JPS5934354B2 - Immobilized thermostable acetate kinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel immobilized thermostable acetate kinase and a method for producing the same.

Adenosine triphosphate (ATP) or its derivatives or adenosine diphosphate (ADP) or its derivatives (
Adenine cofactors (hereinafter collectively referred to as adenine cofactors) are very important as co-substrates for enzymatic reactions.

For example, when D-glyose is converted to glycose 6-phosphate using the enzyme hexokinase, PTPl)S is essential as an auxiliary reaction, and in many other enzymatic reactions, adenine cofactors/ )5 It is well known that bS is essential.

On the other hand, in order to industrially utilize these adenine cofactors as an energy source, it is important to interconvert these cofactors without impairing their activity. It is also industrially important that the method for interconverting adenine cofactors functions stably for a long period of time.

Enzymes that catalyze the interconversion reaction of adenine cofactors, which are important as an energy source in one enzyme reaction, are known as phosphotransferases, and acetate kinase is known to be particularly effective.

Acetate kinase is produced by a number of microorganisms.

For example, Escherichia coli (Escher-i ch.
1acoli), Vei 1lonella alcalescen
Typical examples of microorganisms include Bacillus
Stearothermophilus (Bacillus 5te-
ar□ thermophilus) (J-Bioch
em, .

84.193 (1978), JP-A-52-25088], Clostridium thermoaceticum (C
lostridium therm-oacIeti
cum) (Archiv, Mi crobiol,
.

100.121 (1974)) etc., it is known that thermostable acetate kinase tJS is produced.

These acetate kinases catalyze the following reactions:
DP+acetyl phosphate di-ATP deca-acetic acid-10,000 It is known that when enzymes are used in industrial synthesis reactions, there are various advantages based on their specificity.

For example, there are no side reactions, including stereoisomerism, and therefore no unnecessary environmentally destructive substances are produced, high yields can be achieved l)'5, and the reaction conditions are mild (room temperature, normal pressure). ), which satisfies the energy conservation issue.

However, while the enzymatic reaction has the advantages mentioned above, it is difficult to recover and reuse the vaginal enzyme after the reaction and to perform continuous enzymatic reactions because the enzyme is water-soluble. It has some disadvantages such as:

Furthermore, since enzymes are extremely expensive, it is important to overcome the above drawbacks in order to utilize them industrially.

Tips for improving and overcoming the drawbacks of such enzymes 1
Many studies have already been conducted in this industry and several methods have been proposed.

The most promising method among these is the method of immobilizing enzymes on water-insoluble carriers, and this method is described, for example, in Osker et al.
mmbilized Enzyme) Published by JCRC (1
973) and Chibata, ed. “Immobilized Enzymes,” Kodansha (1.
975) r, (detailed description b5 is available in the following).

It has also been proposed that acetate kinase should be immobilized for industrial use (Enzymes and Industry, Vol. 35, No. 1, pp. 3-10, 1972). .

However, enzymes are extremely unstable under various immobilization conditions such as heat, acid, and alkali, and the properties of enzymes are derived exclusively from the delicate higher-order structure of the enzyme molecule.
Therefore, the higher order structure is inevitably affected by immobilization.

This was another factor that hindered the industrial use of enzymes.

That is, Example 6i where the catalytic function of the enzyme was increased by immobilization.
While this is known, many examples of the opposite are also known.

In addition, cases have been reported in which the immobilization of enzymes causes their inherent properties to be completely lost, or in which their specificity has changed (Enzymes and Industry - Vol. 1, Vol. 35, p. 7, 1977).

Additionally, enzymes have different molecular structures depending on the species of organism that produces them, and this delicate structure easily changes depending on the environmental conditions in which they are placed.

The same goes for acetate kinase 1, for example, Esch
The one produced by erich ia col ib5 has a molecular weight of about 50. ．． Bacillus stearothermoph
illus produces a molecular weight of about 160,000 and 4
Consists of a polypeptide chain (J, Bioi, Che
m,, 8 Sat., 193 (1978)), Veillone
The product produced by S. lla al-calescens consists of two polypeptide chains with a molecular weight of approximately 40,000 (J-Biol, Chem, 251, 3117
(1976)).

In addition, for example, one of the enzyme molecules of Eschrichia coli is thought to contain an SH group (t)S, B.

stearothermophilus does not have an SH group b5 (J, Biol, chem
, 84.193 (1978), l, etc. The molecular structure differs depending on its origin.

Ultimately, at present, no regularity has been found between the immobilization method and the immobilized enzyme, and the properties of the enzyme before immobilization, such as heat resistance, Because it is impossible to predict the properties of immobilized enzymes, such as their catalytic function, specificity, and long-term stability of activity (for example, see "Immobilized Enzymes" above, p. 107), (Whether a stable immobilized enzyme can be obtained or not)
We had to consider each enzyme through trial and error.

In fact, very little research has been done on the immobilization of acetate kinase, and one of the few examples is a method of immobilizing 1-acetate kinase on a polyacrylamide gel containing N-hydroxysuccinimide groups.
Enzymology, 44, 8 8 7 (19
76), 1 is known, but in this method, ``an expensive deactivation inhibitor (dithiothreitol) is coexisting during immobilization, and the acetic acid is This required a delicate and skillful operation in which the kinase was added to a non-oxidizing atmosphere.

Furthermore, recovering and reusing the acetate kinase that was not immobilized was not possible due to its severe deactivation, and was also disadvantageous from an economic point of view.

Moreover, when immobilization is carried out despite the economical and operational disadvantages mentioned above, it has to be carried out under low temperature conditions around 0° C., which is economically disadvantageous.

Furthermore, the present inventor has already developed a BQ Starotherm.
In order to overcome the above-mentioned drawbacks, we established a method for immobilizing the thermostable acetate kinase 1 produced by P. philus (Japanese Patent Application Laid-open No. 154992/1983).

As a result of studying various immobilization methods, the present inventors found that, in the case of acetate kinase in particular, by using acetate kinase that satisfies certain heat resistance requirements, it is possible to maintain it for a long period of time even after immobilization. The present invention was completed based on the discovery that it is possible to obtain a stable immobilized enzyme that maintains its activity over a long period of time.

Therefore, an object of the present invention is to provide a novel immobilized acetate kinase that has a long service life, is economical, and has poor activity.

Another object of the present invention is to improve and overcome the above-mentioned drawbacks and to provide a method for immobilizing acetate kinase that is versatile, economical, and workable.

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

That is, the present invention relates to Clostridium thermoaceticum (
Clostridium thermoacet −
i cum ) or Pseudomonas thermoaminolyticus V-2 (Pseudomonas the
The present invention relates to an immobilized thermostable acetate kinase comprising a thermostable acetate kinase produced by a strain of S. rm-oaminolyticus V 2) and a water-insoluble immobilization carrier.

The thermostable acetate kinase used in the present invention is an acetate kinase produced by microorganism f3s whose optimal growth temperature is within the range of about 50 to 85°C, preferably 60 to 85°C.

"Thermostable" as used in the present invention refers to purified acetate kinase at 50°Cf in a 50mM buffer with a pH of 6.5 to 8.0.
After treatment with a trowel for 15 minutes, the activity was measured [details of the measurement method in J. Biol. Chem., 249, 25
67 (1974)] retains at least about 90%, preferably 95% or more, and optimally 100% of its pre-treatment activity.

Acetate kinase that satisfies such heat resistance requirements includes:
For example, B, stearothermophilu −
s, Clostridium thermoacet
ycum.

pseudomonas thermoaminol
ytic-usV2r, carbon sources such as glycose, sucrose or malic acid and/or peptones, meat extracts,
Nitrogen sources such as amino acids, inorganic salts and/or
Alternatively, it can be easily obtained by culturing the microorganism in a commonly used medium containing vitamins (for example, see Japanese Patent Application Laid-Open No. 52-25088) and purifying it according to a conventional method.

In addition, the water-insoluble carrier used in the present invention refers to a chemical substance that is essentially insoluble in water, such as polysaccharide derivatives such as cellulose, dextran, agarase, and starch; polyacetate cellulose, and polyvinyl alcohol. Derivatives of polystyrene, polypropylene, polyethylene, polyvinyl chloride, poly(methyl methacrylic acid) ester, polybutene, polypentene, polyvinyl dichloride, poly(meth)acrylonitrile, polymethacrylic acid, polyacrylic acid, polyaminostyrene, polybutadiene, polyisoprene , monomethyl polymaleate, crosslinked polyacrylamide, polyacrylamide, polyvinylamine, poly(dialkylaminoethyl methacrylate), poly(dialkylaminomethylstyrene)
, poly(vinylpyridine), poly(vinylpyrrolidone)
, polyacrylic anhydride, polymethacrylic anhydride, polymaleic anhydride, poly(trifluoroethylene) poly(tetrafluoroethylene), poly(divinylbenzene), poly(α-methylstyrene), poly(N-vinylamine) ), poly(tetramethylene glycol sulfinyl ether), polyvinyl sulfone, polyvinyl sulfoxide, polyacrolein, polymethyl vinyl ketone, and other polymers consisting of monomers containing unsaturated carbon; polyphenylene oxide, polymethylene oxy P1 polyethylene oxide , polyethers such as polytetramethylene oxide; polypeptides such as polyalanine, polyphenylalanine; nylon-3, nylon-4, nylon-
6, nylon-7, nylon-11, nylon-12,
Polyamides such as nylon-6,6, nylon-6, to, poly(m-phenylene isophthalamide, poly(p-phenylene-tetraphthalamide)); terephthalic acid,
Polyesters of polycarboxylic acids such as isophthalic acid, adipic acid, maleic acid, fumaric acid, and trimellitic acid and polyols such as ethylene glycol, furopylene gelicol, ethylene glycol, pentaerythritol, bisphenol A/, and C; Polyesters obtained from glycolic acid, lactic acid, hydroxypivalic acid, etc.: 7 recon rubbers such as dimethylpolysiloxane, methylphenylpolysiloxane, methylvinylpolysiloxane, cyanoalkylmethylsiloxane, fluoroalkylmethylpolysiloxane; toluene diisocyanate, xylene diisocyanate, Polyurethanes made of polyisocyanates such as phenylene diisocyanate, ethylene diisocyanate, diphenylmethane diisocyanate, and toluene diisocyanate, and polyols such as polyethylene glycol, polypropylene glycol, and polyesters having OH groups at both ends; phenol, formaldehyde resin, xylene/formaldehyde resin Formaldehyde resins such as urea, formaldehyde resin, and melamine/formaldehyde resin; Polymers containing four-membered rings such as polyimide, polybenzimidazole, and polythiazole; Other synthetic polymers such as polycarbonate and polysulfon; Glass, asbestos, clay, and mica. , hydroxyl apatite, activated carbon, silica gel, alumina, and other inorganic substances and derivatives thereof; and synthetic inorganic polymers such as polyphosphazene.

The immobilization carrier of the present invention can have various shapes, such as granules, fibers, hollow bodies, films, and sheets.

Therefore, the immobilized thermostable acetate kinase of the present invention can be used in a stirred tank,
As packed tanks, fluidized tanks, tubular membrane reactors, etc.
It can be used for mutual conversion of adenine cofactors.

On the other hand, in the present invention 1, the thermostable acetate kinase can be immobilized according to the following method.

That is, it is characterized in that a heat-resistant vinegar-resistant kinase produced by a strain of Clostridium thermoaceticum or Pseudomonas thermoaminolyticus V-2 is reacted with a preactivated water-insoluble immobilization carrier.

Method for immobilizing thermostable acetate kinase.

In general, as methods for immobilizing enzymes, covalent bonding methods, ionic bonding methods, physical adsorption methods, entrapment methods, and the like are known (see "Immobilized Enzymes" above).

Moreover, a novel covalent bonding method using ■-alkyl-2-halopyridinium salts can also be adopted.

First, as a covalent bonding method, for example, a water-insoluble carrier having a carboxyl group is treated with a 1-alkyl-2-halopyridinium salt to make the carrier reactive with amino groups, hydroxyl groups, and carboxyl groups, and this is combined with acetate kinase. A method in which a water-insoluble carrier having an aromatic amine group is used as a diazonium salt and this is diazo-coupled with acetate kinase, a method in which a carrier containing a carboxyl group is subjected to an appropriate treatment to form azide, chloride, carbodiimide, in A method of converting into a derivative such as cyanate and peptide bonding this with acetate kinase, a method of bonding to a carrier having an active leaving group such as a halogen, a method of bonding after treating the hydroxyl group of the carrier with cyanogen halide, at least Bifunctional reagents such as glutaraldehyde, l-alkyl-
Examples include a method of binding the carrier and acetate kinase using 2-halopyridinium salt, epichlorohydrin, or the like.

Further, as an ionic bonding method, for example, carboxymethyl cellulose, diethylaminoethyl Sephadex (product of Pharma-cia), DOWEX-50 [Dow Chemical (1) aw chem,
For example, a method of ionically bonding to an ion exchanger such as a product manufactured by Co., Ltd.) can be mentioned.

In addition, physical adsorption methods include, for example, methods in which adsorption is performed on adsorbents such as activated carbon, alumina, and silica gel, and comprehensive methods include, for example, in polymer gel lattices such as crosslinked polyacrylamide gel and polyvinyl alcohol gel. Alternatively, for example, a method of enclosing it in a film of nylon, polystyrene, collodion, etc. can be mentioned.

These methods can be carried out according to known methods in which an acetate kinase water bath solution and a carrier or its precursor are mixed or polymerized in the presence of a polymerization initiator if necessary, provided that the temperature is room temperature. Is possible.

A notable advantage of the present invention is that the fixation treatment can be carried out under room temperature conditions.

As described above, the immobilized thermostable acetate kinase of the present invention can be produced by various methods, but the covalent bonding method is particularly superior in terms of the binding strength, activity, and service life between the carrier and the acetate kinase.

In particular, at least difunctional reagents, especially l-alkyl-2 - a covalent bonding method using a halopyridinium salt, in particular a carrier obtained by reacting a carrier previously having a carboxyl group with a 1-alkyl-2-halopyridinium salt in the presence of a deoxidizing agent and a thermostable acetate kinase. A method of covalent bonding is preferred.

(1) The covalent bonding method using an alkyl-2-halopyridinium salt can be carried out as follows.

That is, the molar ratio of the water-insoluble carrier having a carboxyl group to the carboxyl group is about 1 to about too much, preferably 2.
~200, optimally 2 to 20 deoxidizers are mixed in an anhydrous organic solvent.

A 1-alkyl-2-halopyridinium salt is then added to the mixture in a molar ratio of 1.2 to about 1 carboxyl group.
200, preferably from 2.4 to 480, optimally from 2.4
24 and reacted.

The reaction temperature is in the range from room temperature to the boiling point of the solvent, and the reaction time is about 10 minutes or more, preferably 1 hour or more.

Washing is performed with an anhydrous solvent that can dissolve the dehydrating agent.

By this, a water-insoluble carrier having enzyme binding ability can be obtained.

The carrier thus obtained is heated to 0 to 50°C, preferably 0 to 30°C.
The desired immobilized heat-resistant acetic acid is obtained by reacting with an acetate kinase aqueous solution at a temperature optimally in the range of 0 to 20°C and under conditions of pH 6 to IO1, preferably 7 to 9, optimally 7 to 8. Get Kinase.

The reaction time is at least 2 minutes, preferably from 5 minutes to 10 hours, optimally from IO minutes to 2 hours.

The immobilization method can also be carried out as follows.

That is, the molar ratio of the carrier having a carboxyl group to the carboxyl group is 1.2 to 240, preferably 2.4.
~120, optimally 2.4 to 48 1-alkyl-2-
Add the halopyridinium salt and the deoxidizer and acetate kinase with a molar ratio to carboxyl groups of 2.4 to 480, preferably 4.8 to 240, optimally 4.8 to 98, directly to the water-organic solvent mixture. It can also be fixed.

In this case, the mixed solvent has a pH of about 6 to 9, preferably 6 to 9.
8. Optimally 6.5-7.5, water:organic solvent mixing ratio l:0.6-6, preferably l:0.8-1.2
It is.

-10,000, reaction temperature and reaction time are 0 to 50°C, respectively.
1 preferably 0-30°C, most preferably 0-20°C and at least 2 minutes, preferably 2 minutes to 5 hours, most preferably 5
It takes from minutes to 2 hours.

In the l-alkyl-2-halopyridinium salt used in the immobilization method, examples of the alkyl group include linear alkyl groups such as methyl, ethyl, n-propyl, n-butyl, and n-dodecyl; i-propyl; Branched alkyl groups such as t-butyl and neopentyl; cyclic alkyl groups such as cyclopentyl and cyclohexyl; aromatic groups such as phenyl and naphthyl as substituents; and/or cyclic alkyl groups such as cyclopentyl and cyclohexyl; and/or vinyl, butenyl, The above-mentioned alkyl groups having an unsaturated alkyl group such as inbutenyl group can be mentioned, and examples of the halogen at the 21-position include fluorine, bromine, chlorine, and iodine.

Examples of the counter ion forming the pyridinium salt include halogen: organic anions such as tosylate and brosylate; and coordination type anions such as fluorosulfate and tetrafluoroborate.

Further, the deoxidizing agent used in the method f does not contain active hydrogen.

Bases such as triethylamine, trimethylamine, methyldiethylamine, pyridine, lutidine: l, 8
-Diazabicyclo(5,4,0)-7-undecene, ■
.

Examples include superbases such as 5-diazabicyclo(4,3,0)-5-nonene.

Furthermore, organic solvents include hydrocarbons such as benzene, toluene, xylene, and cyclohexane; alcohols such as methyl alcohol, ethyl alcohol, amyl alcohol, cyclohexanol, ethylene glycol chrycerol, ethylene glycol monomethyl ether, and diethylene glycol; Ethers such as cyamyl ether, benzyl ether, dioxane, tetrahydrofuran, and ethylene glycol dimethyl ether; Ketones such as acetone, methyl ethyl ketone, and cyclohexanone; esters such as ethyl acetate, propyl propionate, butyl formate, and butyl acetate; chloroform; Halogenated hydrocarbons such as methylene chloride, carbon tetrachloride, and triethylene bromide; Nitro compounds such as nitrobenzene and nitromethane; Nitriles such as niacetonitrile and benzonitrile; Amides such as diethylformamide, dimethylacetamide, and N-methylpyrrolidone. can be used.

Thus, according to the present invention, (1) there is no need to investigate the immobilization method for each acetate kinase by trial and error;
(2) Immobilization does not require delicate and skilled operations, and (3) unimmobilized enzyme that was not immobilized on the carrier during immobilization can be recovered and reused. You can earn a profit.

Since the immobilized thermostable acetate kinase obtained by the present invention stably maintains its activity for a long period of time, it is an object of the present invention to provide a promos that interconverts adenine cofactors extremely effectively in the shape of a reactor as described above. becomes possible.

As a result, it becomes possible to operate a so-called bioreactor system that industrially performs the same biosynthetic reaction as in the living body outside the living body.

For example, an attempt was made to industrially synthesize gramicidin S, a useful antibiotic, using an enzyme.
Eng., 2153 (1974)], it is possible to provide a process for efficiently reproducing ATP consumed as an energy source, thereby making it possible to make such an attempt into an industrially effective process.

This is just one example, and the stable immobilized thermostable acetate kinase of the present invention can be widely used in other industrial reaction systems that require interconversion of adenine cofactors. Become.

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

The enzyme activity is measured using a known reverse reaction system.T=Bio1.
chem-, 249, 2567 (1974)) according to a known method (Methods in Enzymology, 4
4°335 (1976)).

Reference Example 1/Comparative Example I CNBr-activated Sepharose 4B [Pharmacia (Ph
After repeating washing and swelling with 1000 ml of 1mM hydrochloric acid 3 times on a glass filter, the 5S (manufactured by Armacic) was suspended in 50mM boric acid buffer room of pH 8.3, and then suspended in a 50mM boric acid buffer room of pH 8.3. 52-25088)
s B-stear cultured, collected and purified according to
1 d (corresponding to 5000 units) of a solution of acetate kinase produced by P. thermophilus dissolved in the above buffer solution was added to the suspension, and the mixture was allowed to react for 30 minutes at 40° C. with slow shaking.

The reaction mixture was filtered through a glass filter, and the immobilized acetate kinase obtained was then dissolved in 50 mM Tris-HCl buffer solution, pH 8.0, containing 0 and 5 M KCA, respectively.
Washed 3 times with 00 punishment.

Furnace fluids were collected at each stage, and the activities of these furnace fluids and the immobilized acetate kinase obtained were measured.

Furthermore, the same operation was repeated using acetate kinase produced by Escherichia coli (manufactured by Boehringer@Mannheim).

The results were as follows.

Example l Clostridium thermoaceticu
The acetate kinase produced by M.S.
y.

100.121 (1974), No. 5
00 units on a glass filter with a trowel of 1mM hydrochloric acid 300ml
After repeating washing and swelling with rrLl three times, 2 g of activated cH-Sepharose 4B (manufactured by Pharmacia) was added to 50
It was added to a suspension of TfLl in 50mM borate buffer.

This was allowed to react for 1 hour at 30°C with slow shaking.

Thereafter, the same treatment as in Reference Example 1 was carried out to obtain immobilized thermostable acetate kinase.

The activity of the immobilized acetate kinase and the furnace solution was measured, and the immobilized and stabilized acetate kinase was further suspended in 50mM'J acid buffer at pH 7.3 and left at 30°C to observe the change in activity over time. tracked.

The results are as follows.

As is clear from the above results, the method for immobilizing acetate kinase of the present invention has a high immobilization yield, and the unimmobilized enzyme recovered from the P solution has a sufficiently high activity to be reused.

Furthermore, it can be seen that the stability of the immobilized thermostable kinase obtained by the method of the present invention is extremely stable.

Incidentally, in the case of acetate kinase produced by Escherichia coli, there are four cases in which almost all of the activity is lost within one month after immobilization.

Reference example I Pseudomonas thermoaminol
Acetate kinase produced by yti-cusV-2 was cultured, collected, and purified according to conventional methods.

Next, 10 g of CH Sepharose (manufactured by Pharmacia)
The sample was washed and swollen with 5,007 rL of 1 mM hydrochloric acid and three times with 10,100 O of distilled water.

30.000 units of acetate kinase and the CH Sepharose log were suspended in a mixed solution of 250 ml of 50 mM borate buffer and 250 TL of acetonitrile, and then 2.4 mM of t-butylamine and 1-ethyl -2-chloropyridinium bromide 1.2m
M was added and reacted at 20°C for 30 minutes while shaking with a trowel.

The mixture was filtered through a glass filter, and the immobilized acetate kinase obtained was washed with 10,100 O of 500 mM ethanolamino-hydrochloric acid buffer, pH 8.0.
Furthermore, p) (s, o of 0.5MK (50m containing J')
Washed five times with 500 ml each of M Tris-HCl buffer.

Finally, add 500ml each of 50mM'J acid buffer at pH 7.3.
Washed 3 times with 1.

The furnace solution at each stage was collected, and the activity of the furnace solution and immobilized acetate kinase was measured.

Further, the immobilized acetate kinase thus obtained was added at pH 7.0.
The mixture was dispersed in a 50mM α-glycerophosphate buffer solution, left at room temperature with a trowel, and the activity was measured after 6 months.

The results of this reference example are shown below (values are in units).
The immobilized acetate kinase obtained from the acetate kinase produced by S. nolyticus V-2 has also been shown to have extremely high stability.

It can also be seen that the recovered unimmobilized acetate kinase can be reused and that the method of the present invention has a high immobilization yield.

Example 2 Inside a nylon tube with an inner diameter of 0.2 mm, a dichloromethane solution of 7% triethyloxonium tetrafluoroborate was added at 300 C for 3 hours at a flow rate of 0.15 m.
It was cycled at 1/min.

Next, dichloromethane was introduced into the nylon tube at a flow rate of 0.
．． Washing was performed by flowing at 2 rul/min for 15 minutes.

Furthermore, a 25% aqueous hexamethylene diamine solution was added at a flow rate of 0.
After circulating at 30 °C for 1 hour at 15 m1/min, 25
Washed with rnM boric acid value medium (pH 8,0).

Next, a solution of 5% (W/V) dimethyl suberidate dissolved in the same buffer was added at a flow rate of 0.15 ml/min for 20 minutes.
After circulating with a trowel at 30°C for 5 minutes, methanol was flushed for 5 minutes to wash.

Into the thus obtained nylon tube, 500 units of Clo
sridium thermoace-ticum
Borate buffer (pH 8) containing the original thermostable acetate kinase
, 0) at a flow rate of 0. l 5 rul/min for 5 hours, 30
Circulated at ℃.

Next, it was thoroughly washed with borate buffer (pH 8,0) to obtain immobilized heat-stable acetate kinase.

The activity of this product was 300 units.

Reference example 3 0, 12 i 7ml of acrylamide and 6mt;)7
ml of N.

Pseudomon-
20 units of thermostable acetate derived from As thermoaminolytcus V-2 were added.

The mixture was stirred and mixed at 0°C, 0.51 rLl of a 501'v/ml potassium persulfate aqueous solution was added, and the mixture was reacted for 2 hours under irradiation with a 20W fluorescent lamp.

After the generated gel was crushed, it was washed three times with 20 mM phosphate buffer (pH 7,5) L Oml.

The activity of the immobilized thermostable acetate kinase thus obtained was 1
The amount of heat-stable acetate quinase recovered in the washing solution was 4.3 units.

In addition, the immobilized thermostable acetate kinase was suspended in 20mM'J acid buffer (pH 7.5) and left at 30°C for 6 months, and the activity was measured to be 15 units, indicating that it is extremely stable. It is clear that he is worried about his tendency to change.

Claims (1)

[Claims] 1. Clostridium thermoaceticum (Clostridium
ridium thermoaceticum) 1
An immobilized thermostable acetate kinase consisting of a thermostable acetate kinase produced by a bacterial strain and an aqueous immobilization carrier. 2 The thermostable acetate sanase is dissolved in a buffer solution at 50°C for 1
Even after 5 minutes of treatment, 90% of the activity before treatment
The immobilized thermostable acetate kinase according to claim 1, which is an acetate sanase having the above properties. 3. A method for immobilizing heat-stable acetate kinase, which comprises reacting a heat-stable acetate kinase produced by a strain of Clostridium thermoaceticum with a preactivated water-insoluble immobilization carrier. 4 The thermostable acetate kinase l) 5. Even after treatment for 15 minutes in a buffer solution at 50°C, the activity before treatment remained 9
The immobilization method according to claim 3, wherein the acetate kinase is retained at 0% or more. 5. React with a water-insoluble carrier using a reagent having at least two functionalities. The immobilization method according to claim 3. 6 said reagent bS11 having at least two functionalities as described above;
-The immobilization method according to claim 5, which is an alkyl-2-halopyridium salt. 7 A product obtained by reacting a water-insoluble immobilized carrier having a carboxyl group in advance with a -alkyl-2-halopyridium salt in the presence of a deoxidizer and a heat-stable acetate kinase, as claimed in the patent. The immobilization method according to scope 3.