JPS61260894A - Production of physiologically active substance by complex enzyme method - Google Patents

Abstract

PURPOSE:To produce continuously a physiologically active substance by the flow method, by adjusting the concentration of bivalent metallic ions to be fed and the concentration of adenosine 5'-phosphate to be fed within specific ranges in synthesizing the physiologically active substance using a complex enzyme reaction system. CONSTITUTION:The concentration of bivalent metallic ions to be fed is adjusted to >=30mM, and the concentration of adenosine 5'-phosphate to fed is set at a lower value than the concentration of a physiologically active substance precursor in a reaction system containing an enzyme converting adenosine-5'- diphosphate into adenosine-5'-triphosphate and an enzyme synthesizing the physiologically active substance while converting the adenosine-5'-triphosphate into adenosine-5'-diphosphate present in the same reactor. As examples for utilizing the reaction system, synthesis of dihydroxyacetone phosphate from dihydroxyacetone as a precursor with glycerol kinase, synthesis of glucose-6- phosphate from glucose as a precursor with glucokinase, etc., may be cited.

Description

[Detailed description of the invention] (Industrial application field) (Prior art) In recent years, due to a review of the current chemical industry, chemical reactions in living organisms have been attracting attention. Many attempts are being made to reproduce this using a reactor. Originally, many biosynthetic reactions are carried out in living organisms using enzymes as catalysts to maintain life.These reactions not only easily provide compounds that are difficult to synthesize by chemical synthesis reactions, but also are energy-saving and non-polluting. Because it also has elements that meet society's demands, such as this, it is on the verge of becoming an essential technology in the chemical industry. Among these, some have already reached the level of practical use in technical fields such as hydrolysis reactions and isomerization reactions.

However, in carrying out the binding reaction, which is a particularly important reaction among biosynthetic reactions, adenosine-5'-triphosphate (hereinafter referred to as ATP) is required as an energy source or cofactor. At this time, ATP acts as an energy source or a cofactor, and then is decomposed into adenosine-5°-diphosphate (hereinafter referred to as 〇P) or adenosine-5°-monophosphate (hereinafter referred to as AMP), and is consumed. You will end up being rejected. Therefore, in order to reproduce the binding reaction industrially.

Although it is necessary to supply ATP at low cost, in reality ATP is an extremely expensive substance. Therefore, A.T.
An important technology to overcome this problem is to regenerate and convert ADP and AMP, which are the forms of P after consumption, and especially AMP, which has been consumed at the lowest energy level, into ATP.

However, the production of substances through such bonding reactions is
There are few known examples of regenerating P while regenerating P. For example, there is an idea to regenerate or replenish consumed ATP using the glycolytic system of microorganisms.
There are examples of using AMP as a raw material for
7, 1981). However, this AMP is not in the form of ATP after it is consumed, but is added separately as an ATP source, and the result of this attempt was that the conversion efficiency of AMP to ATP was extremely poor (ibid.), i.e. Even when using the glycolytic system of microorganisms, there were only negative speculations regarding the regeneration of ATP from AMP, which is the form after consumption.

Conventionally, from this point of view, the concept of a reactor (bioreactor) for synthesizing useful substances by continuously consuming ATP to AMP has been considered, and the completion of this system has been strongly desired.

In view of these circumstances, the inventors of the present invention have previously conducted intensive studies on the conversion and regeneration of AMP, which is a product decomposed to the lowest energy level, into ATP, and have found that the optimal growth temperature is between 50°C and 85°C. We discovered that AMP can be converted into ATP in a short time and with high yield by using a converting enzyme produced by microorganisms, and that by controlling the mixing ratio of AMP and ATP' under specific conditions to regenerate AMP into ATP, We found that AMP can be converted to virtually 100% ATP stably for a long period of time at low cost and with good operability.As a result of further studies, based on this knowledge, (a) A reaction system that regenerates to ATP, and (b
By connecting a reaction system that synthesizes physiologically active substances using lATP, we discovered that biologically active substances could be synthesized using AMP, which is a product decomposed to the lowest energy level, as a raw material, and we applied for a patent (Unexamined Japanese Patent Application Showa 59-1062
(See Publication No. 96).

On the other hand, G.M. Whiteside (G, M.

Whitesides et al. have reported an example in which a reaction system for regenerating ATP from ADP and a reaction system for synthesizing a physiologically active substance using ATP were used in the same reactor to synthesize a physiologically active substance. [For example, J, Org, Ch
eap, 4L3130 1983).

(Problems to be Solved by the Invention) The physiologically active substance synthesis reaction system described in JP-A-59-106296 and the reaction system for reproducing ATP from AMP are made to coexist in one reactor. Physiologically active substances are synthesized continuously over a long period of time by a method in which raw materials are supplied from one end of one reactor and physiologically active substances are extracted from the other end of one reactor (hereinafter referred to as flow method).

A precipitate was deposited inside the reactor, which clogged the inside of the reactor, and the feeding of the raw material liquid had to be stopped. Furthermore, when the enzyme is immobilized on a water-insoluble carrier, the generated precipitate adheres to the surface of the carrier.

Since the contact between the raw material liquid and the enzyme is hindered, the enzyme activity tends to decrease, and the amount of the target physiologically active substance produced tends to decrease.

On the other hand, the above white size (GoM, Whitesi
In the method of Des) et al., the reaction system for regenerating ATP is ADP.
However, since physiologically active substances were manufactured using a batch method, the productivity was extremely low.

(Means for Solving the Problems) As a result of extensive research into the problems encountered in the continuous production of physiologically active substances using the previous flow method, the present inventors found that By lowering the metal ion concentration to 30 aM or less and lowering the concentration of adenosine-5°-phosphate (hereinafter referred to as AXP) to lower than the concentration of the physiologically active substance precursor, the above-mentioned problems were solved, and the flow method They discovered that physiologically active substances can be continuously produced using the same method, and completed the present invention.

That is, the present invention uses a complex enzyme reaction system in which an enzyme that converts ADP to ATP and an enzyme that synthesizes a physiologically active substance while converting ATP to ADP coexist in the same reactor. Physiologically active substance precursors,
When producing a physiologically active substance by supplying a raw material solution containing adenosine-5'-phosphoric oxide and divalent metal ions and extracting it from the other end of a 1M reactor, only 2 of the metal ions supplied to the reactor are produced. Keep the concentration below 30s+M and AXP
In the present invention, the concentration of physiologically active substance ATP is ADP
Examples of enzymatic reactions (hereinafter referred to as synthesis reactions) that synthesize physiologically active substances by converting NAD into Reaction to synthesize acid, reaction to synthesize glucose 6-phosphate by glucokinase using glucose as a precursor Ribulose 5
- Synthesis of ribulose 1,5-bisphosphate using phosphoribrokinase using phosphoric acid as a precursor, reaction of synthesizing glycerol 3-phosphate using glycerol kinase using glycerol as a precursor, creatine using creatine kinase as a precursor Reaction to synthesize phosphoric acid, reaction to synthesize T-glutamylcysteine by γ-glutamylcysteine synthetase using glutamic acid and cysteine as precursors, reaction to synthesize glutathione by glutathione synthase using T-glutamylcysteine and glycine as precursors 9 A reaction in which AMP is synthesized by adenosine kinase using adenosine as a precursor,
A reaction in which glutamine is synthesized by glutamine synthetase using glutamic acid and ammonia as a precursor, a reaction in which phosphocholine is synthesized by choline kinase using choline as a precursor, and a reaction in which 4'-phosphopantothenic acid is synthesized by pantothenic acid kinase using pantothenic acid as a precursor. Examples include a reaction in which phosphoenobiluvate is synthesized using pyruvate kinase using pyruvate as a precursor.

In the present invention, ATP is consumed in the above synthesis reaction system,
This ADP is converted into ATP by an enzyme that converts ADP into ATP in the presence of a phosphate donor.
Enzymes that convert this 80P into ATP (hereinafter referred to as TP regeneration reaction system) include 6M kinase, carbamate kinase, creatine kinase, 3-
Phosphoglycerate kinase.

Many compounds such as pyruvate kinase and polyphosphate kinase can be used, and examples of phosphate donors include acetyl phosphate, carbamoyl phosphate, creatine phosphate,
3-phosphoglyceroyl phosphate.

Phosphoenolpyruvate, polyphosphoric acid, etc. are used, but in consideration of the price of the phosphate donor, the conversion activity to ATP, the ease of obtaining the enzyme, etc., it is most advantageous to use acetate kinase. In this case, acetyl phosphoric acid is used as the phosphoric acid donor. Acetyl phosphoric acid can be used as a salt such as an ammonium salt, a potassium/lithium salt, a sodium salt, etc., but it is preferable to use a disodium salt from the viewpoint of easy availability.

By using such an enzyme, the produced ADP can be
It becomes possible to synthesize physiologically active substances while regenerating them into TP, but the enzyme that converts ADP to ATP must be produced by microorganisms whose optimal growth temperature is 50°C to 85°C. Examples of such microorganisms include Bacillus stearothermophilus and Bacillus stearothermophilus.
Microorganisms of the genus Bacillus, such as B. plebis, Bacillus coagulans, Bacillus thermobrotheoliticus, and Bacillus acidocaldarius, and microorganisms of the genus Clostridium.

Achromobacter, a microorganism of the genus Thermoactinomyces
microorganisms of the genus Streptomyces, microorganisms of the genus Micropolyspora, microorganisms of the genus Thermus such as Thermus aquaticus, Thermus thermophilus, Thermus flavus, microorganisms of the genus Thermomicrobium, microorganisms of the genus Calburia etc. can be mentioned. It also includes microorganisms that grow at room temperature into which the genes of these microorganisms have been introduced. Among these microorganisms, Bacillus stearothermophilus is particularly suitable for producing acetate kinase.

The enzyme obtained from this microorganism is easy to purify.

Specific activity is high, and at the beginning of the research, the optimal growth temperature was 5.
The use of converting enzymes produced by microorganisms at temperatures between 0°C and 85°C was thought to be unsuitable for ATP regeneration at room temperature, but surprisingly it can be done in a short period of time at room temperature with high yield. It can convert AMP into ATP stably over time, and these effects far exceed those of enzymes produced by normal-temperature organisms.

The reactor used in the present invention includes the above-mentioned enzyme for synthesizing a physiologically active substance and ATP in the same reactor.
Any substance may be used as long as it can coexist with the above-mentioned enzyme for reproducing .

The amount of each enzyme, the concentration of the raw material solution, the pH, and the supply rate.

The size and shape of the reactor may be selected depending on the reaction temperature and the like. As for the shape of the reactor, for example, a membrane type reactor or a column type reactor can be used. A membrane reactor can be used particularly effectively when the physiologically active substance is a low molecular weight substance. In this case, since the enzyme is a polymeric substance, each enzyme can be used while remaining in the reactor.

Next, the column reactor can be used with any physiologically active substance. In this case, each enzyme used may be transported with a light-extending carrier 1 such as a polysaccharide derivative such as cellulose, dextran, agarose, etc., a vinyl polymer such as polystyrene, ethylene-maleic acid copolymer, cross-linked polyacrylamide, etc. derivatives, derivatives of polyamino acids or amides such as L-alanine and monoglutamic acid copolymers, polyaspartic acid, etc., derivatives of inorganic substances such as glass, alumina, hydroxyapatite, etc.,
It may be used by entrapping or adsorbing it and filling it in a column as a so-called immobilized enzyme.

In the present invention, the raw material liquid supplied to the above-mentioned reactor includes 1, for example, a physiologically active substance precursor, a phosphate donor,
Contains AXP, divalent metal ions, etc.
Examples of the physiologically active substance precursor and phosphate donor include the compounds mentioned above.

As AXP, it is possible to use only ADP, but it is also possible to use either ATP alone or a mixture of ADP and ATP, and it is also possible to reuse AXP recovered from the 9 reaction solutions after the physiologically active substance synthesis reaction is completed. It's okay.

In the present invention, the concentration (molar concentration) of AXP in the raw material solution must always be lower than the concentration of the physiologically active substance precursor, and the concentration is 0.01 of the concentration of the physiologically active substance precursor. % to 90%, preferably 0.1%
The range is from 50% to 50%, more preferably from 0.5% to 10%.

Further, the amount of the phosphate donor should be equivalent to or more than the amount of the physiologically active substance precursor to obtain a high yield of the target product.

In the present invention, the concentration of divalent metal ions is 30III
It is necessary to keep it below FI, especially below 205M.・1. Examples of such metal ions, which are preferable from the viewpoint of further preventing the formation of precipitates, include magnesium ions, manganese ions, calcium ions, cobalt ions, and cadmium ions.

Examples include barium ion.

In the present invention, a physiologically active substance is produced by continuously supplying a raw material liquid having the above composition from one end of a reactor and extracting a physiologically active substance from the other end of the nine reactors. The concentration of the liquid is controlled to satisfy the above conditions. As a method for continuously supplying the raw material liquid to the reactor while controlling its concentration, 1. For example, prepare a raw material mixture liquid in which all the raw materials are adjusted to a predetermined concentration in advance, and then continuously supply the raw material liquid to the reactor using a pump etc. Examples include a method of feeding the raw materials, or a method of feeding each raw material solution to the reactor separately without mixing the raw materials in advance, and maintaining the concentration at a predetermined concentration at the inlet of the reactor by adjusting the feeding rate.

(Examples) Next, one embodiment of the present invention will be specifically explained using examples and comparative examples.

Example 1 For acetate kinase and glucokinase, commercially available preparations derived from Bacillus stearothermophilus (Seikagaku Corporation) were used and immobilized on CNBr-activated Sepharose 4B (manufactured by Pharmacia). 20 units of immobilized acetate kinase and 20 units of immobilized glucokinase were packed into one column.

Then 100mM containing 5a+M magnesium chloride
10 mM glucose dissolved in Tris-HCl buffer pH 8.

1mM ADP, 15mM acetyl phosphate disodium salt.

A raw material solution consisting of 20mM mercaptoethanol was supplied to the immobilized enzyme column at a flow rate of 30mM/hour.

Glucose 6-phosphate was continuously extracted at the same rate.

The concentration of glucose 6-phosphate in the extracted column eluate was 8.5n+M, and glucose 6-phosphate was stably produced over the next 20 hours.

The temperature of the column at this time was maintained at 30°C.

Comparative Example 1 The magnesium chloride concentration in the raw material liquid in Example 1 was reduced to 4
The same procedure as in Example 1 was carried out except that the concentration was changed to 0mM.

As a result, a precipitate was formed in the column after 2 hours.
The feed of raw material to the column was stopped.

Comparative Example 2 Comparative Example 2 was carried out in exactly the same manner as in Example 1 except that the ADP concentration in the raw material solution in Example 1 was changed to 20 mM.

As a result, a precipitate was formed in the column after 3 hours.
The feed of raw material to the column was stopped.

Example 2 In the same manner as in Example 1, 160 units of immobilized acetate kinase and 130 units of immobilized glucokinase were packed into one column.

Then 100mM glucose dissolved in 100mM) Lis-HCl buffer pH 8 containing 5mM magnesium chloride.

A raw material solution consisting of 1mM ADP, 120mM 20mM acetyl phosphate disodium salt M mercaptoethanol was supplied to the immobilized enzyme column at a flow rate of 25 val/hour.

Glucose 6-phosphate was continuously extracted at the same rate.

The concentration of glucose 6-phosphate in the extracted column eluate was 10 (1 mM), and glucose 6-phosphate was stably produced over the next 40 hours.

The temperature of the column at this time was maintained at 30°C.

Comparative Example 3 The magnesium chloride concentration in the raw material liquid in Example 2 was reduced to 5
The test was carried out in exactly the same manner as in Example 2 except that the setting was changed to 0s+H.

As a result, a precipitate was formed in the column after 1 hour.
The feed of raw material to the column was stopped.

Comparative Example 4 Comparative Example 4 was carried out in exactly the same manner as in Example 2, except that the ADP concentration in the raw material solution in Example 2 was changed to 150 mM.

As a result, a precipitate was formed in the column after 2 hours.
The feed of raw material to the column was stopped.

Example 3 Methods in Enzymology
inEnzymology) Volume 17A, Page 910,
In 1970, following the method published by Academic Press,
Glutamine synthetase 1 prepared from Enerysia coli
00 units and 100 units of commercially available Enerysia coli-derived acetate kinase (manufactured by Boehringer Mannheim) were dissolved in 100+*M) lithium-hydrochloric acid buffer (pH 7.5) containing 20 mM magnesium chloride to obtain a solution with a molecular weight of 30.00.
The membrane was sealed in a membrane reactor using a 0.0 ultrafiltration membrane.

Then 1100I containing 20mM magnesium chloride
50mM ammonium chloride, 5hML-glutamic acid, 1hM dissolved in II Tris-HCl buffer pH 7.5
A raw material solution consisting of ADP, 80mM acetyl phosphate disodium salt, and 10mM mercaptoethanol was fed at a flow rate of 1.
0mj! /hour, and glutamine was continuously withdrawn at the same rate.

The concentration of produced glutamine in the effluent from the reactor is 35+H
was reached, and glutamine was stably produced over the next 6 hours.

Example 4 Commercially available acetate kinase (manufactured by Seikagaku Kogyo) and choline kinase (manufactured by Boehringer Mannheim) were immobilized on CNBr-activated Sepharose 4B (manufactured by Pharmacia).

Five units of immobilized acetate kinase and five units of immobilized choline kinase were packed into one column.

Then 100mM containing 15mM magnesium chloride
A raw material solution consisting of 5s+M choline, l@M ADP, 8sM acetyl phosphate disodium salt, and 51 dithiothreitol dissolved in Tris-HCl buffer pH 8.5 was
The immobilized enzyme was supplied to the column at a flow rate of -1/hour, and 0-phosphocholine was continuously extracted at the same rate.

The concentration of 00-phosphocholine in the extracted column eluate was 4.5a+M, and the concentration of 0-phosphocholine remained stable over the next 8 hours.
Phosphocholine was produced.

The temperature of the column at this time was maintained at 30'C.

(Effect of the invention) According to the present invention, it is possible to continuously synthesize a physiologically active substance by a flow method without forming a precipitate in the 9 reactors, improving the operability of the reactor and the productivity of the target physiologically active substance. is significantly improved. moreover.

It also prevents the enzymes in the reactor from deactivating, making long-term continuous operation possible.

Patent applicant Yu=F:? 1? ! Ceremony: Kazutomo Imahori Procedural Amendment Form Seal button: February 20, 1985 1. Indication of the incident: Japanese Patent Application No. 1988-103151 2. Name of the invention: Process for producing physiologically active substances by complex enzyme method 3. Person making the amendment Relationship to the incident Patent applicant address 1-50 Higashihonmachi, Amagasaki-shi, Hyogo 541 Address 4-68 Kitakyutabe-cho, Higashi-ku, Osaka Name Unitika Co., Ltd. Patent Department Telephone 06-281-5258 (Dial-in) 4 , Subject of amendment 5, Contents of amendment (1) The scope of claims is amended as shown in the attached sheet.

(2) "Growth" on page 4, line 15 of the specification is corrected to "growth."

(3) "White side" in line 9 of page 5 of the same book is corrected to "white size."

(4) Ibid., p. 14, r3130 1983). ”
r3130 (1983)). ” he corrected.

(5) "Reaction" in the first line of the bottom of page 7 of the same book is corrected to "reaction."

(6) In the first line of page 5 of the same book, "with a compound" is replaced with "a compound is".
I am corrected.

(7) “Mesoft” in line 5 of page 5 of the same book is “method”
I am corrected.

(8) "0-phosphocholine" on page 19, line 10 of the same book is corrected to "O-phosphocholine."

Claims (1) Adenosine-5゛-diphosphoric acid is adenosine-5゛-diphosphoric acid.
- A complex enzyme reaction in which an enzyme that converts to triphosphate and an enzyme that synthesizes a physiologically active substance while converting adenosine-5'-triphosphate to 7-denosine-5'-diphosphate coexist in the same reactor. Using a system, a raw material solution containing a physiologically active substance precursor, adenosine-5°-phosphate, and 2 traces of metal ions is supplied from one end of the reactor, and a physiologically active substance is supplied from the other end of the reactor. When producing physiologically active substances by extracting ions, the concentration of divalent metal ions supplied to the reactor is
0- or less, and the concentration of azo-containing 5''-phosphoric oxide.

Physiologically active substance ■ A method for producing a physiologically active substance by a complex enzyme method, characterized in that the concentration is lower than that of the precursor.

Claims (1)

[Claims] (1) Adenosine-5'-diphosphate is converted into adenosine-5'-diphosphate.
- A complex enzyme reaction in which an enzyme that converts triphosphate and an enzyme that synthesizes a physiologically active substance while converting adenosine-5'-triphosphate to adenosine-5'-diphosphate coexist in the same reactor. Using a system, a raw material solution containing a physiologically active substance precursor, adenosine-5'-phosphate, and divalent metal ions is supplied from one end of the reactor, and a physiologically active substance is supplied from the other end of the reactor. When extracting and producing physiologically active substances, the concentration of divalent metal ions supplied to the reactor is set to 30%.
1. A method for producing a physiologically active substance by a multiple enzyme method, characterized in that the concentration of adenine-5'-phosphorylated is lower than the concentration of a physiologically active substance precursor.