JPS62180787A - Enzyme-containing filter medium and its production and using method thereof - Google Patents

Abstract

PURPOSE:To make the efficient removal of insoluble impurities and removal of dissolved impurities with one stage by using an enzyme-contg. filter medium formed by immobilizing enzyme to a three-dimensional network open-cellular high polymer as a carrier. CONSTITUTION:The enzyme-contg. filter medium is produced by using enzyme such as glucose oxidase, high polymer having a primary amino group such as chitosan gelatin and crosslinking agent such as glutaraldehyde and immobilizing the enzyme to an insoluble polyvinyl formal having the three-dimensional network open-cellular structure as the carrier. This enzyme-contg. filter medium removes the dissolved impurities contained in soln. in a production process of food industry, chemical industry and pharmaceutical industry and is useful to efficiently and inexpensively remove the dissolved materials, for example, the oxygen and hydrogen peroxide to deteriorate the intended material, and glucose, etc., to change flavor in food industry with one stage.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an enzyme-containing filter material in which an enzyme is immobilized using a three-dimensional network continuous porous polymer as a carrier.

More specifically, in the manufacturing process of the food industry, pharmaceutical industry, and chemical industry, enzymes are included in the filter medium to remove μ-containing substances from the solution being processed, thereby removing insoluble impurities and dissolved impurities. This invention relates to an enzyme-containing filter material that can be used simultaneously.

BACKGROUND OF THE INVENTION Conventionally, the removal of insoluble impurities has been an essential step in manufacturing processes in the food industry, pharmaceutical industry, and chemical industry. Additionally, removal of dissolved substances may be necessary as a process.

For example, in the production of milk in the food industry, hydrogen peroxide is sometimes used for sterilization, but in order to remove this hydrogen peroxide from milk after sterilization, there is a method that uses caclase immobilized on carboquine chloride resin. (USp member, 32g
Although trypsin has long been known to be effective in preventing the occurrence of oxidized odor in milk, research is also being conducted using this enzyme immobilized on porous glass (W, 2102). F, 5hipe.

et al., J., Dairy Sci., 3;
! ; Otsu 4t7. (/ri72)].

In addition, if beer is left for a long time, the polyphenols and polypeptides contained in beer combine,
Papain is mainly used in the beer manufacturing process to prevent so-called chill-haze, which results in turbidity. If it is added to the water, protein hydrolysis may proceed too much and have undesirable effects. Therefore, the use of immobilized papain and immobilized polyphenol oxidase is being considered as a method for preventing beer turbidity without such drawbacks (P, R, Witt, et al.
l, Brewers.

Dig,, 0cLober, 10 (/def0)]
.

In addition, in order to prevent mayonnaise from decomposing and spoiling, glucose is removed by the action of glucose oxidase and cuff rase, and no decomposition phenomenon is observed even after storage for 5 months, and no change in flavor is observed. It is said that there is no. (Get, put, , /227zaS5(/
ri 6 O)]. :: In addition, the presence of dextran in the crushing juice of sugar cane causes various problems during the clarification process, and there have been attempts to treat this in advance with dextran-degrading enzymes. (N, W, H.

CheeLhan eL al, Carbobydr
ate Res,, 30 Tata (/def3)
].

Efforts have also been made to remove dissolved oxygen, which causes changes in the flavor of citrus fruit and other soft drinks (T
, Gadfrey, oL al, Ir1dust
real EnrymologyTlre N, LLu
re Press 1129). There are many processes in which it is necessary to remove dissolved impurities. However, currently, industrially, the filtration process, which is a process for removing insoluble impurities, and the process for removing dissolved impurities are carried out as separate processes.

As mentioned above, attempts have been made to enzymatically remove dissolved impurities in the food industry, but these methods are either inefficient, require new expensive equipment, or simply involve the addition of enzymes. Then, the added enzyme is
There are problems such as the formation of inconvenient impurities, and none of them are necessarily satisfactory, and they also do not include a filtration step for insoluble impurities. A membrane with immobilized enzymes is also considered as a membrane that also has the ability to filter insoluble impurities. Because it is used to remove impurities from dissolved polymers such as protein, there are problems with membrane damage due to hard solids, poor efficiency due to clogging, etc. Since polymers are also removed, this does not essentially defeat the purpose of the present invention.

In addition, in the manufacturing process of chemical products and pharmaceutical products in the chemical and pharmaceutical industries, dissolved impurities, such as oxygen, can cause deterioration of chemical products and pharmaceutical products, resulting in a decrease in yield. There has been a desire for inexpensive and efficient removal of impurities.

:Means for Solving the Problems: As a result of intensive studies, the present inventors have found that an enzyme-containing filter material in which enzymes are immobilized using a three-dimensional network continuous porous polymer as a carrier can efficiently remove insoluble impurities and The present invention was completed based on the discovery that the removal of dissolved impurities can be carried out satisfactorily through seven steps.

That is, the present invention uses an enzyme-containing filter material in which an enzyme is immobilized using a three-dimensional network continuous porous polymer as a carrier, and an enzyme/a polymer having an absorbing amino group and a crosslinking agent.
A method for producing an enzyme-containing filter material and a solution to be treated, characterized by immobilizing a three-dimensional network continuous porous polymer as a carrier,
A method of using an enzyme-containing filtration material characterized by removing insoluble impurities and also removing dissolved impurities using an enzyme-containing filtration material obtained by immobilizing an enzyme using a three-dimensional network continuous porous polymer as a carrier. Regarding.

The three-dimensional network continuous porous polymer used in the present invention includes:
For example, water-insoluble polyvinyl formal having a three-dimensional network continuous porous structure is produced and is commercially available for use as a filter medium, and generally has an average pore diameter of S ~ 1000μ, preferably an average pore diameter of 30~
A water-insoluble polyvinyl alcohol resin with a porous structure having a pore size in the range of 100 μm is used. The above-mentioned porous structure has a dendritic structure with completely continuous pores, and is made of a polymer with high air flow rate and low fluid resistance. □The bulk water molecule is preferably one that does not change in quality in water and can withstand heat sterilization temperatures when immobilizing enzyme-containing bacterial cells i Homamoto polymer as a carrier.
It is preferable to use a material with a degree of formalization of about gO% or more.

The water polymer preferably exhibits chemical resistance to weak acids, alkalis, and many organic solvents. When used as a commercially available water-insoluble polyvinyl formal having a three-dimensional network continuous porous structure, it can be used as it is in the form of a sheet, or in an appropriately cut or granular form, or it can be used in an appropriately cut or granular form. It can also be used even if it is processed and molded. Further, as long as the polymer has the properties described above, it is not limited to water-insoluble polyvinyl polymer, and any polymer having a three-dimensional network of continuous porous channels may be used.

・Also, the enzyme used in the present invention is not limited in any way as long as it acts on dissolved impurities contained in the solution to be treated as a substrate, and examples include Curcone J oxidase, catalase, trypsin, papain,
Enzymes such as protease, dextrin-degrading enzyme, and polyphenol oxidase are dissolved impurities to be removed 1
Accordingly, the 7 types selected from the group consisting of the enzymes mentioned above can be used by appropriately selecting two or more types of enzymes. □Example 1: For example, when removing dissolved oxygen and dissolved glucone. When removing dissolved hydrogen peroxide, catalase may be used, and when removing dissolved hydrogen peroxide, catalase may be used. can be protected using dextran and enzymes. Used in the present invention: The above-mentioned enzymes may be commercially available enzymes (or may be necessary microbial cells to be strained and drained, and processed microbial cells). If the body-treated product is immobilized on a carrier according to a known immobilization method, the covalent bonding method may be a diazo method, a peptide method, an alkylation method, or a carrier sericulture method using a cross-linking reagent. I can do this [
Partially edited by Chibata, immobilized enzyme page, published by G5 Sadokorinsha, (
/97! ;) and reference 116].

For example, the enzyme may be immobilized as a three-dimensional network continuous porous polymer carrier using a polymer containing chitosan, seratin, or aminated polyvinyl alcohol, and a crosslinking agent such as g/v taral aldehyde. Furthermore, in immobilizing an enzyme using a polymer having a primary amino group such as chitosan, gelatin, or aminated polyvinyl alcohol, and a glutaraldehyde polymer as a carrier, a three-dimensional network continuous porous polymer is prepared in advance by chitosan, It is coated with a polymer having a seventh amino group such as gelatin or aminated polyvinyl alcohol, and then coated with active esters of dicarboxylic acids such as adipic acid, glutaric acid, and pimelic acid, acid anhydrides, etc., glyoxal, nuxin dialdehyde, and glutaric acid. The enzyme may be immobilized by activation with a crosslinking agent such as a dialdehyde such as an aldehyde.

The quantitative relationship between the carrier used here and the amount of enzyme depends on the ratio of insoluble impurities and dissolved impurities to the treated material, but the amount of enzyme is 0.7 to 10 OOO relative to the amount of carrier/ml.
U may be used, and preferably /~100OU may be used. For example, when removing dissolved hydrogen peroxide as well as removing insoluble impurities, it is sufficient to use 50 to 500 U of catalase per ml of carrier, preferably 100 to 500 U of catalase per ml of carrier.
300U may be used. In addition, when removing dissolved glucose together with removing insoluble impurities, it is sufficient to use 10 to 10 OOU of cyclone oxidase/~10U1 catalase per ml of carrier, preferably 100
~10OOU may be used. In addition, when removing dissolved dextran and removing insoluble impurities, the amount of carrier/
It is sufficient to use 10 to 10 OOU of dextran degrading enzyme per ml, preferably 100 to 10 OOU.

The thus obtained three-dimensional network continuous porous polymer is used as a carrier to immobilize the enzyme, and may be appropriately molded to form an enzyme-containing filter material. All you have to do is cut it to fit the filter you are using.

The present invention uses an enzyme-containing filter material immobilized as a three-dimensional network continuous porous polymer carrier obtained as described above to remove insoluble impurities, and remove dissolved enzyme and/or dissolved hydrogen peroxide. It also includes a method for removing insoluble impurities by removing dissolved dextran and/or dissolved gyrecose and/or dissolved protein, and the filtration flow rate is 007~/(1) m/, depending on the properties of the treated substance. b
It is preferable to use it with r, and shi< is 0/~. ! ;m/
11r may be used. Although the filtration temperature depends on the properties of the substance to be treated, it may be carried out at a temperature of 5 to 00C, and if the stability is poor, the filtration may be carried out at a low temperature. For example, when removing insoluble strained substances and dissolved dextran, preferably 3
0-jOp may be used. However, although the processing substance is safe and will be explained in the following examples, it is not limited to these examples in any way.

(Example/) Porous polyvinyl resin sheet (commercially available)
Rater EB (A) Average pore diameter/30μ, porosity gs%
, /Jyuutsuku Kanebo Co., Ltd.) (hereinafter abbreviated as "porous PVF")] was cut with scissors into an Ox-resistance-resistant porous PVF. 110 sheets of porous PVF with a square size of O x Q mm were immersed in a 7% chitosan solution/l for 7 hours. 7% chitosan solution is 0. / N-formic acid gOOI
10f of commercially available Kuriwaknu CP as chitosan to Rt.
- Otsu 73 (Kurita Industries) 'After understanding IbWl! ;N-N
After adjusting the pH to II5 with aOH, add distilled water to adjust the total volume to /l. The porous PVF soaked in chitosan solution for 7 hours is dried by ventilation drying to obtain dry porous PVF coated with chitosan having a three-dimensional dendritic network continuous structure. Five sheets of the obtained dry porous PVF are 5
% gel tar aldehyde solution R 110 Oml to 6L 棧P
VP After soaking for 30 minutes, remove 49/flf from the geltaldehyde solution and rinse well with distilled water. S% Geltaraldehyde% Geltaraldehyde/Qml 0. /M phosphate buffer (pH 7.5) by mixing with Qml. Commercially available as a 9-base solution of Katara-+z. -Se( Shin-qm.lttsm x
tttigt zzett), and the Kaklase degree is /i2U/m1. //Adjust Qml. The porous PVF/sheet obtained by the above operation is immersed in catalase solution/QQml having each concentration under stirring. Soaking is carried out for 7 hours to obtain catalase-immobilized porous PVF. The enzyme titer obtained per 7 pieces is 1033 U when using 2 U 1 ml of catalase solution, //7
U / ml for Q degree 1073; U and /2
For a concentration of 30U/ml, it was /300U.

It was decided by Kata. Catalase-immobilized porous PVF (107!; U// Milk containing 10 ppm hydrogen peroxide was filtered under pressure using 7 sheets. Filtration required 35 minutes. The concentration of ferron peroxide in the filtered milk was less than O,/ppm.

(Example 2) /33U crucose oxidase and ZOoxi
dase [Aspergi! (derived from Renu niger) and commercially available catalase (same as in Example). Add 3 ml of the obtained enzyme p, 3 ml of 7% chitosan solution (prepared in the same manner as in Example), and a well-mixed solution of commercially available 50% glucuraldehyde 00 Otogo to the porous P of 0X90 chu.
Soak 7 pieces of VF (thickness/chu). The enzyme activity per 17 sheets of porous PVF dried through ventilation after soaking for 7 hours was 311 U for glucose oxidizer and 3 SOOU for catalase. By stirring the /% glucose solution at room temperature, the /% glucose solution with a residual oxygen concentration of 3.5 ppm is added to the glucose oxidase obtained in the above step,
Pressure filtration was performed using catalase-immobilized porous PVF. Filtration required 25 minutes. The dissolved oxygen concentration in the obtained filtrate was O/ppm. The enzyme titer of glucose oxidase was determined by neutralizing the gluconic acid produced using glucose as a substrate and determining the activity as the amount of NaOH required for neutralization.

(Example 3) Add dextran/00 to the catalase enzyme solution of Example/
ml under stirring. Dextranase liquid 100m
1 is obtained by dissolving commercially available Dextoonase 200η (Sigma, derived from benicillinium, 23U/'1G) in 010M phosphate buffer (100ml, pH 7.5). The enzyme titer of dextranase was determined by using dextrin as a substrate and quantifying isomal l--nu produced by enzymatic decomposition of dextran as a reducing enzyme.

The activity per 7 pieces of shiko was 2200U. A solution/l containing 10% dream sugar and dextran/% was prepared, and Dextran T 10 (manufactured by Farmana) was used as the strand. The filtrate contained reducing sugar q,/ as maltose.

Effects of the Invention The enzyme-containing filter material of the present invention, in which an enzyme is immobilized using a three-dimensional network continuous porous polymer as a carrier, can be used in the food industry, chemical industry,
In addition to removing insolubilized impurities contained in solutions in the manufacturing process of the pharmaceutical industry, it is possible to efficiently and inexpensively remove dissolved substances such as oxygen, hydrogen peroxide, which cause deterioration of the target substance, or flavor changes in the food industry. This is a useful product that can remove glucose, etc. that cause oxidation in one step.

Claims (1)

[Claims] 1) An enzyme-containing filtration material in which an enzyme is immobilized using a three-dimensional network continuous porous polymer as a carrier. 2) The enzyme-containing filter material according to claim 1, wherein the three-dimensional network continuous porous polymer is a water-insoluble polyvinyl formal having a three-dimensional network continuous porous structure. 3) The enzyme is glucose oxidase, catalase,
The enzyme-containing filter material according to claim 1, which is one or more selected from the group consisting of trypsin, papain, protease, dextran degrading enzyme, and polyphenol oxidase. 4) A method for producing an enzyme-containing filter medium, which comprises immobilizing an enzyme on a three-dimensional network continuous porous polymer as a carrier using a polymer having a primary amino group and a crosslinking agent. 5) In immobilizing an enzyme as a carrier on a 3D continuous porous polymer using a polymer having a primary amino group and a crosslinking agent, the 3D continuous porous polymer is preliminarily treated with primary amino groups. coated with a polymer having groups, and then
5. The production method according to claim 4, wherein the immobilization is performed by activation with a crosslinking agent. 6) Claim 4 or 5, wherein the polymer having a primary amino group is a polymer consisting of one or two selected from the group consisting of chitosan gelatin and aminated polyvinyl alcohol. manufacturing method. 7) The manufacturing method according to claims 4 and 5, wherein the crosslinking agent is glutaraldehyde. 8) The enzyme is glucose oxidase, catalase,
The production method according to claim 4 or 5, wherein the enzyme is one or more selected from the group consisting of trypsin, papain, protease, dextran degrading enzyme, and polyphenol oxidase. 9) The production method according to claims 4 and 5, wherein the three-dimensional network continuous porous polymer is a water-insoluble polyvinyl formal having a three-dimensional network continuous porous structure. 10) Impurities characterized by removing dissolved impurities as well as insoluble impurities in the treatment solution using an enzyme-containing filter material in which an enzyme is immobilized using a three-dimensional network continuous porous polymer as a carrier. How to remove. 11) The dissolved impurities are dissolved oxygen, dissolved hydrogen peroxide,
11. The method for removing impurities according to claim 10, which is dissolved dextran, dissolved glucose, or dissolved protein.