JPH01132394A - Heat treatment of immobilized cell - Google Patents

Abstract

PURPOSE:To improve initial activity of enzyme and to enable long-term preservation of an immobilized cell without lowering activity of enzyme, by heat-treating the immobilized cell containing a tryptophan synthase under a specific pH condition. CONSTITUTION:A tryptophan synthase producing L-tryptophan from indole and L-serine, such as tryptophan synthase produced by Escherichia coli MT-10232 is immobilized by a conventional procedure and the prepared immobilized cell is heat-treated at pH 6-9, preferably pH 6.5-8.0 at >=about 40 deg.C, preferably about 40-75 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for heat treating immobilized bacterial cells containing tryptophan synthase. More specifically, when immobilized bacterial cells used to produce L-tryptophan from indole and L-serine are stored or used for enzymatic reactions,
Immobilized bacterial cells containing the enzyme, or immobilized bacterial cells cultured after immobilization (hereinafter sometimes referred to as immobilized bacterial cells) are heat-treated to improve the initial activity of the enzyme. The present invention also relates to a heat treatment method for immobilized microbial cells for long-term preservation or enzymatic reaction without reducing enzyme activity.

The present invention improves the initial activity of the enzyme and maintains it for a long period of time without reducing the activity of the enzyme, when immobilized bacterial cells containing an enzyme used in the operation of a so-called bioreactor are stored or used for an enzyme reaction. Used when trying to prove something.

Conventional technology Tryptophan has traditionally been used in medicines such as infusion components and health foods such as sleep-inducing agents, but in recent years demand has been increasing as its effectiveness as a feed additive has been recognized, and it has become available for inexpensive production. The development of a law is awaited. Chemical synthesis methods, fermentation methods, enzymatic methods, and the like are conventionally known as methods for producing tryptophan.

The chemical synthesis method has a long and complicated synthesis route, and the synthesized tryptophan is a racemic compound, which is an isochemical compound of L and D, and it is necessary to perform optical resolution to separate only the effective and non-active compounds. On the other hand, the fermentation method or the enzyme method is advantageous because only effective monomers are produced and there is no need for optical resolution.

However, even in fermentation methods, the product tryptophan is obtained by mixing culture medium components such as sugars or microbial cells in the fermentation liquid, making it extremely difficult and efficient to isolate and purify the target tryptophan. There are disadvantages such as poor performance and time consuming.

Enzyme sources used in the enzyme method include resting bacterial cells obtained by centrifugation from the culture solution of bacterial cells that produce the desired enzyme;
Alternatively, an enzyme isolated from bacterial cells by an extraction method or the like may be used. In addition, in recent years, methods have been developed in which bacteria or enzymes are immobilized on natural polymeric substances such as calcium alginate, or film-like moldings, or bacteria or enzymes suspended in a solution are processed through reverse osmosis membranes or ultrafiltration. Other methods have also been reported, including using a membrane to confine the substance within the system.

In particular, the method of using immobilized cells is useful because it is easy to separate the target product from the immobilized cells or enzymes, or because the immobilized cells or enzymes can be used repeatedly, so the amount used can be reduced. This method has attracted particular attention in recent years because of its advantages.

However, with the immobilization method, there is no problem when using crude or purified enzymes, but when trying to use bacterial cells as an enzyme source, the following problems arise. In other words, if it is necessary to preserve immobilized bacterial cells for a long period of time,
Alternatively, when it is desired to use immobilized bacterial cells for a long period of time in a reaction, it is extremely difficult to maintain the activity of the enzyme in the immobilized bacterial cells for a long period of time without substantially reducing the activity.

In the process of producing valuables using immobilized bacterial cells,
It is no exaggeration to say that the longevity of the enzyme activity in the immobilized bacterial cells determines the success or failure of the entire process, so it is extremely important to maintain the enzyme activity in the reaction field for a long period of time without decreasing it. . There are various possible causes for the decrease in enzyme activity in the bacterial cells, but in the case of the enzyme used in the present invention, it is due to the enzyme effective for the target reaction being degraded by the proteolytic enzymes contained in the bacterial cells. It is estimated that

Now, when the present inventors researched a method for producing L-tryptophan from indole and serine by an enzymatic method using immobilized bacterial cells, we stored the immobilized bacterial cells in a cool, dark place such as a refrigerator. They faced a basic and serious problem: when used in a desired reaction, the activity of the enzyme decreases within a short period of time, making it unusable.

5 Techniques for increasing the fortune to 7 Therefore, the present inventors decided to solve this problem (as a result of intensive research, by heat-treating the immobilized bacterial cells, the enzyme activity could be maintained for a long period of time without decreasing, and We have discovered a method for improving the initial activity of enzymes, and have completed the present invention.

That is, the present invention provides L-serine from indole and L-serine.
- When storing immobilized bacterial cells containing tryptophan synthase that produces tryptophan, or when using the immobilized bacterial cells in a desired reaction, the immobilized bacterial cells should be stored under conditions of pH 6 to 9. This is a heat treatment method for immobilized bacterial cells, characterized by heat treatment at 40''C or higher.

The present invention will be explained in detail below.

As the tryptophan synthase producing bacteria used in the method of the present invention, Escherichia coli MT-10232
(FERM BP-19), Escherichia coli MT-
10242 (FBI?M BP-20), Neusbo Krassa ATCC-14692, etc.

The form of the immobilized microbial cells to be subjected to heat treatment may be immobilized microbial cells obtained by fixing microbial cells collected from a culture solution by centrifugation or the like, or it may be a state in which the immobilized microbial cells are further cultured. .

After heat treatment, the immobilized bacterial cells are stored or used for L-tryptophan synthesis reaction.

There are no particular restrictions on the method of immobilizing the bacterial cells, but - methods of comprehensive immobilization within the shell using calcium alginate, carrageenan, acrylamide polymer, photocrosslinkable resin, etc., nylon, etc. Good methods include enclosing it in microcapsules, bonding it to ion exchange resins, amino acid polymers, etc.Also, there are no particular restrictions on the form in which it is immobilized, but it is usually spherical, cylindrical, rod-shaped, etc.
Alternatively, it may be in the form of a film.

As a method for further culturing the immobilized microbial cells, the usual culture method for obtaining the microbial cells may be used, but depending on the type of immobilization carrier, it may be necessary to devise the medium components.

The temperature during heat treatment is 40°C or higher, more specifically 40~7°C.
It is in the range of 5°C. Since the deterioration of enzyme activity is sensitive to temperature, the heat treatment temperature must be adequately controlled.

In the case of the enzyme according to the present invention, the highest activity is shown at 30 to 40°C, but if the heat treatment temperature is less than 40°C, other enzymes other than the target enzyme, such as proteolytic enzymes, cannot be inactivated. The purpose of the invention cannot be achieved. Furthermore, temperatures exceeding 75°C are undesirable because the original enzyme activity decreases. The temperature of the heat treatment is preferably within the above temperature range; depending on the type of carrier, treatment at high temperatures may cause the carrier to shrink, reducing the mass transfer rate of substrates and products within the carrier during the reaction, which may adversely affect the reaction. There are many, so you need to choose accordingly.

The pH during heat treatment is preferably in the range of 6 to 9, more preferably in the range of 6.5 to 8.0. The enzyme used in the present invention exhibits the highest activity at a pu of 7.5 to 8.5, and the pH during heat treatment must be sufficiently controlled within the above range as well as the temperature.

Commonly used agents for adjusting PU include ammonia, caustic soda, caustic potash, hydrochloric acid, and sulfuric acid.

In addition, the optimal heat treatment time is in the range of 10 to 20 minutes when heat treatment is performed at a temperature of about 45°C, but in order to achieve the purpose of heat treatment without reducing the activity of the target enzyme, the treatment temperature must be adjusted. When the temperature is high, it is preferable to perform the treatment for a short time, and when the treatment temperature is low, it is preferable to perform the treatment for a longer time.

When the heat treatment is performed as described above, the initial activity of the enzyme in the immobilized bacterial cells is 10 to 10% higher than when no heat treatment is performed.
Not only is the enzyme improved by 40x, but when the immobilized bacterial cells are stored or used in a reaction to produce L-tryptophan, the enzyme activity is maintained for a long period of time without decreasing.

As a method for preserving the immobilized microbial cells, the microbial cells may be immersed in a preservation solution and stored in a sealed container made of synthetic resin or metal such as stainless steel, and then stored in a refrigerator.

Also, by enzymatic reaction, indole and L-serine are converted to L-
There are no particular restrictions on the method used to produce tryptophan, but examples include a method in which the immobilized bacterial cells are suspended in a reaction preparation solution, and a method in which tryptophan is used after being immobilized on a membrane, in which a reaction supply solution is used. Examples include a method of flowing on the membrane surface.

Immobilized bacterial cells heat-treated by the method of the present invention not only improve the initial activity of the enzyme, but also can be stored in a refrigerator for a long period of time without reducing the enzyme activity, and can be used for reactions. It has become possible to maintain the activity for a long period of time even in cases where L-tryptophan is used, and it has become possible to industrially produce L-tryptophan using immobilized bacterial cells.

actual evidence Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Escherichia coli MT-10232 (FERM BP-19), which is a tryptophan synthase-producing bacterium, was
Medium Loom I with the composition shown in Table 1 in a 00 ml Sakaro flask.

and cultured at 35°C for 24 hours. This culture solution 20
0mjl! (2 flasks) was inoculated into medium 151 having the composition shown in Table 2 in a 301 jar fermenter, and cultured at 35°C and pH 6.8 (adjusted with 28x ammonia water) for 30 hours.

The culture solution was separated, heat-treated under the conditions shown in Table 3, and then centrifuged to collect the bacteria to measure enzyme activity, and the effects of heat treatment and heat treatment conditions were compared.

Enzyme activity was measured as follows.

Indole, L-serine, pyridoxal-5'-phosphate (PLP) and bacterial cells as dried bacterial cells 0.2g #! 10m of reaction solution! Mix and incubate at 35℃1 in a shaking incubator.
The reaction was carried out by shaking for 1 hour, and the produced L-tryptophan was analyzed by high performance liquid chromatography.

Table 1 Composition of culture medium NaC 15g Distilled water II! (pH 6, 8) Table 2
Composition of growth medium Distilled water II! Table 3: Effect of heat treatment Example 2 Cultured in the same manner as in Example 1 to obtain cells without heat treatment and cells with heat treatment. The heat treatment conditions were p117.0,
The temperature was 55°C and 120 minutes. These bacterial cells were placed in a sealed container and stored in a refrigerator at 3-5''C, and the activity was measured after a predetermined period of time.The results are shown in Table 4.

Table 4: Storage results of bacterial cells The activity after storage was expressed as a relative value to the initial activity.

Example 3 Bacterial cells obtained in the same manner as in Example 1 without heat treatment and p.
An immobilized enzyme was prepared using bacterial cells that had been heat-treated with different amounts of H, and a reaction was carried out to synthesize L-tryptophan from indole and L-serine8, and the lifetime of the enzyme activity was examined. The time required for the yield of L-tryptophan to fall to 50x based on indole was defined as the life of the enzyme, and the results are shown in Table 5.

The immobilized enzyme was prepared as follows.

One part of wet bacterial cells collected by centrifugation and one part of physiological saline were mixed by stirring. On the other hand, 7.76 parts of distilled water and 0.24 parts of sodium alginate (N5PLL manufactured by Kibun Food Chemifa) were mixed with stirring, and the pH was adjusted to 8.5 with caustic potassium.

Stir and mix 2 parts of the suspension of bacterial cells and 8 parts of the above sodium alginate solution, and fill it into a syringe with an inner diameter of 0.5.
It was dropped into the gelatinized liquid from the tip of an approximately 0.8 mm injection needle. The gelling solution used was 50 parts of a 0.5 molar calcium chloride dihydrate aqueous solution whose pH was adjusted to 8.5 with a 6N caustic potassium aqueous solution and kept at 10°C. The particles produced by dropping into the gelling solution were stirred and aged in the solution for about 1 hour, and then taken out from the solution and used as an immobilized enzyme source.

Further, a reaction test was conducted in the following manner to synthesize L-1-lyptophan using the immobilized enzyme prepared above and to determine the life span of the enzyme activity.

In a 500 m 12 glass reactor equipped with a stirrer, add a solution 100II containing only indole from the composition shown in Table 6.
11 of immobilized enzyme and 50 mQ of immobilized enzyme were charged, and the reactor was kept in a hot water bath to keep the temperature constantly at 30°C. Next, the sixth
The reaction feed liquid having the composition shown in the table is continuously supplied to the reactor whose contents are being stirred at a rate of 50 ml/hour, while the reactor is also continuously drained at the same rate to maintain a constant Samples were taken every hour and the concentration of L-tryptophan produced and the concentrations of residual indole and L-serine were determined by liquid chromatography. The reaction results are determined by determining the yield of L-tryptophan produced based on the amount of indole supplied, and
The length of time during which this yield was maintained satisfactorily was investigated. The case where the yield is 100χ is the case where the L-tryptophan concentration in the liquid drained from the reactor reaches 4.36 g/β.

Table 5 Lifespan of immobilized enzyme Enzyme lifespan: Time until yield decreases to 50χ Heat treatment conditions: Temperature 55°C 1 hour 20 minutes Table 6 Composition of reaction feed solution pH 8.5 Example 4 Heat treatment conditions The bacterial cells obtained by treating the cells at pH 7.5 and temperature 55°C for 1 hour and 20 minutes were added to a reaction solution 50% having the composition shown in Table 6 except indole and calcium chloride.
1 at a concentration of Log/Il and maintained at 35°C without stirring in a stainless steel reactor equipped with a jacket and a stirrer. A reaction solution having the composition shown in Table 6 except calcium chloride was added to this reactor.
The L-tryptophan synthesis reaction was carried out by feeding at a rate of 1842 kg/hr. At the same time, a part of the reaction solution is continuously extracted and supplied to the ultrafiltration device to generate L produced in the solution.
- Tryptophan was allowed to permeate into the permeate side of the ultrafiltration membrane, and bacterial cells were left on the non-permeate side. The ultrafiltration device is equipped with a 0.36 m2 GR81PP membrane made by DDS, and the amount of liquid supplied is 8.
It was used under the conditions of 1/min, 35°C and 15Kg/cm2. L4 liptophan dissolved in the permeate was separated and recovered by a conventional method such as an ion exchange resin adsorption method. Further, the non-permeated liquid containing bacterial cells was directly circulated to the reactor and subjected to the synthesis reaction of L-tryptophan. The reactor and ultrafiltration device were operated continuously, and the operation could be continued for 120 hours until the yield of L-tryptophan based on indole decreased to 50χ.

Patent applicant: Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] 1) An immobilized bacterial cell containing tryptophan synthase used for producing L-tryptophan from indole and L-serine, which is heat-treated at 40°C or higher under conditions of pH 6 to 9. heat treatment method.