PLENARY ACCOUNT
Encapsulated Enzyme: a Glucoamylase Copolymer System Steven R. Beck and Howard F. Rase* Department of Chemical Enginesing, The University of Texas at Austin, Austin, Tex. 7871d
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STEVENR . BECKis a Senior Research Engineerf o r Atlantic
Richjield Company in Dallas, Texas. H e received a B.S. in chemical engineering from Kansas State University (1969) and a Ph.D. from The Universitu " of. Texas at Austin (1072).He served as a teaching assistant at the University of TexasandheldanNDEA TitleIVFelluwship.
HOWARD F. RASE is a Professor of Chemical Engineereng at The University of Texas, Austin, Texas. He holds a B.S. degree i n Chemical Engineering from the University of Texas (1048) and M.S.(1049)and Ph.D. (1062) degrees from the University of Wisconsin. His professional career includes seven years of employment as a process engineer for D m Chemical in Freeport, T ~ a s , and Foster Wheeler Corpuratim in New Yorlc, Guest Professor of Chemical Engineering at the Technical University of Denmark, and Chairman of the Chemical Engineering Department at the University of Texas. He is a consultant on catalysis and chemical reactor design, and his research interests are in both heterogeneous and enwmatic catalysis. He has authored three books on chemical engineering design and 69 papers in hisjields of interest.
2W Ind. Eng. Chem. Prod. Res. Develop., Vol. 12. No. 4, 1973
of
the many techniq ues for immmobilizing enzymes for continuous long-term use, encapsulatinIg in a polymer matrix is t h e most generally applicame. lnis method has been studied and proposed for both analytical procedures and industrial processes (Bernfeld, et al., 1969; Bernfeld and Wan, 1963; Chem. Eng. (New York), 1971; Goldstein and Katchalski, 1969; Gruesbeck and Rase, 1972; Hicks and Updike, 1966; Mosbach, 1970; Moshach and Larsson, 1970; Mosbach and Moshach, 1966; Updike and Hicks, 1967a,b). The micropores of the polymer lattice, controllable by the concentration of the cross-linking agent, are adjusted t o prevent the enzyme from "leaking." The method can be applied to any enzyme and no reactive groups on the enzyme are required for attachment as in other immobilizing techniques. Micropore sizes required for effectively trapping the enzyme, however, limit practical use to substrates and products which are small enough to pass through these pores. But since many enzymes are larger macromolecules than t h e substrates or products, a number of systems are amenable to this technique. Large-scale commercial use of any immobilizing enzyme imposes more rigid criteria than the successful laboratory applications which have been described. These necessary criteria include high retention of free enzyme activity as well as high specific activity, which is the activity per unit weight of enzymepolymer complex. Furtber, t h e immobilized enzyme must exhibit long life and the possibility of being reactivated would be most attractive. It should be low in cost, and its physical properties must be eontrollable so t h a t optimum particle size and other characteristics for ease of bandling can be realized. Ideally, the microenvironment should be capable of being tailored to maximize the activity of the enzyme as well as protect it from deliterious changes in t h e bulk liquid. Actual quantitative criteria for each of these requirements will vary with the nature of the process and product. Large tonnages of relatively low-cost product impose particularly severe criteria for economically attractive immobilized enzyme systems. By contrast, specialized uses for immobilized enzymes requiring costly enzymes or unusnal environments become attractive candidates for insolubilized systems even though some properties such as activity are less than ideal. Even in such cases, however, means for improving t h e char-
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Techniques for improving performance of encapsulated enzymes were developed using glucoamylase. A new method for encapsulating the enzyme in a gel b y suspension rather than bulk polymerization has been demonstrated which avoids enzyme-damaging high temperatures and produces a more easily handled product. Further improvement in activity of the encapsulated enzyme was obtained by creating macropores to reduce diffusional resistances and by reactivating the enzyme in the finished gel by sulfhydryl treatment. The final product resulting from a combination of all these techniques retained 98% of the original enzyme activity. An optimum ratio of acrylamide to acrylic acid in the final gel was found which provided a buffering microenvironment that rendered the enzyme system less sensitive to pH changes than that prepared with neutral gels. A method for reactivating the encapsulated enzyme after use was also developed.
acterktics of immobilized enzymes, though less critical, ultimately become essential as user needs and competitive pressures invariably grow. There is, thus, great need and incentive for more detailed studies of various types of immobilizing techniques with the ultimate objective of applying the resulting understandings in developing improved systems. The technique of encapsulation was selected for the study to be described because of its potentially wide applicability together with rather obvious needs for improvements in characteristics. Glucoamylase was chosen as the enzyme to be encapsulated because it is commercially important in the free form for producing glucose from cornstarch, and an encapsulating procedure has been reported in detail by Gruesbeck and Rase (1972). Encapsulated Glucoamylase and Improvements Needed
Glucoamylase has been encapsulated using a cross-linked poly(acry1amide) gel and studied in some detail by Gruesbeck and Rase (1972). The resulting complex retained approximately 22% of the free enzyme activity and had a correspondingly low specific activity. I t \vas much more stable to temperature than the free enzyme and exhibited interesting size select,ivity which prevented large substrate molecules from being hydrolyzed. Life tests up t o 150 hr a t 50' before activity decline were obtained. Longer life a t lower temperatures, though possible, requires more retained enzyme activity to be practical. Clearly, improvements are needed if encapsulated systems such as this are to be considered for commercial processes. This is particularly true in the case of glucoamylase where the low cost of free glucoamylase arid the existence of many fully ammortized, free-enzyme, glucose-producing plants permit low-cost manufacture with once-through enzyme use in direct competition with other sweeteners. Seeded improvements are higher activities, both retained and specific, together with longer life, reactivating methods, improved handling characteristics, and microenvironmental control. Each of these needs was examined theoretically and phenomenologically, in the study to be described, and hypotheses were developed and tested which resulted in rather dramatic improvements as well as suggested avenues for further study and advances. Experimental Methods
Soluble Glucoamylase. d commercial glucoamylase solution was furnished by Clinton Corn Processing Co., Clinton, Iowa. It was produced by submerged fermentation of a strain of Aspergillus niger and contained 36.5 standard glucoamylase units/g (Lloyd, 1971). One glucoamylase unit is t h a t amount of activity which will produce 1 g ol' glucose/hr a t 60' and 11134.3. A single batch, which was stored a t 4', was used for the entire study. No loss in enzymic activity during storage was observed.
Entrapped Enzyme Preparation. The method for entrapping enzyme developed in this study is a new procedure t h a t will be evaluated in detail below. The manipulative procedure is given here as part of the general experimental details. To encapsulate glucoamylase by suspension polymerization, the monomers were first dissolved in distilled water. The solution of acylamide contained 0.04 g/ml; and when amylopectin was used, it was also dissolved in the acrylamide solution. The cross-linking agent was N,N'-methylenebis(acrylamide) u-hich was in an aqueous solution of 0.03 g/ml. The acrylic acid was vacuum distilled and used in a pure form. The initiator used was a mixture of two chemicals, amnionium persulfate in an aqueous solution of 0.50 g/ml of /3dimethylaminopropionitrile. The polymerization was carried out by placing approximately 80 ml of mineral oil in a 100-ml three-necked, roundbottom flask, and 1.0 ml of see-butyl alcohol was added as a dispersing agent. Mineral oil was chosen as the continuous phase after several substances were tried. Organic solvents such as benzene, toluene, and xylene denatured the enzyme as did paraffin hydrocarbons such as hexane. Mineral oil was also chosen because it is edible and trace amounts in the final gel would not be harmful to food products. Many dispersing agents were also tried before sec-butyl alcohol was chosen. It is insoluble in the aqueous phase and soluble in the organic phase. The enzyme was unaffected by see-butyl alcohol. After adding the mineral oil and alcohol, the flask was placed in the water bath, evacuated, and filled with nitrogen t o exclude oxygen which inhibits the polymerization. The monomer solutions were mixed in a test tube with p-dimethylaminopropionitrile. The enzyme was then added to the monomer solution and finally the ammonium persulfate was added and mixed well. The monomer-enzyme mixture was poured into the oil and dispersed by vigorous stirring. The stirrer was then set to the desired speed and a 300-W KO. B E P photoflood lamp turned on to initiate polymeriz a t'ion. Timing began a t this point. The flask was placed close to the wall of the beaker with the light about in. away. The reaction mixture was observed and polymerization considered complete 4 min after the formation of solid particles. Xo evidence of reaction after this time was detected by either temperature rise or change in residual monomer concentration. The polymerized mixture was then poured through a 7.5-g micromesh sieve to separate the gel from most of the oil. The gel and remaining oil were transferred to a 100-ml beaker and 50 ml of see-butyl alcohol was added. see-Butyl alcohol was used as the first wash to remove the remaining mineral oil. I n subsequent washes the see-butyl alcohol was displaced from the gel by water. The gel in alcohol was stirred for 10 min and then vacuum filtered in a Biichner Ind. Eng. Chem. Prod. Res. Develop., Vol. 12, No. 4, 1973
261
funnel. The gel was washed once more for 30 min in 50 ml of distilled water, filtered, and either tested for enzyme activity or treated to improve activity. Batch Reactor. Batch studies were conducted in a 100-ml three-necked, round-bottom flask immersed in a 10-1. stainless steel, temperature-controlled water bath. A variablespeed stirrer was used for agitating the reactor contents, and the side ports of the flask were used for sampling and temperature measurement. To initiate a run in the batch reactor, the enzyme and substrate were first preheated. Because the enzyme was stored a t 4 O , it was first placed in a 30" water bath for 10 min and then in a 60" water bath for 10 min to ensure gradual heating and to prevent thermal shock which could cause denaturation. The substrate was placed in a 60" water bath for 10 min. When both enzyme and substrate were a t 60", they were mixed in the batch reactor. The pH was adjusted to the desired level by adding 0.1 N HCl or 0.1 N NaOH. When the pH reached the desired level, the first sample was taken and timing was started. Throughout the reaction, the temperature and pH of the reacting mixture were monitored. Both the soluble and encapsulated enzyme were evaluated in this manner. The amount of soluble enzyme added t o the reactor was determined by volume. To determine the amount of encapsulated enzyme added to the reactor, the enzyme-gel complex was dried to determine the per cent solids. The percentage of enzyme solid was calculated and then the enzyme content of the gel was calculated. Samples of the reacting mixture were taken periodically and diluted in distilled water a t 0". Such quenching stopped the reaction immediately. The time of quenching was taken as the time of the sample. These samples were later analyzed for glucose to determine reaction kinetics. Flow Reactor. A flow reactor similar to t h a t described by Gruesbeck and Rase (1972) was used t o evaluate the life of the encapsulated enzyme. A Pall metering syringe pump was used as a feed pump. The enzyme-gel complex to be added to the reactor was weighed and suspended in distilled water. A glass wool plug approximately 1 em long was placed in the bottom of the reactor. The dispersed gel was then poured into the reactor and most of the water was allowed t o drain out. Air was forced into the bottom of the reactor to suspend the gel. While the gel was suspended, 3-mm diameter polished glass beads were added to the flocculent gel t o keep the particles well dispersed. The depth of the glass beads was approximately 9 em. The gel particles stayed well dispersed in these beads throughout all runs which was necessary to prevent a high-pressure drop. Once the gel was dispersed, the system was filled with substrate and the feed pump was started. The circulation of water through the jackets of the reactor and feed preheater began a t this time. The system was run a t the reaction temperature for 1 hr before sampling began. Samples were collected in the fraction collector which was set a t a specified time interval. The time of sample mas taken a t the time halfway between uThen the collection of the sample started and when it ended. Throughout each run the feed was changed every 12 hr to prevent mold from entering the system. The syringe on the pump was also changed periodically because it had a tendency t o plug after a long period of service. The system was kept free of air bubbles a t all times. Activity Comparisons. The results of the batch reactor were used to compare the activity of the free enzyme to the encapsulated enzyme. This was done by allowing equivalent 262 Ind.
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Chern. Prod. Res. Develop., Vol. 12, No. 4, 1973
amounts of each to react in starch hydrolyzate of equal concentration and comparing conversion during the first 60 min of reaction. This conversion was defined as activity, Le.
where A is activity, (G), is the glucose concentration at 60 min, (G)o is the glucose concentration a t time 0, and (G)max is the theoretical yield of glucose. -2standard rate equation was used to evaluate the results of the flow reactor, L e .
R
=
FX/W
where R is the rate of reaction (6 of glucose produced/(min) (g of enzyme)), F is the feed rate of starch (g/min), X is the conversion (g of glucose converted/g of starch in feed), and W is the weight of enzyme in catalyst (g of enzyme). By keeping conversion low, the true instantaneous rate is approached. Glucose Analysis. The procedures described by Gruesbeck and Rase (1972) were used. These involve enzymatic oxidation of glucose and colorimetric detection of the hydrogen peroxide formed using a Bausch and Lomb Spectronic 20 with flow-through cuvette. Feed Preparation. All feed solutions used in this study were prepared from a starch hydrolyzate furnished by Clinton Corn Co. This was prepared from cornstarch by a partial hydrolysis with a-amylase. The hydrolysate had an average degree of polymerization of 6 and was received as a syrup containing 69.0% dry substance. This syrup was added to distilled water to prepare the feed solutions. The concentrations of the feed were expressed as a per cent which defines the grams of dry substance per 100 ml of solution; i.e., 10% = 10 g of dry substance/100 ml of solution. I n the pH studies the starch hydrolyzate was dissolved in a 0.05 M citric acid-phosphate buffer and this was used as the substrate. Dialysis Cell. A dialysis cell with a n effective area of 45.6 cm2 was used for studying micropore structure. The membrane for the cell was made by mixing the monomer solutions used in catalyst preparation with p-dimethylaminopropionitrile and ammonium persulfate. This mixture was immediately poured on a glass plate and covered by another glass plate supported by small glass slides approximately 0.2 mm thick. The monomer solution trapped between the two plates was illuminated with a 300-W photoflood lamp ( S o . BEP) for 5 min. The resulting membrane was peeled off the glass plate and inspected for holes under a stereomicroscope a t a magnification of 60 X. If it was found t o be free of holes by microscopic examination, it Tlas placed in distilled water to swell. The swollen membrane was then cut to fit the dialysis cell. The membrane was placed in the dialysis cell being careful always to keep the membrane saturated with water. The dialysis cell was set up with distilled water in the one chamber and an aqueous solution of the substance which was to diffuse through the membrane in the other chamber. The distilled water side was then sampled for the substance a t various times to determine if any diffusion had occurred. Results
A New Encapsulating Procedure. The encapsulating procedure developed in this study and described in detail in the Experimental Methods section constitutes a marked departure from previously used encapsulating techniques such
as described by Bernfeld and Wan (1963), Gruesbeck and Rase (1972), Hicks and Updike (1966), and Mosbach (1970). Usually the enzyme is entrapped in a cross-linked polymer by bulk polymerization. The result is a single gelatinous mass consisting of one large cross-linked polymer molecule with enzyme therein. I n order to render this mass usable the gel must be granulat)ed, which leads t o several undesired results. Particles of widely varying size are produced, and the granulating process must break bonds which will cause loss of enzyme and a corresponding loss of activity. The resulting particles of various sizes and shapes are difficult t o wash, filter, and dry. Bulk polymerization has another distinct disadvantage that can be disastrous for the enzyme present. The reaction is highly exothermic, 19.5 kcal/mol (Joshi, 1962), and the attendant heat buildup due to the poor heat-transfer characteristics of the bulk gel can cause denaturation of the enzyme. The extent of denaturation depends somewhat on the initial t,emperature of the reaction mix, but very little can be done t o control it once the reaction starts. This problem was reasoned to be a major factor in the reported low retained activity of the encapsulated enzyme. The technique developed t o overcome these disadvantages of poor physical and activity characteristics employs a suspension polymerization process. It was hypothesized that by forming polymer in many small drops heat transfer would be improved because of the high surface area, and retained activity would increase accordingly. Also drop size would be more uniform. In processes for producing commercial polymers by suspension polymerization the monomer and growing polymer constitute the dispersed phase and water constitutes the continuous phase. For water-soluble monomers such as used for encapsulating it was necessary to make the aqueous phase the dispersed phase. The aqueous phase containing enzyme, comonomers, cross-linking agent, and initiator was suspended in edible mineral oil using sec-butyl alcohol as a dispersing agent. Other agents were tested, but see-butyl alcohol proved the most effective. Encapsulated enzyme produced by this procedure confirmed both hypotheses. The encapsulated enzyme particles, as shown in Figure 1, retained iiyoof the free enzyme activity in contrast t o 22% observed for the bulk process (Gruesbeck, 1970). The particles were also reasonably uniform in size and easier to handle. Particle size could be varied by altering agitator speed and concentration of dispersing agent. dlthough handling characteristics and mechanical strength were greatly improved over t,hat exhibited by a gel prepared in bulk and then granulated, the gelatinous character common t o all water-soluble polymers persists. The gel swells when placed in mater and tends to adhere to vessel walls. It was found that much of the water could be removed by vacuum filtration, which was facilitated by the narrow particle size range and spheroidal shape. The resulting gel, JThich was not denatured by this procedure, proved easy to handle. The gel could also be frozen or freeze-dried for storage purposes without loss of activity. The freeze-dried particles were freely flowing similar to crystalline solids. Reducing Diffusional Resistances. dlthough 77% retained activity represented a major improvement, reasons for the lack of total activity recovery were sought and techniques for overcoming the problems developed. Reference t o Figure 1 will suggest that the lower initial rate, as indicated by the slope of the encapsulated enzyme, is characteristic of a typical diffusion-limited reaction. Quite logically this follows from the character of the gel particle which consists of a
10 3 % S t a r c h H y d r o l y z a t e ; p H ~ 4 3 6G'C; ; 5 26 x q qlucoamylose/ml s o l u t i o n
50
/
/ o 0
Soluble
- Encapsulated Without Amylopectin Wtthoul C , -MEA
U'
0
I
15
i
1
30
45
,
60
TIME (minl
Figure 1 , Hydrolysis of starch hydrolysate with soluble glucoamylase and encapsulated glucoamylase made without amylopectin and without @-MEA
highly cross-linked molecule containing many micropores through which the substrate and products must diffuse but having no larger macropores as would be the case for a typical supported inorganic catalyst. The procedure for overcoming such a difficulty is to increase the average pore size of the catalyst mass. Such a strategy in the case of a n encapsulated enzyme must be undertaken with care since the micropores or molecular pores of the polymer cannot be changed without losing the enzyme. If numerous macropores, however, can be created throughout each gel particle, the transfer of starch and glucose product t o all parts of the gel would be greatly facilitated. The thought occurred that a complex and bulky starch molecule if included in the encapsulation recipe would penetrate the gel particle in numerous directions. After t h e encapsulated enzyme gel \Tas thus prepared, the bulky molecules could be removed by prolonged contact with free glucoamylase. The enzyme would diffuse gradually along the path created by the starch molecule. Amylopectin, a highly branched starch molecule with a molecular weight of approximately 1,000,000, was selected. Once the encapsulation was completed, the gel mas placed in a solution of glucoamylase which hydrolyzed the amylopectin t o glucose. The glucose was then washed from the gel with water. The washings were analyzed for glucose from which it was calculated that the amylopectin was converted quantitatively to glucose. The gel particles then contained these macropores through which the substrate could reach the inside of the particle. The amount of amylopectin used was varied to determine its effect. A steady and ultimately large decrease in retained activity was seen as the concentration of amylopectin was increased beyond an optimum. Too many macropores apparently allowed excessive amounts of enzyme to escape. Encapsulated enzyme particles containing amylopectin but not treated with free glucoamylase to remove the amylopectin exhibited very low activity, which confirms the conclusion that macropores are formed as the amylopectin is removed from the particle. The optimum amount of amylopectin \?as found t o be around 0.224 g of amylopectin,'g of monomer. The results of hydrolysis of the starch hydrolyzate with this gel are shown in Figure 2. The retained activity is 85%. Treating gel particles made without amylopectin with the free glucoamylase produced no improvement in activity. For many enzymes, the presence of their substrates tends to retard the rate of denaturation. For this reason various Ind. Eng. Chem. Prod. Res. Develop., Vol. 12, No.
4, 1973 263
IO 3 X S t a r c h H y d r o l y z a t e ; p H 5 4 3 ; 60°C, 5 26 x g glucoamylase/ml solution
I
10.3 % Starch Hydrolyzate ; P H = 4 . 3 ; 60°C; 526 x g glucoomylase/ml solution
50
50 z 0
E 40 Y
>
5v
7 0
- Soluble - Encapsulated With Amylopectin Without p - M E A
0
I5
30 45 TIME Iminl
5
1
20 10 0
60
0
Figure 2. Hydrolysis of starch hydrolysate with soluble glucoamylase and encapsulated glucoamylase made with amylopectin and without /3-MEA
I
30
I5
60
Figure 4. Hydrolysis of starch hydrolysate with soluble glucoamylase and encapsulated glucoamylase made with amylopectin and with /3-MEA
I
' I
IO 3 % Starch Hydrolyzate, pH = 4 3 , 6 O o C , 5 2 6 x i O - 3 ~glucoamylase/ml solution
50
30 45 TIME (minl
!
1
!
I
I
35 % S t a r c h H y d r o l y z a t e ; p H ' 4 3 ; 50°C; 2 . 4 2 ~10"g glucoamylase/ml solution
c
-I
5 40 Y
I
s2E i 0
00
15
I
- Soluble
9-
10
1
60
Figure 3. Hydrolysis of starch hydrolysate with soluble glucoamylase and encapsulated glucoamylase made without amylopectin and with P-MEA
substrates were included during encapsulation of glucoamylase. Neither maltose nor the starch hydrolysate improved the retained activity of the encapsulated glucoamylase. Since the amylopectin did improve the retained activity and maltose and the starch hydrolysate did not, it is probable that the observed improvement is attributable solely to the mscropore effect. Just as for inorganic catalysts one might invoke the concept of effectiveness factor to explain the effect of macropores in improving apparent activity. I n this context catalyst size also plays an important role, and declining apparent activities, even when macropores are present, are to be expected as particle size increases. The activities reported here were seen for particle sizes between 100 and 500 p . Above 500 p in size a noticeable drop in apparent activity was noted as is typical for diffusion-limited reactions. Reactivating the Enzyme. Although the polymerization temperature was relatively mild, it is not possible to discount the possibility t h a t some denaturation of the enzyme could have occurred. One mechanism by which heat causes denaturation of some enzymes is oxidation of sulfhydryl groups (-SH) to form disulfide bonds (Haurowitz, 1950). This could occur between the cysteine groups in glucoamylase. I n many enzymes, this can be reversed using a suitable sulfhydryl reagent to reduce the disulfide bonds and restore the sulfhyEng. Chem. Prod. Res. Develop., Vol. 12,
No. 4, 1973
,
IO
I
I
30 45 TIME (minl
264 Ind.
q/,
Encapsulated Without A m y l o p e c t i n Wtth 3 / - MEA
0 0
Figure
1
2 3 TIME ( h r l
4
5
5. Conversion of starch hydrolysate at 50"
dry1 groups. This restores the activity if the sulfhydryl group is crucial in providing the morphology necessary for maximum activity. Since the structure of glucoamylase is not known, one must try this approach without a priori knowledge. A sample of encapsulated enzyme made without macropores produced by amylopectin was placed in a solution of (3-mercaptoethylamine (p-MEA) as a means for reducing the disulfide bonds to restore the sulfhydryl groups. The retained activity of the resulting encapsulated enzyme after washing was 87% (Figure 3). The increase from 77 to 87% strongly suggests that denatured enzyme molecules were restored by this procedure. A sample of soluble glucoamylase was treated with p-AIEA by dialysis and no change in activity was noted. This means that the increase in activity noted in the encapsulated enzyme after treatment with O-MEA is due to restoration of activity lost during encapsulation. Combined Techniques for Enhancing Activity. The various techniques developed for improving the activity of the encapsulated enzyme were combined to determine the total effect. An encapsulated enzyme made with occluded amylopectin which was subsequently removed by glucoamylase treatment and then treated with P-MEA are shown in Figure 4.The retained activity is approximately 98%. To test any independent effect of gel and 0-ME.%, crosslinked polymer gel was made without the enzyme and tested
I
36 'lo S t a r c h H y d r o l y z a t e , pH = 4 3 , 6 O o C , 2 4 4 x IO+^ g l u c o o m y i a s e / m l
IO0
:150 -5 140-
d 9080-
d/
o
- Soluble
u
i
2
w E
30t/!7
J
O n
70601 0
I O % Starch Hydrolyzate
, pH
:
S V = Space Velocity [gm f e e d / [ h r i i g m g l u c o a m y l a s e i
'
20
1
40
I
-
]
-
I
80 IO0 TIME I h r i
60
-
43
120
140
160
Figure 8. Life study of encapsulated glucoamylase a t low substrate concentraticn Figure 6. Conversion of starch hydrolysate a t
60" 2 323- 3 5 % Starch Hydrolyzate , p H = 4 3 5313Temp : 50°C 20'/0 H y d r o l y z e d c 300-
-
I
10 3 Yo S t a r c h H y d r o l y z a t e , p H = 4 3 , 6O0CC, z 24 x 1 0 - 3 9 Concentrated glucoamylase/
100z 900
5 807060-
" 50s 40 -
',?tp/
= -Encapsulated
j
Figure 7. Hydrolysis of starch hydrolysate with concentrated glucoamylase
for activity both before and after @-MEA% treatment. This gel exhibited no activity in the hydrolysis of starch hydrolysate in either case. These observations confirm that the enzyme support did not function as a catalyst. Maximum Attainable Conversion. The activities of the various preparations discussed so far were based on conversion of the starch hydrolysate to glucose during the first 60 min of reaction in a 10.3% starch hydrolysate solution. Although the initial activity is important, the maximum conversion must also be considered. A 35.0% starch hydrolysate solution was used to determine niaximum conversion using soluble and encapsulated glucoamylase a t both 50 and 60'. The results of these runs are shown in Figures 5 and 6. At both temperatures the maximum conversion attained with soluble enzyme is approximately 96% while the maximum conversion using encapsulated enzyme is approximately 937,. The starch hydrolysate is prepared by enzymic hydrolysis of cornstarch LTith &-amylase. This results in a broad molecular weight distribution so that some very large starch molecules remain. The polymer matrix apparently excludes these large molecules and prevents their hydrolysis to glucose. Specific Activity. The encapsulated enzyme part'icles discussed thus far were prepared using a solution produced for a commercial glucose process. The activity of this glucoamylase material based on the testing procedure used for this study was 85 units/g of solution (857, conversion a t 60 min,'g of solution). The gel prepared from this solution
produced a n activit'y of 5.84 units,/g of gel. A% higher specific activity is desired for a commercial process so as to minimize equipment size, and as a step in this direction a more concentrated glucoamylase was prepared which had a n activity of 250 units/g of solution. This concentrated glucoamylase was encapsulated and tested for activity. I t had a specific activity of 21.6 units 'g of gel as showi in Figure 7 . These results suggest that a highly purified glucoamylase could be used to obtain a gel with a much higher specific activity. Based on the known protein impurities in the concentrated enzyme solution, it should be possible to prepare a n encapsulated enzFme by the procedures described which would have an activity of approximately 50 units'g of gel. Even higher activities would be realized if, by purifying the enzyme, nat,ural inhibitors that could be present were removed. Life Studies. The length of service of the encapsulated enzyme was tested in a flow microreactor a t varying temperatures and substrate concentrations. The results of these runs are shown in Figures 8 and 9. The reactor was operated differentially with high throughputs and lorn conversion so that denaturation or other deactivating phenomena would rapidly become apparent as a decline in conversion. The encapsulated enzyme retained its full activity a t 50" for over 125 hr and then gradually denatured, losing approximately 0.17, of its activity,'hr. .kt 60" the encapsulated enzyme retained its full activity for only 14 hr and then denatured a t a rate of 0.9%/hr. Substrate concentration appeared t o have little effect on the length of service of the encapsulated enzyme. At the present time, conimercial hydrolysis of the starch hydrolysate with glucoamylase is done a t 60" to achieve high rates, and in the course of the reaction the enzyme is denatured. Lower temperature operation with a reusable enzyme is certainly feasible if high activity is attained by encapsuInd. Eng. Chem. Prod. Res. Develop., Vol. 12, No. 4, 1973
265
I
I
, 8
8
,
1
10 5 % S t a r c h H y d r o l y z a t e ; 60°C N o 2 H P 0 4 - C i t r i c Acid Buffer(OO5M1, 5 2 6 x 10-3g glucoomylose/ml s o l u t i o n
*
N o 2 H P 0 4 - C i t r i c Acid Euffer10.05M); 5 2 6 x 10-3g glucoomylose/ml solution
*
>
L
'"1 ~
60
s
40
20
'
0-Soluble 0-Soluble 0 - Encapsulated
' "1
\i
s
40
114 9 a c r y l a m i d e / g a c r y l i c a c l d ' I I I I
201 2
Soluble Encapsulated 5.71 g o c r y l o m i d e / g a c r y l i c a c i d 3 0 -
~
3
I
I
4
5 PH
\ 1
6
7
1
Figure 10. pH characteristics of soluble and encapsulated glucoamylase-1 1.4 g of acrylamide/g of acrylic acid
Figure 1 1. pH characteristics of soluble and encapsulated glucoamylase-5.71 g of acrylamide/g of acrylic acid
lating purified enzyme. I n a once-through process, of course, the cost for producing a higher activity free-enzyme solution cannot be justified. Based on the results in Figure 8, it can be seen t h a t a decrease in temperature from 60 to 48" causes only a 14y0 decrease in rate while increasing the life almost 1000yo.Further reducing the temperature while using a higher activity preparation should be possible with correspondingly marked increase in life. Reactivation after Use. J u s t as in practical chemical processes involving inorganic catalysts, a means for reactivating the encapsulated enzyme after even extended use is an important aspect of successful commercial development. Since it is apparent that temperature plays an important role in encapsulated-enzyme life, it is reasonable to hypothesize that the observed decline in activity is caused by heat denaturation. P-MEA by reducing disulfide bonds was shown t o recover activity thought to be lost in the preparative process because of a similar phenomenon and might be useful in restoring activity after long use. Accordingly, a typical sample of the encapsulated glucoamylase was tested for activity after repeated use in the hydrolysis of a 35y0starch hydrolyzate a t 60" and was found to exhibit 37% of its original activity. This sample was treated with (?-MEA by soaking and again tested for activity. The activity was found to have increased to 87% of the original activity. This seems t o confirm the hypothesis that the denaturation during use is, a t least in part, a result of oxidation of sulfhydryl groups and that it can be reversed by reducing the disulfide bonds that are formed by the temperature-sensitive oxidation process. The remaining unrecovered activity must be attributed to some other came the most likely of which might be metal ions. The procedures described thus far employed regular distilled water which certainly contains some copper and other metal ions. To determine if metal ions caused part of the denaturation during use, a sample of the used encapsulated glucoamylase which exhibited 37% of its original activitywas soaked for 24 hr in a solution of (ethylenedinitri1o)tetraacetic acid (0.0674 X ) made with deionized water. The gel was thoroughly washed with deionized water to remove all chelating agent and any complexes containing metal ions. It was again tested and its activity had increased to 47y0 of its original activity. This indicates metal ions cause a small portion of the deactivation of the encapsulated enzyme and that a practical combined treatment with chelate and P-MEA could be developed which would restore most of the original activity. Effect of Microenvironment. Previous investigators
have shown that an immobilized enzyme may see different environmental conditions than exhibited by the bulk solution (Goldstein and Katchalski, 1969; Hornby, et al., 1968; Levin, et al., 1964). If the support is charged, it will attract or repel ions in the bulk solution so that the p H on the surface of the support, which is what the enzyme sees, differs from the bulk pH. The phenomenon accounts for the fact that the apparent p H optimum of the immobilized enzyme may be slirhtly different from that of the free enzyme. The polyacrylamide gels that have been used in previous encapsulation work exhibit this behavior to some degree. The amide groups are weakly basic and tend to attract hydrogen ions so that the p H of the microenvironment is slightly lower than the bulk pH. Consequently the apparent p H optimum of the encapsulated glucoamylase is slightly higher than the free glucoamylase as shown by Gruesbeck and Rase (1972). There exists a real opportunity to control the microenvironment in a positive way so as t o reduce the sensitivity of the encapsulated enzyme to p H fluctuations. By using a weakly acidic monomer with the weakly basic acrylamide, a buffered microenvironment might be produced. The monomer chosen was acrylic acid since it is acidic but still similar to acrylamide in structure. Figure 10 shows the dependence of activity on p H of the free glucoamylase and an encapsulated glucoamylase where the ratio of acrylamide to acrylic acid in the gel was 11.4: 1 on a weight basis. -4s can be seen, the pH optimum shifted slightly but sensitivity t o p H was unchanged. Another sample of encapsulated enzyme was made with a ratio of acrylamide t o acrylic acid of 5.71 : 1 on a weight basis. The p H dependence of this gel is shown in Figure 11. This encapsulated glucoamylase exhibits a high activity over a broader pH range than the free enzyme. This can be explained by assuming that the microenvironment is indeed buffered by the weakly basic and weakly acidic groups on the polymer chain. The lower sensitivity of the encapsulated enzyme to pH changes would be highly advantageous in a continuous commercial process. The control scheme for the process need not be as sophisticated as that required for a sharp optimum. Further work should include a thorough study of various ratios of acrylamide to acrylic acid. I n a polyelectrolyte, such as the copolymer of polyacrylamide and polyacrylic acid, the dissociation constants of the acidic and basic groups are dependent on the chain length between the groups (Edsall and Wyman, 1958). A neutral monomer might be used t o separate the charged groups t o improve the buffering ability of the charged comonomers. Different charged monomers could also
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be tried to increase the p H range over which the encapsulated enzyme is active. Study of Gel Micropores. A reasonable model of a n enzyme encapsulated in a cross-linked polymer matrix suggests t h a t the polymer chains form a three-dimensional porous structure in which the enzyme molecules are trapped, and the substrate and product are free to diffuse. This concept is shown in Figure 12. From the composition of the polyper, the average chain lengte between cross-links is 166 A and the averge pore radius 56 A. The rtdius of gyration of a glucose molecule is approximately 5 A, and a starch hydrolysate molecule with a degrez of polymerization of 6 has a radius of gyration of about 12 A. Both of these molecules should diffuse easily through the polymer matrix. The enzym: molecule has a radius of gyration of approximately 200 A which is much too large to pass through the polymer matrix. I n order to determine directly if the enzyme is physically trapped by the polymer matrix, a membrane of the crosslinked polymer was made. The various substances were then tested for diffusion through the membrane using the dialysis cell described earlier. The results were qualitative, but they showed that the polymer membrane prevents the enzyme from passing while the starch hydrolysate and glucose diffused through the membrane. This does not eliminate unequivocally the possibility that chemical bonds are formed between the enzyme and the polymer, but it does show that the enzyme could be physically trapped within the polymer matrix. Conclusions
Several important techniques have been discovered during the course of this study which have proven most effective in improving physical properties and activity characteristics of a n encapsulated enzyme. Two disadvantages remain when subjected to the economics of the specific process of starch hydrolysis. These are low specific activity and incomplete total hydrolysis because of the presence of some larger starch molecules in the substrate. It is doubtful t h a t this latter disadvantage can be overcome because the polymer mesh size cannot be further increased without loss of enzyme unless the enzyme is bonded to the polymer. Increasing specific activity was shown t o be possible, and higher economically attractive values ought to be attainable with purified enzyme. Most of the techniques developed should be applicable to other enzymes and may result in economical processes
Figure 12. Concept of gel showing encapsulated enzyme molecules
particularly for manufacture of higher cost, and complex products. The suspension polymerization procedure, production of macropores, analogous methods for reactivating the enzyme, and control of the microenvironment are all generally applicable. Acknowledgment
R. V. MacAllister of Clinton Corn Processing Co. supplied the glucoamylase and starch hydrolysate used in this investigation. Literature Cited
Bernfeld, P., Bieber, R. E., MacDonnell, P. C., Arch. Biochem. Biophys., 127,779 (1969).
Bernfeld, P., Wan, J., Science, 42,678 (1963). Chem. Eng. (lVew York), 78 ( l o ) ,39 (1971).
Edsall, J. T., Wyman, J., “Biophysical Chemistry,” Vol. 1, pp 540-545, Academic Press, New York, X. Y., 1958. Goldstein, L., KatchsIlski, E., Z . Anal. Chem., 243,375 ( 1969). Gruesbeck, C., 13h.D. Dissertation, The Universitv of Texa 5, Ma) 1970.
’
Gruesbeck, C., Rase, H. F., I n d . Eng.Chem., P r o d . Res. Develop., 11.74 (1972).
Hau;owitzi F.’, “Chemistry and Biology of Proteins,” p 127, AcademicPress, Yew York, N. Y., 1950. Hicks, G. P., Updike, S. J., Anal. Chem., 38,726 (1966). Hornby, W. E., Lilly, M. D., Crook, E. M., Biochem. J . , 107, 669
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(10fiR) ,A
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Joshi, R. M., J . Polym. Sci., 6 0 , 5 5 6 (1962). Levin, Y., Pecht, M., Goldstein, L., Katchalski, E., Biochemistry, 3,1905 (1964).
Lloyd, M. E., private communication of Clinton Corn Processing MethodGAP-l167,1971.
Mosbach, K., Acta Chem. Scand., 24,2084 (1970). Mosbach. K.. Larsson. P. 0..Biotechnol. Bioeno.. 12.19 (1970). Mosbachj K.; Mosbach, R., Acta Chem. Scand.;2O, 2807 (1966). Updike, S.J., Hicks, G. P., A‘ature (London), 214,986 (1967a). Updike, S. J., Hicks, G. P.,Science, 158,270 (1967b). RECEIVED for review July 5, 1973 ACCEPTED September 4, 1973
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