SU1555362A1 - Method of obtaining ferments - Google Patents

Abstract

The invention relates to biotechnology, in particular, to the production of biologically active substances in a continuous manner and can be used in the microbiological and medical industries. The aim of the invention is to increase the productivity of mycelial microorganisms - producers of enzymes due to the simultaneous release of several target products synthesized by the producer in different phases of growth. Enzymes are obtained by multiphase cultivation in liquid nutrient media, which is carried out simultaneously in several vessels connected in series, operating on the principle of full displacement machines, in which a unidirectional laminar flow of fresh nutrient medium is created. At the same time, from the output of the subsequent vessels, a part of the culture liquid is taken, enriched with oxygen and fed into the previous vessels, and the target products are selected by stages of their maximum formation and separated by known methods, with the parameters optimal for the biosynthesis of each product, maintained in each of the serially connected vessels individually. The proposed method enhances the control of microbial metabolism and leads to an increase in the yield of the target products in comparison with the known methods for the production of enzymes in batch and two-stage battery cultivation. 1 il.

Description

The invention relates to biotechnology, in particular to the preparation of biologically active substances in a continuous manner, and can be used in the microbiological and medical industry.

The aim of the invention is to increase the productivity of crops by

simultaneous release of several target products synthesized by producers in different phases of growth.

The drawing shows the scheme that implements the proposed method.

Cultivation of the producer is carried out simultaneously in several series-connected vessels operating according to the principle of the full displacement apparatus, in which they create a unidirectional laminar flow (Rv12300) of fresh nutrient medium along the surface of a fixed layer of mycelium. At the same time, from the exit of one or several subsequent vessels, a part of the culture liquid is taken, enriching it

OXYGEN AND INCLUDED

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As the culture fluid is directed towards the producer’s mycelium, it is depleted of nutrients and coated, absorbed by metabolic products and carbon dioxide,

Thus, in each subsequent vessel, conditions are created that differ from the previous one in determining the defined physiological activity of the culture and the nature of the biopolymers synthesized by it.

Thus, reintroducing part of the culture fluid from the next vessel into the previous one allows to obtain an increased concentration of the target metabolite in the next vessel, thereby further inducing its biosynthesis in the previous vessel, which leads to an increase in the yield of the target product.

Such reverse flow of part of the culture fluid can be organized both between adjacent (adjacent) vessels, and through one or more vessels. This recycling of a part of the culture liquid makes the whole cultivation process step-controlled, and maintaining the speed of each of these cycles (in each pair of vessels) at a certain level in total with the constant main flow of the culture liquid through all vessels ensures stability. low producer mycelium.

The method is carried out as follows.

Consecutively connected vessels 1-5 with a developed inner surface are filled with a sterile nutrient medium until their geometric volume is completely filled, thermostatic, inoculum is added and the liquid phase is recirculated in the direction from 1 to 5. for 30-40 minutes for homogeneous distribution of inoculum. Then the clerk

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the cultivation is turned off, aeration is enabled with air enriched with oxygen up to 30-40% of the volume, at a rate of 0.4-0.6 l of air / l of medium / min and are cultivated in a periodic manner in the course of 16-72 h depending on producer strain.

After the culture transitions to the growth inhibition phase, fresh nutrient medium is fed to the inlet of the first culture vessel at a dilution rate D in the range of 0.01-0.2 and .. sequentially displaces it and the previous culture liquid from the vessel into the vessel in the direction from 1 to 5. If necessary, the serially connected vessels are thermostatic at different temperatures and additionally enriched with pulsed introduction of oxygen into the liquid phase at a level of from 2 to 5 vessels. In addition, part of the culture fluid from vessel 3 (or 4 or 5), after being enriched with oxygen, is returned, for example, to vessel 2 (or 3, or 4), by partially recirculating the culture fluid in a closed loop.

The removal of the target product is carried out, for example, from one of these vessels in the phases of its maximum yield.

Example 1. The culture of the yeast Endomycopsis fibuliger 5513 as a producer of amylolytic enzymes was grown in a liquid nutrient medium of the following composition,%: corn starch 1.0; KH2P04 0.5; corn extract 1.0.

The cultivation of the producer is carried out in an installation consisting of vessels 1-4, sequentially connected according to the full displacement apparatus principle. Aeration is carried out by blowing oxygen-enriched air at the rate of 0.5 l / l / min. The maintenance of pH is ensured by automatic subtitling of 0.5 NaOH and stabilized in vessels 1 and 2 at the level of 5.5–5.6, –a in

vessels 3 and 4 at the level of 4.5-5.0.

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The vessels are filled with the nutrient medium of the specified composition, thermostatic at 30 ° C; inoculum is introduced and the culture medium is recirculated for 30 minutes in the direction from 1 to 4 to the vessel, then the process of periodic cultivation is carried out for 72 hours.

After that, fresh nutrient medium with D 0.1 h was fed to the inlet of vessel 1 and gradually displaced it into subsequent vessels. From vessel 4, the spent culture liquid enriched with pure oxygen is fed to vessel 3 seconds, 02 hours 1. The activity of glucoamylase and o-amylase is determined in the cultured fluid taken from all four culture phases.

Glucoamylase activity reaches a maximum in vessel 2 of the plant and is 46 µmol / ml, which is 4 times higher than during periodic cultivation, and 3.8 times higher than during two-stage cultivation in fermenters of ideal mixing.

The activity of α-amylase reaches a maximum in vessel 4 of the plant and amounts to 3.4 units / ml, which is 7 times higher than with periodic cultivation 12 times higher than with two-stage cultivation in fermenters of ideal mixing.

Example 2. All operations as in example 1, but the selection of the culture fluid from the vessel 4 and the flow of it into the vessel 3 are missing. Analogously to Example 1, the activity of glucoamylase and c-amylase is determined. The maximum of glucoamylase activity is observed in vessel 2, and the maximum activity of o (α-amylase in-vessel 4). However, the numerical values of these activities are reduced compared with example 1: glu. Example 5. Methods and regimens

coamylase-2.2 times (20.9 μmol / ml), to the same as in example 4 with the only

-amylases - 1.8 times (1.88 units / ml).

Example 3. All operations as in Example 1, with the difference that the culture liquid was taken from vessel 4 and supplied to vessel 3 from 08 h. The maximum glucoamylase activity is observed in vessel 2, and the maximum activity of o (α-amylase in vessel 4), however, the numerical values of these activities are reduced compared with example 1: glucoamylase 2.4 times (19.16 µmol / ml) o ( α-amylases — 3.1 times (1.09 U / ml). Example 4 The culture of Actinomycetes Actinomyces jantinus 118 as a producer of trypsin inhibitor is grown on a liquid nutrient medium of the following composition, wt.%: peptone 5.0j; 30 ml;

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the difference is that the selection of the culture fluid from the vessel 3 and its supply to the vessel 2 is made from, 02.

As a result, the inhibitor activity is J 3 units in the vessel, 2 5 units in the vessel, 3 14 units in the vessel,

Example 6; All methods and modes of Example 4 are repeated; the only difference is that the selection of the culture liquid from the vessel 3 and its supply to the vessel 2 is carried out from 05 h

As a result, the activity of the inhibitor is 1 3 units in a vessel, 2 6 units in a vessel, 3 12 units in a vessel.

Example 7. All methods and modes of Example 4 are repeated with the only difference that vessel 3 is thermostatically controlled at 28 ° C,

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glucose 10.0; NaCl 5.0. Stabilize the pH of the medium with a titration of 0.5 n. NaOH in the zone of 6.8-7.0, cultivation) is carried out in an installation representing three successively connected full displacement vessels with a developed inner surface.

The plant is filled with a nutrient medium of the indicated composition, thermostatically stabilized at 28 ° C, inoculum is introduced and recirculation of the nutrient medium for 30 minutes in a closed loop is started. Periodic cultivation is carried out for 40 hours, and then fresh nutrient medium is fed into vessel 1, and the liquid phase is displaced by flow into subsequent vessels. At that, vessels 1 and 2 are thermostated at 28 ° C and vessel 3 at 34 ° WITH.

The culture fluid from vessel 3 is saturated with oxygen and, 03 h, is additionally introduced into vessel 2 of the plant.

In the culture fluid taken from each vessel, the antitrypsin activity is determined by the change in Trypsin esterase activity, using tosdag-arginine methyl alcohol as a substrate,

In the vessel, the activity of the inhibitor is 3 units. in the vessel 2 8 units. and in the vessel, 3 18 units, which is 4 times higher than the maximum activity, is achieved with periodic cultivation, -

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Example 5. -Technologies and Modes

About the same as in example 4 so only

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the difference is that the selection of the culture fluid from the vessel 3 and its supply to the vessel 2 is made from, 02.

As a result, the inhibitor activity is J 3 units in the vessel, 2 5 units in the vessel, 3 14 units in the vessel,

Example 6; All methods and modes of Example 4 are repeated; the only difference is that the selection of the culture liquid from the vessel 3 and its supply to the vessel 2 is carried out from 05 h I

As a result, the activity of the inhibitor is 1 3 units in a vessel, 2 6 units in a vessel, 3 12 units in a vessel.

Example 7. All methods and modes of Example 4 are repeated with the only difference that vessel 3 is thermostatically controlled at 28 ° C,

As a result, the activity of the inhibitor is 1 3 units in a vessel, 2 8 units in a vessel, 3 10 units in a vessel.

Example 8. Actinomyces Actinomyces Kurssanovii 75 culture as a producer of chitinolytic enzymes is grown on a liquid nutrient medium of the following composition, g / l: P, HP04 3.0; MgSO, 7H20 0.54 yeast extract 0.5, milled chitin (particles of 250 mega) 10.0.

The cultivation of the producer is carried out in an installation consisting of 4-consecutively connected vessels of full displacement with a developed inner surface.

The vessels are filled with the nutrient medium of the specified composition, thermostatically heated at 30 ° C, inoculum is introduced and the medium is recirculated in a closed circuit for 30 minutes. Then for 16 hours, the fungus is periodically cultivated with aeration from the 1st to the 4th vessel. Then fresh nutrient medium is supplied to vessel 1, c, 15 and gradually displaces it in the direction of vessel 4. Part of the culture fluid from the outlet of vessel 4 is enriched with oxygen and c, 05 is fed to vessel 2, and thermostatting of vessel 3 is carried out at 34 C and it additionally serves air enriched with oxygen up to 40% at a rate of 0.1 l / l / min.

In the culture fluid taken from each vessel, the activity of chitinase (CH enzyme) and the activity of the CNA enzyme that breaks down colloidal chitin, as well as the activity of the enzyme that breaks down N-N-diacetylchiobiose and other short chitin oligosaccharides of the chitin series are determined.

Chitinase activity (CH ,, - enzyme) reaches a maximum in vessel 2 and is 22 units or 2.2 times higher than during periodic cultivation, and then decreases.

The maximum of activity (LC-enzyme is reached in vessel 3 and is 32 units or 4 times higher than during periodic cultivation, and the maximum activity of the enzyme that cleaves short oligo-saccharides of the chitin series is increased in vessel 4 and 2.8 times surpasses that found by periodic cultivation.

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Example 9; All methods and modes of Example 8 are repeated with the only difference that the culture liquid taken from vessel 4 and fed to vessel 2 is not enriched with oxygen,

As a result, the maximum activity of chitinase (CH1-enzyme) is observed in vessel 2 and is 12 units, which is 1.8 times lower than in Example 8, the maximum activity of the CH enzyme is observed in vessel 3 and is 15 units, which is 2 , 1 time lower than in Example 8, the maximum activity of the enzyme that cleaves short chitinous oligosaccharides of the chitin series is again observed in vessel 4, however, it is 1.5 times lower than in Example 8.

Example 10. All the methods and modes of Example 8 are repeated with the only difference that the temperature control of vessel 3 is carried out at 3 sec.

As a result, all types of tested activities in vessels, with the exception of the first, decreased by approximately 4–5 times as compared with Example 8.

The proposed method allows controlling the biosynthesis of enzymes at each producer growth phase, separating the yield of the target products in separate stages and increasing their yield several times in comparison with the known methods in batch and two-stage battery cultivation of mycelial forms of microorganisms.

Claims (1)

Invention Formula A method for producing enzymes that involves the multi-phase cultivation of mycelial forms of microorganisms on liquid nutrient media by fixing the producers on the inner surfaces of the fermenter and moving the liquid and gaseous phases of the nutrient medium relative to them, followed by stabilizing the cultures in a given physiological state and optimizing the parameters of the enzyme biosynthesis conditions to maximum accumulation their activity, characterized in that, in order to increase the productivity of crops by simultaneously the yield of several target products synthesized by producers in different phases of growth, the cultivation of lead thirty 35 40 45 50 at the same time in several vessels connected in series, operating on the principle of full displacement devices, under conditions of a unidirectional laminar flow of fresh nutrient medium, while at the outlet of each of the vessels a part of the culture fluid is taken equal to The volume of fresh nutrient medium supplied is aerated and re-fed into the vessel, followed by the selection and release of the target products, at the stage of their maximum formation, and in each of the vessels the parameters that are optimal for the biosia of the corresponding enzymes are maintained. Feeder - Wednesday