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ARTICLES Immobilization of Arthrobacter simplex in Thermally Reversible Hydrogels: Effect of Gel Hydrophobicity on Steroid Conversion Tae Gwan Parkt and Allan S. Hoffman* Center for Bioengineering and Department of Chemical Engineering, FL-20, University of Washington, Seattle, Washington 98195
Arthrobacter simplex cells have been immobilized in a series of thermally reversible hydrogels having different gel hydrophobicities. Steroid conversion from hydrocortisone to prednisolone via the A'-dehydrogenase system was greatly affected by the relative hydrophobicities of the gel matrices, which were prepared by copolymerizing varying ratios of N-isopropylacrylamide to acrylamide. The characteristics of the immobilized cells, such as optimal temperatures, K, values, and the effects of an added artificial electron acceptor, were largely influenced by the gel matrices and their different lower critical solution temperatures (LCST). The data indicate that the microenvironment of the dehydrogenation system is quite different within the different hydrophilic/ hydrophobic gel matrices. The high partitioning of water-insoluble steroids into the hydrophobic regions and the reduced possibility of product inhibition within the more hydrophobic gel matrices may cause the observed higher steroid conversion in these gels. A possible model for immobilized A. simplex cells in such different gel matrices is proposed.
Introduction There have been a great number of studies on the immobilization of biocatalysts, mainly enzymes and cells, within natural and synthetic polymeric matrices. Among them, steroid conversion by immobilized microbial cells has received much attention because of its commercial importance (Kolot, 1982; Miller, 1986). However, there are several limiting factors for microbial steroid transformation, such as the poor water solubility of both substrate and product and the severe product as well as substrate inhibition. Various attempts have been made to increase the mass transport of poorly water soluble steroids to the immobilized cells. For example, watermiscible organic solvents have been used to increase the steroid solubility (Laane et al., 1987; Lilly, 1982; Carrea, 1984;Freeman and Lilly,1987), hydrophobic matrices such as photo-cross-linkable resins and polyurethanes (Fukui and Tanaka, 1984) have been fabricated to enhance the partitioning of substrate from the aqueous phase into the gel matrix, the cells and the substrate have been immobilized together (Kaul et al., 1986),and aqueous two-phase extractive conversion has been carried out (Kaul and Mattiasson, 1986). Fukui et al. reported that steroid transformations are generally enhanced by using hydrophobic gel matrices because of the higher solubility of the steroid in the gel matrix (Fukui and Tanaka, 1984;Fukui et al., 1987). The importance of gel hydrophobicity for water-insoluble substrates has been stressed, but it is difficult to immobilize whole cells within hydrophobic synthetic polymeric materials without the deactivation of the biocatalyst, because one may have to melt the polymer or to use toxic organic t Present address: E25-342,Department of Chemical Engineering, Massachuaetts Institute of Technology, Cambridge, MA 02139.
8756-7938/91/3007-0383$02.50/0
solvents to entrap the cells into the polymeric gel. In the case of Arthrobacter simplex entrapment in urethane prepolymers, it was reported that over 80% of initial activity was lost during the immobilization (Sonomoto et al., 1980). Therefore, most of the immobilization matrices in the past decade have focused on a limited number of hydrophilic polymers, such as polyacrylamide and polysaccharides. It is possible that hydrogels having only moderate water uptake can be good candidates for immobilization of enzymes and cells that transform sparingly water soluble substrates. Hydrogels here are defined as cross-linked hydrophilic polymer networks that swell in aqueous solution. As described in our previous papers (Park and Hoffman, 1990a, 1989), we have immobilized A. simplex cells in a series of thermally reversible hydrogels that exhibit significant differences in water content in response to temperature. These cells convert hydrocortisone to prednisolone as shown in Figure 1. Poly(N-isopropylacrylamide, NIPAAm) and its acrylamide(AAm) copolymeric hydrogels utilized for the cell immobilization exhibit lower critical solution temperature (LCST)behaviors in aqueous solution. These hydrogels swell below the LCST and shrink and collapse above the LCST. Thermal cycling operation around the LCST for these cell-immobilized hydrogels enhanced the steroid conversion rate efficiently relative to isothermal operation at either upper or lower temperature. This was mainly due to a thermal "pumping" action of the hydrogel matrices, which facilitates the mass transfer rates of substrate into and product out of the gel matrices. In this study, we entrapped the A. simplex cell in a series of thermally reversible hydrogel beads having different hydrophobicities through the inverse suspension copolymerization of varying ratios of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) in the presence
0 1991 American Chemical Society and American Institute of Chemical Engineers
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mDR0CDRn-c
7
Figure 1. Steroid conversion by A . simplex cells. of cells. Gel hydrophobicity in the thermosensitive hydrogels can be controlled by careful selection of monomers of the gel to match the desired operating temperature range. The characteristics of the immobilized cells in these hydrogels (e.g., enzyme activity, optimal temperature, K m values, and the effect of artificial electron acceptors) were systematically investigated as functions of gel composition and temperature. In addition, transport parameters such as substrate diffusivities, partition coefficients, and permeabilities have been determined in three hydrogel matrices. On the basis of the experimental results obtained, models for the immobilized cells in the hydrophobic and hydrophilic gel matrices are proposed.
Materials and Methods Cell Cultivation and Immobilization of A. simplex Cells in Temperature-SensitiveHydrogel Beads. The protocols for the preculturing of A. simplex (ATCC 6946) cells before immobilization and following inverse suspension polymerization of aqueous monomer-cell droplets to form immobilized cell-hydrogel microbeads was described in the previous report (Park and Hoffman, 1990a). Briefly, harvested wet cells were suspended in a buffer solution containing varying molar ratios of two monomers (NIPAAm and AAm), cross-linker [N,N'-methylenebis(acrylamide)], and one of two redox initiators (ammonium persulfate). This aqueous phase was suspended in paraffin oil phase containing surfactant, Pluronic L-81, and then polymerized under a nitrogen atmosphere by injecting the other initiator (N,N,N',N'-tetraethylmethylenediamine) into the oil phase. After 30 min of polymerization a t icewater temperature, the immobilized cell beads were separated, washed, and then freeze-dried. The swollen bead sizes ranged from 150 to 300 pm. Enzyme Reaction by Immobilized Cells. For longterm steroid conversion, the substrate hydrocortisone (1 mM) was dissolved in 0.05 M Tris-HC1 buffer (pH 7.4) containing 5% (v/v) methanol. The freeze-dried cells (200 mg) were mixed with 100 mL of the substrate solution, and the flask was closed with a cotton plug and placed on an orbital shaker at various constant temperatures for the isothermal operations. Aliquots (2 mL) of samples were removed with a syringe at various time intervals and filtered to separate the immobilized cells, and the filtrate was analyzed by HPLC for conversion of hydrocortisone to prednisolone. To obtain the initial velocities of the enzyme reactions, 50-mg quantities of freeze-dried immobilized cells were preswollen in the buffer and placed in 10 mL of 1 mM hydrocortisone solution in the same buffer and then reacted in an orbital shaker (180 rpm) at various constant temperatures. After a fixed time, the reaction mixture was taken out and filtered through a 0.2-pm syringe filter and analyzed. In the HPLC method, 1mL of aqueous reaction sample containing the steroids was mixed with 1mL of methylene chloride; the mixture was vortexed for 1 min and then allowed to separate steroids. A 10-pL sample of the methylene chloride phase
was injected into an HPLC system (Waters) with a Lichrosorb Si-60 5-pm column, methylene chloride as a mobile phase containing 5% methanol and 0.55% (v/v) acetic acid, 1mL/min flow rate, and a UV detector at 254 nm (Park and Hoffman, 1990a). Determination of Km Values. A series of substrate solutions having different hydrocortisone concentrations in 5% methanol/95% (v/v) 0.05 M Tris-HC1 buffer (pH 7.4) were prepared. For immobilized cells, 50-mg quantities of freeze-dried, immobilized cell microbeads were preswollen in the buffer and put in 10 mL of each substrate solution. For free cells, 100 mg of a wet cell suspension was used. Steroid conversion experiments were carried out at 30 "C with an orbital shaker (180 rpm). After 30 min of reaction, the reaction mixture was filtered through a 0.2-pm syringe filter and analyzed as described above. Determination of Transport Parameters within Hydrogel Membranes. Equilibrium water uptake by gel membrane discs in 0.05 M Tris-HC1 buffer (pH 7.4) containing 5% (v/v) methanol was used to estimate the water contents of the gel beads. The membranes were prepared with the same compositions as the inverse suspension polymerization. They were polymerized between two glass plates at 4 "C for 5 h by using the same initiator system. Water contents of the gel discs were determined gravimetrically by measuring equilibrium wet weights at 30 "C and dry weights after complete drying under vacuum. Each value of water content reported is an average of at least four samples. Effective diffusivities of hydrocortisone a t 30 "C were obtained by using a SideBy-Side diffusion cell (Crown Glass), which is composed of donor and acceptor reservoirs, each of which is 3.5 mL in volume. After the hydrogel membrane was equilibrated in 5% (v/v>methanol in 0.05 M Tris-HC1 buffer (pH 7.4) at 30 "C, it was mounted in the diffusion cell, which was thermally controlled. Hydrocortisone (1mM) dissolved in the same buffer was added to the donor side. A 2-mL sample was taken at regular time intervals from the acceptor side and 2 mL of fresh buffer solution was replenished in order to maintain a perfect sink condition. The permeated hydrocortisone was analyzed with the spectrophotometer at 242 nm by using our standard calibration curve (Park and Hoffman, 1990a). The effective diffusivities were calculated by the lag time method as follows: lag time = (thickness of membrane)2 + 6(effective diffusivity) where the lag time is defined as the time to reach steadystate solute transport across the membrane. It was determined by extrapolating the linear steady-state permeation curve onto the time scale. Thicknesses of hydrogel membrane equilibrated at 30 OC were measured with a micrometer. Partition Coefficients were measured in a separate experiment where the preequilibrated gel discs in buffer at 30 "C were immersed in 1 m M hydrocortisone solution at 30 "C and then hydrocortisone concentrations in the bulk phase were determined at equilibrium. The partition coefficient ( K ) can be calculated by use of the mass balance equation of hydrocortisone:
K = C,/Cb = (Co - cb)vo/C,v, where CO is the initial hydrocortisone concentration (1 mM), C, is the hydrocortisone concentration in the gel phase, c b is the final hydrocortisone concentration in the bulk medium at equilibrium, VOis the volume of hydrocortisone solution, and V, is the volume of the gel at 30 "C. The permeation coefficient (permeability) can then
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NAO + NA10 + NAU)
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301 25
.
I
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.
I
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.
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Figure 2. Effect of gel composition on gel water content as a function of temperature. Each data point is an average value of at least four samples. The gel discs were equilibrated in 5 vol % methanol, 0.05 M Tris-HC1 (pH 7.4) for 24 h (Park and Hoffman, 1990a).
+ NAO
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be obtained by multiplying the effective diffusivity and the partition coefficient.
Results and Discussion Effect of Gel Hydrophobicity on Steroid Conversion. Most of the matrices used to immobilize A. simplex cells have been based on hydrophilic polymers or waterwettable materials such as polyacrylamide (Ohlson et al., 1978; Koshcheyenko et al., 1983; Silbiger and Freeman, 1987),alginate (Ohlson et al., 1979;Kloosterman and Lilly, 1986), cellulose granules (Krysteva and Grigorova, 1987), collagen (Constantinides, 1980),and glass support (Nozes and Rouxhet, 1984). Since the substrate hydrocortisone is a poorly water soluble steroid, it can be expected that the mass transfer of substrate to entrapped cells in such matrices will be a limiting step in the overall steroid conversion process. In other studies concerned with transformations of water-insoluble substrates, hydrophobic gel matrices such as photo-cross-linkable resins and polyurethanes have been used (Fukui and Tanaka, 1984; Fukui et al., 1987). Improved conversion relative to hydrophilic gels could be obtained due to its high partition of substrate into the hydrophobic matrices. However, in the case of immobilized A. simplex cells in hydrophobic polyurethanes, there appeared to be no direct relationship between gel hydrophobicity and steroid converting activity (Sonomoto et al., 1980, 1984). In these studies, no difference in steroid conversion rates was found in spite of marked differences in partition coefficients of hydrocortisone for a series of hydrophilic/ hydrophobic urethane prepolymers. The copolymerization of NIPAAm with AAm, at different molar ratios and fixed cross-linker concentration, would give different hydrophobicities of the resultant gel matrices because of the hydrophobic isopropyl group on the amide group of the NIPAAm. We prepared a 100% NIPAAm (NA 0) gel as the most hydrophobic and an 80% NIPAAm/20% AAm (NA 20) gel as the most hydrophilic among the three prepared in this study. The effect of gel composition on gel swelling as a function of temperature, as shown in Figure 2, supports the idea of the importance of gel hydrophobicity. Remarkable differencesof the water contents of three gel matrices can be seen as the temperature is raised. Swelling behaviors of these hydrogels
1.o
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I
2
0.8
* NAO * NA10 * NA20
0.6 0.4
b 02 0.0
0
200
400
600
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Time, min.
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Figure 3. Progress of steroid conversion with time at 25,30,and 35 "C.
in response to temperature were described in detail in a previous report (Park and Hoffman, 1990a). The steroid conversions of immobilized A. simplex cells in thermally reversible hydrogels having different hydrophobicities are shown in Figure 3. The increased conversion rates with increase of gel hydrophobicities can be clearly seen. As the gel matrix becomes more hydrophobic, more steroid is converted at any time. However, the enhancement of activity cannot be attributed exclusively to the increase of mass transfer of substrate on the basis of the following reasoning. Generally, if external mass transfer resistance (boundary layer effect) is neglected, the overall mass transfer of substrate from the external bulk phase into the gel phase can be described in terms of the overall permeability coefficient,which is the product of the partition and diffusion coefficients. The former is thermodynamic, and the latter is a kinetic Coefficient.The partition coefficient is related to the chemical composition of the gel matrix and its water content, while the diffusion coefficient is dependent upon the water content (porosity), the pore sizes, and pore interconnections and distribution (tortuosity) as well as the size of the substrate molecule. Normally, these two parameters (partition and diffusion) show opposing trends for the transport of water-insoluble solutes through hydrogel membranes, because of the tendency for such solutes to partition in the polymer phase
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Table I. Physical Properties and Hydrocortisone Transport Parameters of Three Thermally Reversible Hydrogel Membranes at 30 OC hydrogel composition NA 0 NA 10 NA 20
LCST range, O C 32-33 37-38 45-46
physical appearance opaque transparent transparent
hydrogel properties water membrane content, % thickness, mm 75.5 0.98 81.2 1.00 82.9 1.03
hydrocortisone transport parameters effective diffusivity partition permeation coefficient De,"cm*/s (XlO-') coefficient K* D&, cmz/s (XlW) 1.82 3.7 4.92 7.0 2.28 1.60 11.4 61.3 1.86
a CalculatedfromthetimeaxisinterceDt.t.inthetimelarcdiffusionexperiment,i.e.,t = 12/6D,where1 = membranethichmandD, = effective diffusivity. b Measured from a separate- partition experiLent.
and to diffuse mainly through the water pores (Kim et al., 1980; Zentner et al., 1978). It has also been known that the transport of a solute across the hydrogel membrane occurs via two routes, a pore diffusion mechanism through the pore fluid (free water) and a partition diffusion mechanism through the polymer matrix. A hydrophilic hydrogel membrane contains a high water content and associated large pores, so even hydrophobic solutes exhibit large effective diffusivities, even though the partition of those solutes in that hydrogel is low. On the other hand, for a hydrophobic membrane having low water content and small pore sizes, a major portion of the hydrophobic solute partitions in the polymer phase and transports through that phase as well as the pores, so that its overall permeation rate is lower compared to that of hydrophilic membranes. Since the effective diffusivities of hydrocortisone through a series of thermally reversible hydrogels exhibit the same trends as the water contents with temperature (Park and Hoffman, 1990a),the major mechanism for the transport of hydrocortisone through a gel, below its LCST, is pore diffusion through the aqueous pores, with little contribution of diffusion through the polymer matrix (Park and Hoffman, 1990a). However, the contribution of diffusion through the polymer matrix cannot be ruled out, because as the temperature increases, it will be an important transport route. I t is difficult to determine how much the partition diffusion contributes to the overall transport at 30 "C, because the data for porosity and tortuosity of the gel at that temperature are not available. However, it can be presumed that the hydrophobic, NA 0 gel will exhibit more of the matrix diffusion mechanism than any other gels because of its less porous structure. The diffusion, partition, and permeation coefficients in thermally reversible hydrogels are complex functions of gel composition and temperature. The permeation coefficients of hydrocortisone at 30 "C in the three hydrogels studied here illustrate this complexity, as shown in Table I, where physical properties as well as mass transport parameters for three hydrogels a t 30 "C are summarized. The effective diffusivities increase as the amount of hydrophilic AAm monomer in the hydrogel increases, particularly between NA 10 and NA 20. The partition coefficients decrease correspondingly. Overall permeabilities (permeation coefficients) in Table I indicate that the NA 20 gel exhibits the highest mass transfer rate of the substrate. Therefore, the increase of activity with the gel hydrophobicity cannot simply be explained only in terms of the reduced mass transfer resistance as evidenced in the result of the overall permeabilities. A more important factor in determining steroid converting kinetics could be the easy partitionings of the substrate and the product into the local hydrophobic region of the gel matrix in which the immobilized cells are located. Comparing the slightly different water sorption levels in the three hydrogels at 30 "C, ranging from 75.5% to 82.9%, it can be postulated that different micromorphological structures exist in these hydrogels, depending on
the gel composition and temperature. Without cells, the NA 0 gel was opaque and the other two copolymeric gels were transparent at 25 "C, which is below the LCST for all gels. This suggests that the opaque NA 0 gel is a heterogeneous hydrogel containing highly segregated polymer chains in the gel matrix, possibly forming a macroporous structure, and the other two transparent copolymeric gels are more homogeneous, consisting of a less phase-separated microporous structure. A physical appearance of the hydrogel is an important criterion to judge the hydrogel morphology. I t was observed that the transparency degree of the NA 0 gel depends on the polymerization conditions. For example, the opaque gel is often formed when the polymerization is carried out at room temperature, even though the apparent physical properties such as the water content and the LCST, are essentially the same regardless of the gel transparency (Park, 1990). In this sense, an important factor would be the microenvironment around the immobilized A. simplex cell, created by the different gel compositions. In addition, as the temperature is raised to 30 "C, the pore size of the NA 0 gel should decrease more rapidly than those of other gels because that temperature (30 "C) is quite close to the LCST (32-33 "C) of the NA 0 gel. These microstructural differences of the gel matrices may play an important role in the local availability of substrate and related cell turnover kinetics. Therefore, the term "gel hydrophobicity", which is frequently used in this paper, refers to observable macroscopic (e.g., water content) as well as unmeasurable microscopic physicochemical properties of the gel. Figure 4 demonstrates the effect of gel hydrophobicity on the relative activityas a function of temperature, which is defined as a normalized value of the observed activity to the highest activity (XlOO%). The relative activities of immobilized cells decrease with increasing acrylamide content in the gel at any temperature. It can also be seen that the activity in the hydrophobic gel is more sharply decreased with increasing temperature. This effect is due to the different gel hydrophobicities, which are affected by the gel composition as well as the temperature. As reported in a previous paper (Park and Hoffman, 1990a), thermally reversible hydrogels swell and expand below the LCST and deswell and shrink above the LCST. The three hydrogels studied here have LCST ranges that depend on the gel composition, as shown in Figure 2. The LCST ranges for the three hydrogels can be estimated from the steepest region of water content change. When the polymers are in the un-cross-linked state, the NA 0 polymer backbone has its LCST at 32-33 "C, NA 10 ca. 37-38 "C, and NA 20 gel ca. 45-46 "C (Park and Hoffman, 1990a; Dong and Hoffman, 1986). The LCST ranges for the three gels determined from Figure 2 have similar values as those of the un-cross-linked polymers. The degrees of swelling for these three hydrogels are sensitive functions of gel composition and temperature, and the related mass transfer of substrate and product will be similarly sensitive to these variables. This is directly related to the observed
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80'
700'
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a70
30'
21
20
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2
6
~
3
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Figure 4. Effect of gel hydrophobicity on steroid conversion at different temperatures. Acrylamide composition in monomer feed solution represents the degree of gel hydrophobicity. activities. Although the substrate prefers to partition in the more hydrophobic regions of the gel matrices, it will readily diffuse through the open pores of the gel as long as the temperature is below the LCST of gel matrix. Indeed, the activities sharply drop when the temperature reaches the corresponding LCST range of the hydrogel. This is a general phenomenon also observed in immobilized enzymes in our early studies (Park and Hoffman, 1990b; Dong and Hoffman, 1986). For comparison, the temperature-activity profile of the free A. simplex cells is shown in Figure 5, where the optimum temperature is found to be around 35 "C. It is known that the dehydrogenase system, coupled with electron transport system within a cell, is highly labile to temperature (Ohlson et al., 1978). However, at higher temperatures, the decrease of activity of the immobilized cells was more significant than that of free cells. This additional drop of activity is probably due to the increased mass transfer resistances within the gel matrices as the pores collapse within the thermally reversible hydrogels. Km Value Determination. K m values of free and immobilized cells were determined in order to elucidate the possible role of gel hydrophobicity on the entrapped immobilized cells. It has been known that A'-dehydrogenase is a membrane-bound protein associated with an electron transport system along the cell membrane (Koshcheyenko et al., 1983). The AI-dehydrogenase reaction involving two substrates (hydrocortisone and FAD as a cofactor) is coupled with a cofactor regeneration system, where oxygen serves as a key electron acceptor in the electron transport system. In the kinetic analysis here, we assumed that the oxygen concentration is saturated in the vicinity of entrapped cells and its change in concentration can be neglected, because good aeration in the batch reactor was maintained during measurement of the initial reaction velocities (1 h). Thus, the effect of gel hydrophobicity on the mass transfer of oxygen and on the immobilized cell activity will not be considered. The microenvironment of A'-dehydrogenase in a cell entrapped in a thermally reversible hydrogel can be investigated by measurement of apparent K m values. The Lineweaver-Burk plot is shown in Figure 6. In the Lineweaver-Burk plot, parallel slopes can be observed for the three different gel matrices. Since there was no product initially in the substrate solution, the difference of the microenvironments around the enzyme are reflected in the plot. Such parallel lines can be found in uncompet-
45
40
Temperatun, OC
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30
Figure 5. Temperatureactivity profile of free A. simplex cells.
D
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.
, -2
.
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.
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Figure 6. Lineweaver-Burk plots of (a) immobilized and (b) free cells.
itive product inhibition kinetics with increasing inhibitor concentration, in which both K m and V m u values vary. It can be postulated that gel hydrophobicity plays an important role in this system in controlling the product inhibition as well as the mass transfer of the substrate and product. As noted earlier, the A'-dehydrogenase system is inhibited to a large extent by both substrate and product. In the case here, product inhibition should be more severe because the substrate concentration used is far less than the value at which substrate inhibition has been reported to begin (Constantinides, 1980; Kloosterman and Lilly, 1984). Product inhibition in the immobilized A. simplex cells would be influenced more by the gel hydrophobicity than the mass transfer of the substrate, because the local concentration of product should be dependent on the local hydrophobic character of the gel matrices. The exact mechanism of product inhibition is not clear at this point, but it can be deduced from the pattern of the LineweaverBurk plot. Uncompetitive product inhibition may occur in a A'-dehydrogenase system, which is a two-substrate dehydrogenation reaction (Rudolph, 1979; Segel, 1975).
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'*O
0.8
0.6
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& 1
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immobilized cell Km
E
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0.4
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Acrylamide composltlon(%) in gel Figure 7. K , values for immobilized and free cells at 30 "C.
Therefore, higher relative activity in the more hydrophobic gel matrix may have originated from reduced product inhibition rather than the less important mass transfer. The Km values for free and immobilized cells obtained from Figure 6 are illustrated in Figure 7. In a hydrophilic gel, like NA 20, the apparent K m value of immobilized cells is similar to the K m of free cells, implying that entrapped cells in the NA 20 gel are located in a similar aqueous environment as the free cells. On the other hand, the apparent K m value of the most hydrophobic gel, the NA 0 gel, is much higher than that of the NA 20 gel, suggesting that cells in the NA 0 gel are located in a microenvironment different from an aqueous environment. Since the apparent K m value does not directly relate to the amount of cells in the gel matrices, it is improbable to ascribe the different Km values to different cell loading amounts. We assumed that the three hydrogels have the same cell loading amount because of their similar water contents during polymerization. Consideringthe fact that the apparent K m value of an immobilized biocatalyst should decrease as the mass transfer of substrate increases and it increases as the extent of product inhibition becomes lower, it can be concluded that the higher Km value for the NA 0 gel reflects the importance of product inhibition rather than mass transfer. As the product inhibition occurs more, the apparent Kmvalue decreases. Since hydrocortisone and prednisolone have similar molecular structures, partition coefficients of both steroids into the gel matrices are also similar. In the hydrophobic gel, locally segregated polymer segments in the hydrophobic gel may serve as a "depot" for the substrate and the product. However, the degree of product inhibition would be more reduced in the hydrophobic gel, NA 0, which is able to immediately uptake and readily delocalize the local product concentration from the enzyme active site. Even though overall apparent permeability of the substrate for the hydrophilic gel is higher than the hydrophobic one, it can be imagined that a slightly different morphological structure of the hydrogel matrix results in a different effect on the enzyme activity. Particularly, the partitioning of the product through the polymer matrix, even though its contribution is small, may play a crucial role in exhibiting the different enzyme activities. Effect of PMS on Steroid Conversion. The effect of phenazine methosulfate (PMS) on steroid conversionwas also investigated for the three gel-immobilizedcell systems.
with PMS
Figure 8. Effect of phenazine methosulfate (PMS)as an artificial electron acceptor on the steroid conversion for three immobilized cells: substrate 1 mM hydrocortisone in 5% methanol, 1-h reaction at 30 "C.
PMS is known to be an artificial electron acceptor for the Al-dehydrogenase system that facilitates cofactor regeneration and prolongs the stability of immobiliged cells (Kloostermanand Lilly, 1985;Silbiger and Freeman, 1988). The differences of the activities for the three gelimmobilizedcell systems with (0.5mM) and without PMS are compared in Figure 8. It is of interest to note that NA 10 and NA 20 are affected greatly by PMS, but NA 0 is not. This interesting phenomenon can be explained in terms of the gel hydrophobicity, as follows: PMS is an ionically charged, hydrophilic molecule, as seen in Figure 9, that will readily diffuse in the aqueous pores of the more hydrophilic gels. Therefore, the more hydrophilic the gel is, the more PMS should enhance activity of the cell system as seen in Figure 8. Similar observations were reported in hydrophilic and hydrophobic photo-cross-linkable resins for the steroid conversion of androstene3,17-dione to androst-1,4-diene-3,17-dione (Yamane et al., 1979). Models of Immobilized Cells in Hydrophobic and Hydrophilic Gel Matrices. On the basis of the results of this study, such as higher activity in the NA 0 hydrophobic gel, similar Km values for free cells and those in the NA 20 gel, higher Km of cells in the NA 0 gel than that of free cells, and increasing activities of hydrophilic gels in the presence of PMS, a possible model for immobilized A. simplex cells in hydrophobic and hydrophilic gel matrices is schematically presented in Figure 10. In a hydrophobic gel like NA 0, both the substrate and product should partition well into the hydrophobic domains, so there should be higher substrate conversion and lower product inhibition in this gel. On the other hand, the PMS will prefer the hydrophilic aqueous pores. The location of the Al-dehydrogenase system, which is believed to be localized in the cell membrane, may be decided on the basis of gel hydrophobicity. In a hydrophobic gel, most of the Al-dehydrogenase system in the cell should be located preferably in the hydrophobic domains, which are presumably the hydrophobically associated and segregated polymer chains in domains within the gel matrix. Opaque NA 0 cell-free gel membrane indirectly supports the existence of hydrophobic loci in the gel. In a hydrophilic gel like NA 20, the Al-dehydrogenase system should be located in the hydrophilic sites, such as aqueous pores, which are relatively abundant in the gel matrix compared to a hydrophobic gel matrix. However, it is questionable why the enzyme, Al-dehy-
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Figure 9. Chemical structure of PMS (phenazine methosulfate). HYDROPHOBIC GEL
HYDROPHILIC GEL
a
-w~~m---n
Q
H"p0"gjnn
/Ir
H ~ ~ ~ u c p o ~ ~ - ~ x e z i o ~ Fme-mbcub
Figure 10. Schematic diagram of immobilized A. simplex cells in thermally reversible hydrogel matrices having different gel hydrophobicities. The location of entrapped cells is determined by the microstructural differences of gel matrices.
drogenase, locates at different sites depending on the hydrophilic/hydrophobicity of the gel matrix. In the gel matrices of polyurethanes wherein A. simplex cells are immobilized, a similar hypothesis was proposed in order to explain different kinetic behaviors of the enzyme in hydrophilic and hydrophobic polyurethanes (Yamane et al., 1979). It can be speculated that the hydrophobic, phase-separated polymer segments produced during the cell immobilization interact with the membrane-bound enzyme, which has a hydrophobic nature. This will, in turn, decide the cell orientation and its location in the gel matrix. It should be pointed out that the substrate and the product, hydrocortisone and prednisolone, are relatively less hydrophobic than other steroids because they have two hydroxyl groups in their structure. Therefore, the small differences in hydrophobic and hydrophilic gel structures may largely affect the local partitioning of substrate into hydrophobic domains and the locations of A'-dehydrogenase in such domains, which may result in the different steroid conversions. In conclusion,the effect of gel hydrophobicity on steroid conversion of immobilized A. simplex in a series of hydrophilic/ hydrophobic thermally reversible hydrogelshas been demonstrated. Immobilized cells in a hydrophobic gel matrix exhibited higher steroid-converting activity than in a hydrophilic matrix. This may be due to the microenvironmental differences around the enzyme rather than the enhancement of mass transfer of the substrate.
Acknowledgment This work was supported by the WashingtonTechnology Center. Literature Cited Carrea, G. Biocatalysts in water-organic solvent two-phase systems. Trends Biotechnol. 1984,2, 102-106. Constantinides, A. Steroid transformation at high substrate concentrations using immobilized Corynebacterium simplex cells. Biotechnol. Bioeng. 1980, 22, 119-136.
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Registry No. polyNIP, 25189-55-3; NIPAAm, 28500-83-6; hydrocortisone, 50-23-7; prednisolone, 50-24-8; 3-ketosteroid AIdehydrogenase, 9029-04-3.
