Immobilized catalytically active substance and method of preparing the same

An enzyme or microorganism is entrapped within the gel matrix of a sulfated polysaccharide (said polysaccharide containing more than 10 w/w% of a sulfate moiety in its molecule) in the presence of ammonium ion, a metal ion, a water-soluble amine or a water-miscible organic solvent. The immobilized enzyme or microorganism thus obtained shows a high level of catalytic activity for a long period of time and can be used for continuous enzymatic reactions with substrates.

This invention relates to an immobilized catalytically active substance and 
a method of preparing the same. 
Enzymes and microorganisms have been used as catalysts to induce various 
chemical or enzymatic reactions. For example, they have been used as 
catalysts, not only in preparing organic acids, amino acids, 
6-aminopenicillanic acid and so forth, but also in decomposing undesirable 
metabolic products such as urea. When they are used to catalyze reactions 
occuring in aqueous solutions, however, the separation or removal thereof 
from the reaction solution is conducted by boiling or acidifying said 
solution to denature the enzymes or microorganisms, and then filtering off 
the precipitates thereof. Thus, enzymes and microorganisms can be used 
only once and must be discarded thereafter. 
Immobilized enzymes and microorganisms (i.e., enzymes and microorganisms 
bound to carriers) have become of great importance in recent years. Such 
immobilized preparations can be used as heterogeneous catalysts and, after 
the reactions are completed, may be readily removed from the reaction 
mixtures. Further, the immobilized enzymes or microorganisms may be used 
repeatedly or continuously to induce specific chemical changes in large 
amounts of substrates. In this connection, various methods of binding 
enzymes or microorganisms to carriers have been used, including (a) 
covalent binding of the enzymes or microorganisms to water-insoluble 
carriers; (b) ionic binding of the enzymes or microorganisms to carriers; 
(c) physical adsorption of the enzymes or microorganisms to carriers; (d) 
covalent cross-linking of the enzymes or microorganisms by bifunctional 
agents; (e) inclusion of the enzymes or microorganisms within the gel 
lattice of polymers; and (f) microencapsulation of the enzymes or 
microorganisms with semipermeable polymer membranes. Typical examples of 
these methods are seen in Annual Review of Biochemistry, Vol. 35, Part II, 
pages 873-903 (1966); Biochemistry (USSR) (English Translation), Vol. 31, 
pages 337-345 (1966); U.S. Pat. No. 3,278,392; Enzymologia, Vol. 39, pages 
12-14 (1970); and Applied Microbiology, Vol. 27, pages 878-885 (1974). 
However, these immobilized enzymes or microorganisms, when packed in a 
column and used for reactions with substrates, tend to compact and deform 
thereby reducing the flow speed of the substrate solutions in the column. 
Further, it is known that microorganisms are entrapped within the gel 
matrix of Japan agar by cooling an aqueous mixture of said agar and 
microorganisms (Japanese patent application laid open to the public under 
No. 95470/1975). But said Japan agar having microorganisms entrapped 
therein is unable to retain its shape in an aqueous media. When used for 
enzymatic reactions at a temperature higher than 40.degree. C., it loses 
its network structure and is transformed into "sol" within a period as 
short as 60 minutes. 
Apart from the above-mentioned prior art, enzymes and microorganisms may be 
entrapped within the gel matrix of carrageenan (i.e., a sulfated 
polysaccharide) by simply cooling an aqueous mixture of carrageenan and 
enzymes or microorganisms. As in case of Japan agar, however, the gel 
matrix of carrageenan obtained by this method is still too soft and 
unstable to use it in enzymatic reactions with substrates. For example, 
the gel structure of carrageenan obtained by this method, when used for 
enzymatic reactions with substrates in aqueous media, becomes loose and 
results in causes leakage of enzymes or microorganisms therefrom. 
We have now found that a stable, immobilized, catalytically active 
substance can be obtained by entrapping enzymes or microorganisms within 
the gel matrix of a sulfated polysaccharide in the presence of ammonium 
ion, a metal ion, a water-soluble organic amine or a water-miscible 
organic solvent. In other words, when the immobilization or entrappment of 
enzymes and microorganisms with the sulfated polysaccharide is carried out 
in the presence of the above-mentioned ion, amine or solvent, the 
immobilized preparation thus obtained shows a high level of catalytic 
activity for a long period of time and at the same time the gel matrix 
thereof is sufficiently stable to prevent leakage of enzymes or 
microorganisms therefrom. Moreover, the sulfated polysaccharide, having 
enzymes or microorganisms entrapped therein in the presence of said ion, 
amine or solvent does not deform in an aqueous media. Even when used for 
enzymatic reactions by packing in a column, functions effectively so as to 
convert substrates to their conversion products. Further, we have found 
that the carrier-bound enzymes or microorganisms stated hereinbefore may 
also be employed for this immobilization method to improve the quality 
thereof. That is, the catalytic activity of enzymes or microorganisms 
bound to carriers can be maintained and the compacting or deformation 
thereof can be prevented or substantially reduced by entrapping the bound 
enzyme or microorganism within the gel matrix of said sulfated 
polysaccharide in the presence of ammonium ion, a metal ion, amine or a 
organic solvent. 
The term "carrier-bound enzyme or microorganism" as used herein means the 
product produced by binding an enzyme or microorganism to a carrier by any 
of the six mechanisms (a) through (f) mentioned above and described in 
said publications. 
According to the present invention, an immobilized catalytically active 
substance can be prepared by the steps of mixing a catalytically active 
substance with an aqueous solution of a sulfated polysaccharide, and 
contacting the aqueous mixture with ammonium ion, a metal ion, a 
water-soluble organic amine or a water-miscible organic solvent to give 
the gel matrix of the sulfated polysaccharide having the catalytically 
active substance entrapped therein. 
The sulfated polysaccharide employed in the present invention should 
contain more than 10 w/w %, preferably between about 12 and 62 w/w %, of 
the sulfate (-SO.sub.3 H) moiety in its molecule. Representative examples 
of such polysaccharide sulfate ester include carrageenan, furcellaran and 
cellulose sulfate. Carrageenan refers to galactose sulfate esters which 
are obtained by extracting with water the sea weeds belonging to 
Rhodophyceae such as Ginartinacea and Solieriaceae (e.g., Chondrus 
crispus, Gigartina acicularis, Eucheuma cottonii). Among said galactose 
sulfate esters, kappa-carrageenan and iota-carrageenan are especially 
suitable for use in the present invention. Kappa-carrageenan consists 
mainly of .beta.-D-galactopyranosyl-4-sulfate and 
3,6-anhydro-.alpha.-D-galactopyranose, and it contains between about 20 
and 30 w/w % of sulfate (SO.sub.3 H) moiety in the molecule. On the other 
hand, iota-carrageenan is mainly a mixture of 
.beta.-D-galactopyranosyl-4-sulfate and 
3,6-anhydro-.alpha.-D-galactopyranosyl-2-sulfate, and it contains between 
about 20 and 30 w/w % of the sulfate moiety in the molecule. Moreover, 
furcellaran is a sulfated polysaccharide which is obtained by extracting 
the the sea weeds of Furcellariaceae (e.g., Furcellaria fastigiana) with 
water, and consists mainly of D-galactose, 3,6-anhydro-D-galactose and the 
half-ester sulfate of these sugars. Furcellaran contains between about 12 
and 16 w/w % of sulfate moiety in its molecule. Other suitable sulfated 
polysaccharides which may be employed in the present invention include 
cellulose sulfate containing between about 12 and 62 w/w % of the sulfate 
moiety in the molecule. Such cellulose sulfate is available in the market 
under the trade name "KELCO SCS" (KELCO Co., U.S.A.) (sulfate content: 
about 53%) or, if required, may be prepared by conventional esterification 
of cellulose with sulfuric acid. 
In the present invention, a wide variety of enzymes may be employed as one 
of the catalytically active substances. Examples of such enzymes include 
oxidoreductase such as amino acid oxidase, catalase, xanthin oxidase, 
glucose oxidase, glucose-6-phosphate dehydrogenase, glutamate 
dehydrogenase, cytochrome C oxidase, tyrosinase, lactate dehydrogenase, 
peroxidase, 6-phosphogluconate dehydrogenase and malate dehydrogenase; 
transferases such as aspartate acetyltransferase, aspartate 
aminotransferase, glycine aminotransferase, glutamic oxalacetic 
aminotransferase, glutamic pyruvic aminotransferase, creatine 
phosphokinase, histamine methyltransferase, pyruvate kinase, hexokinase, 
.epsilon.-lysine acetyltransferase and leucine aminopeptidase; hydrolases 
such as asparaginase, acetylcholine esterase, aminoacylase, amylase, 
arginase, L-arginine deiminase, invertase, urease, uricase, urokinase, 
esterase, kallikrein, chymotrypsin, trypsin, thrombin, naringinase, 
nucleotidase, papain, hyaluronidase, plasmin, pectinase, hesperidinase, 
pepsin, penicillinase, penicillin amidase, phospholipase, phosphatase, 
lactase, lipase, ribonuclease and renin; lyases such as aspartate 
decarboxylase, aspartase, citrate lyase, glutamate decarboxylase, 
histidine ammonia-lyase, phenylalanine ammonia-lyase, fumarase, fumarate 
hydratase and malate synthetase; isomerases such as alanine racemase, 
glucose isomerase, glucose-phospate isomerase, glutamate racemase, lactate 
racemase and methionine racemase; and lygases such as asparagine 
synthetase, glutathion synthetase, glutamine synthetase and pyruvic acid 
synthetase. The above-mentioned enzymes are not necessarily pure, but 
crude enzyme solutions may be employed in the present invention. For 
example, the extracts of animal or plant tissues and the cell-free 
extracts of microorganisms may be preferably used as the enzyme solution. 
These extracts may be, of course, partially purified prior to using in the 
present invention. A mixture of two or more enzymes mentioned above may 
also be employed for the purpose of the present invention. 
Catalytically active microorganisms such as bacteria, yeast, mold, lichens 
and protozoa are other examples of the catalytically active substances 
which are employed in the present invention. Any microorganisms which 
accumulate at least one of the above-mentioned enzymes within their living 
cells may be used as the catalytically active microorganisms of the 
invention. The microorganisms which can be used include, for example, 
Achromobacter aquatilis, Achromobacter liquidum, Aspergillus oryzae, 
Aspergillus niger, Bacillus megatherium, Bacillus subtilis, Bacterium 
succinium, Brevibacterium ammonia-genes, Brevibacterium flavum, 
Corynebacterium glutamicum, Erwinia herbicola, Escherichia coli, 
Gluconobacter melanogenus, Lactobacillus bulgaris, Micrococcus ureae, 
Penicillium vinaceum, Proteus vulgaris, Pseudomonas aeruginosa, 
Pseudomonas dacunhae, Pseudomonas putidum, Sarcina lutea, Streptomyces 
griseus, Serratia marcescens, Streptomyces phaeochromogenus, and Baker's 
yeast. These microorganisms may not necessarily be intact living cells, 
but may be lyophilized, heat-treated or treated with acetone prior to use 
thereof in the present invention. 
Further, the carrier-bound enzyme or microorganism which is entrapped 
within the gel matrix of the sulfated polysaccharide according to the 
present invention comprises a water-insoluble, hydrophilic carrier having 
a catalytically active enzyme or microorganism bound thereto. The term 
"hydrophilic" as used herein means that the carrier is made wettable or 
swellable in water but is not substantially soluble therein. Any carriers 
having these properties may be utilized herein and the enzyme or 
microorganism may be bound thereto in any known manner, i.e., those 
mentioned hereinbefore. For example, polymers which may be used as 
carriers for the enzymes include diazotized p-aminobenzyl cellulose, 
diazotized p-aminobenzoyl cellulose, diazotized m-aminobenzyloxymethyl 
cellulose, the diazotized copolymer of p-aminophenylalanine and leucine, 
diazotized poly-p-aminostyrene, carboxymethyl cellulose, aminoethyl 
cellulose, carboxymethyl cross-linked dextran, carboxymethyl cellulose 
azide, ethylene-maleic anhydride copolymers, carboxymethyl cellulose 
isocyanate, cyanogen bromide-activated cellulose, cyanogen 
bromide-activated agarose, the aminosillan derivative of porous glass, 
bromoacetyl cellulose, iodoacetyl cellulose, dichloro-s-triazinyl 
cellulose, methacrylic acid-methacrylic acid-3-fluoro-4,6-dinitroanilide 
copolymers, diethylaminoethyl cellulose, triethylaminoethyl cellulose, 
diethylaminoethyl cross-linked dextran, acrylamide-methylenebisacrylamide 
copolymer, polyvinylalcohol, nylon, polyurea and the like. Examples of 
other carriers which may be used include charcoal, porous glass, acid 
clay, kaolinite, alumina and the like. On the other hand, polymers which 
may be used as carriers for the catalytically active microorganisms 
include acrylamide-methylenebisacrylamide copolymer, cellulose triacetate, 
carboxymethyl cellulose, diethylaminoethyl cellulose and the like. This 
listing is no way to be considered as all-inclusive and any other known 
carriers may also be used herein. Binding the enzyme or microorganisms to 
the hydrophilic carriers may be accomplished in a known manner, i.e., 
those described in the aforementioned publications. For example, when a 
carrier containing some groups which react with enzymes is employed, the 
enzymes react with the carrier at a temperature below which the enzymes 
are deactivated. The temperature at which specific enzymes are deactivated 
are well known to those skilled in the art and therefore need not be 
enumerated herein. Suffice to say that generally a temperature below 
75.degree. C., preferably between about 5.degree. C. and 65.degree. C., 
should be used. The reaction is preferably carried out in the presence of 
buffers to control the pH of the reaction mixture at a desired level, the 
particular pH being governed by the particular enzyme being bound, 
according to known techniques. 
In carrying out the method of the present invention, an aqueous mixture of 
the sulfated polysaccharide and the catalytically active substance (i.e., 
the catalytically active enzyme or microorganism of the water-insoluble, 
hydrophilic, catalytically active carrier-bound enzyme or microorganism) 
may be prepared by any appropriate method. For example, the aqueous 
mixture may be readily obtained by dissolving the sulfated polysaccharide 
in water at a temperature of between about 30.degree. C. and 90.degree. 
C., and then mixing the sulfated polysaccharide solution with the 
catalytically active substance. Alternatively, said active substance may 
be employed in the form of a solution or suspension. For example, the 
catalytically active substance may be dissolved or suspended in water, a 
physiological saline solution or a buffer solution, and said solution or 
suspension may be added to the aqueous sulfated polysaccharide solution. 
Buffer solutions such as phosphate buffer solution, carbonate buffer 
solution and acetate buffer solution which are adjusted to a pH of between 
about 1 and 13, especially a pH of between about 4 and 10. These can be 
employed to dissolve or suspend said active substance therein. A suitable 
amount of the sulfated polysaccharide in the aqueous mixture is between 
about 0.05 and 20 w/w %, especially between about 0.4 and 10 w/w %. On the 
other hand, a suitable amount of the catalitically active substance for 
mixture with the sulfated polysaccharide is between about one mg and 50 g, 
especially between about 100 mg and 10 g, per gram of said sulfated 
polysaccharide. In admixing with the catalytically active substance or a 
solution or suspension thereof, it is preferred to use an aqueous 
polysaccharide solution the temperature of which is kept at a temperature 
of between about 30.degree. and 70.degree. C. Moreover, if required, 
proteins such as gelatin, collagen, albumin, globulin, zein, fibrinogen, 
myosin and casein; polysaccharides such as starch, cellulose and dextran; 
gums such as locust been gum, arabic gum, tragacanth gum, guar gum and 
Psylliium seed gum; alcohols such as glycerol, ethyleneglycol and 
polyethylene glycol; or synthetic polymers such as polyvinylalcohol and 
polyvinylpyrrolidone may be added to the above-mentioned aqueous mixture. 
That is, the ability of the sulfated polysaccharide gel to retain its 
shape in an aqueous media can be increased by adding between about 10 and 
250 w/w % (based on the weight of the sulfated polysaccharide) of these 
materials to said aqueous mixture. Water-insoluble, inorganic porous 
materials may also be added to the aqueous mixture of the sulfated 
polysaccharide and the catalytically active substance in order to control 
the macromolecular structure or pore size of the sulfated polysaccharide 
gel matrix. Sellaite, siliceous sand, bentonite and activated charcoal are 
suitable as said water-insoluble, inorganic, porous materials. 
Additionally, if required, glutaraldehyde, tannins and the like may be 
further added to the aqueous mixture of the sulfated polysaccharide and 
the catalytically active substance prior to the subsequent contacting 
step. Addition of glutaraldehyde and tannins may be effective to increase 
the particle size of the enzymatically active substance thereby preventing 
the leakage of said active substance from the gel. 
The aqueous mixture of the sulfated polysaccharide and the catalytically 
active substance is then contacted with ammonium ion, a metal ion having 
an atomic weight greater than 24, a water-soluble organic amine or a 
water-miscible organic solvent. Suitable examples of the metal ion which 
may be employed in the invention include alkali metal ions belonging to a 
group not lower than Series (4) in Mendeleev's Periodic Table (e.g., 
potassium, rubidium, cesium ions), alkali earth metal ions (e.g., 
magnesium, calcium, strontium, barium ions), aluminium ion, lead ion, 
manganese ion, ferric ion and ferrous ion. On the other hand, organic 
amines which are soluble in water may be employed. Such amines include, 
for example, an alkylenediamine of one to 20 carbon atoms (e.g., 
methylenediamine, N-methyl-methylenediamine, N,N-dimethylmethylenediamine, 
ethylenediamine, trimethylenediamine, tetramethylenediamine, 
pentamethylenediamine, hexamethylenediamine, heptamethylenediamine, 
octamethylenediamine, nonamethylenediamine, decamethylenediamine, 
dodecamethylenediamine, eicosamethylenediamine); a phenylenediamine (e.g., 
p-phenylenediamine, m-phenylenediamine, o-phenylenediamine, 
dimethyl-p-phenylenediamine); a hydroxamate, hydrazide, alkyl ester or 
amide of a basic amino acid (e.g., lysine hydroxamate, histidine 
hydroxamate, tryptophan hydroxamate, lysine hydrazide, histidine 
hydrazide, lysine methyl ester, histidine methyl ester, lysine amide); a 
S-aminoalkyl-cysteine (e.g., S-aminomethyl-cysteine, 
S-(2-aminoethyl)-cysteine); an aminoalkylguanidine (e.g., agmatine); 
.delta.-hydroxy-lysine; melanine; an aminoalkyl-imidazole (e.g., 
histamine); an aminoalkyl-indol (e.g., serotonine); a 
guanidinoalkyl-aniline (e.g., 2-guanidinomethyl-aniline); an 
aminoalkyl-pyrrole (e.g., 2-aminomethyl-pyrrole); an aminoalkylpyrroline 
(e.g., 2-aminomethyl-pyrroline); an aminoalkylpyrrolidine (e.g., 
2-aminomethyl-pyrrolidine); an aminoalkylthiazole (e.g., 
2-aminomethyl-thiazole); an aminoalkyl-pyrazole (e.g., 
3-aminomethyl-pyrazole); an aminoalkyl-thiazoline (e.g., 
5-aminomethyl-thiazoline); an aminoalkyl-thiazolidine (e.g., 
2-aminomethyl-thiazolidine); an aminoalkyl-pyridine (e.g., 
2-aminomethyl-pyridine); an aminoalkyl-piperidine (e.g., 
2-aminomethyl-piperidine); an aminoalkyl-pyrimidine (e.g., 
5-aminomethyl-pyrimidine); an aminoalkyl-quinoline (e.g., 
3-aminomethyl-quinoline); an aminoalkyl-morpholine (e.g., 
4-aminomethyl-morpholine); a peptide (e.g., lysyl lysine, histidyl lysine, 
histidyl arginine, histidyl arginyl lysine, polylysine); and a 
polyalkyleneimine (e.g., polyethyleneimine). Further, suitable examples of 
the organic solvent which may be employed in the present invention include 
an alkanone having 3 to 5 carbon atoms (e.g., acetone), an alkanol having 
one to 3 carbon atoms (e.g., methanol, ethanol, propanol), dioxane, 
tetrahydrofuran, dimethylformamide, dimethylsulfoxide, ethyleneglycol and 
glycerin. 
The treatment of the aqueous mixture of the sulfated polysaccharide and the 
catalytically active substance with the above-mentioned ammonium ion, 
metal ion or amine are preferably carried out at a temperature of between 
about - 5.degree. and 70.degree. C., especially at between about 0.degree. 
and 55.degree. C. It is preferred to carry out said treatment by adding 
the aqueous mixture to an aqueous solution of the ion or amine. The ion or 
amine should be employed at a concentration of between about 0.1 mM and 10 
M, especially between about 10 mM and 2 M. On the other hand, the 
treatment of the aqueous mixture of the sulfated polysaccharide and the 
catalytically active substance with the water-miscible organic solvent may 
be preferably carried out at between about -5.degree. and 70.degree. C., 
especially at between about - 5.degree. and 30.degree. C. As compared with 
the preparations solidified with the ion or amine, the catalytically 
active substance immobilized with the polysaccharide in the presence of 
said water-miscible organic solvent may show a higher level of catalytic 
activity per volume of the preparations because the gel matrix of the 
sulfated polysaccharide is partially dehydrated and compacted with the 
solvent during the treatment thereof. Further, when Escherichia coli (one 
of aspartase-producing microorganisms) is immobilized with the sulfated 
polysaccharide in the presence of acetone or ethanol, it shows a catalytic 
activity about 2 to 3 times higher than that of its intact cells. 
By any one of the above-mentioned operations, the sulfated polysaccharide 
is gelled and solidified, and thereby the catalytically active substance 
is entrapped within the resultant gel matrix of said polysaccharide. That 
is, the immobilized catalytically active substance obtained above 
comprises a catalytically active enzyme or microorganism or a 
water-insoluble, hydrophilic, catalytically active carrier-bound enzyme or 
microorganism entrapped within the gel matrix of the sulfated 
polysaccharide containing at least one of ammonium ion, the metal ion, the 
water-soluble organic amine and the water-miscible organic solvent. A 
preferred amount of the ion, amine or solvent which is to be contained in 
said gel matrix of the sulfated polysaccharide is between about 10.sup.-1 
and 10.sup.4 millimoles, especially between about one and 10.sup.3 
millimoles, per g of the sulfated polysaccharide employed. Depending on 
its use, the immobilized catalytically active substance can be shaped as 
particles, beads, plates, rods, tubes, films and fibers. For example, the 
immobilized active substance may be obtained in the form of particles or 
beads when the above-mentioned gellation step is carried out by adding, 
dropwise, the mixture of the sulfated polysaccharide and the catalytically 
active substance to the aqueous ion or amine solution or the 
water-miscible organic solvent. When the immobilized catalytically active 
substance is shaped as particles or beads, the size thereof suitable for 
use in the enzymatic reaction is between about 0.1 and 20 mm, especially 
between about one and 5 mm, in diameter. On the other hand, the fibers of 
the immobilized catalytically active substance may be obtained by 
introducing said aqueous mixture into the aqueous ion or amine solution or 
the water-miscible organic solvent through a small orifice of a container. 
Further, the immobilized active substance may be obtained as films or 
tubes by making a thin layer of the aqueous mixture of the sulfated 
polysaccharide and the catalytically active substance on the surface of 
glass plates, plastic plates, metal plates, glass tubes or plastic tubes, 
and then contacting said mixture with the aqueous ion or amine solution or 
the water-miscible organic solvent on the surface of said plates or tubes. 
The thickness of the membrane of the immobilized catalytically active 
substance thus obtained can be designated to determine the rate of 
diffusion of substrate to the vicinity of the catalytically active 
substance as well as the rate of diffusion of the products out of the 
reaction site. The thickness can be between about 0.01 and 5 mm, 
preferably between about 0.1 and one mm. 
Concomitantly, if required, the immobilized catalytically active substance 
obtained above may be further treated with a gel-hardening agent in order 
to increase the hardness, elasticity and the ability to retain its shape 
in aqueous media. The term "gel-hardening agent" as used herein means a 
compound which can bind, through ionic, coordinate or covalent linkage to 
or be adsorbed by the immobilized catalytically active substance thereby 
increasing its hardness, elasticity and/or ability to retain its shape in 
aqueous media. Examples of such a gel-hardening agent include an aliphatic 
dialdehyde having 3 to 5 carbon atoms (e.g., glutaraldehyde, glyoxal), 
tannins, dihydroxyacetone, epichlorohydrin, ethyl chloroformate, 
hexamethylene diisocyanate, toluene diisocyanate, hexamethylene 
diisothiocyanate, a carbodiimide (e.g., 
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), a Woodward reagent (i.e., 
2-ethyl-5-m-sulfophenyl-isoxazolium hydroxide), and a mixture of ammonia 
or an alkylenediamine having one to 20 carbon atoms (e.g., 
methylenediamine, ethylenediamine, trimethylenediamine, 
tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, 
heptamethylenediamine, octamethylenediamine, nonamethylenediamine, 
decamethylenediamine, dodecamethylenediamine, eicosamethylenediamine) and 
an aliphatic dialdehyde having 3 to 5 carbon atoms (e.g., glutaraldehyde, 
glyoxal). The treatment of the immobilized catalytically active substance 
with the gel-hardening agent may be preferably carried out by dissolving 
the gel-hardening agent in a solvent, and then immersing said immobilized 
substance therein. It is preferred to carry out the treatment at a 
temperature of between about 0.degree. and 50.degree. C., especially 
between about 5.degree. and 37.degree. C. It is also preferred to use a 
solution containing between about 0.01 and one gram (per ml of the 
solvent) of the gel-hardening agent. When the immobilized catalytically 
active substance is hardened by treating it with an aliphatic dialdehyde 
(e.g., glutaraldehyde, glyoxal) in the presence of ammonia or an 
alkylenediamine (e.g., hexamethylenediamine), the immobilized preparation 
obtained in the form of a shiff-base may be further treated with a 
reducing agent such as sodium Since the immobilized catalytically active 
substance treated with the gel-hardening agent has an increased ability to 
retain its shape in aqueous media, it may be suitable for use in a 
long-term continuous enzymatic reactions with substrates. In addition, the 
catalytic activity of the immobilized active substance treated with the 
gel-hardening agent may often be more stable than that of the non-treated 
substance. For example, when aminoacylase, glucose isomerase, Pseudomonas 
dacunhae (L-aspartate .beta.-decarboxylase-producing microorganism), 
Streptococcus faecalis (glucose isomerase-producing microorganism) and E. 
coli (aspartase-producing microorganism) immobilized by the aforementioned 
method of the invention are further treated with the gel-hardening agent, 
they maintain their catalytic activity for at least about 2 times or more 
longer period of time. 
The immobilized catalytically active substance of the present invention 
shows a high level of catalytic activity for a long period of time. The 
enzymes or microorganisms or the carrier-bound enzymes or microorganisms 
are retained firmly in the gel matrix of the sulfated polysaccharide 
whereas molecules of substrates and reaction products, of smaller size, 
will be able to move freely in the polymeric network of said 
polysaccharide. An enzyme or microorganism preparation (hereinafter 
referred to as "the control sample") which is obtained by simply cooling 
an aqueous mixture of the sulfated polysaccharide and enzymes or 
microorganisms is still unsuitable for use in the enzymatic reactions with 
substrates. For example, when an aqueous substrate solution is passed 
through a column charged with the control sample, it deforms within a 
short period of time and does not function effectively to convert 
substrates to their conversion products. As compared with said control 
sample, however, the immobilized catalytically active substance of the 
present invention has a remarkably greater ability to retain its shape in 
an aqueous media and, even when packed in a column, the enzymatic reaction 
thereof with substrates can be continued for a longer period of time 
without repacking or agitating the column. Moreover, the polysaccharide 
gel of the above-mentioned control sample is unstable and easily 
transformed into "sol" (i.e., a colloidal solution) at about 30.degree. to 
40.degree. C. This inevitably causes leakage of the enzymes or 
microorganisms from the gel matrix. Unlike said control sample, however, 
the immobilized catalitically active substance of the invention has 
excellent heat-stability. For example, the polysaccharide gel of the 
catalytically active substance of the invention is not transformed into " 
sol" even at a temperature higher than 40.degree. C. and it can be 
employed for enzymatic reactions at such high temperatures as 60.degree. 
to 70.degree. C. without leakage of the enzymes or microorganisms 
therefrom. Further, the polysaccharide gel of the immobilized 
catalytically active substance of the invention has excellent hardness and 
elasticity and can be shaped into any desired form such as particles, 
beads, films, tubes and fibers according to its intended use. On the 
otherhand, whereas the gel of the above-mentioned control sample is too 
soft to shape it as above. As briefly mentioned hereinbefore, the 
carrier-bound enzyme or microorganism becomes compacted and deformed when 
in use and under such conditions can not function effectively to convert 
substrates to their conversion products. However, the compacting, 
deformation and/or channelling of the carrier-bound enzyme or 
microorganism is materially reduced by entrapping it within the gel matrix 
of the sulfated polysaccharide according to the present invention. This 
may make it unnecessary to repack or agitate the column of the 
bound-enzyme or microorganism during the enzymatic reactions thereof with 
substrates. 
The immobilized catalytically active substance of the invention can be used 
for any of a wide variety of enzymatic reactions which have been conducted 
by the use of enzyme, living microorganisms or carrier-bound enzymes or 
microorganisms. For example, aminoacylase or an aminoacylase-producing 
microorganism (e.g., Aspergillus orizae) entrapped according to the 
present invention may be used in preparing L-amino acids by asymmetric 
hydrolysis of N-acyl-DL-amino acids. The entrapped aspartase or 
aspartase-producing microorganism (e.g., Escherichia coli) can be used, 
instead of the corresponding intact enzyme or microorganism, for the 
enzymatic reaction with fumaric acid and ammonia to produce L-aspartic 
acid. When asparaginase or an asparaginase-producing microorganism (e.g., 
Proteus vulgaris) is immobilized according to the invention, said 
immobilized enzyme or microorganism may be used for enzymatic reaction 
with L-asparagine to decompose it into L-aspartic acid and ammonia. 
L-alanine can be prepared from L-aspartic acid by using the entrapped 
aspartate .beta.-decarboxylase or aspartate .beta.-decarboxylase-producing 
microorganism (e.g., Pseudomonas dacunhae). On the other hand, each one of 
L-arginine deiminase (or a L-arginine deiminase-producing microorganism 
such as Pseudomonas putidum), L-histidine ammonia-lyase (or a L-histidine 
ammonia-lyase-producing microorganism such as Achromobacter liquidum) and 
fumarase (or a fumarase-producing microorganism such as Brevibacterium 
flavum) entrapped according to the present invention can be used for 
enzymatic reactions with L-arginine, L-histidine or fumaric acid to 
produce L-citrulline, urocanic acid or L-malic acid, respectively. 
Moreover, when a L-isoleucine or arginine-producing microorganism (e.g., 
Serratia marcescens, Bacillus subtilis) is immobilized according to the 
present invention, L-isoleucine or arginine are prepared by cultivating 
said immobilized microoganism in a conventional nutrient medium containing 
glucose. Fructose may be prepared from glucose by enzymatic reaction 
thereof with the entrapped glucose isomerase or glucose 
isomerase-producing microorganism (e.g., Streptomyces phaeochromogenes, 
Streptomyces griseus). 6-Aminopenicillanic acid may be prepared from 
penicillins by using the entrapped penicillin amidase or penicillin 
amidase-producing microorganism (e.g., Escherichia coli). Concomitantly, 
urea may be decomposed into ammonia and carbon dioxide by using the 
entrapped urease or urease-producing microorganism (e.g., Sarcina lutea) 
of the invention. The reaction conditions which have been used for the 
enzymatic reactions of intact enzymes or microorganisms with their usual 
substrates can be employed in carrying out the enzymatic reaction of the 
immobilized catalytically active substance of the invention. In carrying 
out said enzymatic reactions of the immobilized catalytically active 
substance, it is generally preferred to add to the reaction solutions a 
small amount (e.g., between about 0.1 mM and 10 M, especially between 
about 10 mM and 5 M) of ammonium ion, metal ion, water-soluble organic 
amine or water-miscible organic solvent used in the gellation step 
mentioned hereinbefore. When the immobilized catalytically active 
substance of the invention treated with the gel-hardening agent is 
employed, however, the enzymatic reaction thereof with substrates may be 
carried out without said ion, amine or solvent. For example, when the 
immobilized glucose isomerase preparation of the invention, pre-treated 
with a mixture of glutaraldehyde and hexamethylenediamine, is employed for 
the enzymatic reaction with glucose, said enzymatic reaction can be 
continued for a period of 120 days or longer in the absence of the ion, 
amine or solvent. Further, the reactions of the immobilized catalytically 
active substance with substrates may be carried out by in a conventional 
manner. For example, the substrate can be dissolved in water. The 
immobilized catalytically active substance is suspended in the aqueous 
substrate solution, and the suspension is stirred. Since the immobilized 
catalytically active substance is insoluble in water, after the reaction 
it can be readily recovered by filtration or centrifugation and the 
reaction products are recovered from the filtrate or supernatant solution. 
Alternatively, the above-mentioned enzymatic reactions may be carried out 
by a column method. The column method enables the reaction to be carried 
out in a successive manner. For example, the immobilized catalytically 
active substance is packed in a column, and an aqueous substrate solution 
is passed through the column at a suitable flow rate. An aqueous solution 
containing the reaction product is obtained as an effluent. In carrying 
out the enzymatic reaction, the rate of conversion of substrates to their 
conversion products mainly depends on the catalytic activity of the 
immobilized active substance, the temperature and the reaction period. In 
case of the column method, however, the optimum reaction conditions for 
complete conversion of substrates to their conversion products may be 
readily obtained by adjusting the flow rate of the substrate solutions.

Practical and presently-preferred embodiments of the present invention are 
shown in the following Examples. 
EXAMPLE 1 
(1) 4 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 5 ml of water. 20 ml of an aqueous 4.4% carrageenan 
(manufactured by The Kopenhagen Pectin Factory Ltd., under the trade name 
"GENU GEL Type WG") solution previously warmed to 45.degree. C. are added 
to the aminoacylase solution, and the mixture is added dropwise to an 
aqueous 2% potassium chloride solution. The resultant gel particles (about 
3 mm in diameter) are collected by filtation, and then washed with an 
aqueous 2% potassium chloride solution. 18.1 g (wet form) of an 
immobilized aminoacylase preparation are obtained. It shows an 
aminoacylase activity of 2.1 units/g. 
[One unit of aminoacylase is defined as the enzymatic activity which 
produces one micromole of L-methionine by reaction with 
N-acetyl-DL-methionine at 37.degree. C. at pH 7.0 for one hour. The 
reaction with N-acetyl-DL-methionine is conducted by adding 15 g of the 
immobilized preparation to 40 ml of an aqueous 0.6 M 
N-acetyl-DL-methionine solution (adjusted to pH 7.0 with potassium 
hydroxide) containing 5 .times. 10.sup.-4 M cobaltous chloride, and then 
shaking the mixture at 37.degree. C. for one hour. The amount of 
L-methionine produced is assayed colorimetrically by the ninhydrin 
method.] 
(2) 15 g of the immobilized aminoacylase preparation obtained in paragraph 
(1) are packed in a column (1.6 cm in diameter and 14 cm in height). An 
aqueous 0.6 M N-acetyl-DL-methionine solution (adjusted to pH 7.0 with 
potassium hydroxide) containing 5 .times. 10.sup.-4 M cobaltous chloride 
is passed through the column at 37.degree. C. at a flow rate of 10 ml/hr. 
The enzymatic activity of the immobilized aminoacylase preparation is 
assayed at intervals. The results are shown in Table 1. The half-life of 
the enzymatic activity of the immobilized aminoacylase preparation is 
about 60 days. As seen from this data, the immobilized aminoacylase 
preparation shows a high enzymatic activity for a long period of time when 
used for the continuous enzymatic reaction. 
On the other hand, just for comparison, immobilized preparations are 
prepared by covalently binding aminoacylase to iodoacetyl-cellulose 
according to the method described in Fermentation Technology Today, pages 
383-389 (1972), or by entrapping aminoacylase within the lattice of 
polyacrylamide according to the method described in the above-mentioned 
literature. The enzymatic activity of these immobilized preparations 
decreases to 50% of their initial activity only after the above-mentioned 
continuous enzymatic reaction for 5 or 30 days. 
Table 1 
______________________________________ 
Aminoacylase 
Potency ratio of 
Operation period 
activity enzymatic activity* 
(days) (units) (%) 
______________________________________ 
1 32 100 
2 30 94 
5 29 91 
7 29 91 
9 29 91 
______________________________________ 
Note: 
*Potency ratio of enzymatic activity is calculated by the following 
formula: 
##STR1## 
EXAMPLE 2 
5 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 5 ml of water. 20 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 45.degree. C. 
are added to the aminoacylase solution, and the mixture is added dropwise 
to an aqueous 0.2 M N-acetyl-DL-methionine solution (adjusted to pH 7.0 
with potassium hydroxide) containing 5 .times. 10.sup.-4 M cobaltous 
chloride. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration. 19.1 g (wet form) of an immobilized aminoacylase 
preparation are obtained. It shows an aminoacylase activity of 4.7 
units/g. 
EXAMPLE 3 
5 mg of aminoacyhlase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 5 ml of water. 20 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 45.degree. C. 
are added to the aminoacylase solution, and the mixture is added dropwise 
to an aqueous 0.2 M N-acetyl-DL-tryptophan solution (adjusted to pH 7.0 
with potassium hydroxide) containing 5 .times. 10.sup.-4 M cobaltous 
chloride. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration. 18.6 g (wet form) of an immobilized aminoacylase 
preparation are obtained. It shows an aminoacylase activity of 3.6 
units/g. 
EXAMPLE 4 
Brevibacterium ammoniagenes IAM (Institute of Applied Microbiology, Tokyo 
University, Japan) 1645 is inoculated into 500 ml of an aqueous medium (pH 
7.0) containing 2.0% of glucose, 0.5% of fumaric acid, 0.2% of urea, 0.2% 
of monopotassium phosphate, 0.05% of magnesium sulfate 7 hydrate and 1.0% 
of corn steep liquor. The medium is cultivated at 30.degree. C. for 24 
hours under shaking. Then, the microbial cells are collected by 
centrifugation. 10 g of the microbial cells are suspended in 100 ml of a 
0.01 M phosphate buffer solution. The microbial cells in the suspension 
are disrupted with a sonicator at 9 Kc for 10 minutes, and then 
centrifuged. Ammonium sulfate is added gradually to the supernatant 
solution, and precipitates which are salted out from partially saturated 
ammonium sulfate solution (40-70% saturation) are collected by 
centrifugation. The precipitates are dissolved in 15 ml of a 0.01 M 
phosphate buffer solution (pH 7.0), and the solution is dialyzed overnight 
against a 0.01 M phosphate buffer solution. The dialyzed solution is used 
as a fumarase solution. 
2 ml of a 0.01 M phosphate buffer solution are added to one ml of the 
fumarase solution. 12 ml of an aqueous 4.2% carrageenan (the trade name 
"GENU GEL Type WG") solution previously warmed to 40.degree. C. are added 
to the fumarase solution, and the mixture is added dropwise to an aqueous 
2% potassium chloride solution. The resultant gel particles (about 3 mm in 
diameter) are collected by filtration. 15 g (wet form) of an immobilized 
fumarase preparation are obtained. It shows a fumarase activity of 
111.6.mu. moles/hr/g. Yield of activity[={(Enzymatic activity of the 
immobilized preparation obtained from one gram of the intact enzyme or 
microbial cells) .div. (Enzymatic activity of one gram of the intact 
enzyme or microbial cells)} .times. 100] : 62%. 
[Fumarase activity is indicated in terms of micromoles of L-malic acid 
which are produced by reaction with potassium fumarate. The reaction with 
potassium fumarate is conducted by adding 15 g of the immobilized 
preparation to 30 ml of a 0.2 M potassium furmarate-0.01 M potassium 
phosphate buffer solution and shaking the mixture at 37.degree. C. for one 
hour. After the reaction, hydrochloric acid is added to the mixture, 
followed by filtering to remove the precipitates of fumaric acid. Then, 
the amount of L-malic acid (i.e., L-malic acid contained in the filtrate) 
is assayed colorimetrically by using 2,7-naphthalenediol as a coloring 
agent.] 
EXAMPLE 5 
Escherichia coli ATCC 11303 is inoculated into 500 ml of an aqueous medium 
(pH 7.0) containing 3% of ammonium fumarate, 0.2% of dipotassium 
phosphate, 0.05% of magnesium sulfate 7 hydrate, 4% of corn steep liquor 
and 0.05% of calcium carbonate. The medium is cultivated at 37.degree. C. 
for 24 hours under shaking. Then, the microbial cells are collected by 
centrifugation. 8 g of the microbial cells are suspended in 8 ml of water. 
The microbial cells in the suspension are disrupted with a sonicator at 9 
Kc for 15 minutes, and then centrifuged. Ammonium sulfate is added 
gradually to the supernatant solution, and precipitates which are salted 
out from partially saturated ammonium sulfate solution (30-50% saturation) 
are collected by centrifugation. The precipitates are dissolved in 5 ml of 
water, and the solution is dialyzed overnight against water. The dialyzed 
solution is used as an aspartase solution. 
2 ml of the aspartase solution obtained above are mixed with 12 ml of an 
aqueous 3.2% carrageenan (the trade name "GENU GEL Type WG") solution at 
37.degree. C. The mixture is added dropwise to an aqueous 2% potassium 
chloride solution. The resultant gel particles (about 3 mm in diameter) 
are collected by filtration. 10.6 g (wet form) of an immobilized aspartase 
preparation are obtained. It shows an aspartase activity of 2,868.mu. 
moles/hr/g. Yield of Activity: 46%. 
[Aspartase activity is indicated in terms of micromoles of L-aspartic acid 
which are produced by reaction with ammonium fumarate at 37.degree. C. at 
pH 8.5. The reaction with ammonium fumarate is conducted by adding 2 g of 
the immobilized aspartase preparation to 30 ml of an aqueous 1 M ammonium 
fumarate solution (pH 8.5) containing 1 mM magnesium chloride, and then 
shaking the mixture at 37.degree. C. for one hour. The amount of 
L-aspartic acid produced is bioassayed by using leuconostoc mesenterioides 
P-60(J. Biol. Chem., 172, 15(1948).] 
EXAMPLE 6 
10 mg of glucose isomerase (223 units/mg) obtained from Streptomyces 
phaeochromogenus are dissolved in 5 ml of water. 20 ml of an aqueous 4.4% 
carrageenan (the trade name "GENU GEL Type WG") solution previously warmed 
to 45.degree. C. are added to the glucose isomerase solution, and the 
mixture is added dropwise added to an aqueous 2% potassium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration, and then washed with an aqueous potassium 
chloride solution. 23.0 g (wet form) of an immobilized glucose isomerase 
preparation are obtained. It shows a glucose isomerase activity of 13.6 
units/g. 
[One unit of glucose isomerase is defined as the enzymatic activity which 
produces one micromole of fructose by reaction with glucose at 37.degree. 
C. at pH 7.0 for one minute. The reaction with glucose is conducted by 
adding 2 g of the immobilized glucose isomerase preparation to 20 ml of an 
aqueous 0.1 M glucose solution (pH 7.0) containing 0.01 M of magnesium 
sulfate, 1 mM cobaltous chloride and 0.1 M sodium sulfite, and then 
shaking the mixture at 37.degree. C. for one hour. The amount of fructose 
produced is measured by the cysteinecarbazole-sulfuric acid method.] 
EXAMPLE 7 
20 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 4 ml of water. 16 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 45.degree. C. 
are added to the aminoacylase solution, and the mixture is added dropwise 
to an aqueous 1 M ammonium chloride solution. The resultant gel particles 
(about 3 mm in diameter) are collected by filtration, and then washed with 
an aqueous 2% potassium chloride solution. 18.8 g (wet form) of an 
immobilized aminoacylase preparation are obtained. It shows an 
aminoacylase activity of 66 units/g. 
EXAMPLE 8 
Pseudomonas dacunhae IAM 1152 is inoculated into 500 ml of an aqueous 
medium (pH 7.0) containing 0.5% of ammonium fumarate, 1.0% of sodium 
fumarate, 0.55% of corn steep liquor, 1.8% of peptone, 0.05% of 
monopotassium phosphate and 0.01% of magnesium sulfate 7 hydrate. The 
medium is cultivated at 30.degree. C. for 24 hours under shaking. Then, 
the microbial cells are collected by centrifugation. 10 g of the microbial 
cells are suspended in 100 ml of a 0.01 M phosphate buffer solution. The 
microbial cells in the suspension are disrupted with a sonicator at 9 Kc 
for 10 minutes, and then centrifuged. The supernatant solution thus 
obtained is used as an aspartate .beta.-decarboxylase solution. 
One ml of the aspartate .beta.-decarboxylase solution obtained above is 
treated in the same manner as described in Example 4. 12 g (wet form) of 
an immobilized aspartate .beta.-decarboxylase preparation are obtained as 
gel particles of about 3 mm in diameter. It shows an aspartate 
.beta.-decarboxylase activity of 36.mu. moles/hr/g. Yield of Activity: 
57%. 
[Aspartate .beta.-decarboxylase activity is indicated in terms of 
micromoles of L-alanine which are produced by reaction with 0.2 M 
L-aspartic acid. The reaction with L-aspartic acid is conducted by adding 
12 g of the immobilized preparation to 30 ml of an aqueous 0.2 M ammonium 
L-asparaginate solution (pH 5.5) containing 10.sup.-4 M pyridoxal 
phosphate, and shaking the mixture at 37.degree. C. for one hour. The 
amount of L-alanine produced is bioassayed by using Leuconostoc 
mesenterioides P-60.] 
EXAMPLE 9 
2 ml of an aspartase solution obtained in the same manner as described in 
Example 5 are mixed with 12 ml of an aqueous 3.2% carrageenan (the trade 
name "GENU GEL Type WG") solution at 37.degree. C. The mixture is added 
dropwise to an aqueous 1 M ammonium fumarate solution (pH 8.5). The 
resultant gel particles (about 3 mm in diameter) are collected by 
filtration. 9.4 g (wet form) of an immobilized aspartase preparation are 
obtained. It shows an aspartase activity of 1,966.mu. moles/hr/g. Yield of 
Activity: 52.4%. 
EXAMPLE 10 
100 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 4 ml of water. 16 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 40.degree. C. 
are added to the aminoacylase solution, and the mixture is added dropwise 
to an aqueous 0.5 M hexamethylenediamine solution (adjusted to pH 7.0 with 
hydrochloric acid). The resultant gel particles (about 3 mm in diameter) 
are collected by filtration, and then washed with an aqueous one % 
potassium chloride solution. 18.9 g (wet form) of an immobilized 
aminoacylase preparation are obtained. It shows an aminoacylase activity 
of 11.9 units/g. 
EXAMPLE 11 
100 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 4 ml of water. 16 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 40.degree. C. 
are added to the aminoacylase solution, and the mixture is added dropwise 
to an aqueous 0.5 M nonamethylenediamine solution (adjusted to pH 7.0 with 
hydrochloric acid). The resultant gel particles (about 3 mm in diameter) 
are collected by filtration, and then washed with an aqueous one % 
potassium chloride soluton. 19.1 g (wet form) of an immobilized 
aminoacylase preparation are obtained. It shows an aminoacylase activity 
of 13.5 units/g. 
EXAMPLE 12 
200 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 4 ml of water. 16 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution containing 5% of gelatin (said 
carrageenan solution being previously warmed to 45.degree. C.) are added 
to the aminoacylase solution, and the mixture is dropwise added to an 
aqueous 2% potassium chloride solution. The resultant gel particles (about 
3 mm in diameter) are collected by filtration, and then washed with an 
aqueous 2% potassium chloride solution. 19.4 g (wet form) of an 
immobilized aminoacylase preparation are obtained. It shows an 
aminoacylase activity of 37.5 units/g. 
EXAMPLE 13 
(1) 100 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae 
are dissolved in 50 ml of water. One g (dry form) of diethylaminoethyl 
cross-linked dextran (manufactured by Pharmacia Fine Chemicals under the 
trade name "DEAE-Sephadex A-25") is added to the solution, and the mixture 
is stirred at room temperature for one hour. DEAE-Sephadex-bound 
aminoacylase thus obtained is collected by filtration. Said 
DEAE-Sephadex-bound aminoacylase is suspended in 5 ml of water. 20 ml of 
an aqueous 4.4% carrageenan (the trade name "GENU GEL Type WG") solution 
previously warmed to 40.degree. C. are added to the suspension, and the 
mixture is added dropwise to an aqueous 0.1 M potassium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration. 29.8 g (wet form) of an immobilized aminoacylase preparation 
are obtained. It shows an aminoacylase activity of 32.9 units/g. 
(2) 14.9 g of the immobilized aminoacylase preparation obtained in 
paragraph (1) are packed in a column (1.6 cm in diameter and 14 cm in 
height). An aqueous 0.6 M N-acetyl-DL-methionine solution (adjusted to pH 
7.0 with potassium hydroxide) containing 5 .times. 10.sup.-4 M cobaltous 
chloride is passed through the column at 37.degree. C. at a flow rate of 
10 ml/hr. The enzymatic activity of the immobilized aminoacylase 
preparation is assayed at intervals. The results are shown in Table 2. The 
half-life of the enzymatic activity of the immobilized aminoacylase 
preparation is about 120 days. As seen from these data, the immobilized 
aminoacylase preparation shows a high enzymatic activity for a long period 
of time when used for the continuous enzymatic reaction. 
Table 2 
______________________________________ 
Aminoacylase Potency ratio of 
Operation period 
activity enzymatic activity* 
(days) (units) (%) 
______________________________________ 
0 25 100 
5 26 100 
10 24 96 
15 25 100 
20 25 100 
______________________________________ 
Note: 
*Potency ratio of enzymatic activity is calculated by the formula shown i 
the foot-note of Table 1. 
EXAMPLE 14 
20 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in one ml of water, and 4 ml of an aqueous 4% tannic acid 
solution (adjusted to pH 7.0 with sodium hydroxide) are added thereto. 20 
ml of an aqueous 4.4% carrageenan (the trade name "GENU GEL Type WG") 
solution previously warmed to 45.degree. C. are added to the 
aminoacylase-tannic acid solution. Then, the mixture is added dropwise to 
an aqueous 0.1 M potassium chloride solution. The resultant gel particles 
(about 3 mm in diameter) are collected by filtration, and washed with an 
aqueous 0.1 M potassium chloride solution. 24.0 g (wet form) of an 
immobilized aminoacylase preparation are obtained. It shows an 
aminoacylase activity of 0.6 unit/g. 
EXAMPLE 15 
200 mg of aminoacylase (20 units/mg) obtained from Aspergillus oryzae are 
dissolved in 4 ml of water. 16 ml of an aqueous 4.4% carrageenan (the 
trade name "GENU GEL Type WG") solution containing 5% of gelatin (said 
carrageenan solution being previously warmed to 45.degree. C.) are added 
to the aminoacylase solution, and the mixture is added dropwise to an 
aqueous one M ammonium chloride solution. The resultant gel particles 
(about 3 mm in diameter) are collected by filtration. The gel particles 
thus obtained are added to a mixture of 20 ml of an aqueous one M ammonium 
chloride solution and 1.6 ml of an aqueous 25% glutaraldehyde solution, 
and the mixture is stirred at 4.degree. C. for 30 minutes. Then, the gel 
particles are collected by filtration, and washed with an aqueous 2% 
potassium chloride solution. 18.6 g (wet form) of an immobilized 
aminoacylase preparation are obtained. It shows an aminoacylase activity 
of 21.5 units/g. 
EXAMPLE 16 
10 mg of glucose isomerase (223 units/mg) obtained from Streptomyces 
phaeochromogenus are dissolved in one ml of water, and 4 ml of an aqueous 
tannin solution (adjusted to pH 7.0 with sodium hydroxide) are added 
thereto. The glucose isomerase solution is allowed to stand at 45.degree. 
C. for 5 minutes, and then 20 ml of an aqueous 4.4% carrageenan (the trade 
name "GENU GEL Type WG") solution previously warmed to 45.degree. C. are 
added thereto. The mixture is added dropwise to an aqueous 2% potassium 
chloride solution. The resultant gel particles (about 3 mm in diameter) 
are collected by filtration, and washed with an aqueous 2% potassium 
chloride solution. 25.4 g (wet form) of an immobilized glucose isomerase 
preparation are obtained. It shows a glucose isomerase activity of 1.5 
units/g. 
EXAMPLE 17 
10 mg of glucose isomerase (223 units/mg) obtained from Streptomyces 
phaeochromogenus are dissolved in one ml of water. 4 ml of an aqueous 
persimmon tannin solution (tannin content: 2.5 mg/ml) are added to the 
glucose isomerase solution. Then, the glucose isomerase-persimmon tannin 
solution is allowed to stand at 45.degree. C. for 5 minutes, and an 
aqueous 4.4% carrageenan (the trade name "GENU GEL Type WG") solution 
previously warmed to 45.degree. C. are added thereto. The mixture is added 
dropwise to an aqueous 2% potassium chloride solution. The resultant gel 
particles (about 3 mm in diameter) are collected by filtration, and washed 
with an aqueous 2% potassium chloride solution. 23.2 g (wet form) of an 
immobilized glucose isomerase preparation are obtained. It shows a glucose 
isomerase activity of 5.2 units/g. 
EXAMPLE 18 
10 mg of vegetable proteolytic enzyme "papain" (3.1.mu. moles/minute/mg of 
protein) are dissolved in 6 ml of a 0.02 M citrate-phosphate buffer 
solution (pH 6.2) containing 0.005 M cysteine and 0.01 M ethylenediamine 
tetra-acetic acid. 25 ml of an aqueous 4% carrageenan (the trade name 
"GENU GEL Type WG") solution previously warmed to 40.degree. C. are added 
to the papain solution, and the mixture is added dropwise to an aqueous 2% 
potassium chloride solution. The resultant gel particles (about 3 mm in 
diameter) are collected by filtration. 30 g (wet form) of an immobilized 
papain preparation are obtained. Yield of Activity: 57%. 
[One unit of papain activity is defined as the enzymatic activity which 
decomposes one .mu. mole of .alpha.-benzoyl-arginine ethyl ester by 
reacting with said arginine ester at pH 6.2 for one minute. The reaction 
with .alpha.-benzoyl-arginine ethyl ester is conducted by adding 5 g of 
the immobilized preparation to 10 ml of a 0.02 M citrate-phosphate buffer 
solution (pH 6.2) containing 0.1 M benzoyl-arginine ethyl ester, and 
shaking the mixture at 37.degree. C. for 10 minutes. The remaining amount 
of said benzyl arginine ester is measured in accordance with the J. R. 
Kimmel et al's method described in J. Biol. Chem., 207, 515 (1954).] 
EXAMPLE 19 
Escherichia coli ATCC 9637 is inoculated into 1,000 ml of an aqueous medium 
(pH 7.0) containing 0.2% of phenoxyacetic acid, 2% of peptone, 0.5% of 
yeast extract, 0.5% of sodium L-gultamate, 0.3% of monopotassium 
phosphate, 0.7% of dipotassium phosphate, 0.02% of magnesium sulfate 7 
hydrate and 0.02% of ferric chloride 6 hydrate. The microbial cells are 
collected by centrifugation. The microbial cells thus obtained are 
suspended in 13 ml of an aqueous physiological saline solution. 52 ml of 
an aqueous 3.2% carrageenan (the trade name "GENU GEL Type WG") solution 
previously warmed to 40.degree. C. are added to the suspension, and the 
mixture is added dropwise to an aqueous 2% potassium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration. 60.7 g (wet form) of an immobilized Escherichia coli 
preparation are obtained. It shows a penicillin amidase activity of 
1,114.mu. moles/hr/g. Yield of Activity: 90%. 
[Penicillin amidase activity is assayed in accordance with the J. 
Bomstein's method described in Analytical Chemistry, Vol. 37, pp. 576-578 
(1965).] 
EXAMPLE 20 
(1) Brevibacterium ammoniagenes IAM 1645 is inoculated into 100 ml of an 
aqueous medium (pH 7.0) containing the same ingredients as described in 
Example 4. The medium is cultivated at 30.degree. C. for 24 hours under 
shaking. Then, the microbial cells are collected by centrifugation. The 
microbial cells thus obtained are suspended in 4 ml of an aqueous 
physiological saline solution. 16 ml of an aqueous 2.5% carrageenan (the 
trade name "GENU GEL Type WG") solution previously warmed to 37.degree. C. 
are added to the suspension, and the mixture is added dropwise to an 
aqueous 2% potassium chloride solution. The resultant gel particles (about 
3 mm in diameter) are collected by filtration. 20 g (wet form) of an 
immobilized Brevibacterium ammoniagenes preparation are obtained. 
10 g of the immobilized Brevibacterium ammoniagenes preparation obtained 
above are added to 30 ml of an aqueous one M potassium fumarate solution 
(pH 7.0) containing 0.3% of ox bile extract. The mixture is allowed to 
stand at 37.degree. C. for 20 hours. After said treatment, the immobilized 
Brevibacterium ammoniagenes preparation is washed with an aqueous one % 
potassium chloride solution. The immobilized preparation thus obtained 
shows a fumarase activity of 5,790.mu. moles/hr/g of microbial cells. 
Yield of Activity: 60% 
(2) 20 g of the immobilized Brevibacterium ammoniagenes preparation 
obtained in paragraph (1) are packed in a column (1.6 cm in diameter and 
12 cm in height). An aqueous 1 M potassium fumarate solution (pH 7.0) is 
passed through the column at 37.degree. C. at a flow rate specified in 
Table 3. The amount of L-malic acid in the effluent is assayed in the same 
manner as described in Example 4, and the percentage conversion of 
potassium fumarate into L-malic acid is calculated therefrom. The results 
are shown in Table 3. 
Table 3 
______________________________________ 
Flow rate Conversion (%) 
(ml/hr) to L-malic acid 
______________________________________ 
3.5 82 
6 80 
9 70 
12 62 
20 40 
45 23 
______________________________________ 
EXAMPLE 21 
(1) Pseudomonas putidum ATCC 4359 is inoculated in 100 ml of an aqueous 
medium (pH 6.2) containing one % of glucose, one % of yeast extract, 0.5% 
of polypeptone, 0.5% of L-arginine hydrochloride, 0.1% of ammonium 
chloride, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate 7 
hydrate, 0.01% of manganous sulfate 4 hydrate, 0.0005% of ferrous sulfate 
7 hydrate and 0.02% of sodium chloride. The medium is cultivated at 
30.degree. C. for 24 hours under shaking. The microbial cells are 
collected by centrifugation. The microbial cells thus obtained are treated 
in the same manner as described in Example 20. 20 g (wet form) of an 
immobilized Pseudomonas putidum preparation are obtained. It shows a 
L-arginine deiminase activity of 1,700.mu. moles/hr/g of microbial cells. 
Yield of Activity: 64%. 
[L-arginine deiminase activity is indicated in terms of micromoles of 
L-citrullin which are produced by reaction with L-arginine. The reaction 
with L-arginine is conducted by adding 10 g of the immobilized preparation 
to an aqueous 0.5 M L-arginine hydrochloride solution (pH 6.0) containing 
0.01% of triethanolamine laurylsulfate, and shaking the mixture at 
37.degree. C. for one hour. The amount of L-citrullin produced is assayed 
colorimetrically by using diacetylmonooxim as a coloring agent.] 
(2) 20 g of the immobilized Pseudomonas putidum preparation obtained in 
paragragh (1) are packed in a column (1.6 cm in diameter and 12 cm in 
height). An aqueous 0.5 M L-arginine hydrochloride solution (pH 6.0) is 
passed through the column at 37.degree. C. at a flow rate of 4.5 or 12 
ml/hr. The amount of L-citrullin in the effluent solution is assayed in 
the same manner as described in paragraph (1), and the percentage 
conversion of L-arginine into L-citrullin is calculated therefrom. The 
results are shown in Table 4. 
Table 4 
______________________________________ 
Conversion (%) of L-arginine into 
Operation period 
L-citrullin 
(days) 4.5 ml/hr 12 ml/hr 
______________________________________ 
1 100 55 
3 100 55 
6 100 54 
8 100 55 
10 100 54 
13 100 54 
15 100 53 
17 100 51 
20 100 50 
22 100 50 
24 100 47 
27 100 48 
29 100 48 
31 100 46 
______________________________________ 
The half-life of the enzymatic activity of the immobilized Pseudomonas 
putidum preparation is about 160 days. 
EXAMPLE 22 
Microbial cells of Escherichia coli ATCC 11303 obtained in the same manner 
as described in Example 5 are lyophilized. 1.1 g of the lyophilized 
microbial cells are suspended in 8 ml of an aqueous physiological saline 
solution. 32 ml of an aqueous 3.2% carrageenan (the trade name "GENU GEL 
Type WG") solution previously warmed to 40.degree. C. are added to the 
suspension, and the mixture is added dropwise to an aqueous 2% potassium 
chloride solution. The resultant gel particles (about 3 mm in diameter) 
are collected by filtration. 32 g (wet form) of an immobilized Escherichia 
coli preparation are thereby obtained. It shows as aspartase activity of 
13,800.mu. moles/hr. Yield of Activity: 13% 
EXAMPLE 23 
Microbial cells of Escherichia coli ATCC 11303 obtained in the same manner 
as described in Example 5 are treated with acetone to give acetone powder. 
576 mg of the thus obtained acetone powder are suspended in 6 ml of an 
aqueous physiological saline solution. 24 ml of an aqueous 3.2% 
carrageenan (the trade name "GENU GEL Type WG") solution previously warmed 
to 40.degree. C. are added to the suspension, and the mixture is added 
dropwise to an aqueous 2% potassium chloride solution. The resultant gel 
particles (about 3 mm in diameter) are collected by filtration. 26.8 g 
(wet form) of an immobilized Escherichia coli preparation are obtained. It 
shows an aspartase activity of 1,118.mu. moles/hr/g. Yield of Activity: 
39%. 
EXAMPLE 24 
Achromobacter liquidum IAM 1667 is inoculated into 100 ml of an aqueous 
medium (pH 7.0) containing one % of glucose, 0.2% of dipotassium 
phosphate, 0.05% of monopotassium phosphate, 0.1% of ammonium chloride, 
0.02% of magnesium sulfate 7 hydrate, 0.1% of yeast extract and 0.02% of 
L-histidine hydrochloride. The medium is cultivated at 30.degree. C. for 
24 hours under shaking. Then, the microbial cells are collected by 
centrifugation. The microbial cells thus obtained are suspended in 4 ml of 
an aqueous physiological saline solution. The suspension is heated at 
70.degree. C. for 30 minutes, and then 16 ml of an aqueous 2.5% 
carrageenan (the trade name "GENU GEL Type WG") solution previously warmed 
to 37.degree. C. are added thereto. The mixture is added dropwise to an 
aqueous 2% potassium chloride solution. The resultant gel particles (about 
3 mm in diameter) are collected by filtration. 20 g (wet form) of an 
immobilized Achromobacter liquidum preparation are obtained. It shows a 
L-histidine ammonia-lyase activity of 434.mu. moles/hr/g of microbial 
cells. Yield of Activity: 62%. 
[L-histidine ammonia-lyase activity is indicated in terms of micromoles of 
urocanic acid which are produced by reaction with L-histidine. The 
reaction with L-histidine is conducted by adding 10 g of the immobilized 
preparation to 30 ml of an aqueous 0.25 M L-histidine solution (pH 9.0), 
and shaking the mixture at 37.degree. C. for one hour. The amount of 
urocanic acid produced in assayed colorimetrically at 277 nm (molecular 
extinction coefficient = 1.88 .times. 10.sup.-4 (pH 7.4)).] 
EXAMPLE 25 
Micrococcus ureae IAM 1010 is inoculated into 400 ml of an aqueous medium 
(pH 7.0) containing the same ingredients as described in Example 24. The 
medium is cultivated at 30.degree. C. for 24 hours under shaking. Then, 
the microbial cells are collected by centrifugation. The cells thus 
obtained are treated in the same manner as described in Example 20. 20 g 
(wet form) of an immobilized Micrococcus ureae preparation are thereby 
obtained as gel particles (about 3 mm in diameter). It shows a L-histidine 
ammonia-lyase activity of 299.mu. moles/hr/g of microbial cells. Yield of 
Activity: 59%. 
EXAMPLE 26 
Streptomyces griseus IFO (Institute for Fermentation, Osaka, Japan) 3430 is 
inoculated into 200 ml of an aqueous medium (pH 7.0) containing one % of 
peptone, 0.25% of yeast extract, 0.5% of meat extract, one % of D-xylose, 
0.05% of magnesium sulfate 7 hydrate, 0.024% of cobaltous chloride 6 
hydrate and 0.5% of sodium chloride. The medium is cultivated at 
30.degree. C. for 48 hours under shaking. Then, the microbial cells are 
collected by centrifugation. The microbial cells thus obtained are 
suspended in 20 ml of an aqueous physiological saline solution, and 80 ml 
of an aqueous 3.2% carrageenan (the trade name "GENU GEL Type WG") 
solution previously warmed to 40.degree. C. are added to the suspension 
under heating (in a water bath, bath temperature: 40.degree. C.). The 
mixture is added dropwise to an aqueous 2% potassium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration. 83 g (wet form) of an immobilized Streptomyces griseus 
preparation are obtained. It shows a glucose isomerase activity of 9.2.mu. 
moles/hr/g. Yield of Activity: 31%. 
EXAMPLE 27 
2.5 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 5 ml of water. 
20 ml of an aqueous 3.1% carrageenan (the trade name "GENU GEL Type WG") 
solution previously warmed to 40.degree. C. are added to the suspension, 
and the mixture is added dropwise to an aqueous one % potassium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration, and then washed with an aqueous one % potassium 
chloride solution. 22.7 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 443 units/g. 
EXAMPLE 28 
(1) Serratia marcescens ATCC 21740 (isoleucine hydroxamate- and 
.alpha.-aminobutyric acid-resistant mutant) is inoculated into 100 ml of 
an aqueous medium (pH 7.0) 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. The medium is cultivated at 30.degree. C. for 24 hours under 
shaking. Then, the microbial cells are collected by centrifugation. The 
microbial cells thus obtained are suspended in an aqueous physiological 
saline solution. 16 ml of an aqueous 3.2% carrageenan (the trade name 
"GENU GEL Type WG") solution previously warmed to 37.degree. C. are added 
to 4 ml of the suspension (microbial cells content: 2 g/4 ml), and the 
mixture is added dropwise to an aqueous 2% potassium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration. 20 g (wet form) of an immobilized Serratia marcescens 
preparation are obtained. It shows a L-isoleucine-productivity of 8.6.mu. 
moles/hr/g of microbial cells. 
[L-isoleucine-productivity is estimated by adding 5 g of the immobilized 
preparation to 20 ml of an aqueous solution (pH 7.5) containing 5% of 
glucose, one % of urea, 2% of DL-threonine, 2% of dipotassium phosphate 
and 0.05% of magnesium sulfate 7 hydrate, and shaking the mixture at 
30.degree. C. for 6 hours. The amount of L-isoleucine produced is 
bioassayed by using Leuconostoc mesenterioides P-60(ATCC 9135).] 
(2) 10 g of the immobilized Serratia marcescens preparation obtained in 
paragraph (1) are packed in a column. 50 ml of an aqueous solution (pH 
7.5) containing 5% of glucose, one % of urea, 2% of DL-threonine, 2% of 
dipotassium phosphate and 0.05% of magnesium sulfate 7 hydrate are passed 
through the column at a flow rate of 250 ml/hr. The effluent is again 
passed through the column at the same flow rate, and this operation is 
repeated for 5 hours. 43.mu. moles of L-isoleucine are accumulated in the 
reaction mixture. 
EXAMPLE 29 
2 g of microbial cells of Serratia marcescens Hd-Mhr ATCC 31026 (FERM P. 
2120) (L-histidine ammonia-lyase-lacking and 2-methylhistidine-resistant 
mutant) are treated in the same manner as described in Example 28-(1). 20 
g (wet form) of an immobilized Serratia marcescens preparation are 
obtained. It shows a L-histidine-productivity of 7.2.mu. moles/hr/g of 
microbial cells. 
[L-histidine-productivity is estimated by adding 5 g of the immobilized 
preparation to 20 ml of an aqueous solution (pH 8.0) containing 5% of 
glucose, 2% of urea, 2% of dipotassium phosphate and 0.2% of magnesium 
sulfate 7 hydrate, and shaking the mixture at 30.degree. C. for 6 hours. 
The amount of L-histidine produced is bioassayed by using Leuconostoc 
mesenterioides P-60 (ATCC 9135).] 
EXAMPLE 30 
2 g of microbial cells of Bacillus subtilis AHr Aur-9 ATCC 31002 (FERM P. 
1998) (arginine hydroxamate- and 6-azauracil-resistant mutant) are treated 
in the same manner as described in Example 28-(1). 20 g (wet form) of an 
immobilized Bacillus subtilis preparation are obtained. It shows a 
L-arginine-productivity of 21.mu. moles/hr/g of microbial cells. 
[L-arginine-productivity is estimated by adding 5 g of the immobilized 
preparation to 20 ml of an aqueous solution (pH 7.5) containing 5% of 
glucose, 2.5% of ammonium chloride, 2% of sodium L-aspartate, 2% of 
dipotassium phosphate and 0.0002% of magnesium 7 hydrate, and shaking the 
mixture at 30.degree. C. for 6 hours. The amount of L-arginine produced is 
bioassayed by using Leuconostoc mesenterioides P-60 (ATCC 9135).] 
EXAMPLE 31 
One g of microbial cells of Escherichia coli ATCC 11303 and 2 g of 
microbial cells of Pseudomonas dacunhae IAM 1152 which are obtained in the 
same manner as described in Example 5 and 8 are suspended in 3 ml of an 
aqueous physiological saline solution. 600 mg of carrageenan (the trade 
name "GENU GEL Type WG") are dissolved in 14.5 ml of water under heating, 
and the solution is mixed with the microbial cells suspension obtained 
above. The mixture is added dropwise to an aqueous 2% potassium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration. 20 g (wet form) of an immobilized microbial cells 
preparation are obtained. It shows a L-alanine-productivity of 340.mu. 
moles/hr/g of microbial cells. [L-alanine-productivity is estimated by 
adding 5 g of the immobilized preparation to 30 ml of an aqueous 1 M 
ammonium fumarate solution (pH 7.0) containing 10.sup.-4 M pyridoxal 
phosphate, and shaking the mixture at 30.degree. C. for one hour. The 
amount of L-alanine produced as assayed in the same manner as described in 
Example 8.] 
EXAMPLE 32 
(1) Escherichia coli ATCC 11303 is inoculated into 500 ml of an aqueous 
medium (pH 7.0) containing the same ingredients as described in Example 5. 
The medium is cultivated at 37.degree. C. for 24 hours. Then, the 
microbial cells are collected by centrifugation. The microbial cells thus 
obtained are suspended in 16 ml of an aqueous physiological saline 
solution, and 64 ml of an aqueous 3.2% carrageenan (the trade name "GENU 
GEL Type WG") solution previously warmed to 40.degree. C. are added to the 
suspension under heating (in a water bath, bath temperature: 40.degree. 
C.). The mixture is added dropwise to an aqueous 1 M ammonium fumarate 
solution (pH 8.5) containing 1 mM magnesium chloride. The resultant gel 
particles (about 3 mm in diameter) are collected by filtration. 60.4 g 
(wet form) of an immobilized Escherichia coli preparation are obtained. It 
shows an aspartase activity of 143.4.mu. moles/hr/g. Yield of Activity: 84 
%. 
(2) 60.4 g of the immobilized Escherichia coli preparation obtained in 
paragraph (1) are packed in a column (4 cm in diameter and 8 cm in 
height), and incubated at 37.degree. C. for 48 hours. After incubation, 
the immobilized preparation shows an aspartase activity of 278, 422.mu. 
moles/hr. Then, 1000 ml of an aqueous 1 M ammonium fumarate solution (pH 
8.5) containing 1 mM magnesium chloride are passed through the column at 
37.degree. C. at a flow rate of 50 ml/hr. The effluent is adjusted to pH 
2.8, and the crystalline precipitates are collected by filtration. 122 g 
of L-aspartic acid are obtained. 
EXAMPLE 33 
Serratia marcescens OUT (Faculty of Technology, Osaka University, Japan) 
8259 is treated in the same manner as described in Example 32-(1). 50.8 g 
(wet form) of an immobilized Serratia marcescens preparation are obtained 
as gel particles (about 3 mm in diameter). It shows an aspartase activity 
of 36.9.mu. moles/hr/g. Yield of Activity: 87%. 
EXAMPLE 34 
Proteus vulgaris OUT 8226 is treated in the same manner as described in 
Example 32-(1). 44.0 g (wet form) of an immobilized Proteus vulgaris 
preparation are obtained as gel particles (about 3 mm in diameter). It 
shows an aspartase activity of 586.9.mu. moles/hr/g. Yield of Activity: 
100%. 
EXAMPLE 35 
Bacterium succinium IAM 1017 is treated in the same manner as described in 
Example 32-(1). 54.8 g (wet form) of an immobilized Bacterium succinium 
preparation are obtained as gel particles (about 3 mm in diameter). It 
shows an aspartase activity of 295.5.mu. moles/hr/g. Yield of Activity: 
100%. 
EXAMPLE 36 
Pseudomonas aeruginosa OUT 8252 is treated in the same manner as described 
in Example 32-(1). 39.8 g (wet form) of an immobilized Pseudomonas 
aeruginosa preparation are obtained as gel particles (about 3 mm in 
diameter). It shows an aspartase activity of 30.1.mu. moles/hr/g. Yield of 
Activity: 87%. 
EXAMPLE 37 
5 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 10 ml of water. 
40 ml of an aqueous 3.1% carrageenan (the trade name "GENU GEL Type WG") 
solution previously warmed to 40.degree. C. are added to the suspension, 
and the mixture is added dropwise to an aqueous 4% ammonium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration, and then washed with an aqueous 4% ammonium 
chloride solution. 57.8% g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 214 units/g. 
EXAMPLE 38 
2 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 4 ml of water. 
16 ml of an aqueous 1.5% carrageenan (manufactured by The Kopenhagen 
Pectin Factory Ltd., under the trade name "GENU VISCO J") solution 
previously warmed to 40.degree. C. are added to the suspension, and the 
mixture is added dropwise to an aqueous 0.1 M magnesium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration. The gel particles are added to a mixture of 10 ml of an 
aqueous 1 M ammonium chloride solution and 4 ml of an aqueous 25% 
glutaraldehyde solution, and the mixture is shaken at 30.degree. C. for 60 
minutes. Then, the gel particles are collected by filtration. 18.7 g (wet 
form) of an immobilized Streptomyces phaeochromogenus preparation are 
obtained. It shows a glucose isomerase activity of 141 units/g. 
EXAMPLE 39 
2 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 4 ml of water. 
16 ml of an aqueous 1.16% furcellaran (manufactured by Litex Co., Denmark) 
solution previously warmed to 40.degree. C. are added to the suspension, 
and the mixture is added dropwise to an aqueous 2% potassium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration, and then washed with an aqueous 2% potassium 
chloride solution. 15.3 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 759 units/g. 
EXAMPLE 40 
2 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 4 ml of water. 
16 ml of an aqueous 1.16% furcellaran (manufactured by Litex Co., Denmark) 
solution previously warmed to 40.degree. C. are added to the suspension, 
and the mixture is added dropwise to an aqueous 4% ammonium chloride 
solution. The resultant gel particles (about 3 mm in diameter) are 
collected by filtration, and then washed with an aqueous 4% ammonium 
chloride solution. 16.4 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 444 units/g. 
EXAMPLE 41 
2 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 4 ml of water. 
16 ml of an aqueous one % furcellaran (manufactured by Litex Co., Denmark) 
solution containing 1.25% of gelatin (said furcellaran solution being 
previously warmed to 40.degree. C.) are added to the suspension, and the 
mixture is added dropwise to an aqueous 2% potassium chloride solution. 
The resultant gel particles (about 3 mm in diameter) are collected by 
filtration, and then washed with an aqueous 2% potassium chloride 
solution. 16.6 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 317 units/g. 
EXAMPLE 42 
2 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 4 ml of water. 
16 ml of an aqueous one % furcellaran (manufactured by Litex Co., Denmark) 
solution containing 1.25% of gelatin (said furcellaran solution being 
previously warmed to 40.degree. C.) are added to the suspension, and the 
mixture is added dropwise to an aqueous 4% ammonium chloride solution. The 
resultant gel particles (about 3 mm in diameter) are collected by 
filtration, and then washed with an aqueous 4% potassium chloride 
solution. 16.9 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 421 units/g. 
EXAMPLE 43 
1.8 g of gel particles (about 3 mm in diameter) obtained in the same manner 
as described in Example 40 are added to 20 ml of an aqueous 1 M ammonium 
chloride solution and 0.8 ml of an aqueous 25% glutaraldehyde solution. 
The mixture is shaken at 30.degree. C. for 60 minutes. The gel particles 
are collected by filtration, and then washed with ice-water. 1.7 g (wet 
form) of an immobilized Streptomyces phaeochromogenus preparation are 
obtained. It shows a glucose isomerase activity of 66 units/g. 
EXAMPLE 44 
(1) 1.66 g of gel particles (about 3 mm in diameter) obtained in the same 
manner as described in Example 41 are added to a mixture of 0.8 ml of an 
aqueous 25% glutaraldehyde solution and 20 ml of an aqueous 2% potassium 
chloride solution. The mixture is shaken at 30.degree. C. for 30 minutes. 
The gel particles are collected by filtration, and then washed with 
ice-water. 1.7 g (wet form) of an immobilized Streptomyces 
phaeochromogenus preparation are obtained. It shows a glucose isomerase 
activity of 318 units/g. 
(2) 10 g of the immobilized Streptomyces phaeochromogenus preparation 
obtained in paragraph (1) are packed in a column (1.6 cm in diameter and 
14 cm in height). An aqueous 50% glucose solution (pH 7.0) containing 0.01 
M magnesium sulfate, 1 mM cobaltous chloride and 0.1 M sodium sulfite is 
passed through the column at 60.degree. C. The glucose isomerase activity 
of the immobilized Streptomyces phaeochromogenus preparation is assayed at 
intervals. The results are shown in Table 5. The half-life of the 
enzymatic activity of the immobilized Streptomyces phaeochromogenus 
preparation is about 180 days. As seen from these data, the immobilized 
Streptomyces phaeochromogenus preparation shows a high enzymatic activity 
for a long period of time when used for the continuous enzymatic reaction. 
Table 5 
______________________________________ 
Glucose isomerase 
Potency ratio of 
Operation period 
activity enzymatic activity* 
(days) (units) (%) 
______________________________________ 
0 3,180 100 
10 3,020 95 
21 2,961 93 
30 2,832 89 
50 2,618 82 
71 2,422 76 
91 2,164 68 
______________________________________ 
Note: 
*Potency ratio of enzymatic activity is calculated by the formula shown i 
the foot-note of Table 1. 
EXAMPLE 45 
1.69 g of gel particles (about 3 mm in diameter) obtained in the same 
manner as described in Example 42 are added to a mixture of 2 ml of an 
aqueous 1 M ammonium chloride solution, 0.8 ml of an aqueous 25% 
glutaraldehyde solution and 17.2 ml of water. The mixture is shaken at 
30.degree. C. for 30 minutes. The gel particles are collected by 
filtration, and then washed with ice-water. 1.7 g (wet form) of an 
immobilized Streptomyces phaeochromogenus preparation are obtained. It 
shows a glucose isomerase activity of 192 units/g. 
EXAMPLE 46 
Microbial cells of Escherichia coli ATCC 11303 obtained in the same manner 
as described in Example 5 are suspended in 8 ml of water. 64 ml of an 
aqueous 3.2% carrageenan (the trade name "GENU GEL Type WG") solution 
previously warmed to 40.degree. C. are added to the suspension, and the 
mixture is added dropwise to acetone (cooled to -1.degree. to 0.degree. 
C.). After dropwise addition, the mixture is stirred for 10 minutes. The 
resultant gel particles (about 3 mm in diameter) are collected by 
filtration, and then washed with an aqueous 4% ammonium chloride solution. 
54.0 g (wet form) of an immobilized Escherichia coli preparation are 
obtained. It shows an aspartase activity of 3,441.mu. moles/hr/g. Yield of 
Activity: 345%. 
EXAMPLE 47 
5 g of heat-treated microbial cells (glucose isomerase activity: 7,740 
units/g) of Streptomyces phaeochromogenus are suspended in 10 ml of water. 
40 ml of an aqueous 3% sodium salt of cellulose sulfate (manufactured by 
KELCO Co., under the trade name "KELCO SCS") solution previously warmed to 
37.degree. C. are added to the suspension, and the mixture is added 
dropwise to an aqueous 2% potassium chloride solution. The resultant gel 
particles (about 3 mm in diameter) are collected by filtration, and then 
washed with an aqueous 2% potassium chloride solution. 48 g (wet form) of 
an immobilized Streptomyces phaeochromogenus preparation are obtained. It 
shows a glucose isomerase activity of 443 units/g. 
EXAMPLE 48 
(1) 8 g of microbial cells of Escherichia coli ATCC 11303 obtained in the 
same manner as described in Example 5 are suspended in 5 ml of a 
physiological saline solution. 80 ml of an aqueous 2.2% carrageenan (the 
trade name "GENU GEL Type WG" solution containing one % of locust been gum 
(said carrageenan solution being previously warmed to 40.degree. C.) are 
mixed with the suspension. 250 ml of an aqueous potassium chloride 
solution are added gradually to the mixture, and said mixture is allowed 
to stand at 4.degree. C. for 30 minutes. The resultant gel is cut into 
cubes of 3 mm in each side, and then washed with an aqueous 2% potassium 
chloride solution. 89.3 g of the gel cubes thus obtained are immersed in 
100 ml of ice-cold ethanol, and glutaraldehyde is added thereto until the 
final concentration thereof becomes 0.49%. The mixture is allowed to stand 
for 15 minutes under ice-cooling. Then, the gel cubes are collected by 
filtration, and then washed with an aqueous 2% potassium chloride 
solution. 86.2 g (wet form) of an immobilized Escherichia coli preparation 
are obtained. It shows an aspartase activity of 32,183.mu. moles/hr/g of 
microbial cells. 
(2) 11.1 g of the immobilized Escherichia coli preparation obtained in 
paragraph (1) are packed in a column (1.6 cm in diameter and 12 cm in 
height). An aqueous 1 M ammonium fumarate solution (pH 8.5) containing 1 
mM of magnesium chloride is passed through the column at 37.degree. C. at 
a flow rate of 6 ml/hr. The enzymatic activity of the immobilized 
Escherichia coli preparation is assayed at intervals. The results are 
shown in Table 6. The half-life of the enzymatic activity of the 
immobilized Escherichia coli preparation is about 113 days. As seen from 
these data, the immobilized preparation shows a high enzymatic activity 
for a long period of time when used for the continuous enzymatic reaction. 
Table 6 
______________________________________ 
Aspartase activity 
Potency ratio of 
Operation period 
(.mu. moles/hr/g of 
enzymatic activity* 
(days) cells) (%) 
______________________________________ 
0 32,183 100 
6 31,413 98 
8 31,625 98 
12 31,600 98 
19 31,906 99 
22 31,036 96 
29 30,574 95 
40 29,932 93 
______________________________________ 
Note: 
*Potency ratio of enzymatic activity is calculated by the formula shown i 
the foot-note of Table 1. 
EXAMPLE 49 
(1) Pseudomonas dacuhae IAM 1152 is inoculated into 120 ml of an aqueous 
medium (pH 7.0) containing 1.4% of sodium glutamate, 0.2% of casamino 
acid, 0.9% of peptone, 0.05% of monopotassium phosphate and 0.01% of 
magnesium sulfate 7 hydrate. The medium is cultivated at 30.degree. C. for 
24 hours under shaking. Then, the microbial cells are collected by 
centrifugation. 2 g of the microbial cells are suspended in 5 ml of a 
physiological saline solution. On the other hand, 600 mg of carrageenan 
(the trade name "GENU GEL Type WG") are dissolved in 15 ml of water. The 
carrageenan solution is mixed with the suspension at 37.degree. C., and 
one ml of an aqueous 1 M hexamethylenediamine solution (pH 7.0) is added 
thereto. The mixture is allowed to stand at 4.degree. C. for 30 minutes. 
The resultant gel is cut into cubes of 4 mm in each side. The gel cubes 
are added to 50 ml of an aqueous 2% ammonium chloride solution containing 
one % of glutaraldehyde, and the mixture is allowed to stand at 0.degree. 
C. for 15 minutes. Then, the gel cubes are collected by filtration, and 
washed with an aqueous 1% ammonium chloride solution. 22 g (wet form) of 
an immobilized Pseudomonas dacuhae preparation are obtained. It shows an 
aspartate .beta.-decarboxylase activity of 3,567.mu. moles/hr/g of 
microbial cells. 
(2) 22 g of the immobilized Pseudomonas dacuhae preparation obtained in 
paragraph (1) are packed in a column (1.6 cm in diameter and 10.5 cm in 
height). An aqueous 1 M ammonium asparaginate solution (pH 5.5) containing 
10.sup.-4 M of pyridoxal phosphate is passed through the column upward 
from the bottom at 37.degree. C. at a flow rate of 13.8 ml/hr. The amount 
of L-alanine in the effluent solution is bioassayed by using Leuconostoc 
citrovorum ATCC 8081. The results are shown in Table 7. The half-life of 
the enzymatic activity of the immobilized Pseudomonas dacuhae preparation 
is about 159 days. As seen from these data, the immobilized preparation 
shows a high enzymatic activity for a long period of time when used for 
the continuous enzymatic reaction. 
Table 7 
______________________________________ 
Aspartate 
.beta.-decarboxylase 
Potency ratio of 
Operation period 
activity enzymatic activity* 
(days) (.mu.moles/ml) 
(%) 
1 517 100 
3 556 108 
4 528 102 
5 576 111 
7 538 104 
12 528 102 
14 528 102 
20 528 102 
24 521 101 
26 489 95 
45 450 87 
______________________________________ 
EXAMPLE 50 
(1) Brevibacterium flavum ATCC 14067 is inoculated into 500 ml of an 
aqueous medium (pH 7.0) containing 2.0% of corn steep liquor, 2.0% of 
malonic acid, 0.2% of diammonium citrate, 0.2% of monopotassium phosphate 
and 0.05% of magnesium sulfate 7 hydrate. The medium is cultivated at 
30.degree. C. for 48 hours under shaking. Then, the microbial cells are 
collected by centrifugation. 8.0 g of the microbial cells are suspended in 
8 ml of a physiological saline solution. 34 ml of an aqueous 5.0% 
carrageenan (the trade name "GENU GEL Type WG") solution previously warmed 
to 50.degree. C. are mixed with the suspension. 250 ml of an aqueous 2% 
potassium chloride solution are added gradually to the mixture, and said 
mixture is allowed to stand at 4.degree. C. for 5 hours. The resultant gel 
is cut into cubes of 3 mm in each side. The gel cubes are washed with an 
aqueous 2% potassium chloride solution. 49.9 g (wet form) of an 
immobilized Brevibacterium flavum preparation are obtained. It shows a 
fumarase activity of 503.mu. moles/hr/g of microbial cells. 
(2) 6.3 g of the immobilized Brevibacterium flavum preparation obtained in 
paragraph (1) are packed in a column (1.6 cm in diameter and 12 cm in 
height). An aqueous 1 M potassium fumarate solution (pH 7.0) is passed 
through the column at 37.degree. C. at a flow rate of 6 ml/hr. The 
enzymatic activity of the immobilized Brevibacterium flavum preparation is 
assayed at intervals. The results are shown in Table 8. The half-life of 
the immobilized Brevibacterium flavum preparation is about 69 days. As 
seen from this data, the immobilized preparation shows a high enzymatic 
activity for a long period of time when used for the continuous enzymatic 
reaction. 
Table 8 
______________________________________ 
Potency ratio of 
Operation period 
Fumarase activity 
enzymatic activity* 
(days) (.mu. moles/ml) 
(%) 
______________________________________ 
1 1,768 100 
2 1,767 100 
4 1,472 83 
9 1,422 80 
13 1,401 79 
18 1,374 78 
20 1,355 77 
25 1,307 74 
30 1,308 74 
______________________________________ 
Note: 
*Potency ratio of enzymatic activity is calculated by the formula shown i 
the foot-note of Table 1.