JPS5933355B2 - Immobilized thermostable acetate kinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized thermostable acetate kinase, a method for immobilizing the acetate kinase, and a subreactor using the immobilized acetate kinase.

In recent years, enzyme reactions have attracted attention for their excellent specificity in reaction, substrate, and optics, as well as for their mild reaction conditions, and enzymes have been widely used as catalysts in industry. There is.

However, at present, most of them do not require an energy source, as seen in amylase, which saccharifies starch, and L-amino acid acylase, which produces L-amino acids from N-acyl-L-amino acids. Limited to hydrolytic enzymes.

However, in living organisms, many synthetic processes are performed mainly by using adenosine diphosphate (ATP) as an energy source and the chemical energy released when it is hydrolyzed into adenosine diphosphate (ADP) and orthophosphoric acid. Biosynthesis is carried out by enzymes.

Therefore, in order to expand the industrial use of enzymes, by extracting these synthetic enzymes or oxidoreductases, immobilizing them, and supplying ATP, etc., it is possible to perform the same production in vitro as in vivo. Attempts have been made to create an entirely new production system for synthesis, commonly referred to as a bioreactor.

FIG. 1 shows a flow diagram of an example of a simple bioreactor consisting of a main reactor and a subreactor.

To construct and implement such a bioreactor,
A stable and large amount of (7) ATP is required as an energy source, and it is necessary to regenerate the used ATP from ADP.

In the above-mentioned bioreactor, the subreactor, which functions as an energy supply system or a coenzyme redox system, plays the role of regenerating ADP into the high-energy substance ATP, and for this purpose, phosphotransferase and The use of enzymes collectively called enzymes is being considered.

The requirements for industrial use of such phosphotransferases are particularly (1) that the equilibrium reaction is AT
(2) It must be extremely stable (can be used for a long time) (3) It must be easy to purify, and it must not contain ATP degrading enzyme or inhibitory factors as impurities. Things, etc.
As an example that satisfies these requirements, the present inventor has already developed a thermophilic bacterium, Bacillus stearothermophilus.
Thermostable acetate kinase (Japanese Patent Application Laid-Open No. 1989-1993) produced by a strain of U.S. stearothermoph-illus)
2-25088) and the highly thermophilic bacterium Thermus Thermophilus.
pyruvate kinase produced by pyruvate kinase
No. 3-9392).

However, merely satisfying the above-mentioned requirements is still not sufficient for industrial use.

This is because such phosphotransferases remain water-soluble (and unstable), making it difficult to recover and reuse the vaginal enzymes after the regeneration reaction.

This is an important problem that must be solved if enzymes are to be used industrially, since enzymes are currently extremely expensive.

In order to overcome these drawbacks of enzymes, various techniques for immobilizing enzymes on water-insoluble carriers, ie, techniques for producing enzyme complexes, have been attempted, and various enzyme immobilization methods have been proposed.

Carrier binding methods, cross-linking methods, and entrapment methods are known as methods for immobilizing enzymes.
A detailed description can be found in Kodansha Scientific (1975), edited by Chibata.

However, it is known that the activity of immobilized enzymes is generally significantly lower than the activity in the free state.

This is thought to be due to the fact that enzymes are unstable to various conditions during immobilization such as heat, acid, and alkali, and the fact that active sites are used during immobilization.

Therefore, the present inventor focused on the fact that the acetate kinase disclosed in JP-A No. 52-25088 has excellent thermal stability, and as a result of immobilizing it, it retains sufficient stability and activity. The present invention was completed based on this discovery.

According to the present invention, an immobilized acetate kinase is provided which is suitable for use in a subreactor for ATP regeneration, has high activity, and can be recovered and reused.

This facilitates the industrial utilization of the enzyme.

Therefore, an object of the present invention is to provide an immobilized acetate kinase that retains high enzymatic activity even after immobilization.

Another object of the present invention is to provide a method for immobilizing the acetate kinase.

Further objects and features of the invention will become apparent from the description below.

That is, the present invention relates to an immobilized thermostable acetate kinase comprising a thermostable acetate kinase and an immobilization carrier.

Furthermore, the acetate kinase used in the present invention is produced by Bacillus stearothermophilus (F3acillus), which produces a heat-stable acetate kinase belonging to the genus Bacillus.
stearothermophilus) can be cultured, and the culture solution, culture product, isolated bacterial cells, isolated bacterial cells, processed products thereof, crude and purified enzymes, etc. can be collected at any stage.

As a purification method, a normal enzyme purification method can be used.

That is, after obtaining bacterial cells by, for example, centrifugation, the cells are disrupted by French press, ultrasonication, etc., cell debris is removed by centrifugation to obtain a cell extract, and this is treated with streptomycin sulfate or Treated with protamine sulfate,
Further, ammonia sulfate precipitation, acetone precipitation, heat treatment, etc. are performed.

For further purification, ion exchange chromatography such as DgAE-cellulose column, adsorption chromatography such as hydroxyapatite column, gel filtration chromatography such as Sephadecnuchromatography, A
It is preferable to perform this in combination with affinitake stomatography such as MP-Sephadex column.

The main amino acid residues contained in the heat-stable acetate kinase thus purified were as follows.

Hinutidine 36 residues (2.2%) lysine
76 residues (5%) glycine, asparagine 300 residues (19.5%) tyrosine
44 residues (2.8%) These values were obtained by hydrolyzing the acetate kinase with hydrochloric acid and then analyzing it using an amino acid analyzer.

Other characteristics of thermostable acetate kinase are described in detail in JP-A-52-25088.

In the present invention, any known carrier for immobilizing acetate kinase can be used.

Examples include porous glass, polysaccharide derivatives, and polyacrylamide.

Specifically, cellulose, Sephadex (5epha
dex), sepharose (5epharose), bio-gel (Blo-Gel), nylon, porous silica glass, alumina, etc.

These carriers are used in various forms depending on immobilization methods; for example, covalent bonding methods require one-phase activation.

Activated Sepharose that can be used in the present invention includes BrCN-activated Sepharose, glutaraldehyde Sepharose, diazotized Sepharose, nitrenated Sepharose, and N-hydroxynuccinimidized Sepharose, among which BrCN-activated Sepharose and N-hydroxysuccinimide sepharose is easily available as a commercial product.

Other activated sepharoses can also be obtained, for example, as follows.

First, diazotized Sepharose is prepared according to the following formula: (where 5eph represents a Sepharose residue). After treating Br0N-activated Cepharose with metaphenylene diamine, it is treated with t-IJium nitrite and hydrochloric acid according to the conventional method. It was obtained as a diazonium salt.

Glutaraldehyde Sepharose was prepared by treating aminehexyl Sepharose (commercially available) with glutaraldehyde.

Nitrenated sepharose can be obtained by reacting the diazonium salt with sodium azide and irradiating it with ultraviolet light as shown in the following formula: However, in this case, the ultraviolet irradiation is carried out in the presence of acetate kinase.

This is because the radical in the above reaction formula is extremely unstable.

The immobilized thermostable acetate kinase of the present invention can be produced by various methods such as those described below.

1. Covalent bonding method a) A diazonium salt is used as a carrier having an aromatic amine group, and this and acetate kinase are diazo-coupled.

b) The carrier having a carboxyl group is an azide, a halide,
After making it into a derivative such as carbodiimide or incyanate,
Peptide bond with acetate kinase.

C) Directly attaching the enzyme to a carrier having an active leaving group such as a halogen.

d) Toluene diisocyanate, epichlorohydrin,
Glutaraldehyde, 2-amino-4,6-dichloro-
The carrier and the enzyme are coupled using a bifunctional reagent such as 8-triazine.

2. Ion bonding method The enzyme is bonded to an ion exchanger such as carboxymethylcellulose, dienalaminosephadex, DOWEX-50, etc.

3. Physical adsorption method The enzyme is adsorbed onto activated carbon, alumina, silica gel, etc.

4. Encapsulation method a) Encapsulation of the enzyme in a gel lattice such as cross-linked polyacrylamide gel or polyvinyl alcohol gel.

b) Encapsulating the enzyme in a film of nylon, polystyrene, collodion, etc.

Care must be taken with the reaction conditions, especially the reaction temperature, as they are related to the problem of inactivation of acetate kinase.

Therefore, the reaction temperature is O~70'C, preferably O~60'C.
'C.

Also, it is necessary to pay attention to the pH of the reaction solution, which is 6.0.
It is preferable to carry out under a single-row condition of 9.5 to 9.5.

For pH adjustment, a buffer having the above p)l range, such as borate buffer or phosphate buffer, is used.

The reaction time can vary widely depending on the particular immobilization method and is not critical.

As a result of the chemical modification described above, this thermostable acetate kinase is
It is believed that the imidazole group, amine group and carboxyl group are involved in its activity.

On the other hand, since no inactivation was observed upon treatment with tetranitromethane, it is thought that tyrosine residues are not so important for the activity of this enzyme.

Therefore, if a method of selectively modifying and immobilizing only tyrosine residues is found, it is thought that the high activity of the enzyme can be maintained as is.

However, such a method is not known at present.

In all of the above immobilization methods, it is thought that the amino group of acetate kinase is mainly involved in binding to Sepharose, and the activity of immobilized acetate kinase is the highest at 16.1%, and in other cases was 6-8%.

From this result, it is presumed that the amine group that is thought to contribute to the activity of acetate kinase is used for immobilization of the acetate kinase.

Furthermore, immobilized acetate kinase can be obtained in various shapes such as granules, membranes, tubes, and fibers.

For example, a membrane-shaped immobilized enzyme can be obtained by applying the above-mentioned carrier binding method to a membrane-shaped carrier, or by molding the enzyme into a membrane shape when encasing the enzyme in a semipermeable membrane-shaped polymer; It can be obtained by partially thawing the inside of a nylon tube, polyaminonunarene tube, etc., and coupling an enzyme thereto using the diazo method.

The immobilized thermostable acetate kinase of the present invention retains most of the activity of free acetate kinase, and is more stable than free acetate kinase, with approximately 100% of the initial activity after being left for 6 months. %, and the activity was maintained at over 90% even after being left for 9 months.

On the other hand, in terms of thermal stability, it was found that the inactivation temperature was at least 10'0 higher than that of free acetate kinase.

An example of the use of the immobilized thermostable acetate kinase of the present invention will be explained with reference to the attached FIG. 1.

First, the target substance B is synthesized from the raw material A in the main reactor 1 by directly or indirectly using the energy of ATP.

Since the purpose part B comes out from the main reactor together with ADP, separator 2 separates it from B(!:ADP).

In this case the separator can be a filter, centrifuge, etc.

The separated ADP is converted into ATP together with acetyl phosphate (abbreviated as CH3C00P) in subreactor 3 containing immobilized acetate kinase according to the following formula: %Formula % and comes out together with acetic acid.

A mixture of ATP and acetic acid obtained from the subreactor is separated into ATP and acetic acid, respectively, by a second separator 4.

The second separator 4 may also be a filter, centrifuge, etc.

The present invention will be further explained below with reference to Examples.

Example 1 Heat-stable acetate kinase 0.121119/ml obtained by culturing Bacillus stearothermophilus purified by electrophoresis, Br0N activated Sepharose (Sepharose-4B), and glutaraldehyde Sepharose (AH-Sepharose), diazotized Sepharose (AP-Sepharose), N-hydroxysuccinimidized Sepharose (OH-Sepharose), carbodiimidized Sepharose (CH-Sepharose) and AH-Sepharose for comparison (however, AH.

AP and CH represent aminohexyl, aminophenyl and carboxymethyl respectively) 50゜pHs,
The reaction was carried out by stirring at room temperature for 2 hours in the boric acid buffer of No. 3.

However, in the case of diazotized Sepharose, the reaction was carried out at 0°C.

In addition, in the case of carbodiimidized Sepharose, the reaction was carried out in pure water.

Furthermore, in the case of nitrenated sepharose, 90W U
The reaction was performed by irradiating with a V (360 nm max) lamp.

Then, the product was separated by furnace using a glass filter, and 0.1Mt
-Lis-〇, 5MN a O// <Zuffer (pH 8
,3) and 25mM phosphate buffer = (pH7,2)
Activity of the product, residual activity of the p-liquid obtained during said filtration and immobilization yield of the obtained immobilized product: Activity of immobilized acetate kinase: 100 Activity of immobilized acetate kinase It was determined.

In measuring the above properties, the activity (potency) was determined as follows: Sodium acetate 0.35 MlA T P 15 mM, Magnesium chloride 30 mM, Chloride chloride 0.35 MlA T P 15 mM in imidazole-hydrochloric acid buffer at pH 7, 2, 7 mM potassium 62.5mM, phosphoenolpyruvate 0.4mM reduced form of nicotinamide adenine dinucleotide (NADH) 0.15mM,
HADH
The titer was measured from the decrease in absorbance at 340 nm per unit time, and 1 micromol of NADH per minute.
The enzyme activity that decreases the absorbance at 340 nm was defined as 1 unit.

(Journal of Logical Chemistry, 2
49, p. 2567, 1974).

(The same applies hereinafter) Furthermore, the storage stability, heat resistance, optimum pH, and Km for ADP and acetyl phosphate were measured for the thus obtained immobilized thermostable acetate kinase.

First, the storage stability of Br0N-activated Sepharose 4B
and thermostable acetate kinase immobilized on N-hydroxysuccinimidized OH-Sepharose (CNBr and succinimide, respectively) and acetate kinase obtained by culturing N. coli for comparison with Br.
Immobilized on 0N activated Sepharose 4B (E co
ll (ONBr)) and free thermostable acetate kinase (
) and 25mM phosphate buffer (pH
7,5), left at room temperature, and measured the change in activity over time.

The results are shown in Figure 2. Thermal resistance was determined using samples other than the acetate kinase originating from N. coli used for storage stability and each immobilized acetate kinase immobilized on diazotized AH-Sepharose (diazo) in 20mM'J acid at pH 6.9. This was determined by dissolving in buffer and monitoring the remaining activity at each temperature.

The results are shown in Figure 3.

As is clear from FIG. 3, the heat resistance was improved by at least 10°C by immobilization compared to free acetate kinase.

The optimum pH was determined by dispersing acetate kinase (BOUND) immobilized by the succinimide method in 25 mM phosphate buffer and monitoring the activity at each pH at room temperature.

The results are shown in Figure 4.

Compared to the free one (FREE), the immobilized one moved to the acidic side by 0.5 pH units, but no significant difference was observed.

According to a conventional method, N-hydroxysuccinimidized CH-
Thermostable acetate kinase (AK) immobilized on Sepharose
The Km for A, DP, and acetyl phosphate was measured by a batch method for the free and free samples.

The results are shown in the table below. Similarly, AK was immobilized on BrCN-activated Sepharose 4B.
m was measured, and similar values of Km were obtained.

Example 2 Fixing to a nylon tube A dichloromethane solution of 7% triethyloxonium tetrafluoroborate salt was placed inside a nylon tube with an inner diameter of 0.2 mm at a flow rate of 0.1 ml/min at 30°C for 3 hours. circulated.

The nylon tube was then washed with dichloromethane for 10 minutes at a flow rate of 0.2 ml and 11 minutes.

Furthermore, a 25% aqueous hexamethylene diamine solution was added at 30°
After circulating at a flow rate of 0.17 rllZ for 1 hour at C, the mixture was washed with 25 mM borate buffer (pl(8,0)).

Next, a solution of 5% (W/v) dimethyl suberimidate dissolved in 25mM borate buffer was added at 30°C for 2 hours.
It was circulated for 0 minutes at a flow rate of 0.1 ml, and washed with methanol for 5 minutes.

Borate buffer containing 1000 units of acetate kinase (p
H8,0) was circulated at 30°C for 5 hours at a flow rate of 0.1 ml/min.

Immobilized acetate kinase was obtained by washing with borate buffer (pH 8,0).

The activity of this product was 730 units.

Example 3 The immobilized acetate kinase immobilized on the Br0N-activated Cephalone 4B obtained in Example 1 was packed into a glass column with an inner diameter of 0.4 cIrL and a length of 5 (ll'771).

Next, ADP 5mM, magnesium chloride 15mM
A 50 mM imidazole buffer solution (pH 7.3) containing 0.25 units/ml of glucose 6-phosphate dehydrogenase and 12.5 units/ml of hexokinase was prepared and added to the above solution. The column was run at a flow rate of 1 ml.

The effluent was introduced into a flow cell, and the change in absorbance at 340 nm was measured to confirm ADP-)ATP conversion.

As a result, this immobilized acetate kinase functioned well as a subreactor for ADP → ATP conversion, and even after continuous use for 3 months, its activity decreased by only 5%. It was found that the kinase stably functions as an ADP-)ATP converting enzyme for at least 3 months.

Example 4 2 g of Sou I-IJ umcarboxymethylcellulose with a degree of etherification of 0.5 and an average degree of polymerization of 100 and a conventional method (Eur,
N-C(6-aminehexyl)carbamoylmethyl) ADP500~ synthesized by J. Biochem, 53.48' (1975)) was dissolved in 50 ml of water.

Next, 5 ml of an aqueous solution containing enal diethylamine propyl luposiimide having a pH of 500 to 100 was added dropwise to the solution over 1 hour at room temperature while keeping the pH at 6.

The obtained reaction product was sufficiently dialyzed using water as an external dialysis solution to obtain carboxymethylcellulose-ADP.

This polymerized ADP 5mM, magnesium chloride 15mM
A 50 mM imidazole buffer solution (pH 7.3) containing 10 mM acetyl phosphate, 10 mM glucose, 0.25 units/ml of glucose 6-phosphate dehydrogenase, and 12.5 units/ml of hexokinase was prepared, and this was brought to room temperature. Example 2
The immobilized acetate kinase obtained in step 1 was passed through the tube.

Conversion of the polymerized ADP to ATP was confirmed in the same manner as in Example 3.

Example 5 o, 12El/ml. of acrylamide, 6ml/ml
(20 containing DN, N'-methylenebisacrylamide)
10 units of thermostable acetate kinase was added to 5 ml of mM phosphate buffer (pH 7.5) at 0°C.

After stirring and mixing, add 50μ of 9/ml potassium persulfate solution.
1 was added thereto, and the mixture was reacted for 2 hours under a 20W fluorescent lamp.

The resulting gel was crushed and washed three times with 10 ml of 20mM'J acid buffer.

The activity of the product was 8.3 units, and 1.5 units of thermostable acetate kinase were recovered in the wash.

This one was also extremely stable.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing a flowchart of a bioreactor, FIG. 2 is a diagram showing the stability of the immobilized thermostable acetate kinase of the present invention, and FIG. 3 is a diagram showing the thermostability of the same acetate kinase. FIG. 4 is a diagram showing the optimum pH of the acetate kinase.

Claims (1)

[Scope of Claims] 1. An immobilized thermostable acetate kinase comprising a thermostable acetate kinase produced by a strain of thermophilic bacterium Bacillus stearothermophilus and an immobilization carrier. (2) The immobilized heat-stable acetate kinase according to claim 1, wherein the immobilization carrier is selected from porous glass, polysaccharide derivatives, and polyacrylamide. 3 A pre-activated immobilization carrier and a thermostable acetate kinase produced by a strain of the thermophilic bacterium Nochthynu stearothermophilus are mixed in a buffer solution having a pH in the range of 6.0 to 9.5. 1. A method for immobilizing heat-stable acetate kinase, which comprises dispersing it in water, stirring at a temperature in the range of 0 to 70°C, and optionally irradiating it with U, V, or light. 4. The method for immobilizing heat-stable acetate kinase according to claim 3, wherein the immobilization carrier is selected from porous glass, polysaccharide derivatives, and polyacrylamide.