of the target product in the medium, and not in the mycelium.

 The introduction of urea into the medium stimulates and stabilizes the biocynthesis of roquefortin, since it allows avoiding repeatedly described abrupt and unpredictable changes in the concentration of roquefortin during the growth of the producer on Abe medium. The control of fermentation is simplified and high reproducibility of the results is ensured.

 Sodium hydroxide is introduced into the medium during its preparation to neutralize succinic acid and create the required pH.

Trace elements are introduced into the medium in the form of a mixture described in book. "Methods of General Bacteriology." Ed. F. Gerhardt and others. T.1. 1983. M.: Mir, 347 p. This mixture has the following composition, g:
EDTA - 50
ZnSO ₄ • 7H ₂ O - 22
CaCl ₂ - 5.54
MnCl ₂ • 4H ₂ O - 5.06
FeSO ₄ • 7H ₂ O - 4.99
(NH ₄ ) ₆ Mo ₇ O ₂₄ • 4H ₂ O - 1.1
CUSO ₄ • 5H ₂ O - 1.57
CoCl ₂ • 6H ₂ O - 1.61
Distilled Water - 1000 ml
The pH was adjusted to 6.0 with KOH.

 The implementation of the method is illustrated by the examples, but is not limited to.

Example 1. A strain of Penicillium roquefortii f39 is grown in an ANKUM-2 fermenter with a volume of 10 l on a medium of the following composition (g / l): mannitol - 65.0; succinic acid - 5.4; urea - 3.0; MgSO ₄ • 7H ₂ O — 0.3; KN ₂ RO ₄ - 1.0; trace element mixture - 2 ml / l; 10% NaOH solution - up to pH 4.5; distilled water.

 The volume of medium in the fermenter is 6.0 liters. The cultivation is carried out with stirring, a temperature of 22-35C, aeration of 30-40% of saturation, for 7 days.

 To determine the concentration of rockfortin, the culture fluid is filtered, the filtrate is extracted twice with equal volumes of chloroform. The chloroform extract was dehydrated, concentrated in vacuo and analyzed by HPLC. By the end of fermentation, the content of rockfortin in the culture fluid is 68 mg / L.

 Example 2. The strain P. roquefortii f39 is grown in an ANKUM-2 fermenter with a volume of 10 l on the medium specified in example 1, but the mannitol content in the medium is 60 g / l, succinic acid is 5.2 g / l, the pH of the medium is 5 , 2. The volume of medium in the fermenter is 7.0 liters.

Cultivation is carried out at a temperature of 22-24 ^o C, aeration of 30-40% of saturation for 8 days.

 By the end of fermentation, the content of rockfortin in the culture fluid is 52 mg / L.

Example 3. The strain P. roquefortii f39 is grown in an ANKUM-2 fermenter with a volume of 10 l on the medium specified in example 1, but the urea content in the medium is 5.0 g / l, the pH of the medium is 4.0; MgSO ₄ • 7H ₂ O — 0.15; KN ₂ RO ₄ - 1.5 g / l. Cultivation is carried out at 25-28 ^o C, aeration of 40-50% of saturation for 7 days.

 By the end of fermentation, the content of rockfortin in the culture fluid is 72 mg / L.

Example 4. The strain P. roquefortii f39 is grown in an ANKUM-2 fermenter with a volume of 10 l on the medium specified in example 1, but the fatty acid content is 2.9 g / l, urea in the medium is 2.0 g / l; pH of the medium is 5.0. MgSO ₄ • 7H ₂ O — 0.3; KN ₂ RO ₄ - 0.5 g / l. Cultivation is carried out at 25-28 ^o C, aeration of 40-50% of saturation for 7 days.

 By the end of fermentation, the content of rockfortin in the culture fluid is 55 mg / L.

Example 5. The strain P. roquefortii f39 is grown in an ANKUM-2 fermenter with a volume of 10 l on the medium specified in example 1, but the urea content in the medium is 3.5 g / l, the pH of the medium is 4.5. MgSO ₄ • 7H ₂ O — 0.2; KN ₂ PO ₄ - 0.8 g / l. Cultivation is carried out at 25-28 ^o C, aeration of 40-50% of saturation for 7 days.

 By the end of fermentation, the content of rockfortin in the culture fluid is 68 mg / L.

 Thus, the method can significantly reduce the duration of the process to 6-8 days with a high content of the target product (about 70 mg / l).

 The proposed method has a high degree of reproducibility, it uses a non-pathogenic strain, which is important for the safety of personnel.

 In addition, a stable level of content of rockfortin is provided, without subtracting the pH of the medium. The target product accumulates in the medium, which does not require destruction of the mycelium and, accordingly, simplifies the subsequent stage of isolation.

Claims (2)

1. The method of producing rockfortin, including cultivating a fungus of the species Penicillium roquefortii on a medium containing carbon and nitrogen sources, and isolating by known methods, characterized in that a strain of Penicillium roquefortii f39 is used from fungi of the species Penicillium roquefortii, cultivating it under conditions of aeration and stirring on a medium containing mannitol, succinic acid, urea, MgSO ₄ × 7H ₂ O, KH ₂ PO ₄ and a mixture of trace elements in the following amounts, g / l: Mannitol 60.0-75.0 Succinic acid 2.9-9.8 Urea 2.0-5.0 MgSO ₄ × 7H ₂ O 0.1-0.5 KN ₂ PO ₄ 0.5-1.5 A mixture of trace elements, ml / l of medium 1-5 Water, l Up to 1 l pH (adjusted with NaOH solution) 3.8-5.5 2. The strain of the fungus Penicillium roquefortii f39 is stored in the collection of mushrooms IBPM them. G.K. Scriabin RAS - producer of rockfortin.

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