JPH08154675A - Production of tyrosinephenol-lyase and stabilization - Google Patents

Abstract

PURPOSE: To mass-produce tyrosinephenol-lyase useful for producing L-tyrosine or L-3,4-dihydroxyphenylalanine, by culturing recombinant Escherichia coli transfected with a tyrosinephenol-lyase gene derived from a kind of bacteria belonging to Enterobacteriaceae. CONSTITUTION: The objective tyrosinephenol-lyase is obtained in highly improved productivity by culturing, at 25-30 deg.C in high concentrations, recombinant Escherichia coli transfected with a tyrosinephenol-lyase gene derived from a kind of bacteria belonging to Enterobacteriaceae [e.g. Eschericthia coli MT-10762 (FERM P-14545)].

Description

Detailed Description of the Invention

[0001]

The present invention relates to L-tyrosine and L-tyrosine.
The present invention relates to a method for producing tyrosine phenol lyase, which is a catalyst for producing 3,4 dihydroxyphenylalanine (hereinafter abbreviated as L-dopa). From another perspective, the present invention is
The present invention relates to a method for producing L-tyrosine and L-dopa.

[0002]

2. Description of the Related Art Tyrosine phenol lyase
phenol-lyase) [EC 4.1.9.2] is a reaction for converting L-tyrosine into pyruvic acid, ammonia and phenol and its reverse reaction, and α and β for decomposing L-dopa into pyruvic acid, ammonia and pyrocatechol. Elimination reaction and its reverse reaction, racemization reaction for converting D-alanine to L-alanine, β-substitution reaction for synthesizing L-tyrosine from L-serine and phenol, L-dopa from L-serine and pyrocatechol It is known as a so-called multifunctional enzyme that catalyzes a β-substitution reaction or the like to be synthesized. In addition, tyrosine phenol lyase is Esherichia intermedia ( Esherichia intermedia ), Citrobacter freundii ( Citrobacter freundii ), Erwinia
Herbicola ( Erwinia herbicola ), Proteus bacteria ( Proteus ) and other Enterobacteriaceae ( Enterobac
Its existence is widely known, centering on microorganisms belonging to teriaceae ).

In recent years, Citrobacter Freundy,
A tyrosine phenol lyase gene has been isolated from Erwinia herbicola and the primary structure of the gene has been clarified. Further, a method for producing a recombinant Escherichia coli into which the gene has been introduced, and culturing the recombinant Escherichia coli to produce tyrosine phenol lyase, in which a tyrosine phenol lyase is highly produced without adding an inducer to the medium Have been found (International Publication Number WO9
0/04031, JP-A-04-218380). Generally, when culturing a recombinant Escherichia coli to produce a target protein, a method of adding glucose to the medium, pH of the medium, culture temperature, dissolved oxygen concentration (hereinafter abbreviated as DO), medium composition (natural product) It is known that the (concentration) influences the growth of the bacterium, the stability of the recombinant gene in the bacterium, and the productivity of the target product. In particular, the culturing temperature in the case of culturing genetically modified Escherichia coli is usually set to 37 ° C. from the viewpoint of microbial growth. However, the present inventors tried to produce tyrosine phenol lyase at a high concentration by culturing recombinant Escherichia coli introduced with the tyrosine phenol lyase gene at a high concentration, and in the absence of selective pressure,
Even if the culture conditions such as pH of the medium, DO and medium components are optimized, the phenomenon that the production amount of tyrosine phenol lyase is not improved compared to the growth of the bacterial cells at the culture temperature of around 37 ° C is observed. It was found that there is a problem that high production of tyrosine phenol lyase cannot be achieved at the optimal culture temperature for the growth of the body.

On the other hand, when industrially producing L-tyrosine and L-dopa using cells that highly produce tyrosine phenol lyase, recombinant Escherichia coli into which the tyrosine phenol lyase gene has been introduced is treated with a polymer substance or the like. When immobilized and used as a reaction catalyst, tyrosine phenol lyase can be repeatedly used in the reaction, and it is considered that the production cost of L-tyrosine, L-dopa and the like can be reduced. The present inventors attempted to immobilize recombinant Escherichia coli into which tyrosine phenol lyase was introduced in high concentration for the above purpose, but the tyrosine phenol lyase activity of the immobilized cells was surprisingly It turned out to be very unstable. That is, for the purpose of utilizing for industrial production of L-tyrosine and L-dopa, recombinant Escherichia coli introduced with tyrosine phenol lyase for high production of tyrosine phenol lyase and stabilization of enzyme in cells And a method for immobilizing the recombinant Escherichia coli have been demanded.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to produce a large amount of tyrosine phenol lyase by culturing recombinant Escherichia coli into which tyrosine phenol lyase gene has been introduced at high concentration, and tyrosine phenol cultivated at high concentration. Method for stably immobilizing the above recombinant Escherichia coli that highly produced lyase and L- using the immobilized cells
It is to provide a dopa production method.

[0006]

[Means for Solving the Problems] As a result of intensive studies conducted by the present inventors, when a large amount of tyrosine phenol lyase was produced by culturing recombinant Escherichia coli having a tyrosine phenol lyase gene introduced therein at a high concentration, It was found that the productivity of tyrosine phenol lyase by the recombinant Escherichia coli is remarkably improved by culturing at 25 to 30 ° C. lower than the temperature range. In addition, the present inventors have examined factors that reduce the tyrosine phenol lyase activity in the immobilized carrier. As a result, in order for the tyrosine phenol lyase activity to be stably maintained in the immobilized carrier, the membrane structure of the microbial cell should be strong, the activity of the protease and the like in the microbial cell should be low, and lysis of the microbial cell or enzyme It has been found that an important factor is that decomposition of tyrosine phenol lyase is unlikely to occur. Then, when the recombinant Escherichia coli into which the tyrosine phenol lyase gene has been introduced is cultured at a high concentration, a bacterium obtained by culturing within 10 hours while flowing the carbon source even after the cell growth reaches the stationary phase Since the body has a strong membrane structure and the decomposition of tyrosine phenol lyase in the microbial cells is unlikely to occur, it was found that by immobilizing the microbial cells, immobilized microbial cells that maintain high tyrosine phenol lyase activity can be obtained. . Further, surprisingly, after immobilizing Escherichia coli producing tyrosine phenol lyase with an immobilization carrier, the immobilized cells are heat-treated at 40 to 60 ° C. for 10 to 120 minutes to obtain a product such as protease. Selectively inactivates factors that reduce tyrosine phenol lyase activity,
It was found that tyrosine phenol lyase can be stabilized. Also, in the production of L-dopa
It was found that the addition of an SH group protecting agent such as mercaptoethanol or dithiothreitol (hereinafter abbreviated as DTT) to the reaction solution is effective in preventing the activity of tyrosine phenol lyase from being reduced by catechol as a substrate. Furthermore, by repeatedly using the immobilized cells, L-
When producing dopa, the tyrosine phenol lyase activity of the immobilized microbial cells is increased by washing the immobilized microbial cells with a buffer solution containing pyridoxal 5'phosphate (PLP), which is a coenzyme of tyrosine phenol lyase, at a certain concentration or more. It has been found that it can be maintained at a certain level or above and can be repeatedly used in the synthesis reaction of L-DOPA. The present invention has been completed based on the above findings.

That is, according to the present invention, recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is cultured at a high concentration to produce tyrosine phenol lyase at a culture temperature of 25.
The present invention provides a method for producing tyrosine phenol lyase, which comprises culturing under controlled conditions within the range of -30 ° C, and recombinant Escherichia coli into which a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae has been introduced. The recombinant Escherichia coli is cultivated at a culture temperature of 25 to 30 ° C. at a high concentration until the growth of the cells reaches a stationary phase, and then the carbon source is flowed down for 3 more times.
Immobilizing the cells obtained by continuing the culture for 10 hours with an immobilization carrier, or fixing the cells to 40-6
It is intended to provide a method for immobilizing the recombinant Escherichia coli in which the tyrosine phenol lyase in the cells is stabilized, which is characterized by performing a heat treatment at a temperature of 0 ° C. Further, the present invention is a method for producing L-dopa from L-serine and catechol using immobilized cells in which recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is immobilized. In the synthetic reaction of L-dopa from L-serine and catechol, a SH group protecting agent is added to the reaction solution, which is a method for producing L-dopa stabilized with tyrosine phenol lyase of immobilized cells. The present invention also provides a method and uses L-serine and L-dopa from catechol using immobilized cells in which recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is used. A method for producing, wherein the immobilized bacterium is washed with a buffer containing PLP and then reused in an L-dopa synthesis reaction. There is provided a method for producing L- dopa to symptoms. Hereinafter, the present invention will be described more specifically.

Recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae , which is a microorganism used in the present invention, is a bacterium belonging to Enterobacteriaceae and having tyrosine phenol lyase activity. It can be obtained by cloning the tyrosine phenol lyase gene, ligating the gene with a vector plasmid expressing in Escherichia coli to prepare a recombinant plasmid, and transforming the host Escherichia coli with the recombinant plasmid. Bacteria that belong to Enterobacteriaceae, which is the source of gene isolation, and have tyrosine phenol lyase activity are Esherichia interme
dia ), Citrobacter
freundii), Erwinia herbicola (Erwinia her
bicola ), Proteus genus bacteria ( Proteus ) and the like, and Citrobacter Freundy MT-10419.
Strain (FERM P-10181) (International Publication Number WO9
0/04031), and Erwinia herbicola MT-10509 strain (FERM BP-3258).
(Japanese Patent Laid-Open No. 4-218380). FE
RM BP and FERM P represent the accession numbers of the Institute of Biotechnology, Institute of Biotechnology, AIST.

In order to collect the DNA encoding the tyrosine phenol lyase gene from the above bacteria, the following may be performed. That is, the above-mentioned bacterium was cultured, chromosomal DNA was extracted from the obtained microbial cell, this was cleaved with an appropriate restriction enzyme, and the cleaved DNA fragment was ligated to a plasmid vector replicable in Escherichia coli. DNA from an Escherichia coli transformant that acquired tyrosine phenol lyase activity by transforming E. coli deficient in tyrosine phenol lyase-producing ability with a recombinant plasmid
Should be extracted. In the present invention, the above-mentioned E. coli transformant can be used, but in order to achieve higher tyrosine phenol lyase productivity, a strong promoter, ribosome binding region, terminator, etc. are linked to the tyrosine phenol lyase structural gene. Then, it is preferable to construct a recombinant Escherichia coli by transforming E. coli with a recombinant plasmid ligated to a plasmid vector capable of replication in an appropriate Escherichia coli. The plasmid vector that can be replicated in E. coli is not particularly limited as long as it can be replicated in E. coli, but a pBR system or a pUC system is preferably used. The host Escherichia coli is not particularly limited, but it is preferable to use tyrosine phenol lyase deficient E. coli from the viewpoint of simplicity of selectivity of the recombinant plasmid in which the tyrosine phenol lyase gene is ligated. In addition, from the viewpoint of biohazard, a selectable marker for the host, for example, an auxotrophic one is preferable, and a tryptophan auxotrophic strain of Escherichia coli K-12 can be used. As such, Escherichia coli ( Escerichia coli ) can be used. ATCC237
18 (trpB ^-) can be mentioned (ATCC is A
Represents the deposit number of the American Typecuture collection). As the recombinant Escherichia coli introduced with the tyrosine phenol lyase gene derived from the bacterium belonging to Enterobacteriaceae constructed as described above, the tyrosine phenol lyase gene derived from Erwinia herbicola MT-10509 strain is used as an expression vector pUC of E. coli.
Recombinant E. coli MT-10519 (F obtained by transforming E. coli MC-1061 strain with the recombinant plasmid pCE26 ligated to the downstream of the lac promoter of 118.
ERM BP-3287) (Japanese Patent Laid-Open No. 04-218380).
Gazette), Citrobacter Freundy MT-10
The tyrosine phenol lyase gene derived from strain 419
The recombinant plasmid pFY12 ligated downstream of the tac promoter of KK223-3 was used to transform E. coli MC-1061.
Recombinant E. coli MT-104 obtained by transforming the strain
88 strains (FERM BP-2616) (International Publication Number W
O90 / 04031), a recombinant Escherichia coli MT-10762 (FERM P-14545) obtained by transforming Escherichia coli ATCC 23718 with pCE26.
And the like. All of these recombinant E. coli have been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology with the above deposit number.

In order to produce tyrosine phenol lyase, recombinant Escherichia coli into which a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae has been introduced is aseptically inoculated into a medium, and a high concentration is obtained at a temperature of 25 to 30 ° C. After culturing, tyrosine phenol lyase may be produced in the culture. As a medium used for high-concentration culture for producing tyrosine phenol lyase, a medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, amino acids, vitamins and the like can be used. Glucose is preferably used as the carbon source. High-concentration culture for producing tyrosine phenol lyase may be performed in a flask or the like, but it is preferable to use a culture tank (jar fermenter) capable of controlling culture conditions. At that time, it is preferable to perform culture (preculture) in which the number of cells to be used for culture is increased in advance, because selective pressure can be applied to the culture by an antibiotic or the like. The temperature of the preculture is 35 ° C. or lower, preferably 25 to 32 ° C., more preferably 28 to 30 ° C. The culture temperature of the high-concentration culture for producing tyrosine phenol lyase is 25 to 30 ° C, preferably 28 to 30 ° C. At 24 ° C or lower and 31 ° C or higher, the activity of tyrosine phenol lyase in the culture system decreases, which is not preferable. The pH of the culture broth of the high-concentration culture for producing tyrosine phenol lyase may be controlled at 5 to 9, preferably 6.5 to 7.5. To control the pH, glucose or ammonia may be added to the culture medium continuously or intermittently. DO in the culture solution of high-concentration culture for producing tyrosine phenol lyase may be controlled so as not to be a rate-determining factor for cell growth and tyrosine phenol lyase production. The high-concentration culture for producing tyrosine phenol lyase is performed at least until the growth of bacterial cells reaches the stationary phase, and the culture time is usually about 5 to 40 hours. The culture thus obtained can be used as it is as an enzyme source for tyrosine phenol lyase, but a crude enzyme extract, purified enzyme, isolated viable cells, a treated product of isolated cells, etc. can also be used as an enzyme source. it can.

When the cultured bacterial cells are used for immobilization, the bacterial cells are grown to the stationary phase by the above-mentioned culture method and then 3-1.
The culture is continued for 0 hours, preferably for 3 to 6 hours while flowing the carbon source. The flow rate of the carbon source may be a constant rate so as not to hinder the growth of the bacterial cells and the production of tyrosine phenol lyase, preferably 1 to 7 g / l · hr, more preferably 3 to 5 g / l · hr. It should be done at the speed of. By culturing as described above, the membrane structure of the obtained microbial cell becomes strong, the activity of protease and the like in the microbial cell is low, and lysis of the microbial cell or decomposition of tyrosine phenol lyase by an enzyme hardly occurs. On the other hand, when the addition rate of the carbon source is higher than the growth phase, the cell membrane structure becomes weak and tyrosine phenol lyase becomes unstable. Further, if the culture time after reaching the stationary phase is longer than 11 hours, the membrane structure of the microbial cell becomes weak, and at the same time, the activity of protease and the like in the microbial cell increases and the tyrosine phenol lyase becomes unstable.

The cells are collected from the culture broth cultured as described above, and a high molecular substance, preferably κ-carrageenan,
The cells can be immobilized by coating with an immobilization carrier such as alginate or polyacrylamide. Further, in order to remove the protease in the immobilized cells as much as possible and stabilize the tyrosine phenol lyase in the immobilized cells, the immobilized cells are heat-treated. This heat treatment is carried out in the temperature range of 40 to 60 ° C., preferably 45 to 55.
It is good to carry out at 120C for 10 to 120 minutes, preferably 30 to 60 minutes. If the temperature of the heat treatment is lower than 40 ° C., protease inactivation is small and becomes insufficient, and if it exceeds 60 ° C., tyrosine phenol lyase is extremely inactivated, which is not suitable. In addition, the heat treatment time is the same, 10
If it is shorter than a minute, the effect of inactivating the protease is small, and if it exceeds 120 minutes, the inactivation of the tyrosine phenol lyase activity becomes serious, which is not suitable.

The tyrosine phenol lyase obtained as described above can be used for the synthesis of L-tyrosine and L-dopa. That is, any of pyruvic acid and ammonia, or L-serine and phenol or catechol can be reacted with tyrosine phenol lyase to synthesize L-tyrosine or L-dopa. In the synthesis reaction of L-tyrosine or L-dopa, the cells obtained by the high-concentration culture, the treated cells, or the immobilized cells are contacted with a substrate in an aqueous medium such as water or a buffer solution. You can do it. Nutrients necessary for the growth of microorganisms, antioxidants, surfactants, coenzymes, metal ions and the like may be added to the aqueous medium. In the synthesis reaction of L-tyrosine or L-dopa, the above-mentioned phenol or catechol as a substrate facilitates inactivation of the protein in the immobilized cells, but tyrosine phenol lyase in the immobilized cells also Affected as well. By adding an SH group protecting agent such as 2-mercaptoethanol or DTT to the reaction solution, the stability of tyrosine phenol lyase against these substrates is significantly improved. In this case, 2-
If the concentration of mercaptoethanol or DTT is less than 1 mM, the effect is small, but if the concentration is too much, it affects the reaction system, so 2 to 200 mM, preferably 5 to 50 m
It is desirable to add M to the reaction solution.

When the immobilized cells were repeatedly used for the production of L-DOPA, L produced by the L-DOPA synthesis reaction.
-A phenomenon was observed in which dopa reacts with PLP, which is a coenzyme of tyrosine phenol lyase, and PLP becomes deficient, resulting in apparent reduction in tyrosine phenol lyase activity. Therefore, in order to restore the tyrosine phenol lyase activity of the immobilized cells, it is necessary to remove the reaction products and byproducts in the immobilized cells and supplement the tyrosine phenol lyase with PLP. Specifically, the tyrosine phenol lyase activity can be recovered by washing with a solution obtained by adding 0.1 to 100 mM, preferably 0.5 to 10 mM of PLP to a potassium phosphate buffer (pH 7.0). In addition, L-tyrosine or L-dopa can be selectively decomposed by utilizing the reverse reaction of the synthetic reaction of L-tyrosine or L-dopa.

[0015]

【Example】

[Example 1] Tyrosine phenol lyase
Effect of culture temperature on gene expression Expression vector pCE26 in which the tyrosine phenol lyase gene derived from Erwinia herbicola MT-10509 strain was ligated downstream of the lac promoter of Escherichia coli plasmid vector pUC118 was used for Escherichia coli ATCC2371.
8 strain MT-10762, which was introduced into (trpB ^over) strain
(ATCC23718 / pCE26) was used as a tyrosine phenol lyase producing bacterium. Recombinant E. coli MT
The -10762 strain has been deposited as FERM P-14545 at the Institute of Biotechnology, Institute of Industrial Science and Technology. Sterile medium 1 in which E. coli MT-10762 strain was added to L medium (polypeptone 1%, yeast extract 0.5%, salt 0.5%) and ampicillin was added at a concentration of 100 mg / l.
Inoculate to 00 ml and in a 500 ml flask at 30 ℃ 1
It pre-cultured by shaking culture for 4 hours. Using this preculture liquid, main culture was performed while controlling the temperature within the temperature range shown in Table 2 (Table 2). In the main culture, 50 ml of the preculture liquid was added to 1 liter of the main culture medium having the composition shown in Table 1 (Table 1) with a 2 liter jar fermenter, and the pH was in the range shown in Table 2 (Table 2). Ammonia and glucose were sequentially added so that the amount of the mixture would be 1 and the aeration rate was 1 vvm and the stirring rate was 600 rpm. The growth of the bacterial cells is carried out by measuring the absorbance (O
D ₆₆₀ ). The L-dopa synthesis activity was measured by the following procedure. 2 mM pyridoxal 5 ′ was added to the cells collected from the culture solution so that the cell concentration became 80 mg / ml.
Phosphoric acid (PLP) -10 mM potassium pyrophosphate (pH
8.5) Suspended in the solution and sonicated. L-serine 4
0 g / l, ammonium acetate 10 g / l, EDTA ・ 2
Na ₂ g / l, Na ₂ SO ₃ 4 g / l, pyrocatechol 1
To 0.5 ml of the substrate solution in which 6 g / l was adjusted to pH 8.0 with ammonia water, 0.5 ml of the ultrasonically disrupted bacterial cell solution was added, and the reaction was stopped at 22 ° C for 1 hour, then the reaction was stopped. Let After the reaction was stopped, the amount of L-dopa produced was quantified by high performance liquid chromatography, and the activity value was calculated. The maximum cell density, the culture time and the tyrosine phenol lyase activity value at that time of the cells thus cultured are shown in Table 2 (Table 2). In Table 2 (Table 2), the maximum cell density of the cells and the value of the tyrosine phenol lyase activity at that time are shown as relative values when the measured values when cultured at 28 to 30 ° C. are each 1.00. . still,
The value of tyrosine phenol lyase activity at this time is 35 U.
/ G wet cell. As a result of the experiment, the tyrosine phenol lyase activity greatly depends on the culture temperature of the main culture,
It was found that 25 to 30 ° C was the best. Also,
It was found that the tyrosine phenol lyase activity, which has a high maximum cell density and a short time until the maximum cell density is reached, does not necessarily give a high tyrosine phenol lyase activity.

[0017]

[Table 1] Table-1 Composition of medium for high concentration ─────────────────────── Component concentration ────────────── ────────── Polypeptone (Nippon Pharmaceutical Co., Ltd.) 7.0 g / l Glucose 4.0 g / l Potassium Phosphate 2.0 g / l Dipotassium Phosphate 2.0 g / l Ammonium Sulfate 1.5 g / l Salt 1.0 g / l Magnesium sulfate 2.0 g / l Ferrous sulfate 40 mg / l Calcium chloride 40 mg / l Manganese sulfate 10 mg / l Aluminum chloride 10 mg / l Cuprous chloride 4 mg / l Cobalt chloride 4 mg / l Zinc sulfate 4 mg / L sodium molybdate 2 mg / l boric acid 0.5 mg / l L-tryptophan 1 g / l ───────────────────────── pH 6.8 to 7 with ammonia Prepared to 0.0 Glucose-added solution ─────────────────────────
─ Glucose 400 g / l ──────────────────────────
─

[0018]

[Table 2]

[Example 2] Tyrosine phenol lyase
Immobilization of Recombinant Escherichia coli Introduced with Gene After the culture reached the stationary phase by the culture method of Example 1, 4 g /
l · hr (medium 1l using glucose solution as glucose)
The above was added at a rate of 4 g per 1 hour), and the culture was continued for 0 to 20 hours while flowing down, and then the culture was stopped. After the culture was completed, the cells were collected by a centrifuge to obtain 50 g of wet cells. To this, 50 g of 50 mM sodium phosphate buffer (pH 7.0, containing 1 mM PLP) was added to suspend the cells. This cell suspension was kept at 50 ° C. for 1 hour. 3.5% κ-carrageenan in physiological saline
250 g of a κ-carrageenan solution that had been added to the solution and was heated and dissolved was cooled to 50 ° C., and then mixed with the cell suspension. While maintaining this mixed solution at 50 ° C., the solution was pumped and dropped from a nozzle into 600 ml of a 0.3 M potassium chloride solution to form a gel. After the dropping was completed, the potassium chloride solution was stirred at 15 ° C. for 2 hours, and then the gel and the potassium chloride solution were separated. L-DOPA was produced using the immobilized bacterial cells thus obtained. The optimal time was determined by considering the time of the end of culture as follows. If the membrane structure of the cells is weakened, the protein in the cells will easily leak into the reaction solution, so the cells in the culture solution at each stage will be partially centrifuged to prepare immobilized cells. Then
It can be measured by quantifying the protein by measuring the absorbance of the protein that appears in the phosphate buffer whose pH is adjusted to near neutral. Also,
Similarly, the protease activity of the bacterial cells was collected by partially centrifuging the bacterial cells in the culture solution, suspending the cells in the above-mentioned phosphate buffer, and sonicating the cells to prepare an intracellular protease solution. , Using casein as a substrate,
It can be examined by quantifying the amount of amino acid that appears in the supernatant by treating with trichloroacetic acid and deproteinizing. Therefore, the strength of the bacterial cell membrane structure and the protease activity of the bacterial cells are determined by the absorbance λ = 2 in each buffer solution.
It was examined by measurement at 75 nm and λ = 595 nm.
The L-dopa synthetic activity was measured by the culture method described in Example 1,
After the culture reaches the stationary phase, 0 hours, 3 hours, 10 hours,
And the cells after 20 hours had passed. L-dopa synthesis reaction is carried out by L-serine 40 g / l, ammonium acetate 10 g / l, EDTA.2Na ₂ g / l, Na ₂ SO ₃
100 g of the immobilized cells were added to 1 liter of a substrate solution in which 4 g / l and 16 g / l pyrocatechol were adjusted to pH 8.0 with aqueous ammonia, and the mixture was subjected to 30 minutes at 30 ° C. The amount of L-dopa in the reaction solution was quantified in the same manner as in Example 1 to calculate the activity value. The strength of the membrane structure (leakage of protein from the immobilized cells), the protease activity, and the activity per 1 g of the immobilized cells were set to 1.0 when the cells that had been cultivated immediately after the stationary phase were immobilized. After 3 hours as a relative value of 6
Table 3 (Table 3) shows the values after 10 hours and 20 hours.
It was shown to.

[0020]

[Table 3] (**) Leakage of protein, protease activity, and activity per 1 g of immobilized microbial cells of microbial cells immobilized with κ-carrageenan using the microbial cells immediately after reaching the stationary phase were 1. Relative value when OO.

Comparative Example 1 Effect of Glucose Flow Down After the culture reached the stationary phase by the culture method of Example 1, the glucose flow was stopped and the culture was continued for another 6 hours.
Immobilized cells were prepared in the same manner as in Example 1, L-dopa was produced, and the tyrosine phenol lyase activity and its stability were examined. Glucose was flowed down to 4 g / l · hr and cultured for 6 hours. The protein leakage from immobilized cells, protease activity, and tyrosine phenol lyase activity per 1 g of immobilized cells were 1.80 times, 1.96 times, and 0.72 times, respectively, compared with those of there were.

[Comparative Example 2] Effect of glucose flow down After the culture reached the stationary phase by the culture method of Example 1, the glucose flow was set to 15 g / l · hr, and the culture was continued for a further 6 hours, and then carried out. Immobilized cells were prepared in the same manner as in Example 2, L-dopa was produced, and the tyrosine phenol lyase activity and its stability were examined. As a result, glucose was flowed down to 4 g / l · hr, and 6 Compared to the case of continuous culture, the leakage of protein from the immobilized cells, the protease activity, and the tyrosine phenol lyase activity per 1 g of the immobilized cells were 1.68 times and 2.26 times, respectively.
And 0.57 times.

[Example 3 and Comparative Example 3] Heat of immobilized cells
After reaching the stationary phase in Treatment Example 2, immobilized cells obtained by immobilizing the cells that had been continuously cultured for 6 hours with κ-carrageenan were treated at 30 ° C, 40 ° C, 50 ° C, 60 ° C and 70 ° C. 5 each
After heat treatment for 10 minutes, 30 minutes, 60 minutes, 120 minutes and 180 minutes, tyrosine phenol lyase activity and protease activity were measured by the same method as in Example 2. As a result, as shown in Table-4.1 (Table 4) and Table-4.2 (Table 5), heat treatment for 10 to 120 minutes in the temperature range of 40 to 60 ° C stabilizes tyrosine phenol lyase activity. It turned out to be effective. In addition, in Table-4.1 (Table 4) and Table-4.2 (Table 5), each activity is tyrosine phenol lyase activity when heat treatment is not performed, protease activity is 1.0, and each condition It was expressed as a relative value of the activity when heat-treated in.

[0024]

[Table 4]

[0025]

[Table 5]

Example 4 Immobilization with PLP-containing buffer
Regeneration of modified cells Using the immobilized bacterial cells heat-treated at 50 ° C. for 60 minutes in Example 3, L-dopa was produced according to the method described in Example 2 using L-serine and catechol as substrates. . After this, the tyrosine phenol lyase activity of the immobilized cells was reduced to 62.5% of the initial activity. Immobilized cells used once in this reaction were mixed with 0.3 MK of 3 times the reaction solution.
Cl-25 mM potassium phosphate buffer (pH 7.0) -1 mM
Upon washing with the PLP solution, the activity recovered to the original value (initial activity value).

[0027] Example 5 and Comparative Example 4 of SH group-protecting agent
Effect The stability against catechol was examined using the immobilized bacterial cells used in Example 4. When 10 mM 2-mercaptoethanol was added to the reaction solution, it was 9 even in the reaction solution containing 15 mM catechol (15 ° C., 12 hours).
It was stable with 5% activity maintained, but those without 2-mercaptoethanol lost 90% activity only by the addition of 10 mM catechol.

[0028]

Industrial Applicability The tyrosine phenol lyase production method according to the present invention is a method that enables a large amount of tyrosine phenol lyase to be accumulated by recombinant Escherichia coli into which tyrosine phenol lyase has been introduced. In addition, the method for immobilizing recombinant Escherichia coli introduced with tyrosine phenol lyase according to the present invention has the highest activity per cell, and the tyrosine phenol lyase activity of the immobilized cell is less likely to decrease. Therefore, it becomes possible to produce L-tyrosine and L-dopa with high efficiency and low cost by using the immobilized bacterial cells obtained by the method. Furthermore, in the method for producing L-dopa using the immobilized cells of the present invention, when the immobilized cells are reused, the lifespan of the immobilized cells is extended because the decrease in tyrosine phenol lyase activity is extremely small, and industrial L-dopa is used. Effective for dopa production.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12N 15/09 C12P 13/22 D 2121-4B // (C12N 9/88 C12R 1:19) ( C12P 13/22 D C12R 1:19 1:01)

Claims (8)

[Claims] 1. When producing a tyrosine phenol lyase by highly culturing recombinant Escherichia coli into which a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is introduced, the culture temperature is controlled within the range of 25 to 30 ° C. A method for producing tyrosine phenol lyase, which comprises culturing at the same time. 2. The method for producing tyrosine phenol lyase according to claim 1, wherein the bacterium belonging to Enterobacteriaceae is Erwinia herbicola or Citrobacter freundii. 3. The recombinant Escherichia coli is Escherichia coli MT-10762.
The production method according to claim 1 or 2, wherein 4. A method for immobilizing recombinant Escherichia coli into which a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae has been introduced, which comprises culturing the recombinant Escherichia coli at a culture temperature of 25 to 30 ° C. High concentration culture until the growth reaches a stationary phase, and then the culture is continued for 3 to 10 hours while flowing a carbon source, and the obtained bacterial cells are immobilized by an immobilization carrier. A method for immobilizing the recombinant Escherichia coli in which tyrosine phenol lyase is stabilized. 5. A method for immobilizing recombinant Escherichia coli into which a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae has been introduced, wherein the immobilized cells are heat-treated at a temperature of 40 to 60 ° C. A method for immobilizing the recombinant Escherichia coli in which tyrosine phenol lyase in the bacterial cell is stabilized, which is characterized in that 6. A method for producing L-dopa from L-serine and catechol, which uses an immobilized bacterium in which recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is immobilized. L-
In a synthetic reaction of L-dopa from serine and catechol, a method for producing L-dopa in which tyrosine phenol lyase of immobilized cells is stabilized, which comprises adding an SH group protecting agent to the reaction solution. 7. A method for producing L-dopa from L-serine and catechol using immobilized cells in which recombinant Escherichia coli introduced with a tyrosine phenol lyase gene derived from a bacterium belonging to Enterobacteriaceae is immobilized. Then, the immobilized cells are washed with a buffer containing pyridoxal 5'phosphate and then reused in the L-DOPA synthesis reaction. 8. The recombinant Escherichia coli is Escherichia coli MT-10762.
The method according to claim 4, wherein