JPH0528109B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to an enzymatic reaction method containing a coenzyme and an oxidoreductase, and more specifically, a coenzyme regeneration reaction and a predetermined enzymatic reaction in the presence of an oxidoreductase, a coenzyme, and a substrate. The present invention relates to an enzyme reaction method in which a reaction product and a coenzyme are separated from each other in a reaction mixture produced by conjugating the same. (Prior art) For example, some enzymatic reactions that do not use coenzymes, such as the production of isomerized sugar using glucose isomerase, have already been put into practical use, but enzymatic reactions that require coenzymes are Coenzymes are significantly more expensive than the reaction target products, and a practical method for separating them from the reaction system has not been established, so they have not yet been put to practical use. Conventionally, the separation of coenzymes from enzymatic reaction systems using coenzymes has been described, for example, in "Microorganisms as Biocatalysts" edited by Saburo Fukui, pages 157-161 (published by Kyoritsu Shuppan Co., Ltd. in 1978). Several methods have been proposed. For example, for the separation of NADP, it is also possible to use a separation membrane that can remove low molecular weight compounds. However, according to this method, not only the coenzyme but also the substrate and reaction product are removed at the same time. If an immobilized coenzyme is obtained by immobilizing a coenzyme on a water-insoluble carrier, it is easy to separate this immobilized coenzyme from a water-soluble reaction product, but production of the immobilized coenzyme is not always possible. In addition to being difficult, immobilized coenzymes generally have lower reaction activity than free coenzymes. Another known method is to supply a low concentration of coenzyme to the reaction system, combine the coenzyme with the enzyme, and separate it as a high molecular weight substance using an ultrafiltration membrane. requires supplying a high concentration of enzyme to the reaction system and is unsuitable as an industrial enzymatic reaction method. Furthermore, it has also been proposed to carry out the required enzymatic reaction while retaining the coenzyme in a negatively charged separation membrane, taking advantage of the fact that coenzymes are generally low molecular weight organic compounds that have a negative charge. However, conventionally, in this method, Tris-HCl buffer or calcium phosphate buffer has been used as the reaction medium, and although the reason for this is not necessarily clear, compared to the case where pure water is used as the reaction medium, Therefore, the retention rate of coenzymes by the separation membrane is extremely low. In addition, hereinafter, in this specification, retaining a coenzyme within a separation membrane means that when a reaction mixture containing a coenzyme is treated with a separation membrane, the coenzyme is not permeated, and the retention rate of the coenzyme is As is well known in the field of membrane technology, the term "removal rate of coenzymes" is the rate of removal of coenzymes when a reaction mixture containing coenzymes is subjected to membrane treatment. The same applies to other solutes. (Objective of the Invention) The present inventors carried out a coenzyme regeneration reaction coupled with a predetermined enzyme reaction in the presence of an oxidoreductase, a coenzyme, and a substrate, and the reaction product in the generated reaction mixture was As a result of intensive research to solve the above-mentioned problems in the enzymatic reaction method for separating coenzymes and coenzymes, we unexpectedly found that a zwitterion buffer was present in the reaction mixture, and this was transferred to a negatively charged reverse osmosis membrane. They have also found that the retention rate of coenzymes can be significantly increased by treatment with an ultrafiltration membrane, and furthermore, when the negatively charged membrane has a predetermined salt removal rate, the retention of coenzymes can be significantly increased. The present invention was developed based on the discovery that the ratio can be increased. Therefore, the present invention provides a method for performing an enzymatic reaction by separating coenzymes and reaction products at a high separation rate from a reaction product of an enzymatic reaction containing an oxidoreductase, a coenzyme, and a reaction product thereof. The purpose is to provide (Structure of the Invention) The present invention involves carrying out a coenzyme regeneration reaction coupled with a predetermined enzyme reaction in the presence of an oxidoreductase, a coenzyme, and a substrate, and combining coenzymes and reaction products in the generated reaction mixture. The enzyme reaction method involves treating the reaction mixture in the presence of a zwitterion buffer with a negatively charged reverse osmosis membrane or ultrafiltration membrane to transfer the enzyme and coenzyme into the membrane. It is characterized by retaining the enzyme reaction product and allowing the enzyme reaction product to pass through the membrane. In the present invention, the coenzyme regeneration system is not particularly limited, and only a single enzymatic reaction may be used;
Two or more types of enzymatic reactions may be used. Usually, it is practical to use three or less types of enzyme reactions in order to avoid complication of the reaction system. Also,
The enzyme used in the present invention is not particularly limited as long as it is an oxidoreductase, and is appropriately selected depending on the desired reaction. This enzyme may be used in a free state or as an immobilized enzyme. Furthermore, the coenzymes and enzymes do not necessarily have to be purified, and may be cell-free extracts of bacteria or yeast. The negatively charged separation membrane used in the present invention may be a reverse osmosis membrane or an ultrafiltration membrane, as long as it has a fractionation property that allows the reaction product of an enzymatic reaction to pass through.
In particular, when a sodium chloride aqueous solution with a concentration of 0.5% is treated at a temperature of 25°C and a pressure of 10 kg/cm ² , the sodium chloride removal rate is at least 3%, and further, when treated at a temperature of 25°C and a pressure of 5 kg/cm ² . When the glucose removal rate is 30% or less, the NADP removal rate is 50% or less.
% or more. In particular, it is preferable to have fractionation properties such that the glucose removal rate is 10% or less and the NADP removal rate is 80% or more. Ultrafiltration membranes have large pores and therefore have poor separation performance. When using an uncharged membrane, since the coenzyme is generally a low molecular weight substance, if the substrate and reaction product are also low molecular weight substances, the substrate and reaction product, especially the reaction product, can be selectively permeated. On the other hand,
Although it is difficult to retain coenzymes within a separation membrane, according to the present invention, when a membrane having the above characteristics and a negative charge is used as a separation membrane, only the so-called sieving effect of the separation membrane is achieved. In addition, the selective retention of coenzymes is also exhibited by the electrostatic repulsion effect between the negative charge of the separation membrane and the negative charge of the coenzyme, and as a result, according to the present invention, the coenzyme can be removed at a high level. It can be retained within the membrane at a high rate. Note that since enzymes generally have high molecular weight, they cannot substantially permeate membranes and are retained within the membrane. According to the method of the present invention, for example, a substrate, an oxidoreductase, and a coenzyme are introduced into a reaction vessel in advance, and a predetermined reaction coupled to a coenzyme regeneration reaction is carried out in the presence of a zwitterionic buffer as a reaction medium. After carrying out the enzymatic reaction, the resulting reaction mixture is treated with the above-mentioned negatively charged reverse osmosis membrane or ultrafiltration membrane to selectively permeate the enzymatic reaction product, while allowing the enzyme and coenzyme to pass through. retained within the membrane, thus
Coenzymes and reaction products can be separated.
In addition, as another method, the space constituted by the above-mentioned separation membrane having a negative charge is used as a reaction system, and the substrate and
After introducing the oxidoreductase and the coenzyme and carrying out the regeneration reaction of the coenzyme and the predetermined enzymatic reaction in a coupled manner in the presence of a zwitterionic buffer as a reaction medium, or after carrying out the above enzyme and the coenzyme, may be retained in a membrane, and the enzymatic reaction product may be separated by permeation through the membrane. After the reaction, a zwitterion buffer may be added to the reaction mixture at the stage of membrane treatment. In the present invention, the method of performing the coenzyme regeneration reaction coupled with a predetermined enzymatic reaction in the presence of an oxidoreductase, a coenzyme, and a substrate may be carried out under the conditions of ordinary enzyme reactions. Therefore, the concentration of the substrate in the reaction system varies depending on the solubility of the substrate, but is usually 10M or less, but especially 1 to 1M.
A range of 5M is preferred. Further, the concentration of the enzyme and coenzyme is usually in the range of 0.01mM to 1M, particularly preferably in the range of 0.1 to 0.5M. but,
It is not limited to these. In the method of the present invention, the form of the separation membrane is not limited at all, and for example, flat membranes, tubular membranes, capillary membranes, hollow fiber membranes, etc. are used. Further, the form of the membrane module equipped with these separation membranes is also appropriately selected depending on the reaction and is not limited in any way. The separation membrane used in the method of the present invention is preferably a separation membrane having a sulfonic acid group as an acidic group. Such separation membranes include, for example, sulfonated polysulfone, sulfonated polyetherketone, sulfonated polystyrene, sulfonated polyquinazolone, sulfonated polyamide, sulfonated polyimide, sulfonated polyamideimide, polyolefin grafted with sulfonic acid groups, and sulfonated polyolefins grafted with sulfonic acid groups. Examples include hyperfiltration membranes and reverse osmosis membranes made of fluorine-based resins and the like. Note that the ultrafiltration membrane means a so-called loose reverse osmosis membrane. In particular, in the present invention, a separation membrane made of a polymer in which sulfonic acid groups account for most of the total acidic groups, preferably 70% or more, particularly preferably 90% or more, is preferred. However, as long as the sulfonic acid group is within the above range among all the acidic groups, the remaining acidic group may be an acidic group other than the sulfonic acid group, for example, a carboxylic acid group. In the present invention, as a separation membrane having a sulfonic acid group that can be particularly preferably used, the repeating unit A A sulfonated polyaryl ether obtained by sulfonating a polyaryl ether consisting of the above, or the above repeating unit A and repeating unit B (However, R represents -CO- or -SO ₂ -, R' is a carbon-carbon bond, or -CO- or -SO ₂ -
Indicates a divalent group containing ) A composite separation membrane can be mentioned in which a skin layer made of a sulfonated polyarylether made by sulfonating a linear polyarylether copolymer made of . Such a composite separation membrane is preferably produced by sulfonating a polyarylether consisting of the above-mentioned repeating unit A or a linear polyarylether copolymer consisting of the above-mentioned repeating unit A and repeating unit B, respectively. Prepare the ether and add it to a small amount of aprotic polar organic solvent,
For example, dimethyl sulfoxide, N-methyl-2
-pyrrolidone, N,N-dimethylformamide,
an alkylene glycol alkyl ether such as ethylene glycol monomethyl ether which may contain N,N-dimethylacetamide and the like;
If necessary, a membrane-forming solution containing a water-soluble and low-volatility organic compound or an inorganic salt as an additive is applied onto the dried support membrane, and then an organic compound is extracted from this membrane-forming solution. It can be obtained by evaporating the solvent. The concentration of the sulfonated polyaryl ether in the membrane-forming solution is related to the thickness of the separation membrane formed by these polymers in the resulting composite separation membrane, but is usually preferably in the range of 0.05 to 10% by weight, particularly 0.1% by weight. A range of 5% by weight is preferred. (Effects of the Invention) According to the present invention, a coenzyme regeneration reaction and a predetermined enzymatic reaction are coupled to each other in the presence of an oxidoreductase, a coenzyme, and a substrate, and the reaction product in the generated reaction mixture is In the enzymatic reaction method for separating coenzymes and coenzymes, the coenzymes and reaction products can be separated at a high rate by treating the generated reaction mixture with a negatively charged membrane in the presence of a zwitterion buffer. It can be separated by In particular, when using a negatively charged membrane with a given salt removal rate,
The separation rate of coenzymes can be further increased. (Examples) The present invention will be described below with reference to Examples and Reference Examples, but the present invention is not limited to these Examples in any way. In the Examples, the solute removal rate was determined by the following formula. Removal rate = (1 - solute concentration in membrane permeate / solute concentration in membrane feed liquid) x 100 (%) Reference example 1 (1) Production of polysulfone copolymer Described in Japanese Patent Publication No. 46-21458 According to the method, formula A ₁ 57 mol% repeating units of formula B ₁ A linear porsulfone copolymer consisting of 43 mol% of repeating units was produced. The polysulfone copolymer thus obtained (yield 100%) was pale yellow granular,
The logarithmic viscosity of this copolymer was 0.84 cm ³ /g. (2) Structure of sulfonated polysulfone copolymer Polysulfone copolymer obtained as above
10 g was added and dissolved in 80 ml of 97% concentrated sulfuric acid, and stirred and reacted at room temperature for 4 hours to obtain a dark brown viscous reaction liquid. This was placed in an ice bath to solidify the sulfonated polysulfone copolymer. After washing with water, 800ml of 0.5N sodium hydroxide aqueous solution
I left it inside overnight. Next, the polymer was washed until the washing solution became neutral, and then vacuum-dried at 60°C for 5 hours. The sulfonated polysulfone copolymer thus obtained had a logarithmic viscosity of 0.84 and a sulfonic acid group content of 1.2 milliequivalents/g. (3) Manufacture of composite semipermeable membrane A polyethylene glycol (average molecular weight 20,000) aqueous solution with a concentration of 0.5% was heated at a temperature of 25°C and a pressure of 3.5 kg/
Repeating unit of the following formula C with a removal rate of 10% when processed at cm ² polysulfone ultrafiltration membrane (molecular weight cut-off
100000) was heat treated by immersing it in hot water at a temperature of 80°C for 1 hour, then immersing it in a 10% 1,4-butanediol aqueous solution at a temperature of 25°C for 1 hour, and then
A dry semipermeable membrane was obtained by leaving it in a dryer at about 60° C. for 5 minutes. Dissolving the sulfonated polysulfone copolymer in ethylene glycol monomethyl ether,
A 1.0% by weight polymer solution was prepared, coated on the dry ultrafiltration membrane, left at room temperature to evaporate almost all the solvent, and then heated at 60°C for 5 minutes. A composite separation membrane was obtained by heating. The performance of this composite separation membrane was 15% removal rate when treated with a 0.5% sodium chloride aqueous solution at 25° C. and 10 kg/cm ² . Reference Example 2 Polysulfone having the repeating unit of formula C (UCC P-1700) was prepared using the method of Noshay et al. (J.
Applied Polymer Sci., 20 , 1887 (1976)), sulfonated polysulfone with a sulfonic acid group content of 0.8 meq/g was obtained. 15 g of this sulfonated polysulfone was dissolved in 82 g of N-methylpyrrolidone, and 3 g of lithium nitrate was added thereto to obtain a homogeneous solution, which was then filtered through a 10 μm filter paper to obtain a membrane forming solution. This membrane-forming solution was applied to a thickness of 285 μm on a glass plate, dried at 130° C. for 1 minute, the solvent was evaporated, and then immersed in water at 5° C. to obtain a negatively charged separation membrane. The performance of this separation membrane is as follows when treated with 0.5% sodium chloride aqueous solution at 25℃ and 10Kg/ ^cm2 .
The removal rate was 3%. Example 1 The separation membrane obtained in Reference Example 1 or 2 above was attached to a batch-type membrane permeation tester equipped with a stirrer and a temperature regulator, and purified water or glycylglycine buffer (100mM,
A coenzyme aqueous solution dissolved in pH 7.5) was heated to 25°C.
The membrane treatment was carried out under a pressure of 5 kg/cm ² . Table 1 shows the coenzyme retention rates. In the example, the membrane of Reference Example 1 was
Furthermore, the comparative example is the result of using the membrane of Reference Example 2, and it is understood that when a separation membrane having a predetermined salt retention rate is used according to the present invention, the coenzyme retention rate is high.

[Table] Example 2 The separation membrane obtained in Reference Example 1 above was attached to a batch-type membrane permeation tester equipped with a stirrer and a temperature regulator.
Coenzyme retention in the presence of various buffers at a temperature of 25
The measurement was carried out at a temperature of 5 Kg/cm ^{2 and a pressure of 5 Kg/cm 2} . As the results are shown in Table 2, it is understood that when the buffer solution is a zwitterion buffer solution, the coenzyme retention rate is high. Example 3 The separation membrane obtained in Reference Example 1 was installed in a batch-type membrane permeation tester equipped with a stirrer and a temperature regulator.

[Table] Enzyme reaction solution with the composition shown below (total volume 8 ml) Hydrogenase (derived from Pseudomonas ruhlandii)
1.4 units lactate dehydrogenase 90.0 units Pyruvate 30.0mM NAD ⁺ 3.0mM glycylglycine (PH7.5) 100.0mM were added and reacted at 35℃ for 12 hours using gaseous hydrogen at a pressure of 2Kg/ ^cm2 as a reducing agent. Lactic acid was produced from pyruvic acid. Thereafter, the reaction solution was passed through under pressure of nitrogen gas at a pressure of 3 kg/cm ² to separate lactic acid. The conversion rate of pyruvic acid was 100%, and the retention rate of NAD ⁺ was 96%. Example 4 In Example 3, alcohol dehydrogenase (derived from baker's yeast) was used instead of lactate dehydrogenase,
Ethanol was produced in the same manner as in Example 2, except that acetaldehyde was used as the substrate. The conversion rate of acetaldehyde to ethanol is
The retention rate of NAD ⁺ was 95%. Comparative Example 1 Lactic acid was produced in the same manner as in Example 3, except that the membrane obtained in Reference Example 2 was used as the separation membrane in place of the membrane in Reference Example 1. The conversion rate of pyruvic acid was 100%, but
The retention rate of NAD ⁺ was 76%. Example 5 The separation membrane obtained in Example 1 was attached to a batch-type membrane permeation tester equipped with a stirrer and a temperature regulator, and an enzyme reaction solution having the composition shown below (total volume 8 ml) was prepared.Hydrogenase (derived from Methanobacterium)
1.4 units F420−NADP ⁺ oxidoreductase (from Methanobacterium) 2.9 units xylose reductase (Candida pelliculosa)
Origin) 90 units Xylose 30.0mM NADP ⁺ 3.0mM F420 0.3mM Glycylglycine 100.0mM was added and reacted at 35℃ for 12 hours using gaseous hydrogen at a pressure of 2Kg/ ^cm2 as a reducing agent to produce xylitol from xylose. did. After this, the reaction solution is under pressure of 3
The reaction solution was permeated under a pressure of Kg/cm ² of nitrogen gas, and xylitol was separated. The conversion rate of xylitol was 100%. Also,
The retention rates of NADP ⁺ , F420 and xylitol were 95.5%, 100% and 0%, respectively. Comparative Example 3 Xylitol was produced from xylose in the same manner as in Example 5, except that 1/15M Tris-hydrochloride buffer was used as the buffer. The conversion rate of xylose was 100%, but the retention rates of NADP ⁺ , F420 and xylitol were 50%, 92% and 0, respectively.
It was %.

Claims (1)

[Claims] 1. In the presence of an oxidoreductase, a coenzyme and a substrate,
An enzymatic reaction method in which a coenzyme regeneration reaction and a predetermined enzyme reaction are conjugated and the coenzyme and reaction product are separated in the generated reaction mixture, the reaction mixture being separated from the reaction mixture in the presence of a zwitterion buffer. An enzyme reaction method characterized in that the enzyme and coenzyme are retained within the membrane by treatment with a reverse osmosis membrane or ultrafiltration membrane having a negative charge underneath, and the enzyme reaction product is allowed to permeate through the membrane. 2. The enzyme reaction method according to claim 1, wherein the zwitterion buffer is a glycylglycine buffer. 3. The enzyme reaction method according to claim 1, wherein the zwitterion buffer is a barbital buffer. 4. The enzyme reaction method according to claim 1, wherein the reverse osmosis membrane or ultrafiltration membrane having a negative charge is a separation membrane having a sulfonic acid group. 5. A patent characterized in that a regeneration reaction of a coenzyme and a predetermined enzymatic reaction are carried out in conjunction with a regeneration reaction of a coenzyme in the presence of an oxidoreductase, a coenzyme, and a substrate in a negatively charged reverse osmosis membrane or ultrafiltration membrane. The enzyme reaction method according to claim 1. 6 If the reverse osmosis membrane or ultrafiltration membrane with a negative charge
When a sodium chloride aqueous solution with a concentration of 0.5% is treated at 25℃ and a pressure of 10Kg/ ^cm2 , at least 3
The enzyme reaction method according to claim 1, wherein the enzyme reaction method has a salt removal rate of %.