JP2517861B2 - Method for producing immobilized enzyme - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an improved method for producing an immobilized enzyme. More specifically, the present invention relates to a highly efficient method for producing a homogeneous and highly active immobilized enzyme used as an element of a bioreactor.

[0002]

2. Description of the Related Art In recent years, with the progress of biotechnology, a system for producing useful substances by a bioreactor using immobilized enzymes and immobilized microorganisms as elements has been rapidly developed. For example, amino acids, organic acids, and nucleic acid-related substances. , Sugar, chemicals, pharmaceuticals, etc., or food production and processing, production of energy substances such as alcohol and methane, as well as analysis and measurement fields, medical fields, and environmental purification fields.

This bioreactor is a chemical reaction system in which enzymes or microorganisms are immobilized and its reaction characteristics as a biocatalyst are utilized. Compared with reactions using conventional chemical reaction catalysts, (1) room temperature, The reaction is carried out under mild conditions under normal pressure, (2) reaction specificity is high and only specific substances are produced, (3) structure specificity or stereospecificity is obtained, (4) energy saving It has features such as

[0004] As a method of immobilizing an enzyme to obtain an immobilized enzyme used as an element of such a bioreactor, conventionally, a carrier binding method, a cross-linking method and an encapsulation method have been mainly used. Each method has advantages and disadvantages and is not always a fully satisfactory method.

For example, the carrier binding method can be broadly classified into a non-covalent binding immobilization method by physical adsorption and ionic binding and a covalent bond immobilization method by chemical reaction depending on the binding mode. However, the noncovalent immobilization method is unavoidable because the binding force between the carrier and the enzyme protein is weak, so that the range of utilization of the obtained immobilized enzyme is limited. Uses the chemical reactivity of the amino acid side chains of the enzyme protein,
Although the bond is extremely stable, it may deactivate the enzyme protein by forming a new chemical bond, and in addition to the amino acid side chain of the same kind (for example, amino group of lysine, aspartic acid) in the enzyme protein. And the carboxyl group of glutamic acid), and these are involved in the immobilization reaction, so that it is extremely difficult to control the immobilization site.

In the immobilization method by the cross-linking method,
Similar to the above-mentioned covalent immobilization method, there is a drawback that it is extremely difficult to control the crosslinking site. Furthermore, although the immobilization method by the inclusion method does not have such a drawback,
Since the conditions for making the gel to be used for encapsulation are severe for the enzyme protein used, there is a risk of destruction, and the permeation of enzyme reaction substrates and products into the encapsulation gel must be considered. I won't.

On the other hand, it is known that the function of the enzyme is hardly affected even if a short amino acid sequence is introduced into the carboxy terminal side of the enzyme protein [JP-A-1-14].
4992, "Methods in Enzymology",
185, 129 (1990)].

[0008]

DISCLOSURE OF THE INVENTION The present invention overcomes the drawbacks of immobilization of an enzyme protein by the covalent carrier-binding method, suppresses the inactivation of the enzyme protein, and easily controls the immobilization site. The purpose of the present invention is to provide a method for efficiently producing a homogeneous and highly active immobilized enzyme by a covalent bond-type carrier binding method.

[0009]

Means for Solving the Problems As a result of intensive studies aimed at achieving the above-mentioned object, the present inventors have found that even if a short amino acid sequence is introduced at the carboxy-terminal side of an enzyme protein, the function of the enzyme is almost the same. Focusing on not being affected, by introducing an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus at the carboxy terminal side of the desired enzyme protein to prepare a modified enzyme protein, It was found that the object can be achieved by binding to the immobilized carrier through the mercapto group of the terminal cysteine residue, and the present invention has been completed based on this finding.

That is, according to the present invention, a modified enzyme protein is prepared by introducing an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus on the carboxy terminal side of the enzyme protein, and then preparing the modified enzyme protein. The present invention provides a method for producing an immobilized enzyme, which comprises binding to an immobilized carrier via the mercapto group in the cysteine residue.

In the method of the present invention, it is first necessary to introduce an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus on the carboxy terminus side of the desired enzyme protein. The method for introducing the amino acid sequence is not particularly limited, and a known method can be used. Modification by introducing the amino acid sequence to the carboxy terminal side of a desired enzyme protein by, for example, a gene recombination method. It is advantageous to prepare the enzyme protein.

[0012] In this gene recombination method, a gene DNA encoding a desired enzyme protein is used, and a sequence portion encoding the carboxy terminal portion of the enzyme protein is replaced with a chemically synthesized DNA, so that the carboxy terminal is Gene D encoding a modified enzyme protein having a short amino acid sequence introduced to form a cysteine residue
After preparing NA, this is incorporated into a vector such as an expression plasmid to construct an expression vector, which is then introduced into a host such as Escherichia coli and expressed, and the modified enzyme protein is separated and purified from the host cells, A modified enzyme protein of interest can be prepared. Therefore, here, a specific example of dihydrofolate reductase will be shown.
Of course, any enzyme protein can be used in the same manner.

In the method of the present invention, the reaction of the mercapto group of the carboxy-terminal cysteine residue of the modified enzyme protein thus obtained is used to bind and immobilize the enzyme protein on the immobilization carrier. By utilizing the reaction of the mercapto group of the cysteine residue, a specific covalent bond can be made under mild conditions ["Biochemical Experimental Method 8, Chemical Modification of SH Group", Academic Society Publishing Center (1978). )]. In particular, the thiol-disulfide exchange reaction is unique to cysteine residues and does not occur with other amino acid side chains. Further, the mercapto group binds to a metal ion (particularly, a mercury ion) to form a mercaptide, and a compound containing an alkyl halide alkylates the mercapto group under mild conditions. Furthermore, N
-Forms an addition compound with a compound having an easily polarizable double bond such as ethylmaleimide. Such a reaction can be specifically caused to a mercapto group by appropriately selecting conditions such as pH. Utilizing these reactions, only the mercapto group of the cysteine residue introduced at the carboxy terminus of the enzyme protein can be linked to the immobilized carrier via a covalent bond. In the present invention, a thiol-disulfide exchange reaction is shown as a specific example of the immobilization reaction.
Of course, the immobilization reaction is not limited to this example.

Proteins generally form a higher-order structure, and the movement of their side chains is restricted. Therefore,
Except for the amino acid side chains present at the termini, their reactivity is generally low. In the present invention, an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus is introduced, for example, Gly-Gly-Gly-Cys is introduced to enhance the motility of the carboxy terminus and particularly enhance the reactivity of the mercapto group. It is one of the characteristics of the present invention that the specificity is increased.

Next, the embodiment of the present invention will be described using an example in which dihydrofolate reductase is used as an enzyme protein. First, a modified dihydrofolate reduction in which a short amino acid sequence having a cysteine residue at the carboxy terminus is introduced. An expression vector is constructed in which a gene DNA encoding an enzyme, that is, a modified dihydrofolate reductase gene DNA is incorporated into a vector such as a plasmid. In the present invention, an Escherichia coli-derived gene is used as the dihydrofolate reductase gene, and the E. coli-derived gene has already been cloned by the present inventors.
In addition, an expression vector in which the dihydrofolate reductase gene DNA has been incorporated has also been prepared (Japanese Patent Publication No. 1-48756).
Issue). Further modified dihydrofolate reductase gene DN
Expression vectors incorporating A have also been prepared (JP-A-63-267276, JP-A-1-252289). Therefore, by utilizing these known techniques, the modified dihydrofolate reductase gene DNA encoding the modified dihydrofolate reductase having a cysteine residue at the carboxy terminus was incorporated from the dihydrofolate reductase gene derived from Escherichia coli. The expression vector can be easily constructed.

Generally, in order to efficiently express a foreign protein gene in a transformed microorganism and mass-produce the target protein, the transcription efficiency and translation efficiency of the foreign protein gene may be increased or the foreign protein gene may be increased. It is necessary to increase the number of copies of. In order to increase the transcription efficiency of the foreign gene, a strong promoter is generally attached upstream of the foreign protein gene. Therefore, the expression vector is preferably one in which a suitable promoter is linked upstream of the modified dihydrofolate reductase gene DNA. Also,
This expression vector is introduced into a host microorganism, and the microorganism into which this expression vector has been introduced is usually selected by culturing the microorganism in a selection medium based on the drug resistance marker of the vector and selecting a microorganism that grows. Because it will be
It is desirable that a drug resistance gene DNA is also incorporated in the expression vector.

Next, the expression vector thus obtained is introduced into a host microorganism such as Escherichia coli by a known method to transform the host microorganism and produce a desired modified dihydrofolate reductase. Produce microorganisms.
The selection as to whether or not the host microorganism has introduced the expression vector may be carried out by culturing the microorganism in a selective medium based on the drug resistance marker of the vector and selecting a microorganism that grows.

The production of this transformed microorganism is carried out by, for example, two 23 nucleotide 5'-GATC obtained by a chemical synthesis method.
CTGGGTGGCTGTTAAC-3 'and 5'-TCGACTTAACAGCCACCGAC-
DNA consisting of 3 '(wherein A means adenine, T means thymine, G means guanine, and C means cytosine) is annealed, while the plasmid pMEK2 (JP-A-1-
252289 gazette, containing a gene DNA encoding a dihydrofolate reductase-methionine enkephalin fusion protein, a promoter, a trimethoprim-resistant and ampicillin-resistant gene DNA, etc., and cleaving it with an appropriate restriction enzyme to obtain a DNA fragment. , This DNA fragment and the above-mentioned annealed chemically synthesized DNA, T4DN
After ligation using A ligase, this was introduced into Escherichia coli (hereinafter abbreviated as E. Coli) according to the transformation method, and then the transformant was transformed with a nutrient agar medium containing ampicillin sodium and trimethoprim. Selected.

It was confirmed that the transformant strain thus produced produced a large amount of modified dihydrofolate reductase having a cysteine residue at the carboxy terminus, and at the same time, the transformant strain was subjected to a known method as shown in FIG. A plasmid pCYS1 having the restriction enzyme cleavage map shown in Figure 3 was prepared.

Restriction enzyme Bg of this plasmid pCYS1
The nucleotide sequence of a DNA of about 700 nucleotides obtained by cleavage with III was determined by a known method, and it was confirmed that the target chemically synthesized DNA was correctly integrated. Furthermore, from the determined nucleotide sequence of the DNA, The amino acid sequence of the modified dihydrofolate reductase was determined.

The modified dihydrofolate reductase gene DN
A is the formula

Embedded image It has the base sequence shown by. The symbols A, T, G, and C in this have the same meanings as described above, and the base sequence is shown in SEQ ID NO: 2 in the sequence listing. The number of the base sequence in this sequence listing is DN, which encodes dihydrofolate reductase.
A of ATG, which codes for methionine at the 1st amino acid in the A sequence
Is counted as number 1. In addition, the portion that encodes dihydrofolate reductase is shown separated by an amino acid code consisting of 3 bases.

The end of this sequence has a recognition cleavage site for the restriction enzyme BgIII and can be incorporated into any vector. The expression plasmid pCYS1 containing the modified dihydrofolate reductase gene was transformed into E. E. coli, which is stably retained in E. coli cells and contains pCYS1. E.coli has been deposited with MIC as FERM BP-3600.

This pCYS1 is the plasmid pMEK2.
BamHI and Xh of (Japanese Patent Laid-Open No. 1-252289)
It is created by replacing the sequence between the oI sites with the double-stranded DNA described above. This pCYS1 is 4637
It consists of base pairs and confers ampicillin resistance and trimethoprim resistance to the host. The position of each gene is shown in the restriction enzyme cleavage map of FIG. 1, and the position of the ampicillin resistance gene is shown by Amp in the figure.
The site indicated by R is the site of the dihydrofolate reductase gene that confers resistance to trimethoprim. BgIII above
The site of the modified dihydrofolate reductase gene sandwiched between the sites is represented by a black arrow in the figure.
ori represents the site required for plasmid replication, and p represents the position of the promoter required for highly efficient expression of the modified dihydrofolate reductase gene.

By expressing the sequence of the modified dihydrofolate reductase gene described above, the expression

Embedded image A modified dihydrofolate reductase having the amino acid sequence shown by can be expressed. The number in the amino acid sequence of this formula is calculated from the first amino acid methionine of dihydrofolate reductase, and
Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cystine, Gln is glutamine, Glu is glutamic acid and Gl.
y is glycine, His histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, Trp is tryptophan, Tyr is tyrosine, and Val is valine. The amino acid unit of is shown.

This modified dihydrofolate reductase is 165
It is an enzyme protein consisting of 4 amino acid residues and catalyzes a reaction of reducing dihydrofolate to produce tetrahydrofolate in the presence of the coenzyme NADPH. Wild-type dihydrofolate reductase consists of 159 amino acid residues,
The sequence is exactly the same as the sequence from 1 to 159 of the above sequence except that the second amino acid residue is a cysteine residue.

There are two cysteine residues in the modified dihydrofolate reductase (85th and 165th positions), but the reactivity of the mercapto group is completely different,
Neutral (pH 7.0) at room temperature (15-30 ° C.)
In the thiol-disulfide exchange reaction using 5'-dithiobis (2-nitrobenzoic acid), the cysteine residue at the 85th position did not react at all, but the cysteine residue at the 165th position rapidly reacted for a few minutes. Completes the reaction. In the wild-type dihydrofolate reductase, two cysteine residues also exist (85th and 152nd), but the reactivity of the mercapto group is low, and the 85th cysteine residue is similar under the same reaction conditions. Is completely inactive,
The reactivity at the 152nd cysteine residue is a fraction of the reactivity at the modified dihydrofolate reductase 165th cystine residue.

Transformation of E. coli into which the expression plasmid vector pCYS1 was introduced. Coli (Microtech Lab.
P-3600) was cultured in the E. It can be carried out in a medium containing nutrients such as carbon sources and nitrogen sources and inorganic components necessary for the growth of E. coli.

Examples of the carbon source include glucose, starch, sucrose, molasses and dextrin, and examples of the nitrogen source include peptone, meat extract, casein hydrolyzate, corn steep liquor, nitrate and ammonium salt. Examples of the inorganic component include salts containing cations such as sodium, potassium, calcium, magnesium, cobalt, zinc, manganese, and iron, and anions such as chlorine, sulfuric acid, and phosphoric acid.

The form of culture is preferably liquid culture, and industrially, it is advantageous to perform deep aeration stirring culture. The culture temperature is E. Coli grows and is appropriately selected within a range where a modified dihydrofolate reductase is produced, but usually 20 to 40
Selected at temperatures in the range of ° C. The culturing time depends on the conditions, but the culturing may be finished at an appropriate time in consideration of the time when the maximum yield of the enzyme is reached.

In the thus obtained culture, the bacterial cells are recovered by means such as filtration or centrifugation, and then the bacterial cells are mechanically disrupted by a ball mill or ultrasonic waves, or enzymatically disrupted by lysozyme or the like. The modified dihydrofolate reductase is solubilized by adding a chelating agent or a surfactant as necessary, separated and collected, and purified. Regarding the purification method, for example, the modified dihydrofolate reductase-containing solution is concentrated under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate, etc., fractionation precipitation with a hydrophilic organic solvent such as methanol, ethanol, acetone, etc. After appropriately selecting and combining to obtain a precipitate, the precipitate containing the modified dihydrofolate reductase is further dissolved in water or a buffer solution, if necessary, and dialyzed with a dialysis membrane to give a lower molecular weight. The impurities may be removed, and it may be purified by ion exchange chromatography using an adsorbent, a gel filtration agent, etc., size separation gel chromatography, affinity chromatography, hydrophobic interaction chromatography and the like. These purification methods may be used alone or in combination of two or more, or
One or more purification methods may be repeatedly used.

In the present invention, the purified and modified dihydrofolate reductase having the cysteine residue thus obtained at the carboxy terminus is immobilized. This immobilization is a function that reacts with the mercapto group of the cysteine residue. It is carried out by utilizing an immobilization carrier having a group. The immobilization carrier used at this time is not particularly limited as long as it has a functional group that specifically reacts with the mercapto group of the cysteine residue, but, for example, commercially available thiopropyl sepharose 6B is used. it can.

This thiopropyl sepharose 6B is mixed with the modified dihydrofolate reductase under neutral conditions,
About 0.3 mg of modified dihydrofolate reductase can be immobilized per 1 g of thiopropyl sepharose 6B.

The modified dihydrofolate reductase immobilized in this manner is active, and when it was used as an enzyme reactor packed in a glass column, it was about 500 mg even at a high flow rate condition of 4 ° C. and a flow rate of 10 ml / min. It was possible to complete the enzymatic reaction almost completely by using a small amount of immobilized gel and confirm its effectiveness.

The enzyme activity of the immobilized modified dihydrofolate reductase was determined by measuring the reaction mixture [0.05 mM dihydrofolate, 0.
06 mM NADPH, 50 mM phosphate buffer (pH
7.0)] was packed in a glass column using a peristaltic pump and continuously fed into an enzyme reactor, and after a suitable time, the eluted solution was collected and the absorbance at 340 nm of the solution was measured and passed through the column. It is determined by calculating the difference from the absorbance at 340 nm of the previous solution. At this time, 340 when reacted with an excess amount of dihydrofolate reductase
All changes in absorbance at nm are used as 100% conversion efficiency.

By using such an immobilization method, it is possible to limit the site that binds to the immobilization carrier to only the carboxy terminus of the enzyme protein, which is an obstacle to conventional immobilization methods. It is possible to eliminate the decrease in the activity of the enzyme itself due to the immobilization reaction and the heterogeneity of the immobilized enzyme.

[0036]

Next, the present invention will be described in more detail with reference to examples.

Example 1 Construction of pCYS1 Two 23 nucleotides chemically synthesized by the phosphoamidite method, that is, 5'-GATCCTGGGTGGCGGCTGTTAAC-3 '.
And DNA consisting of 5'-TCGACTTAACAGCCGCCACCGAC-3 '
About 0.1 ml of each (containing about 0.01 μg of DNA) was taken, and both DNAs were annealed by incubating this at 60 ° C.
(NA1).

About 1 μg of pMEK2 (JP-A-1-252289) was cleaved with BamHI and XhoI, and then DNA was precipitated with ethanol. The precipitated DNA was washed with 70 wt% ethanol, ethanol was removed, and the precipitate was dried under reduced pressure. Each operation relating to recombinant DNA, such as cutting of DNA with a restriction enzyme, is performed by the method of Maniatis et al. [“Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (198
2 years) (Molecular Cloning AL)
Observatory Manual, Cold Spr
ing Harbor Laboratory (19
82) ”]. The cleaved pMEK2 fragment and DNA1 were ligated with T4 DNA ligase and incorporated into E. coli according to the transformation method. Transformants
50 mg / ml sodium ampicillin and 10 mg
Agar medium containing Trimethoprim / ml (medium 11
2g glucose, 1g dipotassium phosphate, 5
g yeast extract, 5 g polypeptone, 15 g agar) were used to select for ampicillin resistance and trimethoprim resistance. Further, the obtained transformant is cultivated, and all proteins produced by the cells are subjected to SDS-polyacrylamide gel electrophoresis ["Natur (Natur
e) ”Vol. 227, pp. 680 (1970)]. As a molecular weight marker, lactalbumin (molecular weight 14,200), trypsin inhibitor (molecular weight 20,100), trypsinogen (molecular weight 2)
4,000), carbonic anhydrase (molecular weight 2
9,000), glyceraldehyde 3-phosphate dehydrogenase (molecular weight 36,000), ovalbumin (molecular weight 45,000) and bovine serum albumin (molecular weight 66,
000) containing a sample containing polyacrylamide at a concentration of 1
It was run on a 0 to 20% gradient gel. As a result, about 80% of the cells express a large amount of modified dihydrofolate reductase.
Was obtained at a frequency of.

From these cells, the method of Tanaka et al. [“Journal of Bacteriology (J. Bacteriol
121), p. 354 (1975)
Year)], and a plasmid was prepared and named pCYS1. For the DNA of about 700 nucleotides length obtained by cleavage of pCYS1 with BglII,
The dideoxy method using M13 phage ["Methods in Enzymology (Methods in Enz
101), 20th page (198).
3 years)], the base sequence is determined according to
It was confirmed that NA was properly incorporated. Also,
From the determined DNA sequence, the amino acid sequence of modified dihydrofolate reductase was determined.

Example 2 Separation and Purification of Modified Dihydrofolate Reductase from Escherichia coli Introduced with pCYS1 The modified dihydrofolate reductase was separated and purified by culturing the cells (1), crushing the cells (3) DEAE. -Toyopearl column treatment, (4) Mesotrixate (MTX) binding affinity chromatography and (5) DEAE-Toyopearl column chromatography.

(1) Culture of bacterial cells E. coli containing pCYS1 was cultured in 6 liters of Y
T + Ap medium (5 g of NaCl in 1 liter of medium,
8 g tryptone, 5 g yeast extract and 50 mg
Liquid medium containing sodium ampicillin) at 37 ° C.
The cells were cultured at the late stage of logarithmic growth. The cultured cells are 5,
The cells were collected by centrifugation at 000 rpm and collected from 6 liters of culture medium to obtain cells with a wet weight of about 17 g.

(2) Crushing of the bacterial cells The bacterial cells obtained by culturing are buffered with twice the wet weight of the buffer solution 1.
(0.1 mM sodium ethylenediamine tetraacetate (E
DTA) containing 10 mM potassium phosphate buffer, pH
The cells were suspended in 7.0) and the cells were disrupted using a French press. The disrupted cell suspension was centrifuged at 5,000 rpm for 10 minutes to obtain a supernatant. In addition, the supernatant was
Ultracentrifuge for 1 hour to obtain a supernatant (cell-free extract).

(3) Treatment with DEAE-Toyopearl column This operation was carried out for the purpose of pretreatment for the subsequent purification step by MTX binding affinity chromatography. The cell-free extract was used as a buffer 1 containing 0.1 M KCl in advance.
Apply to a DEAE Toyopearl column equilibrated with
The column was washed with buffer solution 1 containing M KCl and the enzyme was eluted using buffer solution 1 containing 0.3 M KCl.
The eluate was fractionated by a fixed amount using a fraction collector, and the DHFR activity of the fractionated eluate was measured to collect fractions containing enzyme activity.

(4) MTX Binding Affinity Chromatography The enzyme solution obtained by the above operation was adsorbed on an MTX binding Sepharose affinity column which had been equilibrated with Buffer 1 in advance. After adsorption, it was washed with buffer 2 containing 1 M KCl (10 mM potassium phosphate buffer containing 0.1 mM EDTA, pH 8.5). For washing, the absorbance at 280 nm of the eluate from the column was measured, and the same buffer was kept flowing until the absorbance became 0.1 or less. The elution of the enzyme was performed using a buffer solution 2 containing 1 M KCl and 3 mM folic acid, and the eluate was fractionated by a fixed amount using a fraction collector. The DHFR activity of the fractionated eluate was measured, and the fractions containing the enzyme activity were collected. The obtained enzyme solution was dialyzed against buffer solution 3 times.

(5) DEAE-Toyopearl Column Chromatography D was prepared by equilibrating the dialyzed enzyme solution with buffer solution 1 in advance.
After adsorbing on EAE-Toyopearl column, 0.1M
Washed with buffer 1 containing KCl. For washing, the absorbance at 280 nm of the eluate from the column was measured, and the same buffer was kept flowing until the absorbance became 0.01 or less. The elution of the enzyme was carried out using buffer solution 0.1 to 0.3M KC.
This was carried out using a linear concentration gradient of 1 and the eluate was fractionated in aliquots using a fraction collector. The absorbance at 280 nm and dihydrofolate reductase were measured for the fractionated eluate. The enzyme activity / absorbance value at 280 nm is
A fixed fraction was collected.

By the above operation, highly purified homogenization of dihydrofolate reductase could be performed with good reproducibility. The enzyme activity at the time of purification was as follows: reaction solution [0.05 mM dihydrofolic acid, 0.06 mM NADPH, 12 mM 2-mercaptoethanol, 50 mM phosphate buffer (pH 7.
[0)] was carried out by taking a 1 ml cuvette, adding the enzyme solution thereto, and measuring the time change of the absorbance at 340 nm. 1 unit of enzyme is
It was defined as the amount of enzyme required to reduce 1 micromol of dihydrofolic acid per minute.

The above results are shown in Table 1. Wet weight about 17g
From the microbial cells, about 130 mg of uniformly purified modified dihydrofolate reductase was obtained. The purification steps in Table 1 represent cell-free extract, DEAE-Toyopearl column treatment, MTX binding affinity chromatography and DEAE-Toyopearl column chromatography.

[0047]

[Table 1]

Example 3 Reactivity of mercapto group of cysteine residue in modified dihydrofolate reductase The reactivity of mercapto group of cysteine residue in modified dihydrofolate reductase was determined to be 5,5'-dithiobis (2-
Nitrobenzoic acid).

As the buffer, 10 mM potassium phosphate buffer (pH 8.0) containing 2.5 mM EDTA was used. The protein concentration was 0.7 mg / ml, and 5,5'-dithiobis (2-nitrobenzoic acid) was 2 mg / ml.
The concentration of mM and the temperature of the reaction were 15 ° C. The reaction is 5,
It was started by the addition of 5'-dithiobis (2-nitrobenzoic acid) and measured by the increase in absorbance at 412 nm of 5-thio-2-nitrophenol liberated with the reaction. The result is shown in FIG.

In FIG. 2, when (1) uses the modified dihydrofolate reductase of the present invention [having two cysteine residues (85th and 165th)] as a protein, (2) shows a wild-type protein. Type dihydrofolate reductase [2 cysteine residues (85th and 152nd)]
And (3) has a dihydrofolate reductase mutant [DHFR (C152E) -IQI, JP-A-63-267276, having one cysteine residue (85th)]. The case where it is used is shown.

From this FIG. 2, the reactivity of the dihydrofolate reductase in the present invention is remarkably high [curve (1)],
The 85th cysteine residue does not react at all under this condition [curve (3)], and the reactivity of the 152nd cysteine in the wild-type dihydrofolate reductase depends on the reactivity of the carboxy-terminal cysteine in the modified enzyme protein. In comparison, it is easily understood that it is a fraction or less [curve (2)].
Thus, a highly reactive mercapto group was obtained by introducing an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus.

Example 4 Immobilization of modified dihydrofolate reductase on thiopropyl sepharose 6B As an immobilization carrier, thiopropyl sepharose 6B commercially available from Pharmacia was used. Dry weight is 2
8 mg, 52 mg, 108 mg of each gel,
0.35 mg / ml modified dihydrofolate reductase 2 ml
(10 mM potassium phosphate buffer solution containing 2.5 mM EDTA, pH 8.0) was added, and the mixture was allowed to stand at room temperature for 2 hours, and the enzyme activity in the supernatant was measured. When the amount of modified dihydrofolate reductase was calculated,
It was found that about 0.3 mg of the enzyme protein was bound to 1 g of thiopropyl sepharose 6B gel. Next, 100 ml of modified dihydrofolate reductase was added to 10 g of thiopropyl sepharose 6B and reacted overnight at room temperature, and after separating the gel using a glass filter, 2.5 mM was used.
10 mM potassium phosphate buffer containing EDTA (pH
It was washed with 7.0) and used for the next experiment.

Example 5 Enzymatic activity of immobilized modified dihydrofolate reductase The immobilized gel obtained in Example 4 was packed in various amounts in a glass column (diameter of about 1.5 cm), and a perista pump was used for this. Completely degassed at various flow rates with an active measuring solution [0.
05 mM dihydrofolate, 0.06 mM NADPH,
1 mM EDTA, 50 mM phosphate buffer (pH 7.
0)], and the absorbance of the effluent at 340 nm is measured,
The amount of decrease in absorbance after passing through the column was determined. Using this value, the degree of enzymatic reaction (conversion rate%) was calculated. The enzyme reaction was performed at 4 ° C.

FIG. 3 shows the reaction efficiency when the amount of immobilized enzyme gel was changed using a very high flow rate of 10 ml / min. It is shown that the enzymatic reaction proceeds at an efficiency of almost 100% at a dry gel conversion of about 0.5 g or more, and that a bioreactor can be constructed with a very small amount of gel. Further, below that, the reaction efficiency changes depending on the amount of gel, indicating that the amount of enzyme is rate limiting.

FIG. 4 shows the reaction efficiency when the flow rate was changed using a very small amount of immobilized enzyme gel of 80 mg in terms of dry gel. The reaction efficiency decreased as the flow rate increased.

From the above results, the modified dihydrofolate reductase immobilized with very high efficiency using a very small amount of immobilized enzyme gel at a high flow rate of 10 ml / min and a low temperature of 4 ° C. I understand.

[0057]

EFFECTS OF THE INVENTION According to the covalent carrier binding method of the present invention, inactivation of an enzyme protein can be suppressed and the immobilization site can be easily controlled, so that a homogeneous and highly active immobilized enzyme can be efficiently produced. be able to.

The immobilized enzyme obtained by the method of the present invention is suitably used as an element of a bioreactor used for the production of various useful substances.

[Brief description of drawings]

FIG. 1 is a restriction enzyme digestion map of plasmid pCYS1.

FIG. 2 is a graph showing the reactivity of mercapto groups of cysteine residues in various enzymes.

FIG. 3 is a graph showing the gel dependence of the reaction efficiency in the dihydrofolate reduction reaction by the immobilized modified dihydrofolate reductase.

FIG. 4 is a graph showing the flow rate dependence of the reaction efficiency in the dihydrofolate reduction reaction by the immobilized modified dihydrofolate reductase.

Claims (1)

(57) [Claims] 1. A carboxy-terminal side of an enzyme protein,
A modified enzyme protein was prepared by introducing an amino acid sequence consisting of several amino acid residues having a cysteine residue at the carboxy terminus, and then the modified enzyme protein was bound to an immobilization carrier via the mercapto group at the cysteine residue at the carboxy terminus. A method for producing an immobilized enzyme, which comprises: