JPS637788A - Recovering method for coenzyme - Google Patents

Abstract

PURPOSE:To make it possible to repeatedly use a coenzyme and continuously carry out reaction, by separating the coenzyme from a reaction mixture containing the coenzyme with a pseudo-moving bed and simultaneously separating the reaction product and/or unreacted substances, etc. CONSTITUTION:A reaction solution is fed from a solution feed tank 1 to an enzymic reactor 2, passed through a cushion tank 3 and then to a pseudo-moving bed column 4 prepared by using an adsorption and desorption carrier such as a strong acidic or weak acidic ion exchange resin, e.g. zeolite, silica gel, Sephadex, Amberlite, etc. The coenzyme, reaction product, unreacted substances, etc., are continuously separated with a desorbing solution of the same composition as the solvent in the reaction solution and recycled through an immobilization column.

Description

[Detailed description of the invention] Industrial applications The present invention relates to a method for recovering coenzymes.

Conventional technology With the remarkable development of enzyme and enzyme immobilization technology, in relatively simple reaction systems that do not require coenzymes, we have succeeded in industrializing the production of useful substances using immobilized enzymes or immobilized enzyme groups. It's starting.

-On the other hand, many enzymes considered to be industrially useful require coenzymes. However, coenzymes are expensive, and there is no system that allows them to be used while being repeatedly regenerated at low cost. That is, conventionally, enzymatic reactions involving coenzymes have been carried out in batches, and coenzymes and the like have been disposable. Therefore, the production of useful substances using such enzymes has so far had little success industrially.

Under these circumstances, research is underway to effectively reuse coenzymes. However, all of these studies aim to reuse coenzymes by confining them in the enzyme reaction system, and examples include the following methods.

■ Methods for binding coenzymes to soluble macromolecules (eg, Keimosbach (K, Mo5bach)).

AdvEnzym
ol, ) 4'6.205 (1978) ) ■ Method of immobilization on ion exchange resin (e.g., Enzyme Engineering (Tokyo Kagaku Doujin, published in 1981)) ■ Membrane with a cut-off molecular weight smaller than that of the coenzyme, such as reverse osmosis Methods of immobilization with membranes (for example, R, P, chamber (R, P.

Chamber) et al., Enzyme Engineering (
Enzyme Engineering) Volume 2,
195 (1974), Plenum Publishing.
(Press))■ Immobilization method using a liquid film (for example, Varnish, W. May, et al., Biochemical and Biophysical Research Communications (Biochem, Bio
phys, Res, Comm, )67.786
(1976)).

However, methods (1) and (2) are unsuitable for industrialization because immobilization of the coenzyme requires considerable effort and cost, and the enzyme activity is lower than that of a coenzyme that has not been polymerized. Methods (1) and (2) do not have the disadvantages of (2) and (2), but they have the major disadvantage of extremely limited applicable systems, and the reaction rate is also extremely slow, making them completely unsuitable for industrial use. Not available. Furthermore, it should be noted that in any of the cases (1) to (2), an operation is required to separate the reaction product from unreacted substrates or by-products.

Object and Structure of the Present Invention We have begun to develop an effective coenzyme reuse system that overcomes the drawbacks of the above-mentioned conventional techniques and can withstand industrialization. As a result of intensive research, he realized that there was a problem with the idea of confining coenzymes in the reaction system, and continued research on how to efficiently recover the coenzymes that come out of the reaction system.
We discovered that it is best to continuously separate and recover the product using a simulated moving bed and also recover the coenzyme at the same time.
The present invention has now been completed.

That is, the gist of the present invention resides in a method for recovering a coenzyme, which is characterized by separating the coenzyme from a reaction mixture containing the coenzyme using a simulated moving bed.

When actually adopting the recovery method of the present invention, the steps are as follows:
A system that regenerates coenzymes (hereinafter referred to as system A), an enzyme reaction system that requires coenzymes (hereinafter referred to as system B),
and a simulated moving bed (hereinafter referred to as system C) for recovering coenzymes. These three systems may be combined in any convenient manner depending on the target coenzyme and reaction system.

For example, if system A and system B must proceed simultaneously, or if the reaction conditions of system A and system B are the same or similar (hereinafter referred to as case I), system A and system B are the same. The order or circular permutation of reacting in a reactor and operating System C is preferred.

However, if one or more of the reaction substrates of system A inhibit the reaction of system B, and the inhibitory substances are significantly reduced when the reaction of system A is carried out (hereinafter referred to as
It is called case ■. ), system A, system B, system C or a circular permutation.

Also, if one or more of the reaction substrates or by-products of system A inhibit the reaction of system B and cause the reaction of system A to occur, the inhibitory substance is not significantly reduced or increases (hereinafter referred to as (referred to as case (2)), it is desirable to carry out the permutation of system A, system C, system B, or circular permutation.

Here, the meaning referred to as circular permutation is, for example, system A
This means that operations performed in the order of system C, system B, and system B, system A, and system C can be regarded as the same, and all possible circular permutations can be included.

Further, the reaction solution may be introduced from any of systems A, B, and C, and may be introduced from any convenient location. It is also possible to have different reaction liquid compositions for system A and system B, and it is sufficient to select one suitable for the reaction system.

Also, systems A, B, and C do not need to be single;
It is also easy to use two or more of each. in this case,
This can be easily analogized to all possible permutations using systems A, B, and C.

It is possible to construct a multipurpose bioreactor.

The term coenzyme here refers to a coenzyme that is essential for the existence of enzymatic action. All coenzymes are subject to the present invention, and include, for example, nicotinamide adenine dinucleotide (hereinafter referred to as N
AD), nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP), flavin mononucleotide (hereinafter referred to as FMN), flavin adenine dinucleotide (hereinafter referred to as FAD), thiamine diphosphate, pyridoxal phosphate, coenzyme A, Riboic acid 9 folic acid, glucose-1,6-diphosphoric acid, 2,3-diphosphoglyceric acid, adenosine triphosphate (hereinafter referred to as ATP).

Guanosine diphosphate. Examples include uridine diphosphate glucose.

System A1, that is, a system that regenerates coenzymes, collectively refers to a system that regenerates coenzymes using one or more enzyme groups, or a system that can perform the same thing as using an enzyme without using an enzyme. For the system A, any known method may be followed, and any method may be used. Here, ATP and NAD or NADP, which are particularly important among the known methods, will be mentioned.

Examples of methods for regenerating ATP include methods using glycolytic enzymes, methods using acetate kinase and adenylate kinase, methods using salvage synthesis, methods using the respiratory system, methods using a photophosphorylation system, There are many methods such as electrochemical methods. For example, fermentation and engineering i42,6
67 (1984). Furthermore, NAD or NADP can be roughly divided into three methods: chemical methods, electrochemical methods, and enzymatic methods, and are described in detail in, for example, Enzyme Engineering (Tokyo Kagaku Dojin, published in 1981).

- On the other hand, system B is a system that has one or more or all enzymes that require coenzymes among an enzyme group consisting of one or more enzymes. The enzyme group of system B may be any known enzyme as long as it requires the above-mentioned coenzyme.

Many combinations of system A and system B are known. Among them, a few examples of production systems for industrially important useful substances include glutathione, FAD, CDP-choline, peptides, etc. when using an ATP regeneration system.
S-adenosylmethionine, XMP, gramidin S,
Examples include the synthesis of glucose-6-phosphate (G-6-P) and steroids, and the production of alanine and lactic acid using NAD.

The enzymes of System A and System B may be immobilized according to known immobilization methods (for example, see Enzyme Engineering (Tokyo Kagaku Dojin Co., Ltd.)). Examples include a crosslinking method in which the enzyme is crosslinked with a reagent having a functional group, and an entrapment method in which the enzyme is wrapped in a fine lattice of a polymer gel or covered with a semipermeable polymer film. However, it should be mentioned that in the case of the comprehensive method, it is necessary for the coenzyme to be able to freely enter and exit, and for example, in order to immobilize it by an ultrafiltration membrane, the cost is, for example, 100 to 500,000. More preferably 100
Those having a fraction of molecular insects of 0 to 50,000 are preferred. Examples of immobilization using polymer gel include acrylamide gel, ethyl cellulose, photocurable resin, K
-Carrageenan, calcium alginate gel, agar, agarose gel, gelatin, etc.

Next, regarding system C, a pseudo moving bed contains a solid adsorbent, and the front end and rear end are connected by a fluid passage, so that fluid can circulate within the bed. A desorption liquid fluid inlet and an adsorbate fluid outlet are provided in the packed bed along the flow direction of the fluid in the bed, and each fluid is continuously supplied from each inlet and outlet at regular intervals. Introducing or withdrawing - By sequentially switching each inlet and outlet with the downstream one at regular intervals, the raw fluid is divided into components that are relatively easily adsorbed by the solid adsorbent (adsorbate components) and those that are relatively difficult to be adsorbed. It means something that separates into components (non-adsorbent components), and this itself is publicly known (for example,
-15681).

Examples of the use of such a simulated moving bed technology include a method for producing fructose (see JP-A-53-88335), a method for separating maltose (see JP-A-60-67000), and the like. However, no research has yet been reported on a method for recovering coenzymes using a simulated moving bed.

What is particularly noteworthy about the present invention is that the coenzyme can be recovered and reused at the same time as the reaction product is separated and purified using a simulated moving bed. In other words, even with conventional techniques that prevent coenzymes from escaping, it is necessary to separate and purify the reaction products, and the system as a whole requires separate collection of coenzymes and separation of products. It took a lot of effort. However, the major feature of this process is that it can be done all at once. Furthermore, it should be mentioned that continuous operation is possible by employing a pseudo-moving bed, and the outstanding feature of this system is that it can be operated continuously as a whole, including systems A and B. It should be further mentioned that unreacted materials are recovered and sent to system 1 or B.
operations for recycling or recovery of reaction byproducts,
A major feature of this method is that it can simultaneously and continuously remove reaction inhibitors.

The adsorption/desorption carrier used in the simulated moving bed may be one commonly used, preferably a strongly acidic ion exchange resin, such as one having a So or H group as a functional group (for example, Amberlite (Am -berlite) [R-
200), those having P O-(OH)2W as a functional group (e.g. Daolite C-60,
Those with C0OH group as a functional group (e.g. diaion (
D 1aion) WK 10, chelating resins (e.g. Dowex A-1), or strong or weakly basic ion exchange resins, e.g. Amberlite IR
A-100, Amberlite [RA-401, DOWEX 2, Diaion 5A20, or two-exchange resins such as Amberlite fR-45, ZeoK
arb) 215, snake cage resins such as Retardion 11A8, and the like. Furthermore, porous ion exchange resins such as XAD-
1, XAD-2゜XAD-4, XAD-7, XAD-8
, XAD-9, XAD-11, and XAD-12 may also be exemplified.

Also preferably a cellulose ion exchanger, such as 0M cellulose, P-cellulose, PPM cellulose, SE-
Cellulose, SM-cellulose, DEAE-cellulose, GE-cellulose, ECTEOLA-cellulose, P
Also included are AB-cellulose, AE-cellulose, and derivatives thereof. Furthermore, mention may also be made of dextran ion exchangers. Furthermore, gel filtration agents may be used, such as Sephadex G, Sephadex LH-20, Bio-G
et) P etc. are also preferable. In addition, other adsorbents may be used, and preferably zeolite, sori gel, activated carbon and alumina can be used. Mafko,
Any desorption liquid may be used, but preferably it has the same composition as the solvent of the reaction liquid. Hereinafter, the present invention will be explained in more detail using examples, but it goes without saying that the idea of the present invention is not limited by these examples.

Examples Example 1 After washing 300 g of baker's yeast (manufactured by Kanekabuchi Chemical Industry Co., Ltd.) twice with water, it was dried at 25°C overnight, and then dried in a vacuum dryer for 1 hour.
By drying for 6 hours, 70 g of dried bacterial cells were obtained. Experiments were conducted using the process shown in FIG.

Composition of reaction solution supplied: 1M glucose, 20mM
Adenosine, 500ff 1M phosphate buffer (pH 7)
, 5), 30mM MgSO4, and this reaction solution was supplied to the enzyme reactor at a rate of 1mQ/min.

As the enzyme reactor, an ultrafiltration device with a capacity of ff1l12 was used, an ultrafiltration membrane with a molecular fraction of ff11,000 and a membrane area of 200cm' was used, and the gauge pressure was 5Kg/cn+'', 280cm.
The enzyme group was prevented from leaking out from the immobilized column by ultrafiltration with C. For the enzyme group, 50 g of dried bacterial cells prepared by the above method were added as they were.

The reaction solution (pl-17, 5) coming out of the immobilized column was 1
The simulated moving bed column was fed at a rate of IIIQ/1Ilin.

The simulated moving bed is a column 16 with an inner diameter of 2 cm and a length of 20 cm.
A material made of books was used, and Amberlite IRA-401 was used as the adsorption carrier. 500mM phosphate buffer y-(pi-(7,5), 30mM g
A SO4 solution was used.

XAD as a coenzyme fractionated by a simulated moving bed; unreacted adenone diphosphate (hereinafter referred to as ADP), adenosine-phosphate (hereinafter referred to as AMP) and ADP of adenosine, ATP as a reaction product A total of three components were successively separated, and NAD and ADP were recycled to the immobilization column again. The obtained ATP is AT
As a result of analysis using a P photometer and high performance liquid chromatography, A was obtained from adenosine in a yield of over 95%.
It was found that TP was obtained continuously. Moreover, the recovery rate of NAD in the pseudo mobile phase was 98% or more. It could be reused until the enzymes of Tora Baker's Yeast were deactivated.

Example 2 100 g of bread yeast @ (manufactured by Kanebuchi Kagaku Kogyo (Mai)) was suspended in 30% potassium chloride, and this I! ! A solution of 25 g of acrylamide monomer and 1.25 g of N,N'-methylene-bisacrylamide dissolved in 75 mf2 of water was added to the turbid solution. Add 30ml of 5% β-(dimethylamino)propionitrile to this, and further add 2.0g of potassium persulfate.
A solution of 30mQ of water was added to form a gel, resulting in gel 2.
20g was obtained.

220 g of the gel thus obtained was packed into a column with an inner diameter of 5 cm to prepare an enzyme reactor. Into this column, 20 m each of monoglutamic acid, cyanostine and glycine were added.
M, MgCQI OmM, phosphate buffer (pH 7,5)
The reaction solution of 200mM glucose IM was mixed with a space velocity of 0.
．． 2 was continuously fed.

From the composition of the enzyme reactor outlet liquid, it was found that glutathione (hereinafter referred to as GSH) was produced from amino acids with a yield of 95%.

The outlet liquid was continuously supplied to a simulated moving bed using Amberlite [RA-401 as an adsorption carrier. 20 as desorption liquid
0mM phosphate buffer (pH 7,5).

A 10mM MgCl2 aqueous solution was used. Collect GSH, Nostin and NAD, and combine Nostin and NAD.
was continuously recycled to the enzyme reactor.

As a result, GSH with a cysteine yield of 98% or more was obtained. Furthermore, there were almost no impurities other than phosphoric acid and magnenium, and purification could be performed at the same time.

Moreover, this reaction could be carried out continuously for at least 7 days.

Effect of the invention The effects of the present invention are as follows.

(a) Since a simulated moving bed is used, the separation and recovery of coenzymes, unreacted substrates, and reaction products is easier than in conventional column operations, and can be carried out more frequently.

(b) Since the coenzyme is not immobilized or polymerized, it is easy to operate and is extremely inexpensive.

(c) By employing a simulated moving bed, it is extremely easy to continuously recycle coenzymes and/or unreacted substrates to the reaction system and recover reaction products.

(d) Reaction-hazardous substances can be easily removed and removed continuously from the system.

(e) The selection range of target systems is extremely wide.

Using the present invention, coenzymes can be used repeatedly and can be applied to almost all systems involving coenzymes. Therefore, it can be used in many fields such as food, medicine, cosmetics, and chemical products, and its utility value is extremely high.

[Brief explanation of the drawing]

FIG. 1 is a schematic explanatory diagram of the process used in Example 1.

Claims (1)

[Scope of Claims] 1. A method for recovering a coenzyme, which comprises separating the coenzyme from a reaction mixture containing the coenzyme using a simulated moving bed. 2. A method for recovering a coenzyme according to claim 1, which comprises simultaneously separating a reaction product and/or an unreacted substance and/or a reaction inhibitor when recovering the coenzyme using a simulated moving bed. .