FR2488273A1 - L-Lysine prodn. by fermentation - with control of di:amino-pimelic acid decarboxylase activity - Google Patents

Abstract

Prodn. of L-lysine (I) comprises submerged aerobic cultivation of a suitable microorganism on a conventional nutrient medium. The activity of diaminopimelic acid decarboxylase (A) (or the corresp. (I) concn.) in the cells is determined and when this activity reaches 120-90 picomoles lysine per sec. per mg. protein (units) a nutrient contg. carbon and vitamins (esp. molasses) is added until activity is reduced to 70-60 units. Pref. when the activity has fallen to this level, further addn. of a carbon source and vitamins (esp. sucrose or glucose plus biotin and thiamine) is made to lower the activity to 20-25 units. (I) is used in foods and animal nutrition. By controlling (A) activity, the lysine yield, relative to reducing substances, can be improved to over 43% with absolute yields of 60-80g. per l. Unlike known methods, media of high reducing substance concn. (12-15%) can be converted. Suitable microorganisms are of the genera Brevibacterium and Micrococcus and they are grown on conventional media with pO2 20-25% of saturation.

Description

 The present invention relates to the microbiological industry and in particular relates to a process for the preparation of L-lysine, which finds applications in particular in the food industry and as a fodder element in agriculture.

 A process for the preparation of L-lysine is already known by deep fermentation of a producing strain on a nutritive medium containing a source of carbon, nitrogen, essential mineral salts, amino acids and vitamins, according to which, for increase the yield of final product during fermentation, from the 16th to the 30th hour, the lactic acid concentration must be determined or the activity of the lactate-dehydroge0a determined or else the the activity of isocitrate dehydrogenase in the cells of the producing strain and, taking into account the result, the whole culture process is carried out. Complementary periodic additions of carbon sources such as molasses, acetic acid, sucrose, ethanol, double glucose salt, after reaching a content of reducing substances in the culture liquor equal to 5 to 6%.

On the other hand, a process for the preparation of
L-lysine by culture of a deep producing strain
On a nutrient medium containing a source of carbon, nitrogen, essential mineral salts, amino acids and vitamins, according to which, in order to increase the yield of L-lysine, the growth of the producing strain is carried out by adding periodically a sterile solution of one of the constituents of the nutritive medium, such as molasses, which makes it possible to maintain the content of reducing substances in the culture liquor between 2.5 and 3, '. The yield in
L-lysine in relation to the reducing substances used is 40.2% (USSR author's certificate no. 498 940.1973).

 However, in the event of multiple periodic additions of molasses to the culture medium by the procedures indicated, a determined quantity of L-threonine contained in the molasses is introduced. L-threonine, together with L-lysine, results in multivalent inhibition of the activity of -aspartate kinase, which in turn significantly reduces the activity of the culture in the synthesis of lysine and lowers the yield of the final product. The multiple inhibition of the activity of 2 aspaltate kinase occurs within determined limits of the activity of diaminopimelic acid decarbogylase. The effect is particularly manifest in the case of transformation of concentrated nutrient media: the initial concentration of reducing substances is 12 to 15%., Together with the increase in the L-lysine content of the culture liquor in during the fermentation process, the decarboxylase activity of diaminopimelic acid decreases and the addition of molasses at the end of the process, when the content of reductive substances is 2.5 to 5%, lowers the yield of final product .

It has therefore been proposed to resolve the following problem by regulating the culture conditions and by additional, oriented introduction of certain constituents of the nutrient medium during the culture, increasing the yield of
L-lysine and make possible the transformation of concentrated nutrient media with respect to reducing substances.

 This problem is solved in that the process for preparing L-lysine eonsistgns to carry out a deep culture of its producing strains on a nutritive medium containing sources of carbon, nitrogen, mineral salts, biologically active substances , under aerobic conditions, by addition of the constituents of the nutritive medium during the culture process and subsequent isolation of the culture medium from the targeted product, is characterized according to the invention, in that during the culture, within cells of the strain producing the activity of diaminopimelic acid decarboxylase or the corresponding concentrations of L-lysine, and after its activity has reached a value of 12.10 to 9.10 11 mol of lysine per second and per milligram of protein, we add to the culture medium one of the constituents in the nutritive medium containing carbon and vitamins, such as molasses which is introduced into the culture medium during the pro culture stopped until a decrease in the activity of the diaminopimelic acid decarboxylase equal to 7.101l to 6.1011mo2ede lysine per second and per milligram of protein.

 It is advantageous, to increase the yield of L-lysine, when the activity of the diaminopimelic acid decarboxylase reaches 7.10 11a 6.10 1 mole of lysine per second and per milligram of protein, to introduce additionally into the medium of culture a source of carbon and vitamins which are introduced during the culture process so as to lower the activity of diaminopimelic acid decarboxylase to a value equal to 2.5.10 1 to 2.10 11 mole of lysine per second and per milligram of protein.

 It is preferable to use, as a source of carbon and vitamins, sucrose with biotin and thiamine or glucose with biotin and thiamine.

 The proposed method is implemented in the following manner.

 Microorganisms of the genus Brevibacterium or Micrococcus are used as agents producing L-lysine.

After having carried out the growth of the culture in a seed fermenter, the culture is carried out in industrial ferments on a nutritive medium of carbohydrates, in particular on a molasses medium. During the fermentation during the growth of the producing agent, the aeration is maintained at the level of PO 2 = 20 to 25% of the saturation point. The nutritive medium contains, in addition to the sources of carbon, amino acids and vitamins known as by the producer agent as well as mineral salts: sources of nitrogen and phosphorus. During the culture process one determines (one dose) in cells of the agent producing the activity of diaminopimelic acid decarboxylase
The level of aeration during the culture is lowered to PO2 = 10 to 12% of saturation, maintaining the intensity of agitation of the system Once the level of activity of diaminopimelic acid decarboxylase has reached 12.10 to 9.1O11 mole of lysine per second and per milligram of protein, one begins to add to the fermenter one of the sources of carbon and vitamins, namely sterile molasses. The addition of molasses is carried out continuously or in separate portions at intervals of 1 to 3 hours. The admission of molasses is stopped when the level of activity of the diaeiaopimic acid decarboxylase reaches 7.10 11d 6.10 ? mole of lysine per second and per milligram of protein. During the culture, the temperature is maintained at 31 t 1 C, and the pE, from 7.4 to 7.8. To increase the yield of L-lysine, once the activity of the decarboxylase diaminopineic acid reached 7.10-11 to 6.10-11 mole of lysine per second and per milligram of protein, one adds in addition to the culture medium a source of carbon and vitamins until the activity of the decarboxylase diaminopimelic acid becomes 2.5. 10 1 to 2.10 11 moles of lysine per second and per milligram of protein. As a carbon source, it is possible to use sucrose, glucose, fructose, ethanol, acetic acid or their combinations. As vitamins, thiamine and biotin are used.

 After adding the auxiliary food for the last time, the process is continued for a few hours until the concentration of reducing substances becomes 1 - 1.2%. During the second half of the process, the pH is maintained at the level of 7.5 to 7.8 by means of a nitrogenous body, in particular with ammonia. When the culture is finished, the lysine is isolated from the culture liquor by one of the known methods, or else a feed concentrate of X sine is obtained.

 The activity of diaminopimelic acid decarboxylase is determined by the manometric route according to the quantity of carbon dioxide formed when diaminopimelic acid is used as substrate. The activity of diaminopimelic acid decarboxylase is also determined from the L-lysine concentration in the culture liquor. For each culture it is possible to graphically establish a relationship between the activity of diaminopimelic acid decarboxylase and the concentration of Lysine in the culture medium (a calibration curve is constructed for the relationship between these two quantities). This allows the culture process to be controlled.

 Depending on the concentration of L-lysine during the culture, the activity of the diaminopimelic ace decarboxylase is judged.

 The additional addition of the constituents of the nutrient medium (carbon source and vitamins) during the culture makes it possible to increase the yield of L-lysine. The claimed process makes it possible to control the process as a function of the activity of diaminopimelic acid decarboxylase and to increase the yield of L-lysine relative to the reducing substances used by up to 43.5%, thereby guaranteeing a absolute yield of L-lysine up to 60 to 80.5 gil under industrial conditions.

One of the major advantages of the claimed process is to ensure the possibility of directly transforming the media concentrated at 12 - 15% into reducing substances, which does not allow any of the existing processes.
The manufacturing costs according to the claimed process do not increase, because the volume of the fermentation liquors of the concentrate reduced by evaporation and dried is not modified
Other characteristics and advantages of the invention will be better understood on reading the description which follows of several concrete but non-limiting examples of implementation of the proposed process for the preparation of
L-lysine.

 Example 1.

As the producing agent (L-lysine), Brevibacterium flavium 22 LD is used. The initial culture is multiplied at the start on slanted agars, then in flasks on shakers and then in a seed fermenter with a capacity of 400 l on a nutritious mixture of molasses. The culture is continued for 14 hours at a temperature of 31 - 1 ° C., under aeration of 60 mg of O2 / 1 min, the pH of the medium is maintained between 7.4 and 7.8. The seed material is driven off under pressure in a fermenter with a capacity of 3000 1 with a medium of the following composition
molasses 470 <46% sugar content by weight)
malus extract 50 kg (d dry matter content of
50%)
(NH4) 2 SO 37 kg
4) 2 HPO. 0.75 kg
KH2 PO4, 0.75 kg
sunflower oil 3 I
water up to 1,300 1
Initial concentration of reducing substances 14%, L-lysine content 2.8 g / i after addition of the seed material.

 During the growth of the producing agent, the aeration is maintained at the level p02 = 20 to 25% of the saturation, after 8 to 10 hours of culture, the pH goes to 7.4 7.8 and it is maintained at this level. At the 18th hour of culture, the activity of diaminopimelic acid decarboxylase is 9.10-11 mole of lysine per second and per milligram of protein, the L-lysine content is 18 g / 1, the concentration of reducing substances is 8.8% '.

Is continuously introduced into the fermenter, for three hours, sterile frank sterile at a speed of ik of reducing substances per hour, until the activity of the decarboxylase of acidodiamino-pimelic is reduced to 6.8.10 1 mole of lysine per second and per milligram of protein. Thereafter, the activity of the diaminopimelic acid decarboxylase is determined every 2 to 3 hours.

After the introduction of the supplementary food, the aeration level is lowered to 10 p of saturation. At the 37th hour of culture, the activity of diaminopimelic acid decarboxylase is 5.10'11 mble of lysine per second and per milligram of protein, and carbon sources and vitamins are introduced into the culture medium. ,
As carbon sources, sucrose is introduced in two portions with a period of 2 hours until the activity of the diaminopimelic acid decarboxylase is 3.4. 10-11 mole of lysine per second and per milligram of protein, the concentration of L-lysine at the 48th hour after the introduction of the auxiliary food is 48 l / g Then the culture is continued, at the 52nd hour again introduces sucrose with vitamins (biotin and thiamin) until the activity of diaminopimelic acid decarboxylase is 2.2.10 11 mole of lysine per second and per milligram of protein, the o-concentration in L-lysine then being 58 g / l. Thereafter the culture is continued until the concentration of reducing substances is 1.4%. At the 63rd hour of fermentation, the culture liquor contains 65 g / l of lysine, 24 g / l of cellular material (biomass) of producing agent. The culture liquor is treated by one of the known methods using ion exchange resins and the L-lysine is isolated.

 The yield of L-lysine relative to the reducing substances is 43.6.

 During the process, the pH of the medium is maintained at an optimal level by modifying the aeration as a function of the activity of the lactate dehydrogenase and the isocitrate dehydrogenase. At the end of the process, the pH is adjusted by adding ammonia as a nitrogen bunker.

 Example?

As the producing agent (L-lysine), a culture of Brevibacterium flavum RC-115 is used. The seeding material is prepared in a manner analogous to that described in Example 1. Then the fermentation is continued in a pilot fermenter on a medium of the following composition
molasses 1.65 kg (for a sugar content of 46%)
corn extract 0.52 kg (for a material content
dry 50, ')
(NH4) 2SO4 150 g
KH2P04 2.5 g HP04 HPO, 2.5 g
CaCO3 25 g
sunflower oil 30 g
water 4.6 1.

 The quantity of seeding material is 8 g.

 During the growth of the producing agent, the aeration is maintained at a level pO 2 = 20 to 25%% of the saturation. Agitation is carried out at a speed of 600 rpm. At the 8th - 10th hour of fermentation, the pH increases to a value of 7.6 - 7.8 and is maintained at this level.

At the 14th hour of culture, for a diaminopimelic acid decarboxylase activity of 12.10 11 moles of lysine per second and per milligram of protein, the following are introduced into the culture medium with a period of 2 hours from sterile molasses frank with a consumption of reducing substances of 1% in 2 hours. In this case, between each addition of supplementary food, the activity of diaminopimelic acid decarboxylase is measured. The complementary food is introduced according to the drop in the activity of the diaminopimelic acid decarboxylase up to 7.5 × 10 -11 mole of lysine per second and per milligram of protein. This activity is reached at the 22nd hour of culture, the L-lysine concentration then being 26 g / lA at the 25th hour, the aeration level is lowered to 12 saturation. At the 30th hour of culture, the activity of diaminopimelic acid decarboxylase is 5.10 ~ 11mol of lysine per second and per milligram of protein. The L-lysine concentration is 35 g / l; is continuously introduced into the culture medium of sucrose and a mixture of vitamins 31 and biotin. When the content of
L-lysine is equal to 60 g / l and the activity of diaminopimelic acid decarboxylase is equal to 2.1 × 10 11 mole of lysine per second and per milligram of protein, the introduction of the food is stopped. 'extra. During the fermentation, the pH is adjusted in a manner analogous to that indicated in Example 1. At the 58th hour of fermentation, the L-lysine content is 80.5 g / l, that in matter cell (biomass) of producer agent is 24 g / l, the content of residual reducing substances is 1.4%. The yield of L-lysine relative to the reducing substances used is 43.5%.

 Example 3.

 A culture of Micrococcus glutamicus T-3 is used as the L-lysine producing agent.

 The culture is carried out on a nutritive medium similar to that of Example 1.

 As a source of biological growth factors, a product of hydrolysis of a vitamin-protein concentrate is used.

 At the 22nd hour of fermentation, the activity of diaminopimelic acid decarboxylase is 92.10 11 mole of lysine per second and per milligram of protein. The concentration of L-lysine is 17 g / l. The sterile molasses are introduced continuously into the medium until the activity of the diaminopimelic acid decarboxylase is 6.6 × 1011 mole of lysine per second and per milligram of protein. Such activity is reached at the 32nd hour of culture, the concentration of L-lysine in the culture liquor is 28 g / l. Then the aeration level is lowered from its initial value, which is from 18 to 25% saturation, up to 10 - 12% saturation. At the 48th hour of culture the activity of the decarboxylase of diaminopimelic acid is 4. 10-11 mole of lysine per second and per milligram of protein, glucose and vitamins (thiamine and biotin) are introduced into the culture medium until the activity of decarboxylase of diaminopimelic acid is lowered to the level of 2.5.10-11 mole of lysine per second and per milligram of protein. during the fermentation, the pH is adjusted in a manner analogous to that indicated in Example 1. Then the fermentation is continued until a concentration of reducing substances of 1.4 Sb is reached.

At the 65th hour of fermentation, the culture liquor contains Lysine in an amount of 48.5 gbl, the cellular material (biomass) of producing agent in an amount of 25 g / l.

The yield of L-lysine is 43% compared to the reducing substances.

 Example 4.

A culture of Brevi-bacterium flavum RC-115 is used as the producing agent. The seeding material is prepared in a manner analogous to that described in the example. The fermentation is continued in a pilot fermenter on a nutrient medium of the following composition
molasses 1.72 kg (for a sugar content of 46%)
corn extract 0.52 kg (for a content of substances
50% dry)
(NH4) 2S04 150 g
EH2P04 2.5
2 4 2.5 g
K2H P04 2.5 g
Mg S04 1.0 g
CaC03 25 g
sunflower oil 30 g
water up to 4.6 1.

 The quantity of seeding materials is 8%.

Reducing substances content at the start of the process: 15, '.

During the growth of the producing agent, the addition is maintained at a level of p02 = 2D at 25% of the saturation. Agitation is carried out at a speed of 600 rpm. At the 8th - 10th hour of fermentation the pH increases to 7.8 and it is maintained at this level.

 At the 20th hour of culture, for an activity of the diaminopimelic acid decarboxylase of 9.7 × 10 -11 mole of lysine per second and per milligram of protein, the following are introduced into the culture medium with a period of 2 hours of sterile molasses so that the use of reducing substances is 1 'in 2 hours.

 Between each addition of the supplementary food, the activity of diaminopimelic acid decarboxylase is measured. The introduction of the complementary food is carried out until the activity of the diaminopimelic acid decarboxylase is reduced to 6.4.10 11 mole of lysine per second and per milligram of protein, then this feeding is stopped. . This activity is reached at the 28th hour of fermentation, the concentration of Lysine in this case amounts to 35 g / i.

 The fermentation is continued up to a content of residual reducing substances of 1.6. The pH is maintained during the process by modifying the aeration as a function of the activity of the lactate dehydrogenase and of the isocitrate dehydrogenase. At the end of the process, the pH is adjusted with a nitrogenous substance such as ammonia.

 At the end of the process, at the 69th hour of fermentation, the culture liquor contains 76.4 g / l of L-lysine, 27g / l of cellular material (biomass). The yield of L-lysine relative to the reducing substances used is 43.5%.

 Example 5.

 As a producer agent (L-lysine), a culture of Brevibacterium flavum RC-115 is used.

 The inoculating material is grown and the nutritive medium is prepared in a manner analogous to that described in Example 1. Until the 18th hour of fermentation, the process is carried out in a manner analogous to that of 'example 1. For a Lysine concentration of 18.6 g / lB which corresponds to a diaminopimelic acid decarboxylase activity of 9.1.10-11 mole of lysine per second and per milligram of protein, periodically added to culture liquor (with a two hour period) from sterile molasses. Between each addition of make-up food, the L-lysine concentration is determined and, based on this concentration, the activity of diaminopimelic acid decarboxylase is judged. The addition of auxiliary food is stopped when the L-lysine content of the culture liquor is 30 g / l, which corresponds to a diaminopimelic acid decarboxylase activity of 7.10 -11 mole. of lysine per second per milligram of protein.

 The fermentation is continued until the content of reducing substances in the culture liquor is lowered to 1.4, the pH is maintained during the fermentation by modifying the aeration as a function of the activity of the lactate. dehydrogenase and isocitrate dehydrogenase. At the end of the process, the pH is adjusted with a nitrogenous substance such as ammonia.

At the end of the process, at the 60th hour of culture, the culture liquor contains 65.6 g / l of L-lysine, 24.7 g / l of cellular material (biomass) The yield of L-lysine by ratio to the reducing substances used is equal to 45.7%
Of course, the invention is not in any way limited to the embodiments described and shown which have been given only by way of example. In particular, it includes all the means constituting technical equivalents of the means described, as well as their combinations, if these are carried out according to its spirit and implemented within the framework of protection as claimed.

Claims (4)

 1. Process for the preparation of L-lysine by deep culture of agents producing it on a nutritive medium containing carbon sources, nitrogen, mineral salts, biologically active substances, under aerobic conditions with addition of constituents of the nutritive medium QU during the culture, and with subsequent of the final product with the culture medium, characterized in that during the culture the activity of the producing agent is determined in the cells of diaminopimelic acid decarboxylase or the corresponding concentrations of L-lysine, and when the activity of said decarboxylase reaches 12.10 11 -9.10 11 mole of lysine per second and per milligram of protein, the culture medium is introduced into the culture medium. one of the constituents of the nutrient medium which contain carbon and vitamins, such as molasses, which are introduced into the culture medium during the culture process until the activity of the acid decarboxylase he diaminopimelic has dropped to 7 10 11 ~ 6.10'11 mole of lysine per second and per milligram of protein.  2. Method according to claim 1, characterized in that once united that the activity of the diaminopimelic acid decarboxylase has decreased to 7.10 1 6.10-11 mole of lysine per second and per milligram of protein, additionally introduced into the medium of culture a source of carbon and vitamins, which are introduced throughout the culture process until an activity of diaminopimelic acid decarboxylase equal to 2.5.10-11 - 2.10- 11 moles of lysine per second and per milligram of protein.  3. Method according to one of claims 1 and 2  characterized in that as a source of carbon and  vitamins we use sucrose with biotin and  thiamine or glucose with biotin and  thiamine.  4. L-lysine, characterized in that it is obtained by the process forming the subject of one of claims 1,2 and 3.