SK22006A3 - Streptomyces toxytricini strain producing the lipstatine and the production of the lipstatine by the said strain. - Google Patents

Abstract

Technical solution is related to industry production microorganism Streptomyces toxytricini CCM 7349 and method of lipstatin biotechnology preparation by this strain. Production strain listed above with use of two step vegetative inoculum produce lipstatin in concentration from 3,0 to 3,2 g/kg fermentation broth after 180 till 230 hours of cultivation under temperature at 28°C, under optimal mixing above 450 rpm, aeration 1 till 1,25 vvm and optimal addition of linoleic acid and 8% solution of L-Leucine.

Description

Technical field

The invention relates to the strain Streptomyces toxytricini deposited at the Czech Collection of Microorganisms in Brno under the number CCM 7349, and to the biotechnological preparation of lipstatin with said strain in an amount of 3.0 to 3.2 g / kg of fermentation broth for 180 to 230 hours of cultivation in C.

BACKGROUND OF THE INVENTION

Streptomyces toxytricini strain CCM 7349 is a lipstatin producer under aerobic culture conditions. The basic building block of lipstatin is the β-lactam ring bearing two aliphatic residues of long fatty acids with 6-14 carbon atoms. One of the side chains contains two double bonds and one hydroxy group which is esterified as N-formyl-leucine. By catalytic hydrogenation of lipstatin, tetrahydrolipstatin (orlistat) can be obtained.

The microorganism Streptomyces toxytricini is able to utilize fats or fatty acids up to acetyl-CoA. If lipstatin biosynthesis occurs during fermentation, C14 and C8 fatty acids or long chain fatty acids condense. Linolic acid contains the same number of double bonds as the lipstatin chain and is the basic building block of lipstatin. Claisen condensation of 3-hydroxy-5,8-tetradecadienoyl-CoA and octanoyl-CoA gives the basic intermediate 5-hydroxy-2-hexyl-3-oxo-7,10-hexadecadienoic acid ester. In the next step, catalytic hydrogenation of the ester group occurs, followed by formation of the β-lactam ring. The side chain esterification occurs in the presence of leucine.

Orlistat (tetrahydrolipstatin), with a molecular weight of 495.74 g / mol, is an active substance for weight reduction. It acts at the site of fat breakdown, ie. in the stomach and small intestine. By its action it blocks a pancreatic enzyme called lipase, preventing the absorption of fat from food up to one third. This phenomenon is used in the treatment of obesity, and may also serve to prevent hyperlipemia and arteriosclerosis. The taxonomy of bacterial streptomyces species is described in the International Streptomyces Project in Journal of Fermentation Technology Vol 2, p.52, 1974. The Streptomyces toxytricini strain Preobrazhenskaya & Sveshnikova is described in Bergey's Manual of Determinative Bacteriology, 8th Edition, page 811.

U.S.Patent 4,598,089 and Eur.Pat.Appl. No. 129748 describes a process for the preparation of lipstatin and orlistat by a biotechnological route with a specific strain of Streptomyces toxytricini NRRL 15443. This patent describes the preparation of a two-stage vegetative inoculum required for inoculation of a production step.

The production medium as the main carbon source comprises a mixture of potato starch, glucose, ribose, and glycerin. It mentions peptone, soy flour and ammonium sulfate as the nitrogen source. During the cultivation of the production step, a cultivation temperature of 28 ° C, aeration of 1 vvm and stirring of 150 rpm are maintained. U.S. Patent No. JS2002 / 0110873 A1 discloses lipstatin production under the conditions described in U.S. 4,598,089 and, using the Streptomyces toxytricini NRRL 15443 strain, the production of lipstatin 9106 mg / L in 184 hours of culture.

Weibel et al. describes the fermentation process of lipstatin (Journal of Antibiotics Vol XL, No.8 pp.1086 1091) and E. Houli describes the chemical structure of lipstatin (Journal of Antibiotics Vol XL, No.8 pp.1081 1085).

EP 0 803 567 A2 describes a fermentation process for the preparation of lipstatin using precursors such as linoleic acid, caprylic acid and N-formyl-L-leucine or L-Leucine. The fermentation yield is low because both acids are toxic. In comparison, U.S.Patent 6,844,174 utilizes a production medium containing free fatty acids and oils from the group of natural oils, synthetic oils or a mixture thereof in a fermentation process. The result is the release of fatty acids from the oils during the fermentation process depending on the pH of the production medium and the high residual concentration of oil residues at the end of the fermentation. A related problem is the question of emulsifying the production medium with suitable emulsifiers such as e.g. Lecithin, Triton X-100, Brij 35 S. A disadvantage is that increasing the amount of emulsifier increases the viscosity of the production medium and worsens the transfer of oxygen into the fermentation medium, which has a negative effect on lipstatin production.

Patent Publication No. WO 03/048335 discloses fermentation of lipstatin by cup culture in a production medium comprising soy and cotton flour, glycerin as a carbon substrate, polypropylene glycol as a defoamer, and a lecithin emulsifier. Fermentation lasts 6 to 7 days with a maximum production of 1,700 to 1,800 mg / l. On a pilot scale of 4 liters of fermentation medium, lipstatin production with the same technology achieves 1,500 to 1,600 mg / l after 144 to 168 hours of cultivation.

WO 2004/003212 A1 describes the preparation of lipstatin on various starch substrates with a maximum lipstatin production of 1486 mg / kg substrate.

SUMMARY OF THE INVENTION

The invention relates to a strain of Streptomyces toxytricini CCM 7349 and the biotechnological preparation of lipstatin without the use of an emulsifier in the production medium, and using precursors such as linoleic acid and L-Leucine. The Streptomyces toxytricini CCM 7349 strain produces lipstatin at a concentration of 3.0-3.2 g / kg fermentation broth for 180-230 hours of culture in the production medium at 28 ° C using a two-stage inoculum.

The cultivation of the strain Streptomyces toxytricini CCM 7349 used in the fermentation process to which the patent relates is carried out in four steps:

1. Culture sporulation

2. I. inoculation stage

3. II. inoculation stage

4. Production stage

Sporulácia kultury

Good sporulation is important for the quality of the inoculum (within 5 to 10 days at 28 ° C the culture is sporulated, the colonies are round, slightly convex, wrinkled, the airy mycelium is brownish pink with a white zone at the edge of the colony with a colony size of about 5 to 10 mm) . Sporulation broth with the following composition is used for culture sporulation: dextrin 4g / l, yeast extract 4g / l, malt extract 10 g / l, CaCO ₃ 2 g / l, agar 20 g / l and distilled water up to 1000 ml

I. inoculation stage

Cultivation of the vegetative inoculum of the first stage of inoculation is carried out at 28 ° C for 24 hours. A quality inoculum is characterized by the following properties:

- small pellets

- biomass sediment 14 to 16%

The first stage seed medium contains only one source of carbonaceous substrate having the following composition: soy flour 10 g / l, glycerin 10 g / l, yeast extract 5 g / l, drinking water up to 1000 ml.

II. inoculation stage

Cultivation of vegetative inoculum II. The inoculation step is carried out at 28 ° C for 12 to 16 hours. A quality inoculum is characterized by the following properties:

- Large pellets

- biomass sediment 20 to 26%

Inoculation medium of the 2nd stage contains only one source of carbonaceous substrate with the following composition: soya flour 30 g / l, yeast extract 5g / l, glycerine 20 g / l, (NH ₄ ) ₂ SO ₄ 2 g / l, CaCO ₃ 6 g / l, antifoam Slovaník 1 g / l.

Production stage

a.) On a laboratory scale, a production medium of the following composition is used to test and select the production isolates of the Streptomyces toxytricini CCM 7349 production microorganism: soya flour 48 g / l, glycerine 30 g / l, CaCO ₃ 5g / l, yeast extract 1 g / 1, soybean oil methyl ester 40 g / l, L-Leucine 5 g / l, MgSO ₄ .7H ₂ O 0.05 g / l, drinking water up to 1000 ml.

The production medium contains a non-toxic soybean oil methyl ester as a precursor of lipstatin over linoleic acid, whose concentration above 1 g / l has a toxic effect on the producing microorganism. The cultivation is carried out at a cultivation temperature of 28 ° C at 250 rpm for 72 hours with a production of lipstatin of 0.3 to 0.45 g / kg.

b.) For pilot production of lipstatin with the production microorganism Streptomyces toxytricini CCM 7349, the production medium is as follows: soya flour 48 g / l, glycerine 50 g / l, yeast extract 1 g / l, CaCO ₃ 5 g / l, KH2PO4 0 1 g / l, MgSO ₄ .7H ₂ O 0.3 g / l, FeSO ₄ .7H ₂ O 0.03 g / l, MnSO ₄ .4H ₂ O 0.04 g / l, ZnSO ₄ .7H ₂ O 0.005g / l, soybean oil 40 g / l, Slovanik defoamer 1 g / l.

Cultivation is carried out using a two-stage vegetative inoculum at a culture temperature of 28 ° C hours with a production of delatine of 3.0-3.2 g / kg in 180-230 hours. The pH during cultivation is maintained in the range of 6 to 8, the optimum pH of 6.7 to 7.3 with 28% sodium hydroxide solution or 20% sulfuric acid solution.

In lipstatin synthesis, linoleic acid with a maintenance concentration of 0.2 to 1 g / l of fermentation broth is used as a precursor together with 8% L-Leucine solution at pH 11. The start of dosing of linoleic acid and LLeucine is at the end of the growth phase characterized by utilization of the initial carbonaceous source accompanied by an increased concentration of soluble oxygen in the fermentation broth. Linoleic acid and L-Leucine are dosed continuously at a ratio of 1: 0.8.

The total amount of L-Leucine added during the culture was 3265 g per 40 L fermentation broth. The production medium utilized glycerin and soybean oil as a source of carbonaceous substrate, and soy flour and yeast extract as a source of organic nitrogen and phosphorus. The advantage of cultivation is that it is not necessary to add emulsifiers to the production medium as disclosed in U.S. Patent 6,844,174 to ensure sufficient viscosity. On a pilot scale production stage with a volume of 40 liters of production medium, cultivation takes place in 10 times the volume of WO 03/048335 with transfer characteristics of aeration above 1vvm and stirring above 450 rpm, maintaining a soluble oxygen concentration in the fermentation broth not lower than 30% . These parameters are realistically achievable on a large scale.

The advantage of culturing with Streptomyces toxytricini CCM 7349 is that by eliminating the emulsifier from the base composition of the production medium, oxygen transfer to the producer cells is improved.

Tribe description: Streptomyces toxytricini strain CCM 7349

Morphology: - the substrate mycelium of Streptomyces toxytricini CCM 7349 on sporulation soil is dark brown in color, airy mycelium in pink-pink color. The individual colonies of the strain Streptomyces toxytricini are round, slightly convex to wrinkled, brown-pink in color with a white zone at the edge of the colony, 5-10 mm in diameter.

Optimal pH of cultivation: from 6 to 8

Carbon substrate utilization: well utilizes glycerin, glucose, dextrin, weaker utilizes sucrose, lactose

Optimal culture temperature: 28 ± 1 ° C

Precursors: Linoleic acid, L-Leucine, soybean oil methyl ester

Examples:

Príklad.č.1

(a) Preparation of dispute material

The spore suspension of Streptomyces toxytridni strain is applied to sporulated soil of the following composition: dextrin 4g / l, yeast extract 4g / l, malt extract 10 g / l, CaCO ₃ 2 g / l, agar 20 g / l and distilled water to 1000 ml. The strain is cultured at 28 ° C for 5 to 10 days.

(b) the inoculation stage

Spores of the Streptomyces toxytridni strain are used to inoculate 100 ml of inoculum broth in 500 ml Erlenmeyer flasks. The composition of the seed medium is as follows: soya flour 10 g / l, glycerine 10 g / l, yeast extract 5 g / l, drinking water up to 1000 ml. After sterilization at 120 ° C for 30 minutes, the pH is optimized to 6.8 to 7.2. The inoculum is cultured at 28 ° C for 24-26 hours. The prepared vegetative inoculum is used to inoculate the production step.

(c) production stage

The vegetative inoculum prepared in Erlenmayer flasks is inoculated at 1% to 5% in 60 ml production medium in Erlenmayer flasks of the following composition: soya flour 48 g / l, glycerine 30 g / l, CaCO ₃ 5g / l, yeast extract 1 g / 1, soybean oil methyl ester 40 g / l, L-Leucine 5 g / l, MgSO ₄ .7H ₂ O 0.05 g / l and drinking water up to 1000 ml. The production medium is sterilized for 30 minutes at 120 ° C. The optimum pH is 6.8 to 7.4. After 72 hours of cultivation on a rotary shaker at 28 ° C and at a rotational speed of 180 to 250 rpm, the lipstatin content is determined by HPLC, with a concentration ranging from 0.3 g / kg to 0.45 g / kg.

Príklad.č.2

(a) Preparation of dispute material

The spore suspension of Streptomyces toxytridni strain is applied to sporulated soil of the following composition: dextrin 4g / l, yeast extract 4g / l, malt extract 10 g / l, CaCO ₃ 2 g / l, agar 20 g / l and distilled water to 1000 ml. The strain is cultured at 28 ° C for 5 to 10 days.

(b) the inoculation stage

Spores of the Streptomyces toxytridni strain are used to inoculate 100 ml of inoculum broth in 500 ml Erlenmeyer flasks. The composition of the seed medium is as follows: soya flour 10 g / l, glycerine 10 g / l, yeast extract 5 g / l, drinking water up to 1000 ml. After sterilization at 120 ° C for 30 minutes, the pH is 6.9 to optimal

7.5. The inoculum is cultured at 28 ° C for 24-26 hours. The prepared vegetative inoculum is used to inoculate the production step.

(c) production stage

The vegetative inoculum prepared in Erlenmeyer flasks is inoculated at 1% to 5% to the production stage in a 70 L fermentation tank with a volume of 40 L of the production medium of the following composition: soya flour 48 g / l, glycerin 50 g / l, yeast extract 1 g / 1, CaCO ₃ 5 g / l, KH2PO4 0.1 g / l, MgSO ₄ .7H ₂ O 0.3 g / l, FeSO ₄ .7H ₂ O 0.03 g / l, MnSO ₄ .4H ₂ O 0 , 04 g / l, ZnSO ₄ .7H ₂ O 0.005g / l, soybean oil 40 g / l, Siovanik defoamer 1g / l. Optimal cultivation conditions are maintained during the cultivation. The concentration of saturated oxygen in the soil is maintained above 30% saturation, with aeration of 1 to 1.25 µm, stirring above 450 rpm and temperature of 28 ° C. The optimum pH during cultivation is maintained at 6.8 to 7.2 with 20% NaOH or 28% H ₂ SO ₄ . Linoleic acid and L-Leucine are started to be dosed from 34.hk at a ratio of 1: 0.8. During the cultivation, the optimal concentration of linoleic acid is maintained at values of 0.2 to 1.0 g / l by dosing with linoleic acid. An 8% L-Leucine solution at pH 11 is added to the production medium during the cultivation. Cultivation is complete when the linoleic acid concentration does not fall below 0.2 g / l. After 180 to 200 hours of cultivation, the concentration of lipstatin in the fermentation broth is at a level of 1.8 to 2.3 g / kg.

Príklad.č.3

(a) Preparation of dispute material

The spore suspension of the Streptomyces toxytricine strain is applied to sporulated soil of the following composition: dextrin 4g / l, yeast extract 4g / l, malt extract 10 g / l, CaCO ₃ 2 g / l, agar 20 g / l and distilled water to 1000 ml. The strain is cultured at 28 ° C for 5 to 10 days.

(b) inoculation stage I.

Spores of the Streptomyces toxytricine strain are used to inoculate 100 ml of inoculum broth in 500 ml Erlenmeyer flasks. The composition of the seed medium is as follows: soya flour 10 g / l, glycerine 10 g / l, yeast extract 5 g / l, drinking water up to 1000 ml. After sterilization at 120 ° C for 30 minutes, the pH is 6.9 to optimal

7.5. The inoculum is cultured at 28 ° C for 24-26 hours. The prepared vegetative inoculum is used to inoculate the production step.

(c) inoculation stage II.

The vegetative inoculum from the seed stage I is inoculated in an amount of 1% to 3% into 12 l seed medium in a 30 l fermenter. The composition of the seed lot is as follows: soya flour 30 g / l, yeast extract 5g / l, glycerine 20 g / l, (NH ₄ ) ₂ SO ₄ 2 g / l, CaCO ₃ 6 g / l, antifoam Siovanik 1 g / l . Cultivation takes place under optimal conditions, temperature 28 ° C, aeration 0.5 to 1 µm, stirring 350 rpm, and saturation oxygen concentration in the soil above 60% saturation. The inoculum is cultured at 28 ° C under defined conditions for 12-16 hours.

(d) production stage

Vegetative inoculum II. is inoculated into the production soil as described in Example 2, c).

After 180 to 230 hours of cultivation, the lipstatin concentration in the fermentation broth is 3.0-3.2 g / kg.

Industrial usability:

The strain of the microorganism Streptomyces toxytricini CCM 7349 object of the invention can be used for the biotechnological preparation of the pharmaceutical active substance lipstatin, which by hydrogenation in the isolation process converts to orlistat (tetrahydrolipstatin) which is successfully used in the treatment of obesity, cardiovascular diseases blood pressure, lowering cholesterol, or treating type 2 diabetes.

Claims (6)

What is claimed is: 1. A strain of Streptomyces toxytricini CCM 7349, characterized in that it produces lipstatin. A method for producing lipstatin, comprising culturing a Streptomyces toxytricini CCM 7349 strain according to claim 1 in a production medium under suitable culture conditions. The method of producing lipstatin according to claim 2, characterized in that the production broth contains linoleic acid and L-Leucine as a precursor in a ratio of 1: 0.8. The method of producing lipstatin according to claim 3, characterized in that the linoleic acid and L-Leucine are dosed continuously from 34 hours of cultivation, wherein the concentration of linoleic acid in the production medium is maintained from 0.2 to 1.0 g / l. A method for producing lipstatin according to claim 2 to 4, characterized in that the strain of Streptomyces toxytricini CCM 7349 according to claim 1 is cultivated in a production medium containing glycerol and soybean oil as a carbon source, linoleic acid with L-Leucine as a precursor source. at a cultivation temperature of 28 ° C, a pH in the range of 6.7 to 7.3 producing lipstatin at a concentration of 3.0 to 3.2 g / kg over a period of 180 to 230 hours of culture. A method for producing lipstatin according to claims 2 to 5, characterized in that the production medium does not contain an emulsifier.