Biocatalysts immobilized in a storage stable copolymer gel

Biocatalysts such as cells and enzymes are immobilized in a polymer gel by forming a mixture containing a biocatalyst, an N,N-dialkylacrylamide monomer, a cationic acrylamide monomer and a water-soluble cross-linking monomer, and copolymerizing the monomers to produce a polymer gel entrapping the biocatalyst. Preferably, the N, N-dialkylacrylamide is N,N-dimethylacrylamide or N,N-diethylacrylamide in an amount of about 70 to about 99.8% by weight, the cationic acrylamide monomer is N,N-dimethylaminopropylacrylamide, N,N-dimethylaminopropylmethacrylamide, N,N-diethylaminopropylmethacrylamide, N,N-diethylaminopropylacrylamide and quaternary compounds thereof in a amount of about 0.1 to about 10% by weight and the cross-linking monomer is N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-(1,2-dihydroxyethlene)bisacrylamide, 1,3-diacrylamide methyl-2-imidazolidone or diacrylamide methylene urea in an amount of about 0.1 to about 20% by weight. The polymer gel containing a biocatalyst has excellent storage stability and does not putrefy even after one month of storage at ordinary temperature.

FIELD OF THE INVENTION 
This invention relates to a carrier for use in the immobilization of 
biocatalysts, to an immobilized biocatalyst obtained by entrapping the 
biocatalyst in the carrier, and to a method for immobilizing the 
biocatalyst. 
BACKGROUND OF THE INVENTION 
When biocatalysts are used on an industrial scale, they are usually used in 
an immobilized form with the objectives of preventing elution of 
impurities from the biocatalyst, improving separability of the biocatalyst 
from the reaction product, improving applicability of the biocatalyst to 
repeated use, increasing enzymatic stability of the biocatalyst, and 
carrying out continuous operation of the production steps. 
Immobilization of biocatalysts is effected by a carrier binding method, a 
cross-linking method, an entrapping immobilization method and the like 
(cf. T. Hattori and C. Frusaka, J. Biochem., vol. 48, pp. 831 (1960)), of 
which the entrapping immobilization method is most advantageous in that 
leakage of biocatalysts is small, and a decrease in the biocatalyst 
activity caused by the immobilization process is also small, because the 
biocatalyst and its carrier are not linked to each other. Furthermore, the 
method can be applied to the immobilization of a large variety of 
biocatalysts. 
Examples of known carriers for use in the entrapping immobilization of 
biocatalysts include synthetic, high polymers such as polyacrylamide, 
polyvinyl alcohol, polyurethane, collagen, a photosetting resin and the 
like, and natural, high polymers such as carrageenan, alginic acid, 
agarose, starch, gelatin and the like (cf. U.S. Pat. No. 4,526,867). In 
general, in comparison with the natural, high polymers, the synthetic, 
high polymers are high in strength, excellent in durability and resistant 
to biodegradation. Of these, polyacrylamide is used most frequently 
because it is industrially inexpensive, has high polymer strength and 
causes less inactivation of biocatalysts at the time of polymerization 
(cf. U.S. Pat. No. 4,421,855 and I. Chibata, T. Tosa and T. Sato, Appl. 
Microbiol., vol. 27, pp. 878 (1974)). 
In addition, a process has been proposed in which acrylamide, a cationic 
ethylenic unsaturated monomer, and a water-soluble cross-linking monomer 
are subjected to copolymerization in order to reduce degree of swelling of 
a polyacrylamide base immobilized biocatalyst obtained by the entrapping 
immobilization method and to reduce inactivation of the catalyst at the 
time of the reaction (cf. JP-B-58-35078; the term "JP-B" as used herein 
means an "examined Japanese patent publication"). 
However, such polyacrylamide base immobilized biocatalysts, obtained by the 
entrapping immobilization method, are generally stored by soaking in an 
aqueous solution such as a buffer solution or the like, since their 
activities are apt to decrease when exposed to air oxidation or drying. 
When the storage is continued at room temperature for a prolonged period 
of time (for example, more than 1 month), the storing solution and the 
biocatalyst start to putrefy which causes generation of offensive odors 
from, and turbidity in, the storing solution and causes a decrease in the 
activity to such a level that it cannot be used as a catalyst. As a 
consequence, their storage is effected generally by putting them in a 
refrigerator or adding an antiseptic agent to the storing solution. 
However, the cold storage method requires a huge cost for facilities, 
utilities and the like when biocatalysts are used in an industrially large 
quantity, and the other method, in which an antiseptic agent is added to 
the storing solution, causes permeation of the antiseptic agent itself 
into the immobilized biocatalyst, thereby adversely effecting the quality 
of products for the practical use. 
It is important to overcome these problems especially in the case of the 
industrial application of immobilized biocatalysts. 
SUMMARY OF THE INVENTION 
The inventors of the present invention have conducted intensive studies on 
the development of a biocatalyst-immobilizing method which not only has 
the advantages of the prior art polyacrylamide base carriers but also is 
excellent in storage stability, and as a result, have found that the use 
of a specified copolymer, as a carrier, is markedly effective in 
overcoming the aforementioned problems. The present invention has been 
accomplished on the basis of this finding. 
Accordingly, the present invention comprises a carrier for use in the 
immobilization of biocatalysts which is obtained by copolymerizing a first 
monomer represented by the following general formula (1) with a cationic 
acrylamide monomer and a water-soluble cross-linking monomer, both being 
capable of copolymerizing with the first monomer: 
##STR1## 
wherein each of R.sub.1 and R.sub.2 represents a methyl group or an ethyl 
group, an immobilized biocatalyst which is obtained by entrapping a 
biocatalyst in the carrier for biocatalyst immobilization use as well as a 
method for immobilizing a biocatalyst. 
Other objects and advantages of the present invention will be made apparent 
as the description of the invention progresses. 
DETAILED DESCRIPTION OF THE INVENTION 
Illustrative examples of the compound represented by the aforementioned 
general formula (1) include N,N-dimethylacrylamide, N,N-diethylacrylamide 
and N-methyl-N-ethylacrylamide, which may be used alone or as a mixture of 
two or more compounds. 
Illustrative examples of the cationic acrylamide monomer capable of 
copolymerizing with the monomer of general formula (1) include 
N,N-dialkylaminoalkyl methacrylamides, N,N-dialkylaminoalkylacrylamides 
and quaternary compounds thereof, such as 
N,N-dimethylaminopropylacrylamide, N,N-dimethylaminopropyl methacrylamide, 
N,N-diethylaminopropyl methacrylamide, N,N-diethylaminopropylacrylamide 
and quaternary compounds thereof. 
Illustrative examples of the water-soluble cross-linking monomer include 
N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, 
N,N'-(1,2-dihydroxyethylene)bisacrylamide, 1,3-di-acrylamide 
methyl-2-imidazolidone, diacrylamide methylethylene urea, diacrylamide 
methyl ether, ethylene glycol diacrylate, ethylene glycol dimethacrylate, 
hexahydro-1,3,5-triacyl-S-triazine, 2,2-bis(acrylamide)acetic acid and the 
like. Of these, N,N'-methylenebisacrylamide, 
N,N'-methylenebismethacrylamide, N,N'-(1,2dihydroxyethylene)bisacrylamide, 
1,3-diacrylamide methyl-2imidazolidone and diacrylamide methylethylene 
urea are particularly preferred. 
Based on the total amount of these monomers, the monomer of general formula 
(1) may be used in an amount of from about 70 to 99.8% by weight, 
preferably from 80 to 99% by weight, the cationic acrylamide monomer in an 
amount of from about 0.1 to 10% by weight, preferably from 0.5 to 10% by 
weight, and the water-soluble cross-linking monomer from about 0.1 to 20% 
by weight, preferably from 0.5 to 10% by weight. 
If desired, other water-soluble monomers capable of copolymerizing with the 
monomer of general formula (1) may be used in an amount of about 0.01 to 
10% by weight. 
Examples of the biocatalyst to be immobilized by entrapping in the 
aforementioned immobilization carrier include enzymes, microorganisms, 
organella, animal and plant cells, and they may be used in the purified or 
disrupted forms, with no particular limitation in terms of their origin or 
form. For example, any genus of microorganism including bacteria, 
actinomycetes, yeasts, fungi and the like can be used, such as those 
belonging to the genera Brevibacterium, Corynebacterium, Rhodococcus, 
Gordona, Vibrio, Nitrosomonas, Streptococcus, Lactobacillus, Bacillus, 
Azotobacter, Nocardia, Saccharomyces, Endomyces, Asmergillus, Penicillium, 
Mucor, Rhizopus and the like. 
The immobilized biocatalyst of the present invention can be prepared, for 
example, by adding a mixture of the monomers to a suspension of a 
biocatalyst, further adding a commonly used polymerization initiator and 
accelerator, such as potassium persulfate and 
N,N,N',N'-tetramethylethylene-diamine, to the suspension, and then 
incubating the resulting mixture at a pH value of from about 5 to 10, 
preferably from 6 to 8, at a temperature of from about 0.degree. to 
50.degree. C., preferably from 0.degree. to 35.degree. C., for about 15 to 
120 minutes, thereby effecting polymerization and gelation. 
The biocatalyst content in the polymerized gel varies depending on the 
type, form and the like of each biocatalyst to be used, but the content 
may be in the range of generally from about 0.1 to 40% by weight, 
preferably from 1 to 20% by weight. The content of monomers in the 
polymerization reaction solution may be in the range of generally from 
about 2 to 30% by weight, preferably from 5 to 15% by weight. 
These immobilized biocatalysts may be made into any shape such as granules, 
films, plates and the like. 
The use of the biocatalyst immobilization carrier of the present invention 
renders possible the preparation of immobilized biocatalysts which are 
excellent in storage stability and simultaneously have the strength and 
the like advantages of the prior art high polymer base carriers, 
especially polyacrylamide base carriers. The immobilized biocatalyst of 
the present invention does not putrefy even after one month of storage at 
ordinary temperature and therefore is extremely stable. 
The following examples are provided to further illustrate the present 
invention. It is to be understood, however, that the examples are for the 
purpose of illustration only and are not intended as a definition of the 
limits of the present invention. The terms "part(s)" and as used herein 
are based on weight, unless otherwise indicated.

EXAMPLE 1 
Rhodococcus rhodochruos strain J-1 (FERM BP-1478) was aerobically cultured, 
and the resulting cells were washed and concentrated to prepare a cell 
suspension (15% as dry cells) for use in immobilization. 16 parts of 50 mM 
potassium phosphate buffer (pH 7.0, this is to be repeated in the 
following) and 10 parts of a monomer mixture solution composed of 92% 
N,N-diethylacrylamide, 3% N,N-dimethylaminopropylacrylamide and 5% 
N,N'-methylenebisacrylamide were added to 70 parts of the concentrated 
cell suspension cooled in an ice bath. The mixture was subsequently 
stirred in an ice bath to obtain a uniform suspension. 2 parts of 10% 
N,N,N',N'-tetramethylethylenediamine aqueous solution and 2 parts of 10% 
ammonium persulfate aqueous solution were added, followed by 1 hour of 
incubation at a temperature of 35.degree. C. or lower to effect 
polymerization and gelation. The thus obtained block of immobilized cells 
was cut into small particles and washed with water to be evaluated as a 
sample of immobilized cells. 
When a 20 g portion of the thus prepared sample was soaked in 80 g of 0.5% 
sodium sulfate aqueous solution contained in a polyethylene bottle and 
stored at 30.degree. C. for one month after sealing the bottle, no changes 
in appearance were found and contamination of microorganisms was extremely 
low. 
COMATIVE EXAMPLE 1 
As a comparison, a sample of immobilized cells was prepared using an 
acrylamide base immobilization carrier. 1 part of 50 mM potassium 
phosphate buffer (pH 7.0) and 25 parts of a 40% monomer mixture aqueous 
solution composed of 92% acrylamide, 3% N,N-dimethylaminopropyl 
methacrylate and 5% N,N'-methylenebisacrylamide were added to 70 parts of 
the concentrated cell suspension. 2 parts of 10% 
N,N,N',N'-tetramethylethylenediamine aqueous solution and 2 parts of 10% 
ammonium persulfate aqueous solution were added to this mixture. 
Thereafter, polymerization and washing were carried out in the same manner 
as described in Example 1. 
When the prepared sample was soaked in 0.5% sodium sulfate aqueous solution 
and stored at 30.degree. C. for one month in the same manner as described 
in Example 1, growth of a markedly large number of contaminated 
microorganisms and generation of a strong rotted odor as well as turbidity 
were observed. 
EXAMPLES 2 TO 6 AND COMATIVE EXAMPLES 2 TO 6 
Immobilized biocatalysts were prepared by changing types and concentrations 
of monomers and biocatalysts and evaluated, in the same manner as in 
Example 1 and Comparative Example 1. The results are shown in Table 1. 
In this instance, the immobilized biocatalysts were stored at 30.degree. C. 
for one month by soaking them in 100 mM potassium phosphate buffer (pH 7) 
in the case of Examples 2 to 4 and Comparative Examples 2 to 4 or in 0.9% 
sodium chloride aqueous solution in the case of Examples 5 and 6 and 
Comparative Examples 5 and 6. 
Putrefaction was judged based on the degree of odor generated from, and 
turbidity formed in, the soaking solution. 
In the table, results of the evaluation are shown by 5 degrees where 0 
means no putrefied odor or turbidity of the soaking solution and 4 means 
maximum odor or turbidity. 
TABLE 1 
__________________________________________________________________________ 
Monomers Biocatalysts 
Ratio 
Conc. Conc. 
Putrefaction 
Run No. 
Name (%) (%) Strain (%) Odor 
Turbidity 
__________________________________________________________________________ 
Ex. 2 
N,N-Dimethylacrylamide 
92 
N,N-Dimethylaminopropyl 
5 10 Rhodococcus 
10 0 0 
methacrylamide sp. EA4 
(FERM P-12136) 
N,N'-Methylenebis- 
3 
acrylamide 
Comp. 
Acrylamide 92 
EX. 2 
N,N-Dimethylaminopropyl 
5 10 Rhodococcus 
10 3 4 
methacrylate sp. EA4 
(FERM P-12136) 
N,N'-Methylenebis- 
3 
acrylamide 
Ex. 3 
N,N-Diethylacrylamide 
95 
N,N-Dimethylaminopropyl 
1 10 Corynebacterium 
8 0 0 
methacrylamide quaternary 
sp. N-771 
compound (FERM BP-959) 
N,N'-Methylenebis- 
4 
acrylamide 
Comp. 
Acrylamide 95 
Ex. 3 
N,N-Dimethylaminopropyl 
1 10 Corynebacterium 
8 3 4 
methacrylate sp. N-771 
(FERM BP-959) 
N,N'-Methylenebis- 
4 
acrylamide 
Ex. 4 
N,N-Diethylacrylamide 
92 
N,N-Dimethylaminopropyl 
2 8 Gordona terrae 
5 0 0 
methacrylamide quaternary 
MA-1 
compound (FERM BP-4535) 
N,N'-Methylenebis- 
6 
acrylamide 
Comp. 
Acrylamide 92 
Ex. 4 
N,N-Dimethylaminopropyl 
2 8 Gordona terrae 
5 2 3 
methacrylate MA-1 
(FERM BP-4535) 
N,N'-Methylenebis- 
6 
acrylamide 
Ex. 5 
N,N-Dimethylacrylamide 
8 
N,N-Diethylacrylamide 
5 Nocardia 
sp. N-775 
(FERM BP-961) 
N,N-Dimethylamino- 
5 10 8 0 0 
propylacrylamide 
N,N'-Methylenebis- 
5 
acrylamide 
Comp. 
Acrylamide 90 
Ex. 5 
N,N-Dimethylaminopropyl 
5 10 Nocardia 8 3 3 
acrylate sp. N-775 
(FERM BP-961) 
N,N'-Methylenebis- 
5 
acrylamide 
Ex. 6 
N,N-Diethylacrylamide 
92 
N,N-Diethylaminopropyl 
3 8 Brevibacterium 
4 0 0 
methacrylamide sp. R-312 
(FERM P-2722) 
N,N'-Methylenebis- 
5 
acrylamide 
Comp. 
Acrylamide 92 
Ex. 6 
N,N-Dimethylaminopropyl 
3 8 Brevibacterim 
4 2 3 
methacrylate quaternary 
sp. R-312 
compound (FERM P-2722) 
N,N'-Methylenebis- 
5 
acrylamide 
__________________________________________________________________________ 
While the invention has been described in detail and with reference to 
specific examples thereof, it will be apparent to one skilled in the art 
that various changes and modifications can be made therein without 
departing from the spirit and scope of the invention.