KR920009511B1 - Method for producing l-lysine - Google Patents

Abstract

A L-lysine is prepd. by (a) continuous-culturing corynebacterium sp. TR-3579 (KFCC 10065) at 37-38 deg.C under aerobic condition in the medium contg. 280 ppm or more PO4 ion and (b) collecting L-lysine from the cultured broth. The process is useful for the prodn. of L-lysine with high yield. The obtd. L- lysine is useful for the food additive, medicine additive, feed additive etc..

Description

L-lysine preparation by continuous fermentation method

The present invention is a method for producing L-lysine by a continuous fermentation method using a known microorganism Corynebacterium Species TR-3579 (KFCC-10065) having a lysine generating ability.

L-lysine is an essential amino acid and is used as an additive in food, medicine and feed. Domestic and international demand is increasing rapidly every year.

There are many known methods for producing L-lysine, but not enough to meet the growing demand for mass production. In particular, the production method by continuous fermentation method for mass production has been announced, but it is derived from continuous fermentation for a long time. Due to the deterioration of the microorganisms and the lack of specific techniques for maintaining the activity of the strain, there was a lot of difficulties in the production by continuous fermentation due to the appearance of the return mutants in the continuous culture and the autolytic lysis of the microorganisms.

In order to solve this problem, the present inventors used a microbial Corynebacterium Species TR-3579 (KFCC-10065) having a lysine-producing ability to examine the production method of L-lysine by continuous fermentation. The deficiency of PO ₄ ions induces deterioration of the microorganisms, and the concentration of PO ₄ ions acts as a significant factor in maintaining the non-proliferation rate and activity of microorganisms, as well as in the culture medium during continuous culture. The present invention was completed by increasing the specific growth rate by a method of maintaining a high concentration of PO ₄ ions, accumulating L-lysine at high concentrations in productivity and culture per unit time.

Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC-13286 with lysine-producing ability Corinbacterium Spies (Corynebacterium Species) TR-3579 (KFCC-10065) induced by mutation treatment to be.

As a medium for culturing microorganisms, a synthetic medium containing a carbon source, a nitrogen source, inorganic salts and growth factors is used.

Sucrose, glucose, molasses, and starch hydrolysates are used as carbon sources, and ammonium sulfate, ammonium phosphate, urea, ammonia, corn steep liquor, yeast extract, and peptone can be used as nitrogen sources.

As inorganic salts, monophosphate, diphosphate, polyphosphate, phosphoric acid, magnesium phosphate, ferrous sulfate, and manganese sulfate are used. As growth factors, soybean meal hydrolyzate, cell hydrolyzate, thiamine hydrochloride, biotin, and the like are used.

Seed culture preparation for the continuous culture and the start of fermentation can be carried out by the same method as known batch culture.

Preparation of the fermentation tube is prepared in a two-stage continuous system of the main fermentation tube 30 L, the load fermentation tube 5 L capacity, in the case of the present invention is usually possible if the capacity ratio of the main fermentation tube and the sub-fermentation tube is 6: 1.

이온 농도를 280PPM 이상이 유지되도록 미리 준비된 PO₄이온 용액을 연속적으로 추가한다.Culture conditions are carried out under aerobic conditions such as aeration and agitation. The primary stage fermentation tube is cultured at the end of the log growth period while maintaining 1VVM aeration, 37 ° C fermentation temperature and pH of the culture at 7.2 ± 0.1. From the point of time

The PO ₄ ion solution prepared in advance is continuously added so that the ion concentration is maintained above 280 PPM.

The PO ₄ ion solution was prepared using polyphosphoric acid or phosphoric acid to prepare an aqueous solution having a PO ₄ ion concentration of 18-30%.

In addition, from the point at which the logarithmic growth phase is terminated and the cells reach the normal phase, an additional solution in the range of 24 to ²⁶ % prepared in advance to maintain the sugar concentration in the culture medium is maintained at 2.5 to 3.5%. Automatic fermentation can be performed for more than 400 hours of continuous fermentation.

In the measurement of PO ₄ ion concentration and sugar concentration in the culture, the culture drug recovered from the fermentor was periodically analyzed by a conventional HPLC analysis method.

On the other hand, when the amount of culture in the main fermentation tube reaches 170% of the starting solution, the culture solution is transferred to the secondary fermentation tube for two-stage continuous fermentation.The culture condition of the fermentation tube is 0.7VVM, fermentation temperature 37 ℃, The pH of the culture solution is 6.2 ± 0.1, and the transfer and recovery of the culture solution is automatically operated using a peristaltic pump so that the culture time is 6 hours.

It is important to stop the addition of the PO ₄ ion solution and the additional liquid, and to incubate the pH control of the culture medium by transferring the pH to 6.2 by acetic acid or physical method from the time when the culture medium is transferred from the main fermentation tube.

When the pH is cultured at 7.2 ± 0.1 as in the main fermentation tube, the yield of L-lysine decreases as the cell growth increases, and the production of other amino acids other than L-lysine is induced.

In the fermentation tube, the pH transition in the fermentation tube, the addition of the addition solution and the PO ₄ ion solution were stopped, and the growth of the cells in the fermentation tube was suppressed. In addition to the increase in productivity, the utilization of residual sugars was not only favorable for the next process of lysine recovery and purification, but also significant cost reduction was achieved by the effect of reducing BOD in the wastewater treatment process.

As such, when the lysine is produced by the continuous fermentation method of the present invention, the amount of lysine produced through the entire fermentation period showed an increase in productivity by an average of 2 times or more compared to the batch culture.

As a method of recovering L-lysine from the fermentation broth, it was crystallized with L-lysine hydrochloride using a well-known strong acid cation exchange resin, and lysine analysis was a continuous automatic analysis method using microorganisms producing lysine decarboxylase. It was done.

Hereinafter, a detailed method of the present invention will be described through examples.

Example 1

A medium having the following composition is prepared.

After dispensing 300 ml of the medium into a 2 L shake flask and sterilizing at 121 ° C. for 15 minutes, inoculated with platinum of Corinbacterium Species TR-3579 (KFCC-10065) grown in agar nutrient medium at 35 ° C. Incubate shaking for 16 hours at 140 RPM.

Incubate 600 ml of the culture termination solution in a small fermentation tank of 30 L capacity and sterilize 10 l of the fermentation medium having the following composition at 121 ° C. for 15 minutes.

The temperature during the incubation is controlled to 37 ~ 38 ℃ and the pH is controlled to 7.2 ± 0.1 using ammonia gas.

The aeration rate is 1VVM or more and the rotation speed of the stirrer is maintained at 450 RPM.

From 10 hours after the fermentation to 20 hours after the logarithmic growth period, additional medium having the following composition was continuously added in the dilution range of 0.020 to 0.030H ^-1 to maintain the sugar concentration in the culture solution at 3.0 to 4.0% level. From the 20 hours after the fermentation, the additional medium was continuously added at a dilution rate of 0.030 to 0.035H ^-1 . From this point, the prepared PO ₄ ion solution (containing 25% of PO ₄ ions) was diluted to 0.0004H ^-1 . While adding, the concentration of PO ₄ ions in the culture is maintained above 280 PPM.

From 18ℓ time point, which is 1.7 times of the initial fermentation broth, the fermentation tube operation liquid is fixed to 18ℓ and from this time, the culture medium of the main fermentation tube is transferred to the secondary fermentation tube. Continuous fermentation is carried out while carrying out a continuous transfer and recovery at the same time.

It is important to transfer the pH to 6.2 ± 0.1 as a culture condition of the secondary fermentation tube.

As a method of transferring the pH of the secondary fermentation tube to 6.2, the use of acetic acid is most effective at the initial stage of transferring fermentation broth from the primary fermentation tube to the secondary fermentation tube.

In addition, the operating amount of the fermentation tube is 3.5L level, and the aeration rate is 0.7VVM, the incubation temperature is 37 ℃, and the stirrer speed is 400RPM.

When continuous fermentation was performed for 400 hours as described above, L-lysine production amount, yield and productivity increase rate of the primary fermentation tube and the secondary fermentation tube according to the dilution rate were shown in Table 1 and 2.

TABLE 1

TABLE 2

Example 2

PO ₄ ion concentration of the main fermentation tube in the method of Example 1 of the preceding paragraph was adjusted to the concentration shown in Table 3 below, and continuous fermentation was performed at a dilution rate of 0.0325 H ⁻¹ .

Fermentation time was PO ₄ ion concentration of 50, did 210PPM for 100 hours, after 180 hours, respectively, will not cause any further cell growth and produce L- lysine, PO ₄ ion concentration of the more than 400 hours continuous fermentation, respectively over the 280PPM It was possible.

The lysine production concentration, yield per yield, and productivity increase rate are shown in Table 3 along with the continuous incubation time at each PO ₄ ion concentration.

Table 4 shows the results of measuring the number of return strains by taking the culture solution at intervals of about 100 hours during the continuous incubation at each PO ₄ ion concentration.

TABLE 3

TABLE 4

As shown in Tables 3 and 4, the method of culturing the PO ₄ ion concentration above 280 PPM is significantly improved in productivity, and the frequency of occurrence of return mutants by continuous fermentation is significantly lower than the method of culturing below the PO ₄ ion concentration of 280 PPM. By showing the level it was possible to continuous fermentation for more than 400 hours.

Example 3

The culture time of the side fermentation tube in the method of Example 1 of the preceding paragraph was adjusted to the range shown in Table 5, and continuous fermentation was performed for 400 hours.

Lysine production concentration and yield per each incubation time are shown in Table 5.

TABLE 5

As shown in Table 5, there was no increase in L-lysine production and yield per 6 hours after the incubation time, and the use of residual sugar no longer occurred, and the final residual sugar was identified as non-fermentable sugar.

Example 4

In the method of Example 1 of the preceding paragraph, the culture time of the fermentation tube was 6 hours, and the pH was adjusted to the range shown in Table 6, followed by 400 hours of continuous fermentation.

L-lysine production concentration and yield per each range of pH are shown in Table 6, where cell growth did not occur at the control level of pH 6.2 ± 0.1 and showed the highest productivity.

TABLE 6

Example 5

In Table ¹ , the dilution rate of the main fermentation tube was 0.0325H- ¹ and the concentration of PO ₄ ion was 280PPM, and the culture conditions of the fermentation tube were pH 6.2 and the culture time was 6 hours. Continuous fermentation was performed up to one hour.

As a result of continuous fermentation until each hour, the average L-lysine concentration, yield per yield and yield increase rate of the final fermentation broth are shown in Table 7.

TABLE 7

Claims (2)

In the continuous fermentation of L-lysine by the microorganism Corynebacterium Species TR-3579 (KFCC-10065) having L-lysine production ability, the concentration of P0 ₄ ion in the main fermentation broth culture was higher than 280 PPM. L-lysine production method by a continuous fermentation method characterized in that the continuous fermentation of the addition of additional medium and P0 ₄ ions solution to maintain the L-lysine in the culture medium to recover L- lysine from the culture . The L-lysine according to the two-stage continuous fermentation method according to claim 1, wherein in the continuous fermentation of the culture medium transferred from the main fermentation tube to the side fermentation tube, the pH is transferred to 6.2 ± 0.1 and the culture time is 6 hours. Production method.