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INTRODUCTION AND GENERAL HISTORY OF IMMOBILIZED ENZYMES
Chapter 1 INTRODUCTION AND GENERAL HISTORY OF IMMOBILIZED ENZYMES Ralph A. Messing I. HISTORY OF ENZYMES Enzyme technology is truly an ancient art. Pri mitive herdsmen discovered in prehistoric times that storing milk in the stomachs of animals re sulted in a tasty solid food, cheese. This is one of the earliest recorded applications of enzymes to the processing of foods. The enzyme, rennin, clots milk by limited hydrolysis which results in the formation of solid cheese. Primitive tribes of tropical regions for centuries have practiced the art of meat tenderization by employing the leaves and the fruit of plants for processing meat. Among the enzymes identified in those plants which were employed by primitive natives for tenderizing meat are papain, bromelain, and ficin. These pri mitive people, in fact, recognized the effect of utilizing plant and animal materials for process ing food, but they did not appreciate or under stand the means by which the food was modified. The properties and reactions of enzyme cataly sis were first recognized by G.S.C. Kirchhoff in 1811; however, the actual word "catalysis" was coined in 1838 by Berzelius. The word enzyme was proposed by Kuhne in 1878. Although a number of enzyme reactions were studied and utilized dur ing the 19th century, the fruits of this early en zyme research were not harvested until the arrival of the 20th century. Modern enzyme chemistry was heralded by the proposed hypothesis for enzyme re actions authored by Michaelis and Menten (1) and the isolation of an enzyme, urease, by J.P. Sumner (2). M
M
II. HISTORY OF IMMOBILIZED ENZYMES One of the earliest reports of immobilized en zymes was that of Nelson and Griffin in 1916 (3). These researchers reported the adsorption of invertase on charcoal and on alumina and demonstrated that these immobilized enzymes retained their acti vity. Approximately 40 years elapsed before the 1
RALPH
A.
MESSING
champions of immobilized enzyme technology appeared on the scene. Among the early workers who gained renown in this area were E. Katchalski, M.A. Mitz, A.D. McLaren and C.A. Zittle. These leaders in immobilized enzyme technology became quite active in the period between 1954 and 1961. McLaren (4) and Zittle (5) were preoccupied with adsorption of enzymes on inorganic carriers. Katchalski (6), on the other hand, took the tack of immobilizing en zymes by covalent attachment to organic copolymers, and Mitz (7) directed his attention towards cova lent attachment to cellulose. It should be noted in these pioneering efforts that adsorption work was primarily performed upon inorganic carriers, while the covalent attachment was reserved for or ganic carriers. III. THE PROLIFERATION OF THE TECHNOLOGY Increasing emphasis has been placed on the im mobilization of enzymes during the past 10 years. A variety of immobilization techniques and carriers have been employed. From the wealth of literature available, the only conclusions that can be drawn are that there is no ideal or universal immobiliza tion technique, and there is no ideal or universal carrier. It has, however, become increasingly apparent that the method of attachment and the carrier must be chosen by the dictates of the ap plication, the enzyme, and the use. IV. REASONS FOR IMMOBILIZING ENZYMES We have now travelled from prehistoric times to modern enzyme chemistry, during which time the stage was set for the employment of immobilized enzymes in continuous processing. Now, let us spe cifically address the question Why bother immo bilizing enzymes? . Enzymes are catalysts which perform the function of inducing and governing re actions, as well as increasing reaction rates. Unlike most inorganic catalysts, enzymes are gen erally soluble and unstable, thus these organic catalysts can be used but once in free solutions. The advantages of immobilizing enzymes may be summed up as follows: 1. Multiple or repetitive use of a single batch of enzymes. 2. Ability to stop reaction rapidly by removlf
11
2
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
ing the enzyme from the reaction solution. 3 . In many cases, the enzyme is stabilized by bonding (maintenance of tertiary structure, antiturbulence) . 4. The processed solution is not contaminated with the enzyme (immune responses in the body, food and pharmaceutical applications, protein se quence and structural determinations). 5. Analytical purposes - long half-life, pre dictable decay rates, elimination of reagent pre parationEach one of the above points contribute to the total utilization of enzyme technology. Enzymes, on a milligram basis of pure enzyme protein, are perhaps the most expensive and difficult materials to obtain in reasonable quantities. Therefore, any procedure that can economically extend the life time of these biologically active molecules should be considered. If it is possible to immobilize an enzyme without appreciable losses of activity dur ing the immobilization procedure, then not only reuse of the enzyme is gained, but also the abil ity to process on a continuous basis, is achieved. Beyond the economic advantages that may be achieved by the immobilization of an enzyme, an additional control can be exercised upon the extent of con version of substrate to products. When soluble enzymes are employed in a reaction, the reaction can be stopped only by destroying the enzyme or changing the environment (pH, salt concentrations). When the enzyme is immobilized, the extent of the reaction can be modified either by flow rates of the substrate through the immobilized enzyme or by removing the enzyme from solution. These controls can be exercised fairly rapidly. Thus, it is ap parent that the immobilized enzyme may be more pre cisely and more rapidly controlled than the enzyme in solution. Recently the FDA has become more concerned with the actual content of foods and pharmaceuticals. The requirement for labelling and informing the customer of the total content of his purchase has placed an additional burden upon the manufacturer. These requirements would probably become more exacting with time. The processor will be re quired to report even trace contaminants within his products. A process employing a well immobil3
RALPH
A.
MESSING
ized enzyme may offer the advantage of having an enzyme free product, while employing the catalytic effect of the enzyme itself. This may be of parti cular importance to the pharmaceutical manufac turer. The presence of an enzyme, a foreign pro tein, in an injectable may cause an immune re sponse. This immune response could lead to ana phylactic shock and death. Thus, for both humani tarian reasons and legal protection, it is abso lutely essential that appreciable enzyme is not present in the final product. It has been recognized that long half-lives, predictable decay rates, and the elimination of the preparation of enzyme solutions have been ad vantageous for the employment of immobilized en zymes for analytical purposes. These same attri butes offer values to continuous processing and argue for the employment of immobilized enzymes in manufacturing operations. A predictable decay rate will allow close process control and a more uniform product. The dissolution of the soluble enzyme employed in the batch process requires periodic and irregular consumption of labor. An economic edge may be gained in the employment of labor by utilizing the immobilized enzyme. Another problem that may be encountered in utilizing the soluble enzyme is that of dusting during the pre paration of these solutions. Many employees find themselves afflicted with an allergic response to the enzyme as they are being prepared for the pro cess. The immobilized enzyme can be prepared in an area remote from the processing area and the employees who exhibit this allergic response. V. IMMOBILIZATION TECHNIQUES The literature has recently proliferated with a variety of methods for immobilizing enzymes. Al though none of these procedures are distinctly de void of all other methods and, in fact, they are probably combinations of two or more of the bonding techniques, they may be classified as five differ ent approaches to immobilization. These immobili zation techniques are as follows: 1. the crosslinking of enzyme to enzyme without the benefit of carrier. 2 . crosslinking of enzymes within carriers or on the surface of carriers. 4
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
3. covalent attachment to carriers. 4. adsorption on or in carriers. 5. encapsulation or entrapment. In fact, it is rather obvious that during the pro cess of crosslinking within the carrier, some ad sorption to the surface of the carrier occurs. During the covalent coupling to carriers, it is probable that some of the enzyme is adsorbed on the carrier surface and in addition, some crosslinking may occur between the enzyme molecules. To carry this further, it is possible that during adsorption immobilization, not only cross-linking between molecules may occur, but also some cova lent bonds may be established at the surface of some carriers and between the enzyme molecules. Encapsulation and entrapment may involve not only physical and chemical adsorption, but also crosslinking and covalent attachment to the surfaces. When we describe a particular bonding technique during the course of our discussions, we merely imply that the proposed attachment is predominate ly of one type of bonding. VI. PROLIFERATION OF CARRIERS The carriers utilized for the immobilization of enzymes may be broadly classified into two groups: 1.) organic; 2.) inorganic. This broad classification of carriers does not adequately describe the versatility and differences which may be brought to bear in the immobilization techni ques. In order to more clearly understand the carrier, we must understand the parameters and the configuration of the carrier. These points will be further discussed and elaborated upon later in the chapter devoted to carriers. An appreciation of the diversity and variety of carriers that are currently available for immobilization may be par tially achieved by examining just a small sampling of the materials utilized for this purpose that follows : 1.) Inorganic carriers a. Kaolinite (8) b. Colloidal silica (9) c. Glass particles (10) d. Controlled pore glass (11) e. Alumina (12) f. Controlled pore alumina (13) 5
RALPH
A.
MESSING
g. h. i. j.
Controlled pore titania (14) Nickel oxide (15) Controlled pore zirconia (16) Zirconia coated controlled-pore glass (17) k. Carbon (charcoal) (18) 1. Hydroxyapatite (19) m. Iron oxide (20) 2.) Organic carriers a. Cellulose (21) b. Agarose (22) c. Collodion (23) d. Starch (24) e. Polyacrylamides (25) f. Dextran (26) g. Nylon (27) h. Collagen (28) i. Organic copolymers (maleic anhydride, ethylene) (29) j. DEAE cellulose (30) VII. REACTORS There is no ideal or universal reactor. The reactor design and operation for immobilized en zymes, however, is a most critical factor in this technology. Three basic reactor configurations have been proposed for utilization with immobilized enzymes for continuous processing. These are: 1.) the continuous stirred tank reactor; 2.) the fluidized bed reactor; 3.) the packed bed or plug flow reactor. In addition to making a decision with re spect to the type of reactor, one must also consi der the temperature conditions, flow rates, pH con trol and adjustment, ionic strength, substrate con centration, diffusional effects, etc. These points will be explored in detail in the chapter devoted to reactors. VIII. SCOPE OF THE TECHNOLOGY To date, most of the work performed with im mobilized enzymes has not been synthetic in nature. Immobilized enzymes have been utilized for hydrolyzing or breaking down proteins to amino acids, de grading or hydrolyzing starch to glucose, isomerizing glucose to fructose, hydrolyzing lactose to glucose and galactose and converting milk to cheese. Perhaps the most important future applications of 6
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
enzymes are in synthetic processes. In virtually all synthetic processes employing enzymes, an en zyme cofactor is required. We must learn to emplcy these cofactors in continuous enzyme reactors be fore we can fully utilize this technology for syn thetic reactions. In addition to synthetic reac tions, if we aspire to use the immobilized enzyme for energy transferring systems, we must under stand and employ the enzyme cofactors. IX. ECONOMIC CONSIDERATIONS AND PRECAUTIONS There are certain points that should be under scored as early as possible. If it is feasible to perform a process with a very inexpensive enzyme preparation, i.e., a crude, impure culture fil trate, it may be unreasonable to employ the en zyme in its immobilized form. One may actually be chasing a rainbow by pursuing an immobilized en zyme to be utilized in a continuous reactor. Fur ther, if large quantities of products from this re action are not required, then it may actually be preferred to perform this operation by a single batch reaction with a soluble enzyme. Another trap that has engulfed some researchers in immobilized enzymes is that of extrapolating half-lives from limited data. The projection of a one-year half-life from a one month reactor perfor mance is very hazardous. The economic data that can be achieved from such information may lead to a disastrous process. X. ENZYME ^ PURITY Appreciable discussion has evolved with respect to the purity of the enzyme preparation employed for immobilization. Generally, a relatively crude preparation is far more stable than a highly puri fied enzyme. Thus, the crude enzyme can withstand some of the rigors that are inevitable consequences of immobilization. In fact, the enzyme may actu ally undergo purification during the immobiliza tion procedure. If a bonding process selects the enzyme protein over the impurity which may be car bohydrate, salt, or non enzymic protein, it would be of advantage to choose the crude preparation. However, most immobilization techniques are not that selective. The impurity occupies some of the binding sites on the carrier that may more econo7
RALPH
A.
MESSING
mically be utilized for the enzyme. Under these circumstances, it would be of advantage to utilize a purified enzyme or perform a purification process prior to the immobilization. This requirement for purity should be examined carefully with respect to each immobilized enzyme before the final process is established.
8
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
REFERENCES : 1.
L. Michaelis, M. L. Menten, Biochera. Z., 49, 333 (1913).
2.
J. B. Sumner, J. Biol. Chem. 69, 435
3.
J. M. Nelson, E. G· Griffin, J. Am. Chem. Soc. _38, 1109 (1916).
4.
A. D. McLaren, J. Phys. Chem. 58, 129
5.
C
6.
A. Bar-Eli, Ε. Katchalski, Nature 188, 856 (1960) .
7.
M. A. Mitz, L. Summaria, Nature 189, 576 (1961) .
8.
A. D. McLaren, E. F. Estermann, Arch. Biochem. Biophys., 61, 158 (1956).
9.
R. Haynes, K. A. Walsh, Biochem. Biophys. Res. Commun., _36, 235 (1969).
10.
J. P. Hummel, B. S. Anderson, Arch. Biochem. Biophys., JL12, 443 (1965).
11.
R. A. Messing, (1969).
12.
T. Tosa, T. Mori, N. Fuse, I. Chibata, Enzymol., 31, 214 (1966).
13.
R. A. Messing, R/D, _25,
14.
R. A. Messing, Biotechnol. Bioeng., 16, 897 (1974).
15.
H. H. Weetall, L. S. Hersh, Biochem. Bio phys. Acta, 206, 54 (1970).
16.
R. A. Messing,
17.
H. H. Weetall, Ν. B. Havewala, Biotechnol. Bioeng. Symp., _3, 241 (1972).
A. Zittle, Adv. Enzymol.
(1926).
14, 319
(1954)i (1953).
J. Am. Chem. S o c , _91, 2370
R/D,
9
32
25.'
(1974) .
32
(1974) .
RALPH
A.
MESSING
18.
J. M. Nelson, D. Hitchcock, J. Am. Chem. Soc. 43, 1956 (1921) .
19.
A. Traub, E. Kaufmann, Y. Teitz, Anal. Bio chem. , 28, 469 (1969) .
20.
G. Gellf, J. Boudrant, Biochim. Biophys. Acta, 334, 467 (1974) .
21.
K. P. Wheeler, B. A. Edwards, R. Whittam, Biochim. Biophys. Acta, 191, 187 (1969) .
22.
D. Gabel, B. v. Hofstein, Eur. J. Biochem., 15, 410 (1970) .
23.
R. Goldman, H. I. Silman, S. R. Caplan, 0. Kedem, Ε. Katchalski, Science, 150, 758 (1965) .
24.
Ε. Κ. Bauman, L. H. Goodson, G. G. Guilbault, D. N. Kramer, Anal. Chem. 32, 1378 (1965) .
25.
S. J. Updike, G. P. Hicks, Science, 158, 270 (1967) .
26.
R. A x è n
27
W . phys
E . .
Hornby Acta
J . F.E.B.S
H .
Jul iard
28 29
. . .
30.
,
J .
.
,
Porath
Let .
,
, H . 0
2 , ,
Nature
Fil p us on 34 3
,
C . Godinot 1 4 , 81 5
0791(
)
210
, (1971)
,
, .
D .
Y . Levin , M . Pecht , L . Goldstein chalski, Biochemistry _3, 1905
36
7
Biochim
.
(196 )
C . .
. Bio
Gautheron
, Ε. Kat (1964) .
G. Kay, M. D. Lilly, A. K. Sharp, R. J. H. Wilson, Nature, 217, 641 (1968).
10
,
M
II. HISTORY OF IMMOBILIZED ENZYMES One of the earliest reports of immobilized en zymes was that of Nelson and Griffin in 1916 (3). These researchers reported the adsorption of invertase on charcoal and on alumina and demonstrated that these immobilized enzymes retained their acti vity. Approximately 40 years elapsed before the 1
RALPH
A.
MESSING
champions of immobilized enzyme technology appeared on the scene. Among the early workers who gained renown in this area were E. Katchalski, M.A. Mitz, A.D. McLaren and C.A. Zittle. These leaders in immobilized enzyme technology became quite active in the period between 1954 and 1961. McLaren (4) and Zittle (5) were preoccupied with adsorption of enzymes on inorganic carriers. Katchalski (6), on the other hand, took the tack of immobilizing en zymes by covalent attachment to organic copolymers, and Mitz (7) directed his attention towards cova lent attachment to cellulose. It should be noted in these pioneering efforts that adsorption work was primarily performed upon inorganic carriers, while the covalent attachment was reserved for or ganic carriers. III. THE PROLIFERATION OF THE TECHNOLOGY Increasing emphasis has been placed on the im mobilization of enzymes during the past 10 years. A variety of immobilization techniques and carriers have been employed. From the wealth of literature available, the only conclusions that can be drawn are that there is no ideal or universal immobiliza tion technique, and there is no ideal or universal carrier. It has, however, become increasingly apparent that the method of attachment and the carrier must be chosen by the dictates of the ap plication, the enzyme, and the use. IV. REASONS FOR IMMOBILIZING ENZYMES We have now travelled from prehistoric times to modern enzyme chemistry, during which time the stage was set for the employment of immobilized enzymes in continuous processing. Now, let us spe cifically address the question Why bother immo bilizing enzymes? . Enzymes are catalysts which perform the function of inducing and governing re actions, as well as increasing reaction rates. Unlike most inorganic catalysts, enzymes are gen erally soluble and unstable, thus these organic catalysts can be used but once in free solutions. The advantages of immobilizing enzymes may be summed up as follows: 1. Multiple or repetitive use of a single batch of enzymes. 2. Ability to stop reaction rapidly by removlf
11
2
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
ing the enzyme from the reaction solution. 3 . In many cases, the enzyme is stabilized by bonding (maintenance of tertiary structure, antiturbulence) . 4. The processed solution is not contaminated with the enzyme (immune responses in the body, food and pharmaceutical applications, protein se quence and structural determinations). 5. Analytical purposes - long half-life, pre dictable decay rates, elimination of reagent pre parationEach one of the above points contribute to the total utilization of enzyme technology. Enzymes, on a milligram basis of pure enzyme protein, are perhaps the most expensive and difficult materials to obtain in reasonable quantities. Therefore, any procedure that can economically extend the life time of these biologically active molecules should be considered. If it is possible to immobilize an enzyme without appreciable losses of activity dur ing the immobilization procedure, then not only reuse of the enzyme is gained, but also the abil ity to process on a continuous basis, is achieved. Beyond the economic advantages that may be achieved by the immobilization of an enzyme, an additional control can be exercised upon the extent of con version of substrate to products. When soluble enzymes are employed in a reaction, the reaction can be stopped only by destroying the enzyme or changing the environment (pH, salt concentrations). When the enzyme is immobilized, the extent of the reaction can be modified either by flow rates of the substrate through the immobilized enzyme or by removing the enzyme from solution. These controls can be exercised fairly rapidly. Thus, it is ap parent that the immobilized enzyme may be more pre cisely and more rapidly controlled than the enzyme in solution. Recently the FDA has become more concerned with the actual content of foods and pharmaceuticals. The requirement for labelling and informing the customer of the total content of his purchase has placed an additional burden upon the manufacturer. These requirements would probably become more exacting with time. The processor will be re quired to report even trace contaminants within his products. A process employing a well immobil3
RALPH
A.
MESSING
ized enzyme may offer the advantage of having an enzyme free product, while employing the catalytic effect of the enzyme itself. This may be of parti cular importance to the pharmaceutical manufac turer. The presence of an enzyme, a foreign pro tein, in an injectable may cause an immune re sponse. This immune response could lead to ana phylactic shock and death. Thus, for both humani tarian reasons and legal protection, it is abso lutely essential that appreciable enzyme is not present in the final product. It has been recognized that long half-lives, predictable decay rates, and the elimination of the preparation of enzyme solutions have been ad vantageous for the employment of immobilized en zymes for analytical purposes. These same attri butes offer values to continuous processing and argue for the employment of immobilized enzymes in manufacturing operations. A predictable decay rate will allow close process control and a more uniform product. The dissolution of the soluble enzyme employed in the batch process requires periodic and irregular consumption of labor. An economic edge may be gained in the employment of labor by utilizing the immobilized enzyme. Another problem that may be encountered in utilizing the soluble enzyme is that of dusting during the pre paration of these solutions. Many employees find themselves afflicted with an allergic response to the enzyme as they are being prepared for the pro cess. The immobilized enzyme can be prepared in an area remote from the processing area and the employees who exhibit this allergic response. V. IMMOBILIZATION TECHNIQUES The literature has recently proliferated with a variety of methods for immobilizing enzymes. Al though none of these procedures are distinctly de void of all other methods and, in fact, they are probably combinations of two or more of the bonding techniques, they may be classified as five differ ent approaches to immobilization. These immobili zation techniques are as follows: 1. the crosslinking of enzyme to enzyme without the benefit of carrier. 2 . crosslinking of enzymes within carriers or on the surface of carriers. 4
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
3. covalent attachment to carriers. 4. adsorption on or in carriers. 5. encapsulation or entrapment. In fact, it is rather obvious that during the pro cess of crosslinking within the carrier, some ad sorption to the surface of the carrier occurs. During the covalent coupling to carriers, it is probable that some of the enzyme is adsorbed on the carrier surface and in addition, some crosslinking may occur between the enzyme molecules. To carry this further, it is possible that during adsorption immobilization, not only cross-linking between molecules may occur, but also some cova lent bonds may be established at the surface of some carriers and between the enzyme molecules. Encapsulation and entrapment may involve not only physical and chemical adsorption, but also crosslinking and covalent attachment to the surfaces. When we describe a particular bonding technique during the course of our discussions, we merely imply that the proposed attachment is predominate ly of one type of bonding. VI. PROLIFERATION OF CARRIERS The carriers utilized for the immobilization of enzymes may be broadly classified into two groups: 1.) organic; 2.) inorganic. This broad classification of carriers does not adequately describe the versatility and differences which may be brought to bear in the immobilization techni ques. In order to more clearly understand the carrier, we must understand the parameters and the configuration of the carrier. These points will be further discussed and elaborated upon later in the chapter devoted to carriers. An appreciation of the diversity and variety of carriers that are currently available for immobilization may be par tially achieved by examining just a small sampling of the materials utilized for this purpose that follows : 1.) Inorganic carriers a. Kaolinite (8) b. Colloidal silica (9) c. Glass particles (10) d. Controlled pore glass (11) e. Alumina (12) f. Controlled pore alumina (13) 5
RALPH
A.
MESSING
g. h. i. j.
Controlled pore titania (14) Nickel oxide (15) Controlled pore zirconia (16) Zirconia coated controlled-pore glass (17) k. Carbon (charcoal) (18) 1. Hydroxyapatite (19) m. Iron oxide (20) 2.) Organic carriers a. Cellulose (21) b. Agarose (22) c. Collodion (23) d. Starch (24) e. Polyacrylamides (25) f. Dextran (26) g. Nylon (27) h. Collagen (28) i. Organic copolymers (maleic anhydride, ethylene) (29) j. DEAE cellulose (30) VII. REACTORS There is no ideal or universal reactor. The reactor design and operation for immobilized en zymes, however, is a most critical factor in this technology. Three basic reactor configurations have been proposed for utilization with immobilized enzymes for continuous processing. These are: 1.) the continuous stirred tank reactor; 2.) the fluidized bed reactor; 3.) the packed bed or plug flow reactor. In addition to making a decision with re spect to the type of reactor, one must also consi der the temperature conditions, flow rates, pH con trol and adjustment, ionic strength, substrate con centration, diffusional effects, etc. These points will be explored in detail in the chapter devoted to reactors. VIII. SCOPE OF THE TECHNOLOGY To date, most of the work performed with im mobilized enzymes has not been synthetic in nature. Immobilized enzymes have been utilized for hydrolyzing or breaking down proteins to amino acids, de grading or hydrolyzing starch to glucose, isomerizing glucose to fructose, hydrolyzing lactose to glucose and galactose and converting milk to cheese. Perhaps the most important future applications of 6
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
enzymes are in synthetic processes. In virtually all synthetic processes employing enzymes, an en zyme cofactor is required. We must learn to emplcy these cofactors in continuous enzyme reactors be fore we can fully utilize this technology for syn thetic reactions. In addition to synthetic reac tions, if we aspire to use the immobilized enzyme for energy transferring systems, we must under stand and employ the enzyme cofactors. IX. ECONOMIC CONSIDERATIONS AND PRECAUTIONS There are certain points that should be under scored as early as possible. If it is feasible to perform a process with a very inexpensive enzyme preparation, i.e., a crude, impure culture fil trate, it may be unreasonable to employ the en zyme in its immobilized form. One may actually be chasing a rainbow by pursuing an immobilized en zyme to be utilized in a continuous reactor. Fur ther, if large quantities of products from this re action are not required, then it may actually be preferred to perform this operation by a single batch reaction with a soluble enzyme. Another trap that has engulfed some researchers in immobilized enzymes is that of extrapolating half-lives from limited data. The projection of a one-year half-life from a one month reactor perfor mance is very hazardous. The economic data that can be achieved from such information may lead to a disastrous process. X. ENZYME ^ PURITY Appreciable discussion has evolved with respect to the purity of the enzyme preparation employed for immobilization. Generally, a relatively crude preparation is far more stable than a highly puri fied enzyme. Thus, the crude enzyme can withstand some of the rigors that are inevitable consequences of immobilization. In fact, the enzyme may actu ally undergo purification during the immobiliza tion procedure. If a bonding process selects the enzyme protein over the impurity which may be car bohydrate, salt, or non enzymic protein, it would be of advantage to choose the crude preparation. However, most immobilization techniques are not that selective. The impurity occupies some of the binding sites on the carrier that may more econo7
RALPH
A.
MESSING
mically be utilized for the enzyme. Under these circumstances, it would be of advantage to utilize a purified enzyme or perform a purification process prior to the immobilization. This requirement for purity should be examined carefully with respect to each immobilized enzyme before the final process is established.
8
IMMOBILIZED
ENZYMES
FOR INDUSTRIAL
REACTORS
REFERENCES : 1.
L. Michaelis, M. L. Menten, Biochera. Z., 49, 333 (1913).
2.
J. B. Sumner, J. Biol. Chem. 69, 435
3.
J. M. Nelson, E. G· Griffin, J. Am. Chem. Soc. _38, 1109 (1916).
4.
A. D. McLaren, J. Phys. Chem. 58, 129
5.
C
6.
A. Bar-Eli, Ε. Katchalski, Nature 188, 856 (1960) .
7.
M. A. Mitz, L. Summaria, Nature 189, 576 (1961) .
8.
A. D. McLaren, E. F. Estermann, Arch. Biochem. Biophys., 61, 158 (1956).
9.
R. Haynes, K. A. Walsh, Biochem. Biophys. Res. Commun., _36, 235 (1969).
10.
J. P. Hummel, B. S. Anderson, Arch. Biochem. Biophys., JL12, 443 (1965).
11.
R. A. Messing, (1969).
12.
T. Tosa, T. Mori, N. Fuse, I. Chibata, Enzymol., 31, 214 (1966).
13.
R. A. Messing, R/D, _25,
14.
R. A. Messing, Biotechnol. Bioeng., 16, 897 (1974).
15.
H. H. Weetall, L. S. Hersh, Biochem. Bio phys. Acta, 206, 54 (1970).
16.
R. A. Messing,
17.
H. H. Weetall, Ν. B. Havewala, Biotechnol. Bioeng. Symp., _3, 241 (1972).
A. Zittle, Adv. Enzymol.
(1926).
14, 319
(1954)i (1953).
J. Am. Chem. S o c , _91, 2370
R/D,
9
32
25.'
(1974) .
32
(1974) .
RALPH
A.
MESSING
18.
J. M. Nelson, D. Hitchcock, J. Am. Chem. Soc. 43, 1956 (1921) .
19.
A. Traub, E. Kaufmann, Y. Teitz, Anal. Bio chem. , 28, 469 (1969) .
20.
G. Gellf, J. Boudrant, Biochim. Biophys. Acta, 334, 467 (1974) .
21.
K. P. Wheeler, B. A. Edwards, R. Whittam, Biochim. Biophys. Acta, 191, 187 (1969) .
22.
D. Gabel, B. v. Hofstein, Eur. J. Biochem., 15, 410 (1970) .
23.
R. Goldman, H. I. Silman, S. R. Caplan, 0. Kedem, Ε. Katchalski, Science, 150, 758 (1965) .
24.
Ε. Κ. Bauman, L. H. Goodson, G. G. Guilbault, D. N. Kramer, Anal. Chem. 32, 1378 (1965) .
25.
S. J. Updike, G. P. Hicks, Science, 158, 270 (1967) .
26.
R. A x è n
27
W . phys
E . .
Hornby Acta
J . F.E.B.S
H .
Jul iard
28 29
. . .
30.
,
J .
.
,
Porath
Let .
,
, H . 0
2 , ,
Nature
Fil p us on 34 3
,
C . Godinot 1 4 , 81 5
0791(
)
210
, (1971)
,
, .
D .
Y . Levin , M . Pecht , L . Goldstein chalski, Biochemistry _3, 1905
36
7
Biochim
.
(196 )
C . .
. Bio
Gautheron
, Ε. Kat (1964) .
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