JPS6332478A - Enzymic reaction apparatus - Google Patents

Abstract

PURPOSE:To suppress occurrence of clogging and make it possible to sustain enzymic activity for a long period, by forming an enzyme-carrying separation membrane immobilizing the enzyme in the substrate layer thereof having a dense layer and coarse layer. CONSTITUTION:An enzyme-carrying separation membrane 1 is a tubular module and a liquid containing a substrate is passed through the interior of the tube having the internal and external peripheries respectively formed into a dense layer and coarse layer. The thickness of the membrane substrate is preferably about 0.5-1.5 mm. The pore diameter of the dense layer is <= 0.01mum and thickness of the layer is preferably about 1/1000-1/2000 based on the total thickness.

Description

Detailed Description of the Invention (Field of Application of the Invention) The present invention relates to an enzyme reaction device for producing, separating and recovering a target substance from a solution containing a predetermined substrate by utilizing enzymatic reactions such as hydrolysis and isomerization. It is something.

(Background of the Invention) As is well known, enzymes are often effectively used as catalysts in various reactions, but because they are generally expensive, they are difficult to use, especially when considering the use and application of enzymes on an industrial scale. Therefore, it is necessary to devise ways to improve the utilization rate.

The conventional way of using enzymes is usually in the form of an immobilized enzyme fixed on some kind of carrier and used by filling a column which is a reaction vessel (hereinafter referred to as column method).

It has been pointed out that the Shikaji column method is prone to clogging due to the contamination of fine particles, and the probability of contact between the immobilized enzyme in the column and the substrate is not necessarily high, and for these reasons, the effective utilization rate of the enzyme is low. is the current situation. Further, the following problem is pointed out as another drawback of the column method. In other words, in column-type devices, it is common to change the enzyme activity by changing the temperature as a control element for the reaction amount (reaction rate), but since there is a time lag in the temperature dependence of the enzyme activity, it is difficult to The problem is that it is difficult to control the amount of reaction.

Therefore, in recent years, enzymes have been immobilized on membranes instead of the column-type devices, and this membrane has been used to produce target substances.
Enzyme reaction devices that perform separation and recovery have been considered.

A hollow fiber membrane module is usually used as the membrane for this enzyme reaction device, and the enzyme is held on the shell side of the module and the substrate is passed through the lumen side.The reaction principle in this device is as follows. The substrate passes through the membrane and an enzymatic reaction occurs on the shell side, and the reaction product passes through the membrane and returns to the lumen side, where it is taken out from the module.

However, this method has the disadvantage that the reaction rate is slow because it is dominated by diffusion.

There is also a device using the same hollow fiber membrane module, in which the enzyme is held on the shell side, the substrate is allowed to flow from the shell side, and the reaction product is taken out from the lumen side. However, this method has the problem that clogging is likely to occur because the substrate is supplied in a dead-end manner.

(Object of the Invention) The present invention has been made from the above-mentioned viewpoints, and its object is to produce a target substance from a substrate by enzyme activity and to separate and recover it without causing problems such as clogging. It is an object of the present invention to provide an enzyme reaction apparatus that enables continuous reaction over a long period of time, and therefore enables extremely efficient operation of the actual capacity N on an industrial scale.

Another object of the present invention is to provide an enzyme reaction device that allows the amount of enzyme reaction to be controlled with high responsiveness and precision by a simple operation of adjusting the applied pressure for substrate flow. .

Still another object of the present invention is to provide an enzyme reaction apparatus that has a high enzyme utilization rate and can therefore be operated with reduced running costs.

(Summary of the Invention) A feature of the enzyme reaction apparatus of the present invention, which has been made to achieve the above object, is that an enzyme acting on the substrate is contained in a passage through which a liquid to be treated containing a substrate flows. An enzyme reaction device in which a supported separation membrane is disposed to divide the liquid passageway into an upstream side and a downstream side, the separation membrane having a layered dense layer facing the upstream side of the liquid passageway. The enzyme is constructed by immobilizing an enzyme inside a base membrane having an asymmetric structure in the thickness direction and having a dense and dense layer facing the downstream side of the liquid passage.

The reason why the above configuration is adopted in the present invention is as follows.

In other words, when a target liquid containing a substrate is passed through an enzyme reaction device to perform separation and recovery of the target substance, not only the target substance but also other substances (fine particles, polymeric substances) are present as impurities in the target liquid. It is usually included.

In an enzyme reaction apparatus using a conventional membrane (separation membrane), as mentioned above, when these impurities come into contact with the membrane as the liquid flows through the separation membrane, they enter the membrane and cause clogging. As a result, pressure loss rapidly increases and continuous membrane separation operation becomes impossible.

Therefore, in the present invention, the framework of the separation membrane is configured to allow the substrate and the target substance to be separated and recovered to pass through, but to prevent polymeric substances or fine particles with a larger molecular weight from entering the membrane. In the base membrane, a dense layer with a sufficiently small pore diameter is formed in a layered manner facing one side (the side facing the upstream side of the flow of the liquid to be treated containing the substrate), and the other side is In order to satisfy the requirement for adhesion and fixation of enzymes having activity against substrates inside the separation membrane (inside the base membrane), on the other side opposite to the one side, there is a mechanism for allowing the enzyme to enter the inside of the membrane. The membrane has an asymmetric structure that allows the formation of dense and dense layers.

The separation membrane used in the present invention is composed of a substrate (hereinafter referred to as an original substrate) as a porous body supporting an enzyme, and an enzyme adhered and immobilized inside the membrane substrate.

The separation membrane of the present invention has a structure in which the enzyme distribution is asymmetrical in the thickness direction.

The substrate used as a carrier supporting an enzyme in the present invention satisfies the condition that a dense layer and a sparse layer are formed with a layered distribution in the thickness direction in order to adhere and fix the enzyme inside the membrane. The other materials are not particularly limited, and may be either an inorganic film (ceramic film, glass film) or an organic polymer film. The thickness of the primordia is
Although it is not particularly limited, it is generally 0.1 to 3 mm, preferably 0.5 to 1.5 mm from the standpoint of handling strength.
In many cases, it is better to set it to a certain degree, but in general, it is appropriately designed depending on the implementation scale of the enzyme reaction apparatus.

Enzyme adhesion and immobilization provided by being distributed in layers inside the separation membrane has a dense layer portion facing one side in the thickness direction and having a pore size large enough for the enzyme to pass through but not for the substrate to pass through, In addition, on the other side, an enzyme solution is flowed from the coarse layer side to the base membrane, which has a layered coarse and dense layer portion with a pore size that allows entry of the enzyme to be adhered and immobilized inside the membrane. It is done.

Therefore, the pore diameter of the dense layer portion of the protosubstrate is selected and determined depending on the molecular weight of the enzyme to be adhered and fixed, but is generally 0.04 μm or less, preferably 0.01 μm.
In many cases, it is better to make it less than m.

The thickness of the dense layer is usually 1/1000 to 1/1000 of the total thickness of the base film.
About 1/5, preferably 1/100 to 1/200
Although it is generally considered as a degree, it is not particularly limited to this.

The coarse and dense layer provided together with the dense layer has a relatively large pore diameter with respect to the dense layer, and is used for physical adhesion and fixation provided as a result of the penetration of the polymeric enzyme into the membrane substrate. A pore size large enough for this purpose is required.

The pore size of this coarse-dense layer satisfies the above conditions, and
They are selected taking into account the structural strength of the proto-substrate, the difficulty of creating the proto-substrate, the release of the immobilized enzyme, etc., and generally a size range that allows the enzyme to be adhered and immobilized to easily pass through is sufficient. It is better to set it small.

The distribution in the thickness direction of the dense layer and coarse-dense layer of the protosubstrate varies stepwise or continuously in a linear or curved manner from one end surface to the other end surface of the protosubstrate. Good too.

A base film having such a required distribution can be created according to a known method, for example, by dissolving the polymer in a solvent, casting it, evaporating the solvent, performing heat treatment, and then applying a thin film of the dense layer material. j＜I can.

Among the inorganic membranes that can be used as base membranes,
Typically, MEMBR-ALOX;
Cerahe-JL/manufactured by), Carbocep (CAR-BO)
5EP, 5FEC), Seraph 0-((:ER
Examples include ceramic membranes such as AFLO (manufactured by NORTON'I Jr.) and Dynaceram (manufactured by TDK). In addition, as an organic polymer film, NTU film (manufactured by Nitto Denko Corporation)
, GR film (manufactured by DDS), DIY film (manufactured by Daicel)
For example, the following membranes may be used. Note that the base film is not particularly limited to those exemplified above.

The enzyme to be immobilized on the base membrane is selected in relation to the substrate or the target separation and recovery substance of production 1, and is physically adhered and immobilized inside the base membrane by the flow of the enzyme solution.

As mentioned above, the method of adhesion and fixation to the base membrane is carried out by flowing a solution containing the enzyme from the coarse layer side of the base membrane to the dense layer side, but the added pressure of this flow is , - numerically 1 kg/cm 2 ~ lOkg
/cm2.

The enzyme used reacts (
It is not particularly limited as long as it has an active action). Examples of this include invertase, glucose isomerase, fructosyltransferase, β-galactosidase, melibiase, and the like.

The separation membrane having an immobilized enzyme according to the present invention has a flat membrane shape,
Conventionally known membrane modules such as hollow fiber, tubular, and spiral shapes can be constructed and used.

The enzyme reaction device of the present invention, which has a separation membrane in which enzymes are distributed in layers in the thickness direction, has a relationship between the layered distribution of enzymes in the membrane and the flow of the liquid to be processed containing the substrate to be separated. Except as specified above, it can be constructed in substantially the same manner as an enzyme reaction device using a numerical membrane.

Specific fields to which the apparatus of the present invention is applied can be categorized and exemplified in terms of substances to be separated and recovered (target valuable substances), such as recovery of invert sugar and isomerized sugar, production of various oligo rings, etc. , production of amino acids and other seasonings, etc.

(Example of the invention) The present invention will be described below based on embodiments shown in the drawings.

Figure 1 shows an outline of the structure of the enzyme reaction apparatus according to the present invention - an example of the apparatus flow. The dense layer is formed facing the outer circumferential surface of the membrane.

The substrate is passed from the substrate tank 5 to the inside of the membrane of the membrane module 1 through the pump 2, and the substrate flows from the inside to the outside of the membrane in accordance with the feeding pressure of the liquid to become a permeate. When this substrate passes through the membrane, it undergoes the activity of the enzyme supported and fixed on the membrane to produce the desired reaction product (target substance).
can be obtained. Note that 3 is a temperature control coil for maintaining the substrate in the substrate tank 5 at a predetermined temperature.

FIG. 2 shows a device for supporting and immobilizing enzymes inside the membrane of the membrane module 1, and is applied to a tubular base membrane in which the dense-coarse layered distribution is given in the thickness direction from the inside to the outside. An enzyme solution is passed through the pump 2 from the enzyme solution tank 4 from the outside of the membrane, and the enzyme is supported and fixed according to the coarse and dense layered structure of the base membrane.

Hereinafter, specific embodiments of the present invention will be described according to examples carried out using the apparatus shown in the above drawings.

Example 1 O-Sato 1 Membrane substrate: @1 mm thick alumina l1i (trade name r ME
MBRALOX”.

Toshiba Ceramics Corporation) Dense layer (pore diameter 0.04 μm) (layer thickness 5 μm) Dense layer (pore diameter 0.2 to 15 μm) (layer thickness 0.995 mm) Immobilized enzyme (invertase) Operations during enzyme immobilization: 100 mg/It at temperature 10℃
The enzyme solution was flowed for 4 hours at a flow pressure of 3 kg/cm2.

Processing liquid to be subjected to membrane separation operation: ShoW10 zero solution Target substances for separation and recovery Niglucose, Fructose Operation conditions: The above sucrose solution was flowed at a temperature of 50°C and the flow pressures shown in Table 1 for 30 minutes at each pressure. .

The test results conducted above are shown in Table 1. Table 1
As is clear from the results, it was found that the reaction amount can be controlled by pressure, and that glucose and fructose can be taken out of the system at a sufficient reaction rate.

Furthermore, when switching the flow pressure in Table 1, the time required for the reaction amount to reach a steady state after switching is as follows:
The reaction time was several minutes, which was extremely short compared to when the reaction was controlled by temperature.

Comparative Example 1 A test was conducted in the same manner as in Example 1 using the apparatus shown in the drawing above, except that the flow direction of the sucrose solution was reversed. The results are shown in Table 2.

From Table 2, it was found that when the substrate was flowed from the dense layer side, both the reaction rate and reaction amount were lower than in the above example.

Table 1 Table 2 Example 2 Using the same apparatus as in Example 1, a 10% solution of ShiF111 to which 1% of glass beads of several microns was added was used as a raw material.

In this example, as in Example 1, the apparatus could be operated at a high reaction rate, and glucose and fructose could be taken out continuously.

Comparative Example 2 The same raw materials as those shown in Example 2 were used in the same manner as in Comparative Example 1, with the sucrose solution flowing in one direction from the outside of the separation membrane (dense layer side). The glass beads became clogged, making it impossible to operate.

(Effects of the Invention) As described above, the enzyme reaction device according to the present invention can process target substances (substrates) without causing problems such as clogging.
This has the advantage that the separation can be carried out continuously over a long period of time and therefore can be carried out very effectively on an industrial scale, and the quantity control of the enzymatic reaction can be carried out by adjusting the applied pressure. This simple operation allows the process to be carried out with good responsiveness and high accuracy, which has the advantage that it can be carried out more suitably on an industrial scale.

Furthermore, the present invention has a high effective utilization rate of enzymes, and therefore can be carried out with reduced running costs, and is extremely useful.

[Brief explanation of drawings]

Figure 1 shows the configuration of the enzyme reaction apparatus according to the present invention.
FIG. 2, a diagram showing an example, shows an example of a method for immobilizing an enzyme on a base membrane in order to create a separation membrane used in the present invention. 1: Membrane module 2: Pump 3: Temperature control coil 4: Enzyme tank 5: Substrate tank

Claims (4)

[Claims] (1) An enzyme reaction device in which a separation membrane supporting an enzyme that acts on the substrate is placed in a liquid passage through which a liquid to be treated containing a substrate flows, and the liquid passage is divided into an upstream side and a downstream side. The separation membrane is a substrate having an asymmetric structure in the thickness direction, having a layered dense layer facing upstream of the liquid passageway and a laminated coarse layer facing the downstream side of the liquid passageway. An enzyme reaction device comprising a membrane with an enzyme immobilized inside the base membrane. (2) A patent claim characterized in that the separation membrane is made of a porous ceramic membrane, a glass membrane, or an organic polymer membrane as a base membrane, and an enzyme is attached and fixed inside the base membrane. Enzyme reaction apparatus according to item (1) within the scope of (3) Claim (1) characterized in that the separation membrane is one in which an enzyme is adhered and fixed by flowing an enzyme solution from the coarse layer side to the dense layer side of the base membrane. or the enzyme reaction device described in paragraph (2) (4) The enzyme according to any one of claims (1) to (3), characterized in that the enzyme is invertase, glucose isomerase, fructosyltransferase, β-galactosidase, or melibiase. enzyme reaction device