Method for immobilizing biocatalyst in granular form with a photo-crosslinkable resin

A biocatalyst is immobilized with a graft product of a polymer and saponified polyvinyl acetate containing a stilbazolium group as a photo-crosslinking group. The polymer is preferably gelatin, collagen, starch, cellulose, gum arabic, tragacanth gum, carrageenan, mannan, dextrin or alginic acid. The graft product is prepared by bonding the polymer to the polyvinyl acetate, either directly through functional groups of the polymer and polyvinyl acetate or through a crosslinking agent. The polymer provides affinity for the biocatalyst and by grafting the polymer to the saponified polyvinyl acetate, damage to cells upon immobilization is reduced. A liquid composition containing a biocatalyst and the graft product is added to an aqueous solution of an inorganic salt or an organic salt to form a granular gel and the gel is irradiated with actinic rays to cure the gel.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method of immobilizing a biocatalyst in 
the form of a granular formed body by using a water-dispersible 
photo-crosslinkable resin. 
2. Description of the Related Art 
As the biocatalyst to be immobilized, there can be mentioned animal cells, 
mammalian cells, plant cells, microbial cells and enzymes. 
Attempts have been made to produce valuable substances by immobilizing 
biocatalysts by various polymers, but since the number of immobilizing 
polymers having a high affinity with biocatalysts is limited, a practical 
utilization of this technique is difficult. 
The conventional technique will now be described, with reference to an 
animal cell as an example. 
Intensive investigations have been made into the producing of many valuable 
substances by culturing animal cells in liquid culture media and some 
thereof have been industrially utilized. 
As the method of culturing an animal cell, there can be mentioned not only 
a culturing method in which an animal cell is merely suspended in a liquid 
medium but also a gel entrapping immobilization culturing method in which 
an animal cell is entrapped in an aqueous gel and the immobilized cell is 
cultured in a liquid culture medium to effect a propagation of the cell in 
the gel. The latter method is advantageous in various points. For example, 
the animal cell which is mechanically weak or brittle against changes of 
the liquid properties can be protected by a gel layer during the 
culturing, and a solid-liquid separation between the cell and liquid, 
which is necessary for an exchange of the culture medium or recovery of 
the intended product after the culturing, can be performed very 
advantageously. Furthermore, an industrial production system can be easily 
established. For example, the intended substance can be continuously 
obtained by filling the gel which includes the immobilized cell in a 
bioreactor. Therefore, serious research has been made into materials for 
and methods of immobilizing animal cells. 
Since animal cells are weak and brittle, natural substances such as alginic 
acid, carrageenan, mannan and gelatin are used as the immobilizing gel, 
but since the durability of the gel strength is poor, these gels are not 
industrially utilized, and research has been made with a view to improving 
the durability thereof. For example, there can be mentioned a method in 
which the kind of the salt is changed, to improve the durability of the 
gel strength at the entrapping immobilization of an animal cell with 
alginic acid, a method in which an animal cell-immobilizing gel is caused 
to flow in a reactor, to prevent a breaking of the gel, and a method in 
which, after the inclusion of an animal cell and an alginic acid gel, a 
synthetic polymer film is formed on the gel surface. Under this 
background, the development of a method in which a gel capable of 
entrapping an animal cell by an industrially applicable simple means, 
which can be used stably, can be formed, is urgently required. 
SUMMARY OF THE INVENTION 
It is therefore a primary object of the present invention to solve the 
foregoing problems of the conventional methods and provide a gel 
entrapping method in which an intended product is produced in a high yield 
over a long period without deactivation of cells in an animal cell colony 
formed by propagation in a gel. 
In accordance with the present invention, there is provided a method of 
immobilizing a biocatalyst in a granular form, which comprises dropping a 
liquid composition comprising a graft product of a natural polymer and 
saponified polyvinyl acetate, containing a stilbazolium group as the 
photo-crosslinking group, and a biocatalyst into an aqueous medium 
containing an inorganic salt or an organic salt to gelatinize the liquid 
composition in the granular form, and irradiating the obtained granular 
gel with actinic rays to cure the photosensitive graft product of the 
natural polymer and saponified polyvinyl acetate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention will now be described in detail. 
Preparation of Liquid Composition 
The main skeleton of the stilbazolium photo-curable resin used in the 
present invention is a graft product of a natural polymer and saponified 
polyvinyl acetate. The introduction ratio of the natural polymer is 
preferably 1 to 2000 parts by weight per 100 parts by weight of saponified 
polyvinyl acetate. 
As the natural polymer, there can be mentioned collagen, gelatin, casein, 
starch, cellulose, gum arabic, tragacanth gum, carrageenan, mannan, 
dextrin, alginic acid and derivatives of these natural polymers. Of these 
natural polymers, gelatin and collagen derived from animals are preferably 
used. 
A natural polymer has an inherent good affinity with a biocatalyst, and by 
grafting the natural polymer to saponified polyvinyl acetate, damage to 
cells upon the immobilization of the biocatalyst can be reduced, and the 
propagation of the cell in a gel support by culturing greatly enhanced. 
Accordingly, if the introduction ratio of the natural polymer is too low, 
the above effect is reduced, and if the introduction ratio of the natural 
polymer exceeds 2000 parts by weight, the content of saponified polyvinyl 
acetate is relatively reduced and the concentration of the stilbazolium 
group that can be added to saponified polyvinyl acetate is reduced, and 
therefore, the density of the photofunctional group is low at the 
formation of a gel by irradiation with actinic rays, and a 
biocatalyst-included gel having a high strength cannot be obtained. 
As the method of grafting and integrating the natural polymer and 
saponified polyvinyl acetate, there can be adopted a method in which the 
natural polymer is directly bonded to saponified polyvinyl acetate by 
utilizing functional groups thereof, such as hydroxyl, carboxyl and amino 
groups, a method in which the natural polymer is bonded to saponified 
polyvinyl acetate by using a crosslinking agent such as glutaraldehyde, or 
a method in which a polymerizable monomer such as vinyl acetate is 
polymerized in the presence of a mixture of the natural polymer and 
saponified polyvinyl acetate, as the protecting colloid to effect the 
bonding integration indirectly. 
The amount of the photo-crosslinking stilbazolium group to be added to the 
photo-curable resin is preferably 5 to 100 parts by weight per 100 parts 
by weight of saponified polyvinyl acetate contained in the graft product 
of the natural polymer and saponified polyvinyl acetate as the main 
skeleton of the photo-curable resin. If the addition ratio of the 
stilbazolium group is low, the ratio of reaction by irradiation with 
actinic rays is reduced, and therefore, the gel strength is reduced and 
unreacted low-molecular-weight resin composition remains in the gel and 
has an adverse influence on the entrapped biocatalyst. If the addition 
ratio of the stilbazolium group is too high, the properties of the 
photo-curable resin are changed and an economical disadvantage arises. 
The thus-synthesized stilbazolium type photo-curable resin can be purified 
by solvent fractionation or can be directly used for the immobilization of 
the biocatalyst. When the solvent fractionation or the like is adopted, it 
is necessary to sufficiently remove the used solvent so that the solvent 
does not remain in the photo-curable resin, and an adverse influence 
cannot be imposed on the biocatalyst. 
The kind of the biocatalyst that can be immobilized according to the method 
of the present invention is not particularly critical, and as the 
biocatalyst, there can be mentioned animal cells, mammalian cells, plant 
cells, microbial cells and enzymes. Normal cells and tumor cells can be 
used as the animal and plant cells, or hybridoma having an enhanced 
substance productivity of cells per se by cell fusion, cell technology or 
genetic engineering can be used. As the microbial cell, there can be used 
yeasts, bacteria, molds, actinomycetes, basidiomycetes and all of other 
microorganisms. As specific examples of the yeast, there can be mentioned 
baker's yeast, wine yeast, sake yeast, Shizosaccharomyces pombe and 
Rhodotorula clutinis. 
As the enzyme, there can be mentioned, for example, hydrolases such as 
amylase, protease, cellulase, hemicellulase, lipase, pectinase, lysozyme, 
naringenase, hesperidinase, anthocyanase, aminoacylase, urease, invertase, 
melibiase, dextrase, peptitase, ribonuclease and lactase, oxidoreductases 
such as glucose oxidase, uricase, catalase, lipoxygenase and cytochrome c 
peroxidase, isomerases such as glucose isomerase, transferases such as 
cyclodextrin glucocyl transferase and transaminase, and lyases such as 
aspartase, hyaluronidase, chondroitinase and pectin eliminase. 
A liquid composition is prepared by mixing a biocatalyst as mentioned above 
with the stilbazolium type photocurable resin. An aqueous solution of 
sodium alginate can be added to the liquid composition, whereby an effect 
of enhancing the granule-forming property can be attained when the liquid 
composition is dropped into an aqueous medium containing a salt. The 
amount added of sodium alginate is preferably 1 to 50% based on the solids 
of the photo-curable resin. The cell concentration in the initial 
biocatalyst in the thus-obtained liquid composition is preferably 10.sup.4 
to 10.sup.7 cells/ml. 
Granulation 
The liquid composition prepared from the graft product of the natural 
polymer and saponified polyvinyl acetate, in which the stilbazolium group 
is introduced as the photo-crosslinking group, and the biocatalyst is 
dropped into an aqueous medium containing an inorganic salt or organic 
salt, whereby the liquid composition is gelatinized in the granular form. 
If the inorganic or organic salt is present in the aqueous medium at a 
concentration of 0.001 to 5 moles/1, preferably 0.05 to 0.5 mole/1, the 
kind or valency of the salt is not particularly critical. 
As specific examples of the inorganic salt, there can be mentioned halides, 
carbonates, bicarbonates, sulfates and nitrates of alkali metals such as 
sodium and potassium, alkaline earth metals such as calcium and barium, 
aluminum and trivalent iron; among these, alkali metal salts are 
preferably used. These inorganic salts can be used alone or in the form of 
a mixture of two or more thereof. Furthermore, natural sea water or 
artificial sea water can be used. 
As specific examples of the organic salt, there can be mentioned onium 
salts such as tetra-n-butyl ammonium bromide, and aniline hydrochloride, 
piperidine hydrochloride, hydrazine sulfate and phenylhydrazine sulfate. 
Where a liquid composition comprising the photo-crosslinkable graft product 
of the natural polymer and saponified polyvinyl acetate, sodium alginate 
and the biocatalyst is dropped into an aqueous medium containing an 
inorganic salt to gelatinize the liquid composition into the granular 
form, an aqueous medium containing an inorganic salt as mentioned above 
can be used, and an alkaline earth metal salt is preferably used. 
Dropping of the above-mentioned liquid composition into an aqueous medium 
containing an inorganic or organic salt as mentioned above is 
accomplished, for example, by a method in which the liquid composition is 
dropped from the top end of a tube having a sharp edge, such as a hydermic 
needle, a method in which the liquid composition is scattered in the form 
of granules by utilizing the centrifugal force, or a method in which the 
liquid composition is atomized from the top end of a spray nozzle. The 
size of drops of the liquid composition can be changed according to the 
particle size desired for the final granular immobilization product, but 
for the reasons set forth above, the diameter of the liquid drops is 
preferably 0.5 to 5 mm. 
Instead of the method of immobilizing the biocatalyst on the granulation 
immobilization carrier, there can be effectively adopted a method in which 
the biocatalyst is immobilized on a membrane comprising the graft product 
of the natural polymer and saponified polyvinyl acetate having the 
stilbazolium group as the photo-crosslinking group according to the 
present invention. 
Photo-curinc 
If the thus-formed granular gel is irradiated with actinic rays while 
dispersed in the aqueous medium or separated therefrom, 
crosslinking-curing is accomplished by the stilbazolium group added to the 
grafting product of the natural polymer and saponified polyvinyl acetate 
contained in the granular gel, whereby the granular gel is converted to a 
water-soluble granular immobilization product. 
Preferably, the wavelength of the actinic rays used for the photo-curing is 
in a region not imposing an adverse influence on the biocatalyst. Namely, 
rays having a wavelength shorter than about 320 nm are preferably 
eliminated. As examples of the light sources emitting such rays, there can 
be mentioned a low-pressure mercury lamp, a high-pressure mercury lamp, a 
fluorescent lamp, a xenon lamp, and sunlight. The irradiation time should 
be changed according to the ray intensity of the light source, the 
distance from the light source and the like, but in general, the 
irradiation time can be 1 to 5 minutes. Furthermore, the actinic rays must 
be applied to the formed granular gel as uniformly as possible. 
After the irradiation treatment, the treated granular gel can be washed 
with water or an aqueous buffer solution and then stored, or can be 
directly used for culturing. 
EXAMPLES 
The present invention will now be described in detail with reference to the 
following examples, that by no means limit the scope of the invention. 
EXAMPLE 1 
In 100 g of an aqueous solution of an emulsion of polyvinyl acetate 
obtained by polymerizing vinyl acetate according to customary procedures 
by using, as the protecting colloid, a mixture of gelatin [jelly strength 
(6.66% aqueous solution) of 259 g, viscosity (6.66% aqueous solution) of 
42 mps, molecular weight of 100,000] and saponified polyvinyl acetate 
(saponification degree of 88 mole %, polymerization degree of 1400) was 
dissolved 2 g of N-methyl-4-(p-formylstyryl)pyridinium methosulfate, and 
addition condensation was carried out to synthesize a photosensitive 
emulsion. In the polyvinyl acetate emulsion aqueous solution, the 
gelatin/saponified polyvinyl acetate weight ratio was 15/85, the solid 
weight ratio of the protecting colloid in the total solids was 50%, and 
the solid concentration was 15%. To 20 parts by weight of the 
photosensitive emulsion was added 10 parts by weight of a cell suspension 
of PRM18204 derived from human leukocytes to form a liquid mixture having 
a cell concentration of 7.times.10.sup.5 cells/ml. The liquid mixture was 
charged into a syringe and was dropped into a 0.05M aqueous solution of 
sodium chloride while shaking the syringe, whereby granulation was 
effected. Then, the mixture was irradiated with rays having a main 
wavelength of 300 to 400 nm for 2 minutes to obtain a granular gel having 
a particle size of 1.5 mm. Thereafter, twenty immobilized animal cells 
were sampled and added to 10 ml of a culture medium formed by adding 10% 
of FCS to a PRM11640 culture medium, and culturing was conducted at 
37.degree. C. When the culturing was carried out for 2 days, the glucose 
concentration in the culture medium was reduced to 0.8 g/1 from 1.8 g/1. 
Example 2 
To 20 parts by weight of the photosensitive emulsion prepared in Example 1 
was added 40 parts by weight of a 1% aqueous solution of sodium alginate, 
and monoclonal antibody-producing mouse hybridoma strain 16-3F was added 
to the mixed resin liquid to form a liquid mixture having a cell 
concentration of 2.times.10.sup.6 cells/ml. The liquid mixture was dropped 
into a 0.1M aqueous solution of potassium chloride from a syringe to 
effect granulation. The liquid mixture was irradiated with rays having a 
main wavelength of 300 to 400 nm. The formed granular immobilized cells 
were transferred into a 0.lM aqueous solution of sodium citrate and stored 
in a refrigerator overnight. Then, 24 granular immobilized cells were 
sampled and added to 8 ml of DF/ITES culture medium, and exchange of the 
culture medium was performed every other day. After 22 days' culturing, 
the cell concentration in the granular gel became 1.times.10.sup.7 /ml of 
the gel, and the antibody value of the obtained culture liquid was 200. 
Comparative Example 1 
A photosensitive emulsion was synthesized by dissolving 2 g of 
N-methyl-4-(p-formylstyryl)pyridinium methosulfate in 100 g of an aqueous 
solution of an emulsion of polyvinyl acetate formed according to customary 
procedures by polymerizing vinyl acetate in the presence of saponified 
polyvinyl acetate (saponification degree of 88 mole %, polymerization 
degree of 1400) as the protecting colloid [the solid weight ratio of the 
protecting colloid based on the total solids was 50% and the solid 
concentration was 15%] and carrying out the addition condensation 
according to customary procedures. To 20 parts by weight of the 
photosensitive emulsion was added 10 parts by weight of a cell suspension 
of PRM18204 derived from human leukocytes to form a liquid mixture having 
a cell concentration of 7.times.10.sup.5 cells/ml. The liquid mixture was 
charged in a syringe and dropped into a 0.05M aqueous solution of sodium 
chloride while shaking the syringe, whereby granulation was effected. The 
liquid mixture was irradiated with rays having a main wavelength of 300 to 
400 nm for 2 minutes to obtain a granular gel having a particle size of 
1.5 mm. Then, 20 immobilized animal cells were sampled and added to 10 ml 
of a culture medium formed by adding 10% of FCS to RPM11640 culture medium 
and culturing was conducted at 37.degree. C. Even when culturing was 
carried out for 2 days, the glucose concentration in the culture medium 
was little changed from the initial level of 1.8 g/1. 
Comparative Example 2 
In 100 g of an aqueous solution of an emulsion of polyvinyl acetate 
obtained by polymerizing vinyl acetate according to customary procedures 
by using as the protecting colloid the same gelatin as used in Example 1 
(the solid weight ratio of the protecting colloid to the total solids was 
50% and the solid concentration was 15%) was dissolved 2 g of 
N-methyl-4-(p-formylstyryl)-pyridinium methosulfate, and the addition 
condensation was carried out according to customary procedures. The 
reaction liquid had no photo-crosslinkability. 
Comparative Example 3 
A 2% aqueous solution of sodium alginate was dropped into an aqueous medium 
containing 1% of potassium chloride from a hypodermic needle to form a 
granular gel having a diameter of about 2 mm. A bioreactor having an inner 
volume of 100 ml, into which germ-free air could be introduced from the 
bottom, was charged with 70 ml of BME culture medium, and about 10 ml of 
the granular gel was added into the reactor and fluidized by aeration. 
When the gel was fluidized at a temperature of 37.degree. C. for 24 hours, 
the granular gel was dissolved in the BME culture medium.