Reversibly precipitable immobilized enzyme complex and a method for its use

A process for using and preparing a reversibly soluble enzymatically active polymer enzyme product which consists of an enzyme covalently bonded to a water soluble organic polymer selected from polyacrylic acid, dextran, carboxy methyl cellulose, and polyethylene glycol which have carboxyl or amino side groups that impart to the complex its reversible solubility.

The object of the present invention is a process for the use of an enzyme 
in order, by enzymatic reaction, to transform an organic substance into at 
least one different organic substance, in which the enzyme is attached, by 
covalent bonds, onto an organic polymer which is soluble in aqueous 
medium, so as to form an active enzymatic complex which is soluble in 
aqueous medium, and the complex is maintained, in dissolved state, in an 
aqueous solution of the substance to be transformed, for a period of time 
and at a temperature sufficient to permit the obtaining of the desired 
degree of transformation of said substance. 
The use of enzymes in order to transform an organic substance into at least 
one other organic substance is well known in industry, particularly in the 
food industry. 
The traditional processes for the use of enzymes to effect an enzymatic 
reaction consist in introducing the enzyme, in dissolved state, into a 
reaction medium, generally a liquid, which contains the substance 
(so-called "substrate") which it is desired to transform, and maintaining 
said reaction medium at a temperature and for a period of time sufficient 
to permit the obtaining of the desired degree of transformation of said 
substance. 
Upon the carrying out of these processes, it is not possible, at the end of 
the reaction, to recover the excess, if any, of unconsumed enzyme. For 
this reason the remaining amount of enzyme is generally destroyed in situ 
in order to avoid the presence of enzyme in mixture with the reaction 
product. 
On the other hand, since the cost of enzymes is generally high, it is 
possible in practice, for reasons of an economic nature, to use only small 
quantities thereof, which results in a low enzymatic reaction velocity, 
which velocity in most cases is insufficient to permit the use of enzymes 
of an industrial scale. It is only in the case of enzymes which are of 
relative low price, such as the proteases and amylases, that industrial 
utilization of these processes can be contemplated. 
In accordance with more recent processes, use is made of "immobilized 
enzymes", that is to say enzymes which are fixed by covalent bonds on a 
suitable organic polymer, so as to constitute an active enzymatic complex. 
These last mentioned processes make it possible to recover any excess of 
enzyme after the enzymatic reaction. These processes therefore make it 
possible to use a larger amount of enzyme than the amount theoretically 
necessary for the transformation of the substance subjected to the 
enzymatic reaction, which results in an increase in the velocity of this 
reaction and therefore in the hourly yield. 
Moreover, the attaching of an enzyme to a polymer, in the form of an active 
enzymatic complex, generally has the effect of increasing the stability of 
the enzyme and, on the other hand, in certain cases makes it possible to 
increase the useful pH range of the reaction medium and facilitate the 
adjustment of the course of the enzymatic reaction. 
Finally, the use of an enzyme in "immobilized" state in the form of an 
active enzymatic complex makes it possible to obtain the product of the 
enzymatic reaction directly in a state which is free of enzyme. 
The enzyme may be attached either to a water-insoluble polymer so as to 
form an active enzymatic complex in solid state which is insoluble in 
aqueous medium, or to a water-soluble polymer so as to form an active 
enzymatic complex which is soluble in aqueous medium. 
In the former case, the insoluble active enzymatic complex can be used in 
the form of particles of powder in a column similar to solid-phase 
chromatography columns through which the reaction medium is passed, or 
else in suspension in a vessel containing the reaction medium. 
Due to the fact that the active enzymatic complex is in solid state in the 
reaction medium, this complex can easily be separated inexpensively from 
the reaction medium and recovered at the end of the enzymatic reaction. 
However, this manner of procedure, when a column is used, has the drawback 
of the danger of the clogging of the column or of the production of 
so-called "channeling" (flow of the reaction medium in the form of liquid 
streams which are limited to only a portion of the cross section of the 
column). 
When the enzymatic reaction is carried out in a tank, the use of a solid 
active enzymatic complex has the drawback of poor efficiency of contact 
between the particles of said complex and the substance which it is 
desired to transform, due to the difficulty, or impossibility, of said 
medium penetrating into these particles. 
The use of an active enzymatic complex dissolved in the reaction medium 
makes it possible to carry out the enzymatic reaction in a homogeneous 
liquid medium, which improves the effectivensss of the contact between the 
enzymatic complex and the substance to be transformed and therefore 
increases the yield of the reaction. 
Moreover, the percentage attachment of the enzyme to an organic polymer 
which is soluble in aqueous medium is generally greater than that of the 
attachment of this same enzyme to an insoluble organic polymer. Moreover, 
the use of a polymer which is soluble in aqueous medium in order to attach 
the enzyme makes it possible to obtain an enzymatic complex whose specific 
activity, expressed in units of enzyme per units of mass of the complex is 
generally higher than that of the enzymatic complexes obtained by 
attaching the enzyme to an insoluble polymer. 
(An enzyme unit is defined as the quantity of enzyme which makes it 
possible to cause the complete transformation of one micromol of substrate 
per minute under optimum reaction conditions.) 
On the other hand, for given operating conditions, the activity of an 
enzyme with respect to a substrate of high molecular weight is generally 
greater when said enzyme is attached to a polymer which is soluble in the 
reaction medium than when said enzyme is attached to an insoluble polymer. 
The use of an active enzymatic complex in solution in the reaction medium 
also has the advantage over the use of an insoluble solid active enzymatic 
complex that it makes it possible to avoid the proliferation of 
micro-organisms, which frequently takes place in the case of the solid 
enzymatic complexes. For this purpose, it is sufficient to effect a 
filtration of the solution of soluble enzymatic complex through a 
microfilter, for instance a filter having pore sizes of the order of 0.2 
microns, so as to retain the microorganisms and obtain a sterile solution 
of active enzymatic complex as filtrate. 
However, the use of the active enzymatic complexes soluble in aqueous 
medium which have been proposed up to the present time presents the 
drawback that an ultra-filtration of the reaction medium is required in 
order to separate the reaction product from the enzymatic complex and to 
recover the latter for its further reuse. Such ultra-filtration requires 
the use of costly equipment and is furthermore a cause of a decrease in 
the overall yield of the process. This drawback therefore stands in the 
way of the industrial use of the active enzymatic complexes which are 
soluble in aqueous medium. 
The object of the present invention is to combine the advantages of the use 
of an insoluble active enzymatic complex with those of the use of a 
soluble enzymatic complex, without having the drawbacks thereof. 
For this purpose, the process of the invention is characterized by the fact 
that the organic polymer is selected from polymers which form an active 
enzymatic complex which is reversibly precipitable and retains its 
enzymatic activity after redissolving, and that, after the desired degree 
of transformation of the substance to be transformed has been obtained, 
the enzymatic complex is precipitated and removed from the reaction 
medium. 
In the present specification the expression "reversibly precipitable" means 
that the enzymatic complex is capable of being redissolved after it has 
been precipitated, and that it can be again precipitated from the solution 
thus obtained, which successive precipitating and redissolving operations 
can be repeated an indefinite number of times, without change in the 
physical-chemical and enzymatic properties of the complex, either in 
dissolved state or in the state of a precipitate. 
It should be noted that the phenomena which may be involved in the 
precipitation and redissolving of the enzymatic complex are not 
necessarily "reversible" in a thermodynamic sense of the term, that is to 
say they do not exclude heat exchanges with the outside environment. 
As water-soluble organic polymer one can use, for instance, a derivative of 
polyacrylic acid, such as polyacrylomide or else a dextran, 
carboxymethylcellulose, polyethyleneglycol, etc., this polymer having 
chemical groups which impart to it the property of reversibly 
precipitating or flocculating in aqueous medium as a result of a 
modification of at least one physicochemical parameter of said medium, 
such as the temperature, pH, concentration of solute, etc., or else by 
adding to such medium ions, such as bivalent or trivalent metallic ions, 
which are capable of bringing about the precipitation of the enzymatic 
complex, combined with the further use of at least one complexing agent 
for said ions in order to cause the redissolving of the active enzymatic 
complex. 
In particular, as organic polymer one can employ a derivative of 
polyacrylamide bearing --COOH side groups which impart to it the property 
of precipitating quantitatively and reversibly by decrease of the pH of 
the medium below a value of the order of 4.5 to 5, while being soluble in 
this same medium at a pH higher than this value. 
More particularly, one can employ organic polymers having acid side groups 
of the benzoic or isophthalic type. Such polymers have the advantage that 
they are precipitable at a pH of less than 4.5 in a manner which is 
practically quantitative since the proportion of complex remaining in 
solution after the precipitation is generally less than 1 ppm referred to 
the initial quantity.

Example 1 
A. Preparation of the active enzymatic complex: 
Acrylic chloride and p-amino benzoic acid are reacted in accordance with 
the equation: 
##STR1## 
whereupon the monomer thus obtained is polymerized in aqueous medium in a 
nitrogen atmosphere and in the presence of a small amount of 
N,N'-methylene-bis-acrylamide and of ammonium persulfate, the latter 
compound serving as polymerization catalyst. 
In this way there is obtained a water-soluble polymer derived from 
polyacrylamide, composed of interconnected macromolecular linear chains 
having the recurrent unit: 
##STR2## 
An aqueous solution of this polymer is filtered by passing it through a 
filter having pores of 0.22 microns, whereupon the polymer is precipitated 
in the filtrate by lowering the pH to 4.5 by means of a dilute aqueous 
solution of citric acid. Thereupon the precipitate is collected and dried. 
Molecules of glucose isomerase are attached to this polymer by proceeding 
in the following manner: 
the polymer precipitate obtained in the manner described above is placed in 
suspension in dioxan (in a proportion of 10 ml of dioxan per g of 
precipitate) whereupon 1.1 ml of n-tributylamine per 10 ml of the 
suspension is added; 
the suspension is cooled to 0.degree. C and 0.4 ml of ethyl chloroformate 
is added; 
the suspension is maintained at 0.degree. C for 10 minutes whereupon 10 ml 
of aqueous glucose isomerase solution (20 g/liter) is added for every 10 
ml of the suspension; 
the mixture which has thus been formed is maintained for 15 minutes at 
0.degree. C, evaporated to dryness, and the solid residue obtained is 
dissolved in water; 
the enzymatic complex thus obtained is precipitated by lowering the pH to 
4.5 by means of a dilute aqueous solution of citric acid; 
the precipitate is washed with an aqueous solution of sodium chloride (0.1 
M) until no further enzymatic activity appears in the liquid, whereupon it 
is subjected to a final washing with distilled water and dried. 
In this way one obtains an enzymatic complex which is soluble in water (at 
a pH of more than 4.5) and has an enzymatic activity corresponding to 1500 
enzyme units per g of complex (an enzyme unit being defined by the amount 
of fructose, expressed in micromols, produced in 30 minutes, at 50.degree. 
C, from an 0.66 M glucose solution containing this enzymatic complex in 
solution. 
B. Use of the enzymatic complex to effect the transformation of glucose 
into fructose: 
Reaction medium: 250 ml of an aqueous glucose solution of 12% by weight. 
Reaction temperature: 50.degree. C. 
pH of the reaction mixture during the enzymatic reaction: 7.0. 
Two tests are carried out, one using 0.18 g of active enzymatic complex and 
the other using 1 g thereof (the complex having been previously dissolved 
in a small amount of water). 
The partial transformation of glucose into fructose (51% by weight 
fructose; 49% by weight glucose) is obtained at the end of a time of 
reaction of 40 hours when using 0.18 g of enzymatic complex and 9 hours 
when using 1 g of enzymatic complex. 
After reaction, the enzymatic complex is precipitated by lowering the pH of 
the reaction medium to 4.5 by means of an aqueous solution of citric acid, 
whereupon this precipitate is separated from the reaction medium by 
settling. 
This precipitate is then washed with an aqueous solution of citric acid 
having a pH of 4.5 and then dissolved in water; the resultant solution is 
filtered through a filter having pores of 0.22 microns and the enzymatic 
complex is precipitated by lowering the pH to 4.5, whereupon finally it is 
dried. 
The powdered enzymatic complex thus obtained is ready to be used again in 
the same manner as just described, with its enzymatic activity unchanged. 
EXAMPLE 2 
The same procedure is employed as in Example 1 but instead of causing the 
precipitation of the enzymatic complex after the reaction by lowering the 
pH of the reaction medium to 4.5, this precipitation is produced by adding 
to the medium 2 ml of a 1M solution of calcium chloride, CaCl.sub.2, of a 
pH of 7.0, the pH of the reaction medium being maintained at a value of 
7.0. 
One thus obtains the precipitation of 98% of the active enzymatic complex. 
The precipitate thus obtained is separated from the reaction medium by 
centrifuging, whereupon it is introduced into an aqueous solution of 
ethylenediamine tetraacetic acid (E.D.T.A., a well-known complexing agent 
for calcium ions). The redissolving of the active enzymatic complex is 
thus effected and the E.D.T.A./calcium complex is separated from the 
aqueous redissolving medium by dialysis. The aqueous solution of enzymatic 
complex which is free of calcium ions which has thus been obtained and 
which has the same enzymatic activity as it initially had can be reused 
for a new operation of transforming glucose into fructose, as described in 
Example 1. 
EXAMPLE 3 
The same procedure is employed as in Examples 1 and 2, except that the 
precipitation of the enzymatic complex after the reaction is brought about 
by the simultaneous addition to the reaction medium of 2 ml of a 1M 
solution of calcium chloride with a pH of 7.0 and of 50 ml of ethanol, the 
pH of the reaction medium being maintained at 7.0. In this way 99.7% of 
the active enzymatic complex is precipitated. 
EXAMPLE 4 
A. Preparation of the active enzymatic complex 
A methyl ester of polyacrylic acid formed of linear macromolecules having 
the recurrent unit: 
##STR3## 
and having a molecular weight of 80,000 is reacted with hydrazine at 
90.degree. C so as to form a polyacrylamide hydrazide (polymer formed of 
linear macromolecules having the recurrent unit: 
The polyacrylamide hydrazide is then transformed into the corresponding 
azide derivative by reaction at 0.degree. C in the presence of a mixture 
of hydrochloric acid and sodium nitride, in accordance with the reaction: 
##STR4## 
in which n represents the number of recurrent units per molecule of 
macromolecular substance. 
Thereupon, glucoamylase molecules are attached to the polymer thus obtained 
by reacting said enzyme with this polymer at 0.degree. C in an aqueous 
medium having a pH of 9.4. 
In this way, an active enzymatic complex is obtained dissolved in aqueous 
medium which however does not have the property of being reversibly 
precipitatable which is required for the enzymatic complexes which enter 
into consideration for the carrying out of the invention. In order to 
obtain a soluble, reversibly precipitable enzymatic complex, the copolymer 
is formed between the nonprecipitable soluble complex obtained in the 
manner just described and the monomer derived from acrylamide having the 
formula 
##STR5## 
prepared in the manner described in Example 1. 
For this purpose, an aqueous solution is prepared which contains, in 
mixture, this complex and this monomer in dissolved state, and the monomer 
is polymerized in this solution in the presence of ammonium persulfate, in 
a nitrogen atmosphere. 
In this way there is obtained an active enzymatic complex which is soluble 
in water at a pH of more than 4.5 and reversibly precipitable at a pH of 
less than 4.5 and which has an enzymatic activity corresponding to 4800 
enzyme units per gram (one enzyme unit corresponding to the amount of 
glucose, expressed in micromols, produced in one minute at 60.degree. C 
from an aqueous solution of enzymatically "liquified" starch containing 
30% by weight solids (commercial product sold by the A.E. Staley 
Manufacturing Co.). 
B. Use of the enzymatic complex to effect the transformation of starch into 
glucose: 
To 250 ml of an aqueous solution of "liquified" starch containing 30% by 
weight solids, maintained at 60.degree. C and the pH of which is brought 
to 5, there are added 2 g of the soluble, precipitatable active enzymatic 
complex the preparation of which has been described above, and the 
reaction medium thus formed is maintained at 60.degree. C. 
At the end of 12 hours the starch is practically entirely transformed into 
glucose. The reaction medium is then allowed to cool to room temperature 
whereupon its pH is lowered to 4.5 so as to cause the precipitation of the 
enzymatic complex, and the latter is removed from the reaction mixture by 
centrifuging. 
The enzymatic complex thus recovered may be reused repeatedly in the same 
manner as just described, it exhibiting just as high an enzymatic activity 
as upon its initial use. 
EXAMPLE 5 
A. Preparation of the active enzymatic complex: 
Proceeding in the manner described in Example 4, the azide derivative of 
polyacrylamide hydrazide is prepared, whereupon molecules of glucose 
isomerase are attached to this polymer derivative, proceeding in the 
manner described in Example 4, in the case of the attachment of 
glucoamylase to this same polymer. In this way an enzymatic complex is 
obtained. 
On the other hand, an acrylic monomer of the formula 
##STR6## 
is prepared by reaction between acrylyl chloride and ethylene diamine. 
The monomer thus obtained is mixed in aqueous solution with the said 
enzymatic complex of glucose isomerase and the polymerization of said 
monomer is effected in said solution in a nitrogen atmosphere in the 
presence of ammonium persulfate. 
In this way there is obtained an enzymatic complex which comprises glucose 
isomerase molecules attached via their amino groups, -to an acrylic 
copolymer having side chains terminated by amino groups. 
This last-mentioned enzymatic complex is an amphoteric polymer which is 
soluble in aqueous medium but precipitated by bringing the pH of this 
medium to 7.6 (isoelectric point of this complex). 
B. Use of enzymatic complex to effect the transformation of glucose into 
fructose: 
The same procedure is used as in Example 1, except that the precipitation 
of the enzymatic complex after the reaction is brought about by bringing 
the pH of the reaction medium to a value of 7.6. 
EXAMPLE 6 
A. Preparation of the active enzymatic complex: 
Carboxymethylcellulose hydrazide is transformed into the corresponding 
azide derivative by reaction at 0.degree. C in the presence of sodium 
nitrite and hydrochloric acid, whereupon the azide thus formed is reacted, 
still at 0.degree. C, in an aqueous medium of a pH of 8.7 with a mixture 
of orthoaminobenzoic acid and lactose (Miles). For 1 g of initial 
hydrazide derivative 0.5 g of lactase and 0.2 g of orthoaminobenzoic acid 
are used. 
In this way there is obtained an active enzymatic complex which is soluble 
in water with a pH of more than 4.5 and reversibly precipitable at a pH of 
less than 4.5, formed of a polyanhydroglucose macromolecular chain bearing 
side groups derived from orthoaminobenzoic acid and other side groups 
formed of lactase molecules, all these groups being bound to the 
polyanhydroglucose chain by groups of the formula: --O--CH.sub.2 
--CO--NH--. 
This enzymatic complex has an enzyme activity corresponding to 520 units of 
lactase per gram. 
B. Use of the enzymatic complex to effect the transformation of lactose 
into galactose and glucose: 
To 250 ml of an aqueous solution containing 5% by weight lactose, of a pH 
of 6.6, which is maintained at 40.degree. C there is added 1 g of the 
enzymatic complex obtained in the manner described above. 
The enzymatic reaction is allowed to proceed for 2 hours, whereupon the 
enzymatic complex is precipitated by lowering the pH of the reaction 
medium to 4.5 by a dilute solution of citric acid. The precipitate thus 
obtained is separated by settling, from the reaction medium. The enzymatic 
complex thus recovered can be used again several times, after purification 
by redissolving in aqueous medium at a pH of 7.0 and filtering through a 
filter having, for instance, pores of 0.22 microns, without losing its 
original enzymatic activity. 
The analysis of the reaction medium after separation of the enzymatic 
complex indicates that 30% of the initial quantity of lactose has been 
converted into galactose and glucose. 
The method which has just been described is capable of very different 
industrial applications, in particular in the following fields: 
Utilization of lactose serum by hydrolysis of the lactose. 
Isomerization of glucose by means of glucose isomerase in order to obtain 
glucose and fructose syrup of high sweetening power. 
Production of invert sugar from sucrose, by means of invertase. 
Degradation of starch into glucose by means of alphaamylase and 
amyloglucosidase. 
Production of maltose from starch by means of betaamylase and 
amyloglucosidase. 
Production of highly fermentable syrups for the beer manufacturing industry 
.