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FIELD OF THE INVENTION
The present invention relates to a process for the production of a
homogeneous hydrophilic complex by lyogel or xerogel which has desirable
compatibility and affinity with a biological substance and is useful as a
supporting matrix for microbial cells and as an excellent pharmaceutical
material. The invention also comprises a process for immobilization of
microbial cells possessing enzymatic activities by entrapping them inside
the hydrophilic complex gel matrix under quite mild conditions, which is
produced from a water-soluble-polymer and silicate.
Enzymes are biological catalysts possessing extraordinarily high efficiency
and specific properties. They may be used to catalyze almost any type of
chemical reaction. Enzymatic reactions are accomplished under milder
conditions than chemical reactions and do not produce any harmful
substances. In recent days serious attention has been focused on the
important problem of environmental pollution in the chemical industry.
Accordingly, it is very advantageous to apply enzymes to the chemical
industry. Industrial application of enzymes or microorganisms has been
accomplished by using intact microorganisms or soluble enzyme
preparations. But such biocatalysts can be used just for only a single
batch reaction or fermentation. If enzymes and microorganisms can be
stabilized and recovered inexpensively without inactivation, it will be
possible to use biocatalysts more widely in the industry.
Immobilization of the catalyst offers a means of achieving objectives.
Immobilized enzymes are coming into use for technological purposes as well
as for medical and analytical purposes. Most immobilization studies
concern cell-free enzymes, but in recent years increasing attention has
been directed towards the use of immobilized whole microbial cells. These
systems obviate the need for cell separation, enzyme extraction and enzyme
purification steps prior to immobilization. Immobilized cells are also
more applicable to catalyzing sequential reactions and provide a means of
regenerating in situ the necessary cofactors. Further, if it should be
possible to continuously utilize a multiple enzyme system in the
immobilized state, it would mean that the traditional fermentation process
could be replaced with one employing immobilized cells.
The disadvantages associated with immobilized cells are the cost of the
supporting matrix and the loss of catalytic activity or the difficulty to
maintain the integrity of the cells during immobilization. In particular,
most microbial cells and enzymes are so unstable that subjecting them to
the immobilization process causes a decrease in their enzymatic activities
and changes in the specificity of their activities. It is very difficult
to develop a practical immobilizing process without the above mentioned
disadvantages. Therefore, few easily and widely utilized immobilizing
processes have been developed.
The present invention offers a method for immobilization of microbial cells
having enzymatic activity under quite mild conditions without decreasing
the activity thereof, and describes some desirable chemical, physical and
biological properties of the prepared immobilized microbial cells.
PRIOR ART
Method for immobilization of microbial cells are classified into four
types: support-binding method, crosslinking method, gel-entrapping method,
and microcapsule-entrapping method. Among these types, the gel-entrapping
method, by which microbial cells are immobilized in the gel matrix, is
widely used in practice. The conventional gel entrapping method, however,
have the following defects. By the gel formation processes of entrapping
microbial cells, their enzymatic activities are apt to be decreased
remarkably because the activities are frequently affected by environmental
factors such as temperature, pH, ionic strength, pressure and the like.
Therefore, a novel gel formation method which does not have any harmful
effect on the stability of enzymes has been desired. Many investigations
on crosslinking and gelling have been carried out with various polymer
compounds from this point of view. However, physical methods for gelling
polymers such as lowering the temperature or adding salts or non-aqueous
solvents have not been widely employed since it is generally difficult to
obtain permanent gels by these reversible gelling reactions. For example,
in Japan Kokai Patent Showa 50-52276, it has been disclosed that enzymes
can be entrapped inside the gel matrix of polyvinylalcohol polymers by a
method wherein enzymes and polyvinylalcohol polymers are solubilized in
water and solidified at preferably -25.degree. C. to -80.degree. C. and
melted at room temperature to form the gel entrapping enzymes. But the gel
prepared by the abovecited patent disclosure were unstable at
comparatively high temperatures such as 60.degree. C. or 70.degree. C.,
which is the reaction temperature range of glucose isomerase. Furthermore,
chemical methods for gel matrix formation by crosslinking reagents are too
strong and destructive for utilization with biological substances. This is
because of the high reactivity of the crosslinking reagents and the high
temperature and the extremely high or low pH of the gelling reaction. For
example, when polyacrylamide gel-entrapping method is employed, acrylamide
monomer is polymerized with N,N'-methylene-bis-acrylamide in the presence
of a catalyst such as ammonium persulfate. In this case, the enzyme is
often inactivated by the highly reactive catalyst. Therefore the range of
pH and temperature of the gelling reaction must be carefully chosen for
the maintenance of the enzymatic activities (S. S. Wang: Biotech & Bioeng,
15, 93, (1973)). And further, the gels obtained must be prohibited from
the application in the pharmaceutical and food industries because of the
possibility of remaining toxic acrylamide monomer. In Japan Kokai Patent
Showa 50-53583, it has been disclosed that enzymes can be entrapped inside
the gel matrices of polyvinylalcoholic polymers by method wherein enzymes
and polyvinylalcoholic polymers are dissolved in water and mixed with
boric acid or sodium borate to form the gel-entrapping enzymes. According
to the above-cited patent disclosure, enzymes can be immobilized without
remarkable denaturation at gelling temperatures below 45.degree. C.
However, as the gels are formed only at alkaline pH, this method can be
applied only to alkaline-stable-enzymes. The toxicity of boric acid must
also be considered. Other crosslinking methods using such high-energy
radiation as .gamma.-ray, electron ray or X-ray are difficult of practical
application because they require large equipment and precautions must be
taken to prevent the physiological effects associated with high-energy
rays (ref. H. Maeda; Biotech. & Bioeng., 15, 607 (1973).
The immobilized enzymes or microorganisms prepared by the conventional
gel-entrapping methods are physically weak and are especially low in
mechanical strength when in wet condition. So it is difficult to use such
gels in continuous reactions over a long period. When the gels are packed
in a column for continuous reaction, they are crushed by water pressure
and a satisfactory flow rate of the reaction mixture cannot be obtained.
Other prior art in this field is summarized in F. Katchalski & I. Silman,
"Effect of the microenvironment on the mode of action of immobilized
enzymes": Advance in Enzymology, 34, 445 (1971).
The present inventors have thoroughly studied the formation of homogeneous
complex gels from silica and various kinds of organic compounds because
silica is a compound which is harmless to bioorganisms and inexpensive as
well. When silica sol and silica gel manufactured by conventional methods
were mixed with water-soluble-polymer compounds under various conditions
no homogeneous complex lyogel containing much water, homogeneous
transparent complex lyogel or transparent complex xerogel could be
obtained, although the nature of the polymer compounds was considerably
changed. For example, when silica sol is added into the aqueous solution
of polyvinyl-alcohol (abbreviated as PVA hereinafter), the separation of
two layers is observed (5 to 20% silica, 1 to 5% PVA). Above pH 5 the
mixture is completely separated into two layers, one containing silica and
the other PVA. Under pH 5 the silica disperses into the PVA layer and
coacervate is formed. The desired homogeneous transparent sol or gel is
not formed at any pH. Furthermore, inorganic silicate salts such as water
glass cannot form a homogeneous solution with PVA by acid-hydrolysis, so
it is impossible to obtain the desired homogeneous transparent complex
gel.
SUMMARY OF THE INVENTION
According to the present invention there is provided a process for
preparing a hydrophilic complex gel by mixing a water-soluble-polymer and
an organic silicate, particularly, tetraalkoxysilane, and gelling under
mild conditions, whereby is obtained a hydrophilic complex gel having no
harmful effect on the activity of biological substances such as microbial
cells or enzymes and useful for the immobilization of microbial cells
possessing enzyme activity and for use as a pharmaceutical material.
It has been found that a tetraalkoxysilane such as tetraethoxysilane is
acid-hydrolyzed in an aqueous solution of water-soluble-polymer such as
polyvinylalcohol, gelatin, carboxymethylcellulose, starch and sodium
alginate, to form a homogeneous complex sol. It has been further found
that the formed homogeneous complex sol is gelled to become a
water-insoluble xerogel through the drying process. And it has been still
further found that microbial cells possessing enzymatic activity can be
immobilized by entrapment inside the gel matrix without decrease of the
enzymatic activity because the above-mentioned complex sol is gelled under
quite mild conditions.
Accordingly, it is the most important object of this invention to provide a
hydrophilic gel which is insoluble in water and inexpensive to produce,
and has excellent compatibility and affinity with a biological substance.
It is another object of the present invention to provide a hydrophilic gel
matrix which is readily applicable to immobilization of microbial cells
possessing enzymatic activity.
It is another object of the present invention to provide a novel means for
immobilizing microbial cells while maintaining at least more than 50% of
th enzymatic activity which was originally shown by the untreated
microbial cells.
It is another object of the present invention to provide immobilized
microbial cells which can be employed for use in a continuous process with
a column reactor or in a batch process with stirring.
Other and further objects will be evident from the following detailed
description of the present invention.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 gives the relation between the conversion rate of D-glucose to
D-fructose and the number of uses of the enzyme preparation. The ordinate
axis represents the conversion rate expressed in "% in weight" and the
abscissa axis represents the number of uses expressed by "times". Curves 1
and 2 show the results obtained with immobilized cells and untreated
cells, respectively.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The hydrophilic complex gel according to the present invention is produced
by reacting a water-soluble-polymer and a tetraalkoxysilane to form a
homogeneous sol and subsequently by gelling under quite mild conditions.
The tetraalkoxysilane is acid-hydrolyzed in an aqueous solution of the
water-soluble-polymer to form a homogeneous sol. Microbial cells
possessing enzyme activity are added to the sol thus obtained. The mixture
is gelled by pH adjustment to give a complex lyogel, or dried after the pH
adjustment to give a water-insoluble-xerogel in which lyogel or xerogel
are entrapped without decreasing the enzymatic activity.
The water-soluble-polymer compounds employed in this invention have many
polar groups such as --OH group, --COOH group, --NH.sub.2 group, .dbd.NH
group and so on, which form strong hydrogen bonds with acidic --OH groups
of silicate by hydrolysis. The water-soluble-polymer compounds shown in
Table 1 below can be used.
TABLE 1
(a) natural polymer compounds and their derivatives
1. Celluloses; carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose.
2. Starches; hydroxyethylstarch, carboxymethyl-starch.
3. Other polysaccharides; mannan, dextran, chitosan, pullulan, guar gum,
locust bean gum, tragacanth, xanthangum, agar, sodium arginate.
4. Proteins; gelatin, albumin.
(B) Synthetic polymer compounds
Polyvinylalcohol, polyethyleneglycol, polyethyleneimine.
The preferred water-soluble-polymers employed in the present invention may
be a polyvinylalcohol that has an average polymerization degree between
500 and 2,000, and a saponification degree within the range of 70 to 100%;
a commercial gelatin of edible grade that has a jelly strength of more
than 200 g shot/5 second in Bloom Gelometer; or a carboxymethylcellulose
of commercial edible grade that has a carboxylation degree between 0.4 and
0.8 and a sodium content within the range of 7.0 to 8.5%.
The water-soluble-polymer compound is solubilized in water at a
concentration having a viscosity below 10,000 centi poise, preferably
below 3,000 centipoise for full agitation. The above mentioned viscosity
is measured by No. 3 rotor of a B type viscosity meter at 40.degree. C.
The tetraalkoxysilanes which may be used in the present invention are those
of the structural formula: Si(OR).sub.4, in which R is alkyl group of up
to 12 carbon atoms, e.g., methyl, ethyl, propyl, butyl, octyl and lauryl
groups. Compounds in which R represents an alkyl group of up to 3 carbon
atoms are preferably utilized for the effective formation of the
homogeneous complex sol. Tetraethoxysilane is the most suitable compound.
The water-soluble-polymer compound is mixed with the above-mentioned
tetraalkoxysilane, and then the pH of the mixture is adjusted below 3 with
acid or acidic salt which exhibits no harmful effect on the enzymatic
activities of microbial cells. The acids or the acidic salts which may be
used in the present invention are shown in Table 2.
TABLE 2
(a) inorganic acids: HCl, HNO.sub.3, H.sub.3 PO.sub.4, H.sub.2 SO.sub.4.
(b) organic acids: Acetic acid, glutamic acid, lactic acid, maleic acid,
succinic acid, ascorbic acid, citric acid, tartalic acid.
(C) Inorganic or organic acidic salt: AlCl.sub.3, citrate-1-ammonium.
The amount of tetraalkoxysilane necessary for the above-mentioned mixing
with the aqueous solution of water-soluble-polymer varies according to the
kind of silane compound and the properties of the water-soluble-polymer
employed. The amount of SiO.sub.2 contained in the tetraalkoxysilane,
calculated based on SiO.sub.2 formed by acid-hydrolysis, should be 5 to
300% (w/w) of the dry weight of the water-soluble-polymer. The preferable
amount of SiO.sub.2 is in the range of between 50% and 200%. When the
amount of SiO.sub.2 is below 5%, the solubility in water of the formed gel
increases significantly, whereas, in case of it being above 300%, the gel
becomes fragile. The property of the gel depends on the SiO.sub.2 content
of the tetraalkoxysilane and/or the chemical structure of the
water-soluble-polymer. For example, PVA having an average polymerization
degree between 500 and 2,000 and a saponification degree within the range
of 70 to 100%, may be used, but the preferred amount of SiO.sub.2 is above
20%, more preferably above 50%, in case of completely saponified PVA,
while a smaller amount of SiO.sub.2 is required for a PVA having a lower
saponification degree.
In the initial stage of the reaction process, the mixture of
water-soluble-polymer compound and tetraalkoxysilane is separated into two
layers because of their mutual insolubilities. After adjusting the pH
below 3 by the addition of acid or acidic salt, the mixture is stirred
well at room temperature with heating below 80.degree. C., if necessary,
to be hydrolyzed completely. The lower the pH and the higher the
temperature is, the faster the hydrolytic reaction is, but the conditions
are chosen according to the nature of the desired gel. The aqueous
solution of PVA and tetraethoxysilane are mixed at such ratio that the
SiO.sub.2 amount of the tetraethoxysilane is 100% (w/w) of the dry weight
of the PVA. The mixture is completely hydrolyzed at pH 3 and room
temperature over two hours to form a colorless transparent homogeneous
complex sol. The completion of the hydrolysis may be determined by the
fact that the boundary of the two separated layers disappears and a
homogeneous and transparent solution is obtained. The original specific
smell of tetraalkoxysilane changes to an alcoholic perfume.
The formed sol can be preserved for a long period below room temperature.
In particularly, no property of the sol is changed at 5.degree. C. even
after one month.
As previously mentioned, various kinds of water-soluble-polymer compounds
may be used as one of the components of the complex sol. The complex sol
can be easily gelled by the pH adjustment or the drying process. The
complex gels formed under different conditions from the same
water-soluble-polymer compound have various degrees of turbidity. For
example, the complex sol of gelatin and tetraethoxysilane is converted to
a transparent gel below pH 4. The most turbid gel is obtained at pH 5, and
above pH 7, the gel becomes semi-transparent.
The complex gels formed are normally classified into the lyogel type which
contains much water inside and the xerogel type which contains little
water. In the latter type, the moisture of the gel is almost completely
removed through the drying process. According to this invention, the
components of the sol are not separated, but maintain the homogeneous
complex state throughout the processes of the sol formation.fwdarw.gel
formation.fwdarw.xerogel formation. The xerogels prepared are insoluble or
hardly soluble in water, but exhibit hydrophilic property.
As shown in the Examples described hereinafter, the properties of the
formed xerogel depend on the nature of the water-soluble-polymer employed
and also on the ratio of SiO.sub.2 to the polymer. Generally the gel
becomes more insoluble in water, and harder and more fragile in accordance
with the increase of SiO.sub.2 amount. In contrast, it becomes more
flexible with the increase of the polymer. As for the effect of the
water-soluble-polymer (PVA, for example), the properties of the xerogel
vary according to the saponification degree of the PVA employed. In the
case of completely saponified PVA, the smallest amount of SiO.sub.2
required is 50% (w/w) based on the PVA used. But, for the formation of a
similar xerogel with partially saponified PVA (87% saponification), the
required amount of SiO.sub.2 is smaller, but must be at least more than
20% (w/w).
According to the present invention, microbial cells possessing enzymatic
activities can be immobilized under quite mild conditions by entrapping
them inside the gel matrix of the above-mentioned hydrophilic complex gel
produced from the water-soluble-polymer compound and silicate. The
microbial cells are added into the above-mentioned homogeneous sol without
any pH adjustment or basic salts as shown in Table 3.
TABLE 3
(a) bases: NaOH, KOH, Ca(OH).sub.2, NH.sub.4 OH.
(b) basic salts: Na.sub.2 CO.sub.3, CH.sub.3 COONa, NaHCO.sub.3, K.sub.2
CO.sub.3, K.sub.2 HPO.sub.4, Na.sub.2 HPO.sub.4, CH.sub.3 COOK.
the microbial cells which may be used in the present invention are dried
cells, wet cells harvested from broth by centrifugation or filtration and
the cultured broth itself. These microbial cells are classified into the
following 5 groups, bacteria, actinomycetes, fungi, yeast and algae.
Bacteria of the first group, belonging to Class Shizomycetes
taxonomically, are Genus: Pseudomonas, Acetobacter, Gluconobacter,
Bacillus, Corynebacterium, Lactobacillus, Leuconostoc, Streptococcus,
Clostridium, Brevibacterium, Arthrobacter, Erwinia and so on.
Actinomycetes of the second group, belonging to Class Shizomycetes
taxonomically, are Genus: Streptomyces, Nocardia, Mycobacterium and so on.
(R. E. Buchran & N. E. Gibbons, "Bergey's mannual of Determinative
Bacteriology" 8th edition, (1974); The Williams & Wilkins Company). Fungi
of the third group, belonging to Class: Phycomycetes, Ascomycetes, Fungi,
imperfecti, and Bacidiomycetes taxonomically, are Genus: Mucor, Rhizopus,
Aspergillus, Penicillium, Monascus, Neurosporium and so on (J. A. von ARX,
"The genera of Fungi sporulating in pure culture" Verlag von J. Cramer,
and H. L. Barnett & Barry B. Hunter, "Illustrated genera of Imperfect
Fungi" 3rd edition, (1970); Burgess publishing company). Yeast of the
forth group, belonging to Class Ascomycetes, taxonomically, are Genus:
Saccharomyces, Zygosaccharomyces, Pichia, Hansenula, Candida, Torulopsis,
Rhodotorula, Kloechera and so on (J. Lodder, "The yeast A taxonomic study"
2nd edition (1970); North-Holland publishing company). Algae of the fifth
group are single-cell Genus: Chlorella and Scedesmus in green-algae, and
single-cell Genus Spirulina in blue-green-algae (H. Tamiya, "Studies on
microalgae and Photosynthetic Bacteria" (1963); The University of Tokyo
press).
Most of these microbial cells are single-cell-grown organism. The cell
sizes of individual microorganisms belonging to the above mentioned groups
are different from one another, but the diameter or width of each cell
employed in the present invention is within the range of 1-20.mu.. The
microbial cells can be dispersed in aqueous solvents. Some kinds of
Actinomycetes of the second group and Fungi of the third group are more
than 20.mu. in length. Long sized microbial cells (average length:
50-100.mu.) and/or pellet-shaped microbial cells can be entrapped inside
the above mentioned gel matrix, but the amount of entrapped cells is
comparatively decreased. Therefore, such microbial cells are preferably
shredded to be less than 20.mu.. For this purpose, mechanical
homogenization in water may be employed.
Microbial cells used in the present invention, selected from the above
mentioned five groups, possess more than one enzymatic activity. The
enzymes are classified into the following five groups.
TABLE 4
(a) oxido-reductases: Glucose oxidase, nitrite reductase, catalase, phenol
oxidase, monoamine oxidase, cytochrome c reductase.
(B) Transferases: Glutamate-oxaloacetate-transaminase, 16-membered
macrolide 3-acyltransferase, ATP: nucleoside-5' monophosphate
pyrophosphotransferase.
(C) Hydrolases: Proteases, glucoamylase, .alpha.-amylase, isoamylase,
lipase, lactase, penicillin amidase, alkaline phosphatase, amino acylase,
urease, cellulase.
(D) Isomerases: Glucose isomerase, alanine racemase.
(E) Lyases: .beta.-Tyrosinase, histidine decarboxylase, tryptophanase.
The enzymes used in the present invention are the intracellular enzymes
existing inside the microbial cell or mycelium, or inside the organella of
bioorganisms. In the present invention, "activity" means not only
enzymatic activity, but also other biological activities, such as for
example, activities of inhibitors, co-enzymes, antibiotics, antigens,
antibodies and the like.
The pH of the mixture of the complex gel and the microbial cells should be
chosen in consideration of the enzyme stability or the properties of the
formed gel. Usually, it is adjusted to pH 4-8, preferably 5-7. In the
immobilization of microbial cells, the adjustment of pH is not needed in
most cases because the microbial cells usually have buffer activity in
themselves. The pH of the above mentioned mixture usually falls within 5
to 6.5 after the addition of the cells.
The dry weight of the microbial cells to be added is less than 1,000 (w/w),
preferably 20-500% (w/w), based on the dry weight of the homogeneous
complex sol. After adding the microbial cells to the homogeneous complex
sol, the mixture is stirred well to disperse them homogeneously. The
gelling reaction of the sol containing the microbial cells takes place at
any temperature. However, as the enzymes are unstable at the higher
temperature, the gelling reaction is carried out at a temperature between
0.degree. C. and 70.degree. C., preferably between 10.degree. C. and
40.degree. C. The gelling reaction is usually completed within 10-30
minutes. For example, the sol is fully gelled at pH 6.0 by continuous
stirring at room temperature for 10-20 minutes.
One of the most important advantages of the present invention is the mild
conditions for the immobilization, under which the enzymatic activities
can be maintained throughout the whole processes without remarkable
inactivation.
The formed complex gel entrapping microbial cells is dried and converted to
the desired shape. The drying process is carried out below 75.degree. C.
In the case of yeast cells, the fermentation activity is comparatively
stable and the whole process of the present invention may be conducted at
room temperature. But, as far unstable bacterial cells such as those used
for enzymatic L-tyrosine synthesis, eliminate the whole process is
performed below 5.degree. C. in order to maintain the enzymatic
activities, and especially, freeze-drying is preferred in the drying
process. As long as the temperature is maintained within the above
mentioned range, almost no inactivation of the enzyme is observed. For the
conversion of the above mentioned lyogel immobilizing microbial cells into
various shapes, the conventional methods are employed in the present
invention. The molding process can be carried out before or after the
drying. Especially, hydrophilic or hydrophobic organic solvents in which
the complex lyogel is almost insoluble can be employed for this molding
process. The following organic solvents as shown in Table 5 may be used.
TABLE 5
(a) alcohols: Methylalcohol, ethylalcohol, n-propylalcohol, n-butylalcohol,
ethyleneglycol, glycerol.
(B) Ketones: Acetone, methylethylketone.
(C) Ethers: Dioxane, tetrahydrofuran.
(D) Alkanes: n-heptane, n-paraffin.
(E) Aromatics: Benzene, toluene, xylene.
(F) Others: Methylenechloride.
In the present invention, the shapes of the gel-immobilized microbial cells
may be the granular-type having a round section and in particularly may be
spheres, granules, pellets, filaments and so on. When the molded
immobilized microbial cells are packed in a column reactor, they give good
results in the continuous reaction. In some cases, the molded immobilized
microbial cells may also be the film-type or the strip-type or in the
other cases, irregular-granules or powder. The average diameter and/or the
thickness of the molded gel containing the microbial cells may be between
0.2 and 5 mm, preferably between 0.4 and 1.0 mm. With the molded gels of
this thickness, a desirable contact between the immobilized cells and
their substrate can be obtained. Furthermore, a desirable flow rate of the
reaction mixture through the column reactor may also be obtained.
The desired shape may be obtained by the method of slit-casting because the
complex lyogel entrapping the microbial cells is a soft dough. For
example, the lyogel according to the present invention is converted into a
long cylinder by slit-casting in an organic solvent or the air, and then
dried. And also, the lyogel is formed into round granules by dropping into
an organic solvent or air, and subsequently by drying. Thus prepared gels
have high specific surface area.
Spray-drying may also be employed for the preparation of the desired
shapes. The prepared granules have high specific surface area and good
properties for utilization in a column reactor. This method may be
advantageously used on an industrial scale from the economical viewpoint.
In the present invention, good results may also be obtained by employing
the freeze-drying method. The enzymatic activities of the prepared
granules according to the method are satisfactorily maintained even if the
method is applied to the heat-unstable microbial cells as shown in Example
16 and 17 described hereafter, where the bacterial cells capable of
enzymatic L-tyrosine synthesis were used.
Another preferable shape of the gel is the film-type, which is prepared by
spreading the sol over the surface of a plate. This type of the gel may
also be advantageously applied to industrial use because it has a high
specific surface area and is easily produced. The granules of the
immobilized microbial cells may also be obtained by pulverization process.
Usually more than 80%, and at least 50%, of the enzymatic activities of
the untreated microbial cells can be maintained in the glanules prepared
by the method.
The above-mentioned immobilized microbial cells prepared according to the
present invention are stable and maintain this activities for a long
period, at least for a year when stored at 10.degree. C., and can be
applied to continuous and batch-repeated reactions. For example, the
immobilized microbial cells of glucose isomerase can be used without any
loss of the activity in a continuous reaction using a column reactor for
30 days. Throughout the long-period continuous process, pressure drops can
be ignored even with comparatively high flow rate and with various degrees
of ionic strength.
The immobilized microbial cells according to the present invention can be
utilized as the catalysts for biochemical conversion of various substrates
not only in the column reactor system, but also in the batch-repeated
reactor system. In using the usual batch-repeated reactor system with
stirring, no pronounced leaking of the microbial cells from the gel or
destruction of the molded gel is observed. Further, the
water-soluble-polymer compound can be recovered from the gel immobilizing
microbial cells and used repeatedly when the activities have decreased
after a long period of use. The water-soluble-polymer compound or the
complex sol can be recovered by dissolving the gel in a hot alkaline
water, such as aqueous ammonia, removing the cells by centrifugation or
filtration, and subsequently adjusting the pH.
The following examples illustrate methods of carrying out the present
invention but it is to be understood that they are given for purposes of
illustration and not of limitation.
EXAMPLE 1
50 g of 10% PVA (polymerization degree: 2,000 and completely saponified)
aqueous solution was mixed with 20 g of water, 5 g of tetraethoxysilane,
and 1 g of 1 N HCl and stirred. The turbid mixture gradually became a
colorless, transparent homogeneous complex sol at room temperature in two
hours. The prepared homogeneous complex sol was neutralized by adjusting
the pH to 7.0 with 1 N NH.sub.4 OH, and a colorless, transparent
homogeneous complex lyogel was formed without any separation of the lyogel
components such as PVA or tetraethoxysilane. The homogeneous sol was also
gelled without neutralization by storing for a long period to form complex
lyogel, which also was transparent and homogeneous and insoluble in water
of room temperature. The prepared homogeneous complex lyogel was dried by
ventilation at room temperature to form a transparent homogeneous complex
xerogel, which was insoluble in tap water. The complex xerogel thus
obtained was swelled without any separation of the components, when
treated in boiling water for an hour. In case of the complex xerogel
prepared after neutralizing and drying by ventilation mentioned above, the
solubilized amount was 50% of the initial weight of the complex xerogel,
and the swelling degree was about 30 fold in boiling water. In case of the
complex sol without neutralization, the solubilized amount was 30% and the
swelling degree was about 12 fold in boiling water.
EXAMPLE 2
By the same procedure as described in Example 1, except for adding 15 g of
tetraethoxysilane, homogeneous complex lyogel was formed. The reaction
process, intermediately produced sol and the appearance of resulting
lyogel were almost the same as those in Example 1. The complex xerogel
formed with or without neutralization was insoluble in tap water. The
solubilized amount was less than 3% and the swelling degree was less than
3 fold by treatment in boiling water.
EXAMPLE 3
Employing the same procedure as described in Example 1, except for adding
20 g of tetraethoxysilane instead of 5 g, homogeneous complex lyogel was
again formed and was found to have similar appearance (i.e. was colorless
and transparent) to that of Example 1. Subsequently formed complex
xerogel, with or without neutralization, was harder and more brittle to
physical forces than that of Example 1. The gel was insoluble in tap
water, the solubilized amount being about 2% and the swelling degree about
2 fold in boiling water.
EXAMPLE 4
By the same procedures as described in Example 1, except for adding 2 g of
tetraethoxysilane into 5 g of 10% PVA (polymerization degree: 1,700 and
saponification degree: 87) aqueous solution, the homogeneous complex
lyogel was formed and was found to have very similar appearance to that of
Example 1. The complex xerogel formed with or without neutralization was
insoluble in tap water and the solubilized amount was less than 9% and the
swelling degree was less than 6 fold in boiling water.
EXAMPLE 5
A similar reaction was proceeded as in Example 4, using 10 g of
tetraethoxysilane and 5 g of 10% PVA (polymerization degree: 1,700 and
saponification degree: 87) aqueous solution. The reaction characteristics
and the appearance of the complex sol and resulting homogeneous complex
lyogel were almost the same as that of Example 1. The final complex
xerogel obtained with or without neutralization was insoluble in tap water
and its solubilized amount was about 4% and the swelling rate about 2.5
fold in boiling water.
EXAMPLE 6
2 g of tetraethoxysilane was added with stirring at room temperature to a
mixture of 20 g of 5% PVA (polymerization degree 1100, completely
saponified) aqueous solution and 0.5 g of aluminum chloride. Stirring was
continued for about 2 hours and a colorless, transparent and homogeneous
complex sol was obtained. The sol thus formed was dried under ventilation
to give a film of transparent homogeneous complex xerogel. This film was
insoluble even by extraction in boiling water and maintained its original
film shape. The above mentioned sol was also neutralized with 1 N NH.sub.4
OH causing the sol to turn white-turbid. It was then dried in ventilation
to give a semi-transparent xerogel. The final gel was insoluble in tap
water and maintained its original shape in boiling water.
EXAMPLE 7
100 g of 5% gelatin aqueous solution was diluted into 66 g of water,
stirred well with heating, and added with 15 g of tetraethoxysilane. After
adjusting the pH to 3 or below with diluted HCl, the mixture was stirred
continuously at room temperature for 3 hours after which the mixture
turned transparent. The homogeneous complex sol so formed was left to
stand at room temperature to obtain a transparent homogeneous complex
lyogel. After the pH of the lyogel was adjusted with diluted NH.sub.4 OH,
the complex gel became turbid at about pH 4.5 and very turbid at about pH
5 to 6, but again returned to be semi-transparent at above pH 7.
Homogeneity was maintained and no water layer was not observed. The pH
value influenced the standing period for formation of the complex lyogel.
By drying homogeneous complex sols and homogeneous complex lyogels, there
were obtained homogeneous complex xerogels having various degrees of
turbidity according to the pH variation (transparent in acidic range,
whitely turbid at about pH 5 to 6, and semi-transparent above pH 8). The
xerogel was insoluble in water at room temperature and swelled in boiling
water without loosing its original shape.
EXAMPLE 8
100 g of 5% soluble starch aqueous solution was diluted into 66 g of
H.sub.2 O, and after stirring homogeneously with heating, 15 g of
tetraethoxysilane was added. After adjusting the pH to 3 with diluted
H.sub.3 PO.sub.4 solution, the mixture was stirred at room temperature for
about 2 hours until a transparent homogeneous complex sol was formed. This
sol was neutralized with diluted NaOH and after about 30 minutes storage
homogeneous complex lyogel was obtained. Either drying under ventilation
of the homogeneous complex sol without neutralization, or drying under
ventilation of the homogeneous complex lyogel formed after neutralization,
led to formation of whitely turbid xerogel which was brittle to physical
force. Only a small amount of starch was extracted from the xerogel in tap
water and 50% was extracted in boiling water. The original shape was
preserved in both cases and swelling occurred particularly in boiling
water.
EXAMPLE 9
100 g of 5% carboxymethylcellulose aqueous solution was added into 66 g of
water the the mixture was stirred well at 45.degree. C., and then 20 g of
tetraethoxysilane was added to the mixture. The pH of the mixture was
adjusted to 3 with 1 N HCl. The mixture was continuously stirred for about
two hours until a transparent homogeneous complex sol was radially formed.
Further, by adjusting the pH of sol to 6.0 with 1 N NH.sub.4 OH and drying
under ventilation, a transparent homogeneous complex xerogel was obtained.
The homogeneous complex xerogel was insoluble and did not swell in water
at room temperature. Even in boiling water, it was almost insoluble and
the swelling degree was about 2 fold.
EXAMPLE 10
15 g of tetraethoxysilane was added to 100 g of 5% aqueous polyacrylate
solution diluted with 66 g water and the mixture was stirred at room
temperature for about 2 hours. The mixture gradually became a transparent
homogeneous complex sol, which was neutralized with 17 g of 1 N NH.sub.4
OH and stored for the formation of semi-transparent complex lyogel. The
homogeneous complex sol formed without neutralization and the homogeneous
complex lyogel formed by neutralization were dried to give respective
white semi-transparent xerogels. About a half part of the xerogel thus
obtained was dissolved in boiling water and the other half remained as the
soft hydrogel insoluble in boiling water.
EXAMPLE 11
15 g of tetraethoxysilane was added to 100 g of 5% polyethyleneglycol
(#4,000) aqueous solution diluted with 66 g of water and the mixture was
stirred at room temperature. By addition of 1 g of 1 N HCl, over 3 hours,
the mixture became a colorless and transparent homogeneous sol. This sol
was neutralized with 1 N NH.sub.4 OH and stored for 3 hours to form a
colorless transparent and homogeneous complex lyogel. By drying the
lyogel, a colorless transparent and homogeneous complex xerogel was
obtained. In boiling water, 50% of the formed xerogel was dissolved to
leave a turbid gel. This solid xerogel was rather susceptible to
destruction.
EXAMPLE 12
100 Parts of 10% PVA aqueous solution (polymerization degree: 1,700 and
saponification degree: 99.5) were mixed with 231.5 parts of distilled
water, 28.5 parts of tetraethoxysilane and 1 part of 1 N HCl, and stirred
for more than two hours at room temperature to give a transparently
homogenized sol at about pH 3, containing 5% of solid part (abbreviated as
PVA-SiO.sub.2 complex sol hereinafter). One part of commercially available
dried baker's yeast (Saccharomyces cerevisiae) was suspended into 2 parts
of water and was then mixed with PVA-SiO.sub.2 complex sol. After the
yeast was dispersed homogeneously by stirring, the pH was adjusted to 7.0
with 1 N NH.sub.4 OH solution. The gel was poured into a petri dish and
dried spontaneously at room temperature. A yellowish brown film containing
the yeast cells was obtained. A strip of the film immobilizing 1 g of the
yeast cells was taken out and incubated in 50 g of 5% glucose aqueous
solution at 30.degree. C. to carry out the fermentation test. A large
amount of gas production began in a few minutes and in thirty minutes a
large amount of gas production was observed from the whole surface of the
film. The film swelled a little and decolorized, but the shape showed no
change, and the bubbling continued for 30 hours. The reaction was
estimated by the measurement of the weight decrease of the medium by
liberation of CO.sub.2 gas. The reaction was carried out in proportion to
the reaction time. A slight fragrant smell peculiar to alcoholic
fermentation was detected. Increase in the turbidity of glucose solution,
which is considered to come from the leaking yeast cells from the film,
was hardly observed, and hence, the mixture was almost transparent. As a
control, the same reaction was conducted by using 1 g of dried yeast cells
not immobilized. At the very initial stage of reaction, a slightly large
amount of CO.sub.2 was generated by using the control cells than the
immobilized cells but, after 30 hours incubation, the rate of CO.sub.2 gas
liberation from both media became almost equal. Accordingly, this fact
indicated that the unit activity in both cases, native and immobilized
cells, was almost comparable to each other, and hence, inactivation of
enzymes in the immobilized cells did not occur during the immobilizing
process of yeast cells. As a matter of course, since the non-immobilized
cells were suspended in the medium, mild agitation was required for
securing the progress of reaction. From the above results, it was
demonstrated that the microbial cells were firmly immobilized by the
present process while maintaining fermentation activity equal to that of
the native cells.
EXAMPLE 13
The same PVA-SiO.sub.2 complex sol as described in Example 12 was used. 4
parts of commercially available dried yeast (Saccharomyces cerevisiae) was
suspended in 5 parts of water. The suspension was mixed in 20 parts of the
PVA-SiO.sub.2 complex sol and the gel was spread on a plate and dried by
ventilation to obtain a yellowish brown film containing yeast cells. A
strip of the film containing 1 g of immobilized cells was shred, and
incubated in 50 g of 5% glucose aqueous solution at 30.degree. C. to carry
out fermentation as in Example 12. A large amount of CO.sub.2 gas was
liberated even in the early stage of incubation as observed in the control
experiment. After terminating the fermentation, the film was taken out of
the medium and soaked in another 50 g of 5% glucose aqueous solution
again, liberation of CO.sub.2 gas was again observed as much as in the
previous test with native cells. This fermentation test was repeated 5
times, and the film in glucose solution showed almost the same
fermentation activity every time. The | | |
