Method of immobilizing enzymatically active materials

This invention discloses a method of immobilizing an enzymatically active material to a water-insoluble polyanion which comprises bringing a polycation-containing aqueous medium and the enzymatically active substance into contact with the water-insoluble polyanion having any desired form. This method is characterized in that the enzymatically active substance is adsorbed on the surfaces of the water-insoluble polyanion and, at the same time, a strong complex is formed by the interaction of the polycation and the polyanion to produce a water-insoluble film. As a result, the enzymatically active substance so adsorbed is securely fixed to the surfaces of the water-insoluble polyanion and scarcely released therefrom.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a novel method of immobilizing enzymatically 
active materials and, more particularly, to a method of immobilizing an 
enzymatically active material to a water-insoluble polyanion which 
comprises bringing a polycation-containing aqueous medium and the 
enzymatically active material into contact with the water-insoluble 
polyanion. 
2. Description of the Prior Art 
Conventionally, a number of techniques for immobilizing enzymatically 
active materials are well known which include, for example, the method of 
chemically combining an enzymatically active material with another 
enzymatically active material or an insoluble carrier by covalent or ionic 
bonding and the method of entrapping an enzymatically active material 
within a water-insoluble substance or microcapsules. Among the rest, the 
adsorption method in which an enzymatically active material is fixed on 
the surfaces of an inert carrier by ionic or other physical force has 
several advantages over other immobilization methods. For example, the 
adsorption method permits an enzymatically active material to be 
immobilized under very mild conditions and bound loosely to a carrier. 
Thus, the enzymatically active material suffers little damage from the 
immobilization procedure and, after being once immobilized, it undergoes 
only a slight degree of deactivation. Moreover, the adsorption method 
permits the enzymatically active material to be fixed in the vicinity of 
the surfaces of the carrier. Accordingly, when a substrate is brought into 
contact with the immobilized preparation of the enzymatically active 
material, the substrate readily spreads over the enzymatically active 
material, so that it can exhibit high activity even in the immobilized 
state. Furthermore, it is possible to prepare and pretreat the carrier in 
the absence of enzymatically active material. Thus, as compared with the 
preparation and pretreatment of a carrier in other immobilization methods, 
greater latitude is given in determining the type of carrier used and the 
method of preparation. This makes it possible to select a carrier highly 
suitable for the intended purpose. A further advantage of the adsorption 
method is that the immobilization procedure can be carried out in a 
culture vessel or the like. This serves not only to largely decrease the 
risk of contamination with undesired microorganisms during the procedure 
for immobilizing an enzymatically active material, but also to simplify 
the immobilization procedure to a remarkable degree. 
With all these advantages, the adsorption method still has many 
disadvantages and cannot be regarded as a satisfactory method of 
immobilizing enzymatically active materials. Specifically, as compared 
with the above-described chemical and physical immobilization methods, the 
adsorption method is disadvantageous in that the immobilization procedure 
has low efficiency and requires a long period of time. Moreover, since the 
adsorption depends largely on the surface condition of the carrier the 
enzymatically active material is liable to desorption due to the change in 
the surface conditions of the carrier during use. Furthermore, the 
adsorptive power of the enzymatically active material to the carrier is so 
low that, during the immobilization of the enzymatically active material 
or during the use of the resulting immobilized preparation, the 
enzymatically active material tends to be released from the carrier under 
the influence of mechanical shock and the like. In addition, when living 
microbial cells and the like are used as the enzymatically active 
material, most of the newly grown cells are released from the carrier. 
This makes the adsorption method unsuitable for the immobilization of 
growing microbial cells. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a novel method of 
enzymatically active materials which comprises bringing a 
polycation-containing aqueous medium and an enzymatically active material 
into contact with a water-insoluble polyanion. 
According to the method of the present invention, a polycation-containing 
aqueous medium and an enzymatically active material are brought into 
contact with a water-insoluble polyanion having any desired form, whereby 
the enzymatically active material is immobilized, simultaneously with the 
formation of a film, in the vicinity of the surface of the water-insoluble 
polyanion. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
When compared with the prior art adsorption method, the method of the 
present invention has the following advantages: 
(a) The rate of adsorption of an enzymatically active material is markedly 
increased. 
(b) An immobilized preparation can be obtained in high yield and with good 
reproducibility. 
(c) Once an enzymatically active material is immobilized, it is scarcely 
released. 
(d) Where living microbial cells are immobilized, newly grown microbial 
cells are also immobilized. 
(e) An enzymatically active material can be immobilized without regard to 
the surface condition of the water-insoluble polyanion. 
(f) The life of the enzyme activity is prolonged. 
(g) This method is unlimitedly applicable to a wide variety of 
enzymatically active materials. 
(h) Little damage is caused to the enzymatically active material. 
In addition, the method of the present invention also has the advantages 
possessed by the prior art adsorption method. Specifically, the method of 
the present invention is also characterized, for example, good contact 
between the substrate and the enzymatically active material, simplicity of 
operation, little risk of contamination with undesired microorganisms, and 
the like. 
Furthermore, when compared with the prior art covalent bonding method, the 
method of the present invention is characterized by less damage to the 
enzymatically active material and more simplicity of operation. When 
compared with the prior art entrapping method, the method of the present 
invention is characterized by less release of the enzymatically active 
material, a longer life of the enzyme activity, less risk of contamination 
with undesired microorganisms during the immobilization procedure. 
No particular limitation is placed on the type of polycation used in the 
present invention, provided that it can react with a polyanion as 
described hereinbelow to form a complex. The polycations which can be used 
in the present invention are exemplified by the following polymers. 
(I) Polymers containing a quaternary ammonium ion group in the backbone 
Typical examples of such polymers are: 
(a) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR1## 
where R.sub.1 and R.sub.2 are hydrogen atoms or alkyl radicals having not 
more than 4 carbon atoms, and R.sub.3 and R.sub.4 are alkyl radicals 
having not more than 4 carbon atoms; 
(b) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR2## 
where R.sub.1 is a hydrogen atom or an alkyl radical having not more than 
4 carbon atoms, R.sub.2 and R.sub.3 are alkyl radicals having not more 
than 4 carbon atoms, and m is a whole number of 3 or more; and 
(c) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR3## 
where R.sub.1 and R.sub.2 are alkyl radicals having not more than 4 
carbon atoms or allyl radicals. 
(II) Polymers containing a quaternary ammonium salt group in the side 
chains 
Typical examples of such polymers are: 
(a) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR4## 
where R.sub.1, R.sub.2 and R.sub.3 are hydrogen atoms or methyl radicals, 
R.sub.4 and R.sub.5 are alkyl radicals having not more than 4 carbon 
atoms, R.sub.6 is an alkyl radical having not more than 4 carbon atoms, a 
benzyl radical, an allyl radical, an alkoxyl radical or a carbonamide 
group, m is a whole number of 1 to 30, and n is a whole number of 0 to 5; 
(b) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR5## 
where R.sub.1 is a hydrogen atom or a methyl radical, R.sub.2, R.sub.3 
and R.sub.4 are hydrogen atoms, methyl radicals or hydroxyl groups, 
R.sub.5 and R.sub.6 are alkyl radicals having not more than 4 carbon 
atoms, R.sub.7 is an alkyl radical having not more than 4 carbon atoms, an 
allyl radical or an alkoxyl radical, and n is a whole number of 1 to 4; 
(c) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR6## 
where R.sub.1 is a hydrogen atom or a methyl radical, R.sub.2 and R.sub.3 
are alkyl radicals having not more than 4 carbon atoms, R.sub.4 is an 
alkyl radical having not more than 4 carbon atoms or a benzyl radical, and 
n is a whole number of 1 to 4; 
(d) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR7## 
where R.sub.1 is a hydrogen atom or a methyl radical, and R.sub.2, 
R.sub.3 and R.sub.4 are alkyl radicals having not more than 4 carbon 
atoms; 
(e) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR8## 
where R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen atoms or methyl 
radicals, and R is an alkyl radical having not more than 5 carbon atoms; 
and the like. 
(III) Polymers containing an amine group or a salt thereof in the backbone 
Typical examples of such polymers are: 
(a) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR9## 
or a salt thereof, where R.sub.1, R.sub.2 and R.sub.3 are hydrogen atoms 
or alkyl radicals having not more than 4 carbon atoms; 
(b) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR10## 
or a salt thereof, where R is an alkyl radical having not more than 4 
carbon atoms; and the like. 
(IV) Polymers containing an amine group or a salt thereof in the side 
chains 
Typical examples of such polymers are: 
(a) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR11## 
or a salt thereof, where R.sub.1 is a hydrogen atom or a methyl radical, 
and R.sub.2 and R.sub.3 are hydrogen atoms or alkyl radicals having not 
more than 4 carbon atoms; 
(b) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR12## 
or a salt thereof, where R.sub.1, R.sub.2 and R.sub.3 are hydrogen atoms 
or methyl radicals, R.sub.4 and R.sub.5 are hydrogen atoms, alkyl radicals 
having not more than 4 carbon atoms, benzyl radicals, allyl radicals or 
alkoxyl radicals, m is a whole number of 1 to 30, and n is a whole number 
of 0 to 35; 
(c) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR13## 
or a salt thereof, where R.sub.1 is a hydrogen atom or a methyl radical, 
R.sub.2 and R.sub.3 are hydrogen atoms, alkyl radicals having not more 
than 4 carbon atoms, benzyl radicals or alkoxyl radicals; 
(d) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR14## 
or a salt thereof, where R.sub.1 is a hydrogen atom or a methyl radical, 
and R.sub.2 and R.sub.3 are hydrogen atoms or alkyl radicals having not 
more than 4 carbon atoms; 
(e) Homopolymers, copolymers and graft polymers having structural units of 
the general formula 
##STR15## 
or a salt thereof, where R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are 
hydrogen atoms or methyl radicals; 
(f) Amino sugars (such as chitosan) and salts thereof; and the like. 
In the practice of the present invention, polymers falling under any one of 
the above categories I to IV may be used, either alone or in admixture, as 
the polycation. It is also possible to use polymers falling under two or 
more of the above categories I to IV. 
In the above categories I and II, x.sup..crclbar. is an atom or atomic 
group constituting an anion. Where x.sup..crclbar. is a monovalent anion, 
typical examples thereof include halogen anions, nitrate anion, nirite 
anion, sulfuric monoester anions (such as monomethyl sulfate and monoethyl 
sulfate anions), cyanide anion, formate anion, acetate anion, propionate 
anion and the like. On the other hand, where x.sup..crclbar. is a 
divalent, typical examples thereof include sulfate anion, carbonate anion, 
thiosulfate anion and the like. In the case of a divalent anion, it is 
used in an amount equal to one-half that of a monovalent anion. 
In the above categories III and IV, a salt of an amine group is a salt 
formed by the reaction of a group derived from a primary, secondary or 
tertiary amine with an acidic substance. Typically, inorganic acids such 
as hydrochloric acid, sulfuric acid, nitric acid, etc. and organic acids 
such as formic acid, acetic acid, propionic acid, etc. are used as the 
acidic substance. 
In the polycation used in the present invention, the cationic structural 
units containing a quaternary ammonium ion group, amine group, a salt of 
an amine group, or the like are present in an amount of 1 to 100% and 
preferably 10 to 100% based on the total structural units of the 
polycation. If the proportion of the cationic structural units is too 
small, the complex formed by the interaction of the polycation and the 
water-insoluble polyanion tends to show a decrease in strength. On the 
contrary, if the proportion of the cationic structural units is too high, 
the method becomes uneconomical. 
The polycation used in the present invention usually has a molecular weight 
in the range of 1,000 to 10,000,000. If the molecular weight is too low, 
the formed complex shows a descrease in strength. On the contrary, if the 
molecular weight is too high, the viscosity of the aqueous medium having 
the polycation dissolved or suspended therein is increased to impair the 
operating efficiency. 
In the practice of the present invention, the polycation may be dissolved 
in water and used as an aqueous solution. If at least a part of the 
polycation is insoluble in water, it may be used as a suspension. 
The aqueous medium used in the present invention may comprise water, an 
aqueous solution or an aqueous suspension according to the intended 
purpose. For example, a buffer solution, an aqueous solution of the 
substrate, a culture medium, and an aqueous solution of a water-soluble 
organic compound such as ethyl alcohol and the like can be used as the 
aqueous medium according to the need. Moreover, an aqueous suspension of 
an enzymatically active material insoluble in water or the like can also 
be used as the aqueous medium according to the need. It is not precluded 
that the aqueous medium further contains one or more substances which do 
not interfere with the practice of the present invention. Where a 
polycation falling under the above category I or II is added to the 
aqueous medium, no particular limitation is placed on the hydrogen ion 
concentration of the aqueous medium. However, where a polycation falling 
under the above category III or IV and containing a salt of an amine 
groups is added to the aqueous medium, it is preferable to use an acid pH 
lower than 7. Since the aqueous medium usually comes into contact with an 
enzymatically active material as described hereinbelow, it is necessary to 
select appropriate conditions (e.g., hydrogen ion concentration, the type 
of concentration of the solute and the suspensoid, and temperature) which 
do not deactivate the enzymatically active material. In the practice of 
the present invention, the concentration of the polycation in the aqueous 
medium is generally in the range of 0.01 to 70% by weight and preferably 
0.1 to 50% by weight. If the concentration is too low, the yield of the 
immobilized preparation of enzymatically active material is reduced. On 
the contrary, if the concentration is too high, the viscosity of the 
aqueous medium is increased to make the method impractical. 
The enzymatically active materials which can be used in the present 
invention include enzymes, microorganisms, cell fractions and the like. 
Among them, no particular limitation is placed on the type of enzyme used. 
Typical examples of useful enzymes are given in the following. 
Oxidoreductases 
Amino acid oxidases, uricase, catalase, xanthine oxidase, glucose oxidase, 
glucose-6-phosphate dehydrogenase, glutamate dehydrogenases, cytochrome c 
oxidase, tyrosinase, lactic dehydrogenase, peroxidases, 6-phosphogluconate 
dehydrogenase, malate dehydrogenase and the like. 
Transferases 
Aspartate acetyltransferase, aspartate aminotransferase, amino acid 
aminotransferases, glycine aminotransferase, glutamic-oxaloacetic 
aminitransferase, glutamic-pyruvic aminitransferase, creatine 
phosphokinase, histamine methyltransferase, pyruvate kinase, fructokinase, 
hexokinase, s-lysine acetyltransferase, leucine aminopeptidase and the 
like. 
Hydrolases 
Asparaginase, acetylcholinesterase, aminoacylase, arginase, L-arginine 
deiminase, invertase, urease, uricase, urokinase, esterases, kallikrein, 
chymotrypsin, trypsin, thrombin, naringinase, nucleotidases, papain, 
hyaluronidase, plasmin, pectinase, hesperidinase, pepsin, penicillinase, 
penicillin amidase, phospholipase, phosphatases, lactase, lipase, 
ribonucleases, rennin and the like. 
Lyases 
Aspartate decarboxylases, aspartase, citratelyase, glutamate 
decarboxylases, histidine ammonia-lyase, phenylalanine ammonia-lyase, 
fumarase, fumarate hydrase, malate synthetase and the like. 
Isomerases 
Alanine racemase, glucose isomerase, glucosephosphate isomerase, glutamate 
racemase, lactate racemase, methionine racemase and the like. 
Ligases 
Asparagine synthetase, glutathione synthetase, glutamine synthetase, 
puruvate synthetase and the like. 
No particular limitation is placed on the type of microorganism used in the 
present invention, provided that it serves as a source of enzyme. The 
microorganisms which can be used in the present invention include, for 
example, bacteria, fungi, slime molds, lichens, algae, protozoa and other 
microrganisms that contain enzymes as enumerated above. These 
microorganisms may comprise either living cells or a preparation obtained 
by subjecting living cells to freeze-drying, freezing and thawing, acetone 
treatment, heat treatment or the like. The cell fractions which can be 
used in the present invention include microbial cell wall, mitochondria, 
microsomes, organelles, rami and soluble fraction that contain enzymes as 
enumerated above, as well as mixtures of the foregoing. The enzymatically 
active material used in the present invention may comprise either a single 
enzyme or a complex enzyme system. Moreover, two or more enzymatically 
active materials can be used in combination. 
No particular limitation is placed on the type of water-insoluble polyanion 
used in the present invention, provided that it has any desired form 
suitable for the intended use of the resulting immobilized preparation of 
enzymatically active material and contains an anionic group which can 
react with the above-described polycation to form a strong complex. 
Although the water-insoluble polyanion can have any desired form suitable 
for the intended purpose, it is usually used in the form of a solid mass, 
a hydrogel, a suspension of a solid or liquid polymer, or the like. In 
order that the water-insoluble polyanion of the present invention may 
react with the above-described polycation to form a strong complex, it 
must contain an anionic group which can be, for example, a carboxylic acid 
group or a salt thereof, a sulfonic acid group or a salt thereof, a 
sulfuric ester group or a salt thereof, or a phosphoric ester group or a 
salt thereof. Specific examples of useful polyanions are given in the 
following. 
(a) Polymers containing a carboxylic acid group or a salt thereof 
Homopolymers, copolymers and graft polymers containing, as a monomer, a 
carboxylic acid (such as acrylic acid, methacrylic acid, maleic acid, 
fumaric acid, itaconic acid, etc.) or a salt thereof, as well as 
carboxyl-containing polysaccharides (such as alginic acid, pectin, 
carboxymethyl celulose, etc.) and salts thereof. 
(b) Polymers containing a sulfonic acid group or a salt thereof 
Homopolymers, copolymers and graft polymers containing, as a monomer, a 
sulfonic acid (such as 2-acrylamido-2-methylpropanesulfonic acid, 
2-acrylamidoethanesulfonic acid, 2-methacrylamidoethanesulfonic acid, 
vinylsulfonic acid, allylsulfonic acid, styrenesulfonic acid, etc.) or a 
salt thereof. 
(c) Polymers containing a sulfuric ester group or a salt thereof 
Sulfuric ester compounds (such as carrageenan, furcellan, agar, ce-lulose 
sulfate, starch sulfate, sulfated starch, sulfated polyvinyl alcohol, 
etc.) and salts thereof. 
(d) Polymers containing a phosphoric ester group or a salt thereof 
Homopolymers, copolymers and graft polymers containing, as a monomer, a 
phosphoric ester (such as acid phosphoxyethyl methacrylate, acid 
phosphoxypropyl acrylate, acid phosphoxypropyl methacrylate, etc.) or a 
salt thereof, as well as phosphated polyvinyl alcohol, starch phosphate 
and salts thereof. 
In the practice of the present invention, for example, a water-insoluble or 
water-insolubilized form of polymers as described above can be used as the 
water-insoluble polyanion of the present invention. For this purpose, such 
polymers may be used alone or in admixture. The term "water-insoluble 
polyanion" as used herein denotes water-insoluble polyanions obtained, for 
example, by crosslinking a polymer as described above according to 
conventional procedure so as to render them insoluble in water, 
water-insoluble polyanions obtained, in the practice of the present 
invention, by keeping a polymer as described above or an aqueous solution 
thereof at a temperature lower than its solidifying point, and the like. 
In the above-described water-insoluble polyanion, the anionic functional 
group is generally present in an amount of 1 to 70% by weight and 
preferably 5 to 60% by weight. 
As stated before, the water-insoluble polyanion of the present invention 
can be used in either solid or liquid form. Specific examples of 
water-insoluble polyanions in solid form include a hydrogel having a water 
content of, for example, not less than 100% by weight, a solid mass having 
a water content of less than 100% by weight, and the like. Water-insoluble 
polyanions in solid form can be prepared according to the method of 
crosslinking through ionic bonds (for example, by bringing a water-soluble 
salt of alginic acid into contact with an aqueous solution, such as an 
aqueous solution of calcium chloride, which can cause gelation of the 
salt) or the method of crosslinking through covalent bonds by means of 
radical reaction, condensation reaction or the like (for example, by 
mixing acrylic acid with a radical crosslinking agent and then subjecting 
the mixture to radical polymerization). It is to be understood that 
water-insoluble polyanions obtained by introducing an anionic group into 
crosslinked polymers (for example, water-soluble polyanions obtained by 
introducing the sulfonic acid group into crosslinked polymers prepared 
from styrenedivinylbenzene or phenol-formaldehyde) can also be used in the 
present invention. Further practicable methods for obtaining 
water-insoluble polyanions in solid form are, for example, to cool 
.kappa.-carrageenan to a temperature lower than its solidifying point, to 
keep a polyethylene resin having the sulfonic acid group introduced 
thereinto at a temperature lower than its solidifying point, and the like. 
Where a water-insoluble polyanion in solid form is used in the present 
invention, any desired shape can be given thereto according to the 
intended purpose. For example, a granular water-insoluble polyanion can be 
prepared by adding a water-soluble salt of alginic acid dropwise to a 
gelling solution therefor. Moreover, a sheet-like or filmy water-insoluble 
polyanion can be prepared by spreading a mixture of acrylic acid and a 
radical crosslinking agent on a shallow dish and then polymerizing the 
mixture. Thus, according to the intended purpose, the water-insoluble 
polyanion of the present invention can be shaped, for example, into 
sheets, films, granules, fibers, tubes, hollow fibers, nets and the like. 
In the practice of the present invention, the water-insoluble polyanion can 
also be used in the form of a suspension. Such a suspension can be 
prepared, for example, by suspending a water-insoluble form of a solid or 
liquid polymer as described above in water or by dissolving or suspending 
it in a water-immiscible solvent and then suspending the resulting 
solution or suspension in water. 
In immobilizing an enzymatically active material according to the method of 
the present invention, the enzymatically active material is brought into 
contact with a water-insoluble polyanion. Where the water-insoluble 
polyanion is used in solid form, the enzymatically active material can be 
brought into contact with the surfaces of the water-insoluble polyanion. 
Where the water-insoluble polyanion is present in the liquid phase, the 
enzymatically active material may be suspended in the liquid phase. 
Alternatively, a solution or suspension of the enzymatically active 
material in water or an aqueous solution may be brought into contact with 
the water-insoluble polyanion. Subsequently to this step, the 
water-insoluble polyanion in contact with the enzymatically active 
material is brought into contact with a polycation-containing aqueous 
medium to complete the immobilization procedure. In another embodiment, 
the immobilization procedure can be carried out in one step by dissolving 
or suspending the enzymatically active material in a polycation-containing 
aqueous medium and bringing the resulting solution or suspension into 
contact with the water-insoluble polyanion. It is to be understood that, 
during the above-described immobilization procedure, one or more 
substances which do not interfere with the practice of the present 
invention may be present in the enzymatically active material, the aqueous 
solution or suspension thereof and the polycation-containing aqueous 
medium. 
No particular limitation is placed on the manner in which the enzymatically 
active material is brought into contact with the water-insoluble 
polyanion, provided that they can substantially contact each other. For 
example, the enzymatically active material may be sprinkled on the 
water-insoluble polyanion. In the embodiment in which a solution or 
suspension of the enzymatically active material in water or an aqueous 
solution and the polycation-containing aqueous medium are brought into 
contact with the water-insoluble polyanion, it is preferable to immerse or 
suspend the water-insoluble polyanion in the polycation-containing aqueous 
medium. In the above-described reaction, the water-insoluble polyanion and 
the polycation-containing aqueous medium are used in such a proportion 
that the ratio of the equivalents of the cationic group of the latter to 
the equivalents of the anionic group of the former is in the range of 0.01 
to 100 and preferably in the range of 0.1 to 10. If the amount of the 
cationic group is too small, the gel does not harden to a full degree. On 
the contrary, if the amount of the cationic group is too large, the method 
becomes uneconomical. 
The above-described contact reaction is carried out at a temperature in the 
range of -20.degree. to 100.degree. C. and preferably in the range of 
0.degree. to 80.degree. C. In the practice of the present invention, the 
pH of the water, aqueous solution or aqueous medium used to dissolve or 
suspend the enzymatically active material may vary widely, depending on 
the nature of the enzymatically active material. However, it is generally 
in the range of 1 to 12 and preferably in the range of 2 to 9. 
According to the present method of immobilizing enzymatically active 
materials, the immobilized preparation of enzymatically active material 
resulting from the above-described reaction may further be brought into 
contact with an aqueous medium containing a polyanion and, if desired, the 
enzymatically active material, whereby a novel immobilized preparation of 
enzymatically active material is obtained. Specifically, an enzymatically 
active material can be immobilized in layers by using a 
polyanion-containing aqueous medium and a polycation-containing aqueous 
medium alternately. 
The immobilized preparation of enzymatically active material obtained by 
the method of the present invention can be stably stored by washing it 
with a buffer solution, if necessary, and keeping it at a temperature 
ranging from -20.degree. C. to room temperature. 
Since the immobilized preparation of enzymatically active material obtained 
by the method of the present invention is insoluble in water, the 
enzymatic reaction can be continuously carried out for a long period of 
time by packing it into a column and passing therethrough a solution of 
the substrate. Also by using it batchwise, the same enzymatic reaction can 
be carried out repeatedly. Moreover, since the method of the present 
invention allows the surface of an electrode to be coated with a film of 
an enzymatically active material and serves to immobilize an enzymatically 
active material on the outer or inner surface of a tubular body, its 
practical application in the fields of enzyme electrodes, medical 
analyzers and artificial organs is anticipated. Furthermore, the 
immobilized preparation of enzymatically active material obtained by the 
method of the present invention may be utilized in the field of 
fermentation to facilitate continuous and effective fermentation reaction. 
It is applicable to various types of fermentation. For example, where 
foaming is involved, it can be used in a fluidized-bed fermenter as well 
as a fixed-bed fermenter.

The present invention is further illustrated by the following examples. 
However, these examples are not to be construed to limit the scope of the 
invention. 
EXAMPLE 1 
A solution was prepared by dissolving 2 g of 
poly(vinylbenzyl-trimethylammonium chloride) in 100 ml of physiological 
saline. After this solution was sterilized by steam at 120.degree. C. for 
15 minutes, 50 loopfuls of J.B.A. (Japan Brewer's Association) No. 6 yeast 
was added thereto and suspended therein. 
On the other hand, a solution was prepared by dissolving 10 g of sodium 
2-acrylamido-2-methylpropanesulfonate and 1 g of 
N,N'-methylenebisacrylamide in 89 g of water, and nitrogen was bubbled 
therethrough at 5.degree. C. for an hour. To this solution were added 1 ml 
of a 0.05% aqueous solution of ammonium persulfate and 0.04 ml of 
N,N,N',N'-tetramethylethylenediamine. After nitrogen was bubbled through 
the aqueous solution for 30 minutes, polymerization reaction was effected 
at 30.degree. C. for 30 minutes and then 70.degree. C. for an hour. Ten 
grams of the polymer obtained in the form of a hydrogel was cut into cubes 
of 3 mm size and then sterilized at 120.degree. C. for 15 minutes. 
Then, these hydrogel cubes were aseptically added to and immersed in the 
aforesaid yeast suspension in the poly(vinylbenzyltrimethylammonium 
chloride) solution, which was kept at 20.degree. C. for an hour. At the 
end of this period, the yeast suspension was separated and the hydrogel 
cubes were washed twice with previously sterilized physiological saline. 
Thereafter, they were added to and immersed in 100 ml of a culture medium 
for yeast which had been sterilized at 120.degree. C. This culture medium 
contained 10% of glucose, 0.15% of yeast extract, 0.25% of ammonium 
chloride, 0.1% of sodium chloride, 0.55% of dipotassium phosphate, 0.01% 
of magnesium sulfate, 0.001% of calcium chloride and 0.3% of citric acid. 
After its temperature was maintained at 30.degree. C. for 40 hours, the 
culture medium had an ethanol concentration of 2.5%. Moreover, yeast 
colonies were observed in the vicinity of the surfaces of the hydrogel 
cubes. 
EXAMPLE 2 
A solution was prepared by dissolving 2 g of sodium alginate (manufactured 
and sold by Kamogawa Kasei Co. under the trade name of "Duck Algin NSPM") 
in 98 g of water. After being sterilized at 120.degree. C. for 15 minutes, 
this solution was aseptically added dropwise to a 5% aqueous solution of 
calcium chloride which had been sterilized at 120.degree. C. for 15 
minutes, so that granular calcium alginate gel was obtained. This gel was 
aseptically packed into a jacketed glass column having a diameter of 2 cm 
and a height of 10 cm. 
On the other hand, a nutrient medium containing 0.5% of glucose, 1.25% of 
yeast extract, 1.0% of peptone, 0.5% of meat extract and 0.5% of sodium 
chloride (pH 7.0) was inoculated with Serratia marcescens and then shaken 
at 30.degree. C. for 16 hours. In 500 ml of the resulting culture medium 
having microbial cells suspended therein was dissolved 1.0 g of 
polyamine-sulfone (manufactured and sold by Toyobo Co. under the trade 
name of "PAS-H-40") which had previously been sterilized at 120.degree. C. 
for 15 minutes. 
This solution was passed through the aforesaid column from the bottom at a 
flow rate of 8 ml/hr, its internal temperature being kept at 30.degree. C. 
After 24 hours, the passage of the solution was discontinued. On the other 
hand, 2% of "PAS-H-40" was dissolved in a fresh medium having the same 
composition as described above but containing no microbial cells. This 
solution was sterilized at 120.degree. C. for 15 minutes and then passed 
through the column at a flow rate of 8 ml/hr, its internal temperature 
being kept at 30.degree. C. After 60 hours, the effluent emerging from the 
outlet of the column had an isoleucine concentration of 2.3 mg/ml. 
Moreover, colonies were observed both in the vicinity of the surfaces of 
the gel and the interior of the gel. 
EXAMPLES 3 
A solution was prepared by dissolving 4 g of .kappa.-carrageenan 
(manufactured and sold by Sansho Co. under the trade name of "Genugel-WG") 
in 96 g of water. After being sterilized at 120.degree. C. for 15 minutes, 
this solution was kept at 50.degree. C. and aseptically added dropwise to 
a 5% aqueous solution of calcium chrloride (at 20.degree. C.) which had 
been sterilized at 120.degree. C. for 15 minutes, so that granular calcium 
alginate gel was obtained. This gel was aseptically packed into a jacketed 
glass column having a diameter of 2 cm and a height of 10 cm. 
On the other hand, a culture medium having Serratia marcescens suspended 
therein was prepared in the same manner as described in Example 2. In 500 
ml of the resulting culture medium was dissolved 20 g of 
poly(4-vinyl-1-methylpyridinium chloride) which had previously been 
sterilized at 120.degree. C. for 15 minutes. 
This solution was passed through the aforesaid column from the bottom at a 
flow rate of 8 ml/hr, its internal temperature being kept at 30.degree. C. 
After 24 hours, the passage of the solution was discontinued. On the other 
hand, 4% of poly(4-vinyl-1-methylpyridinium chloride) was dissolved in a 
fresh medium having the same composition as described in Example 2 but 
containing no microbial cells. This solution was sterilized at 120.degree. 
C. for 15 minutes and then passed through the column at a flow rate of 8 
ml/hr, its internal temperature being kept at 30.degree. C. After 60 
hours, the effluent emerging from the outlet of the column had an 
isoleucine concentration of 2.0 mg/ml. Thereafter, a fresh medium having 
the same composition as described in Example 2 but containing no 
polycation was passed through the column. After 60 hours, the effluent 
emerging from the outlet of the column had an isoleucine concentration of 
2.5 mg/ml.