Biotechnol. Prog. 1992, 8, 197-203
197
Highly Enantioselective Hydrolysis of (R,S)-Phenylalanine Isopropyl Ester by Subtilisin Carlsberg. Continuous Synthesis of (S)-Phenylalanine in a Hollow Fiber/Liquid Membrane Reactor Edith E. Ricks, Maria C. Estrada-ValdQs,Thomas L. McLean, and Guillermo A. Iacobucci' Corporate Research and Development Department, The Coca-Cola Company, P.O. Drawer 1734, Atlanta, Georgia 30301
The resolution of racemic phenylalanine esters with esterases has been reexamined in relation to the development of a continuous process based on the use of hollow fiber/ liquid membrane (SLM) reactors. The requirement for high enantioselectivity was met by phenylalanine isopropyl ester, whose (R)-enantiomer was found refractory to the action of subtilisin Carlsberg, in water, pH 7.5, a t 25 "C. The continuous feeding of (R,S)-Phe-O-iPr-HCla t 2 mM/h to 0.7 g/L subtilisin resulted in steady-state reaction conditions that gave (S)-Phe-OH of 95% enantiomeric excess (ee) a t the theoretical rate. The unreacted (R)-Phe-0-iPr, which permeated out continuously, had 80% ee and was racemized quantitatively by heating with a salicylaldehyde catalyst in refluxing toluene. Doubling the feeding rate (4 mM/h) created excessive accumulation of (R)Phe-0-iPr in the reactor, which inhibited the enzyme through nonproductive binding, lowering the (S)-Phe-OH resolution rate to 68% of the theoretical rate. Similar experiments with (R,S)-Phe-OMe-HCl gave (SbPhe-OH of 9-4896 ee, due to the enzymatic hydrolysis of (R)-Phe-OMe experienced during the runs. When a-chymotrypsin was used for the continuous hydrolysis of (R,S)-Phe-0-iPr, the isolated (S)Phe-OH showed 67% ee, indicating that substantial hydrolysis of the @)-ester had occurred.
Introduction The use of esterases for the enantioselective hydrolysis of chiral amino acid esters is a subject of current interest in the literature (Chen et al., 1986; Mirviss et al., 1990) and is of particular importance for the preparation of (5')phenylalanine from synthetic (R,S)-phenylalanine as an alternative approach to fermentation processes already in use (Aida et al., 1986). The availability of (S)-phe-OH at low cost is critical to the manufacture of the sweet dipeptide (S)-aspartyl-(S)-phenylalaninemethyl ester (aspartame), whose production in 1984had alreadyreached 4000 tons (Meienhofer, 1984) and has since more than doubled. Notwithstanding the innovative efforts of Klibanov (1990) and others (Laane et al., 1987) to broaden the usefulness of enzymes as catalysts for organic reactions in nonaqueous media, the fact remains that subtle manifestations of enzyme specificity such as enantioselectivity are highly dependent on the conformational integrity of the hydrated macromolecule and are better attained in water (Sakurai et al., 1988). Preliminary hydrolysis studies done by us on (S)-and (R)-Phe-OMe using three esterolytic proteases, chymotrypsin, subtilisin, and aminoacylase I, have confirmed previous observationsthat both enantiomersare substrates for these enzymes, although the @)-ester hydrolyzes at a rate substantially higher than that of the @)-enantiomer. (S)I(R) kinetic ratios of 1099, 87, and 47, respectively, were measured for the three enzymes in 0.1 M phosphate buffer, pH 8.0, with a substrate to enzyme ratio of 10, at 25 "C. This degree of stereodiscrimination is sufficient for use in batch resolutions, in which the reaction is quenched by necessity at 50% completion (Chen et al., 1982). For the design of time-extended operations, however, when the 8756-7938/92/3008-0197$03.00/0
racemic substrate is continuously fed and is in contact with the enzyme for longer periods while the unreacted (R)-ester is continuously removed from the system, the need for the highest possible enantiomeric discrimination becomes critical for securing high optical purity of the (5')-phenylalanine. Continuous processing offers important advantages over batch, particularly in the economics of reactor productivity (output per unit time) and enzyme recovery. Forty years ago it was observed (Wretlind, 1950) that a crude enzyme preparation from pancreas, acting on (R,S)-phenylalanineisopropyl ester in water at 23 "C, was able to hydrolyze selectively the @)-ester to (S)-Phe-OH, leaving the (R)-esterunchanged. The apparent resistance of the (R)-Phe-0-iPr to hydrolysis was later confirmed with purified chymotrypsin (Purdie and Benoiton, 1970). In this report, we are demonstrating the superior enantioselectivity of subtilisin Carlsberg (EC 3.4.21.14) toward (S)-Phe-0-iPr during continuous hydrolysis, a behavior that, contrary to those prior assertions, is not quite shared by a-chymotrypsin under similar conditions.
Experimental Procedures Materials and Methods. Melting points are uncorrected. Polarimetric measurements were done on a PerkinElmer Model241polarimeter. The 'H NMR and 13CNMR spectra were acquired in the FT mode, using a Varian Gemini 200 spectrometer at the frequencies of 200 and 50.30MHz, respectively. Chemical shifts are given in parts per million d o d i e l d from Mersi. The abbreviations used are s, singlet; d, doublet; t, triplet; q, quartet; br s, broad singlet; mult, multiplet. HPLC analyses were done on a Perkin-Elmer system consisting of a 410 LC pump, an LC 235 diode array detector set at 210 nm, and an LCI-100 laboratory computing integrator for data analysis. The
0 1992 American Chemical Society and American Institute of Chemical Engineers
lee
Biotechnol. Prog., 1992, Vol. 8, No. 3
Scheme I
gl
90
.-a
70
Subllllsln
60
(Sbamlno a d d
0
IO
60
30
(Rkamlno ester
90
Minutes
Figure 1. Effect of ester structure on the rate of hydrolysis of (R)-and (23)-phenylalanineesters by subtilisin Carlsberg, in 0.2 M bicarbonate buffer, pH 8.5,25 O C , with a substrate to enzyme ratio of 10,by weight. Symbols: 0 , (S)-Phe-OMeHCl,108mM; A, (R)-Phe-OMe.HCl,141 m M 0 (S)-Phe-O-iPr.HCl,170 mM; A, (R)-Phe-O-iPr.HCl,121 mM. analytical column was an Alltech 150-mm Adsorbosphere HS CU (5 pm) cartridge. The mobile phase was a v/v mixture of 20% CH3CN in 0.1% KH2P04, pH 4.2, with a flow rate of 0.5 mL/min. Retention times for Phe-0-iPr and Phe-OH were 10.35 and 2.91 min, respectively. The amino acids (R)-Phe-OH,(SI-Phe-OH, and (R,S)-Phe-OH and their methyl esters were purchased from Sigma. The corresponding isopropyl esters were prepared according to the standard procedure described below. The determination of enantiomeric excess (ee) was done by HPLC analysis of phenylalanine samples, using a Bakerbond chiral phase Crownpack CR(+) column supplied by J. T. Baker, containing an 18-crown-6chiral crown ether as the enantioselective agent. The column (150 X 4 mm i.d., 5-pm particle size)was operated a t 25 'C using aqueous HC104, pH 1.9, as the mobile phase, at a flow rate of 0.8 mL/min. Peaks were detected at 210 nm on a PerkinElmer LC 235 diode array detector. The recorded retention times were 8.31 min for (A)-Phe-OH and 10.24 min for (S)-Phe-OH. The ee percentage values were calculated from the recorded chromatograms, by measuring peak areas (A) for each enantiomer and using the expression %ee=- As-AR x 100 AS + for @)-enantiomer excess. For the analysis of phenylalanine esters, samples (50 mg) were hydrolyzed in 0.5 N HCl(5 mL) at 80 "C for 5 h, using 10-mL glass-stoppered test tubes. Adequate dilutions of the hydrolysate were used for analysis. Purified subtilisin Carlsberg (from Bacillus licheniformis) and a-chymotrypsin type I-Swere purchased from Sigma Chemical Co. (1990 catalog), products no. P5380 and no. C7762, respectively. Hollow fiber modules were manufactured by Bend Research Inc. (Bend, OR 97701), using Celgard fibers supplied by Celanese Chemical Corp., New York, NY. Each module provided 900 cm2 of an SLM made of 33% NJVdiethyldodecanamidel67 5% dodecane. This choice of SLM composition was adequate for the selective permeation of (R)-Phe-0-iPr. A flux value of 0.5 mg/(cm2-h)at 25 'C was measured for this ester, at the concentration of reactants used in the resolution experiments described below. (R)-PhenylalanineIsopropyl Ester Hydrochloride. To 6 L of ice-cooled2-propanol was added 420 g of HCUg), followed by 696 g of (R)-Phe-OH, and the mixture was heated a t reflux for 8 h. After cooling,the suspension was
filtered, and the solid was resuspended in 1 L of 2-propanol and filtered again, washed with 2-propanol,and dried in vacuo. Yield: 987 g (90% ), white crystalline solid. [ c Y I ~ ~ D = -36.7' (c, 2.0; EtOH). mp: 223-224 'C. lH NMR (CD3OD, TMS, 6): 7.25-7.45 (5H, mult,-Ar), 5.05 (1H, septet, J = 6.3 Hz, -CH=), 4.89 (2 H, br s, -NH2), 4.28 [l H, t, J = 7.0 Hz, -CH(NH2)(COz)l, 3.15-3.35 (2 H, mult, ArCH2-), 1.26 and 1.17 (each 3 H, d, J = 6.3 Hz, -CH3). 'WNMR (CDsOD,TMS, 6): 170.00 (-C02), 135.80,130.93, 130.39, 129.17 (Ar), 72.09 (OCH=), 55.39 [-CH(NH2)(COz)], 37.60 (ArCH2-); 21.91 and 21.80 (-CH3). Anal. Calcd for C12Hle02NCl: C, 59.13; H, 7.39; N, 5.75; C1,14.58. Found: C, 59.30; H, 7.45; N, 5.70; C1, 14.51. Continuous Resolution of (&,S)-Phe-O-iPr.HClunder Steady-State Conditions. A solution of 400 mg of subtilisin Carlsberg in 600 mL of deionized water, pH 7.5, was placed between the reaction vessel A (250 mL) and the intermediary vessel B (350 mL), which made up the reaction phase (Figure 2). This reaction phase was circulated with a peristaltic pump at a flow rate of 100 mL/min through the shell side of a hollow fiber module, containing 900 cm2 of an SLM made of 33% v/v NJVdiethyldodecanamide in dodecane. A second pump was used to circulate the reaction mixture between vessels A and B at 140 mL/min, through a T-tube that split the flow into two streams, whose relative fluxes could be adjusted with clamps V1 and V2 to a ratio V2/V1 = 6. This setup provided 12 min of retention time to the added ester, ample to secure the completion of the hydrolysis before the solution entered the SLM module. The product phase in vessel C consisted of 200 mL of deionized water, pH 3.5, that was circulated through the tube side of the module at 100 mL/min, countercurrent to the shell phase. The separation was run at room temperature, keeping the pH of both phases constant at 7.5 and 3.5 by pH-stat monitoring, using 0.2 N NaOH and 0.2 N HC1 as titrants. The reaction was started by adding to vessel A, by means of a Milton Roy micropump, a solution of 1.17g (4.8 mmol) of (R,S)-Phe-0-iPr.HC1 in 60 mL of deionized water, pH 7.5, a t the rate of 15 mL/h (1.2 mmo1/600 mL.h, or 2 mM/ h), while keeping both phases under circulation through the hollow fiber module. The course of the resolution was followed by HPLC, on samples taken from vessels A, B, and C at 15-min intervals during the first hour and at every hour for the remaining time. For plotting purposes (Figure 4), the values of A and B for each component were added in order to describe the changes in the reaction phase as a whole, as concentrations in both vessels were found to be similar. A t the end of 4 h, both phases were recovered and the products were isolated as follows. (a)Product phase: the solution (200 mL), pH 3.5, was shell frozen and freeze dried. Yield: 515 mg of (R)-Phe0-iPr.HC1, white solid. mp: 198-200 'C. [(Yl2'D = -29.0' (c, 2; EtOH). 81% ee.
Bbtechnoi. Pmg., 1992, Vol. 8, No. 3
199
HCI (pH Stat) "2
--
Tube phase Water pH 3.5
c (R,S)-pheWPro*HCI NeOH (pH Stat)
1
",I.;
1I
SuW II sI n
Figure 2. Apparatus for the resolution of (R,S)-Phe-0-iPr.HC1with subtilisin, using a hollow fiber SLM module.
Figure 3. Effect of pH on the partition coefficient of (R,S)-Phe-O-iPr.HCl between toluene and water, at 25 "C. The crystallized product was collected, washed with 0.5 mL of iced water, and recrystallized from water. (8)-PheOH recovered (285 mg) had [ ( u I ~ ~=D-32.2' (c, 2; H2O) and 95% ee. Effect of Subtilisin on the PermeationRate of (R)Phe-0-iPr. Measurement of KR.The binding of (R)Phe-0-iPr to subtilisin decreases the concentration of free ester available for permeation. The decrease of the escape velocity V,,,, with respect to a control without enzyme allows for the calculation of [ERI in the following equilibrium:
:p 1 1
0
0
30
80
90
120 150 m1nutes
180
210
240
Figure 4. Continuous resolution of (R,S)-Phe-O-iPr.HCl in an SLM reactor. Rate of the addition of substrate: 2.0 mM/h. Subtilisin concentration: 0.7 g/L in water, pH 7.5,25 OC. SLM: 900 cm2,33% NJV-diethyldodecanamide in dodecane. Symbols: 0, (S)-Phe-OH (reaction phase); A, (R)-Phe-O-iPr.HCl (permeate phase); . , (R,S)-Phe-O-iPr.HCl (reaction phase); (- -), theoretical rate, 1 mM/h.
-
(b) Reaction phase: the solution (650 mL), pH 7.5, was placed in an Amicon 2000 ultrafiltration cell fitted with a YM5 membrane (MW cutoff 5 kDa) and ultrdiltered to 100 mL under 50 psi of N2. The subtilisin-free diafiltrate (500mL) was adjusted to pH 6.0 with 1 N HC1, evaporated in vacuo to 10 mL, and left overnight at 4 "C.
KR
E + R F? E-R A total of 200 mL of 10 mM (R)-Phe-O-iPr.HCl solution in 0.1 M phosphate buffer, pH 7.5 (vessel A, Figure 2), placed in a constant temperature bath at 25 "C, was circulated through the shell side of an SLM module at the rate of 180 mL/min. Countercurrent to it was circulated 200 mL of 0.1 M citrate/phosphate buffer, pH 3.5 (vessel C), through the lumen of the hollow fibers, and the permeation of (R)-Phe-0-iPr was followed by UV absorbance measurements of the tube phase at 260 nm. This
200
Blotechnol. Prog,, 1992, Vol. 8, No. 3
Table I. Continuous Resolutions of (It,@-Phenylalanine Esters in an SLM Reactor, with Subtilisin (0.7 g/L), pH 7.5, 25 OC: Effect of Ester Structure on the ODtical Purity and Observed Rates of the Enantiomers
X isopropyl
substrate (R,S)-Phe-OX-HCl (8)-Phe-OH (reaction phase) addition rate (mM/h) VW (mMlh) re1 rate ( % ) [a]: (deg) een(%) V,,, 2.0 4.0 4.7 9.4
methyl a
0.93 1.36 2.00 5.86
93 68 85 125
-32.2 -31.8 -4.0 -12.1
95 97 9 48
(R)-Phe-OX-HCl(permeate phase) (mM/h) re1 rate (%) (deg) eea (7%) 1.02 2.05 1.91 3.72
102 103 81 79
-29.0 -26.1 -32.4 -33.2
81 73 97 95
By chiral HPLC analysis (cf. Experimental Procedures). mM 8 7
:wl, ,
I
I
,
300
360
I ,
1
0
0
60
120
180 240 minutes
Figure 5. Continuous resolution of (R,S)-Phe-0-iPr.HC1in an SLM reactor. Rate of the addition of substrate: 4.0 mM/h. Subtilisin concentration: 0.7 g/L in water, pH 7.5,25 "C.SLM: 900 cm2,335% Nfl-diethyldodecanamide in dodecane. Symbols: 0 , (S)-Phe-OH (reaction phase); A, (R)-Phe-0-iPr.HC1 (permeate
.
phase); B, (R,S)-Phe-0-iPr.HC1 (reaction phase); (- -): theoretical rate, 2 mM/h.
experiment was repeated in the presence of 0.04 mM subtilisin Carlsberg, and after 30 min of equilibration time at 25 "C, the V, was measured again as before. The results are given in Table 111. Kinetics of Phe-0-iPr-HC1Hydrolysis with Subtilisin Carlsberg. Measurement of Ks and To a solution of 100mM (S)-Phe-O-iPr.HClin 0.1 M phosphate buffer, pH 7.5 (20 mL), immersed in a water bath kept at 25 "C, was added subtilisin Carlsberg (20 mg), and the course of the hydrolysis was monitored by HPLC by following the decrease of substrate concentration during 30 min of time. The initial velocity of hydrolysis (Vs) was deduced from the plotted data, and the values KS = Vs/2 and kz were calculated (Table IV). From a similar experiment done with the racemic ester, the corresponding kcatwas obtained, and the value of KM was calculated from the expression (Fersht, 1985)
&.
KM =
+ (Ks/KR)]]Ks
using the experimental values of Ks and KRfound above. Distribution of (R,S')-Phe-0-iPrbetween Toluene and Water as a Function of pH. A total of 2.0 g of (R,S)-Phe-0-iPr.HC1was dissolved in 50 mL of deionized water at 25 "C, 50 mL of toluene was added, the pH was adjusted to 8.5 with 1 N NaOH under vigorous stirring, and the dispersed system was kept under stirring for 15 min. After equilibration, the phases were allowed to separate, the pH was recorded, and 1-mL aliquots were removed from both phases for HPLC analysis. Mixing was restarted, and the pH was reduced at 0.5-unit intervals using 1N HC1, repeating the sampling procedure at each pH value down to pH 4.0. For HPLC analysis, each sample was evaporated to dryness in vacuo and the residue was dissolved in 1 mL of MeOH and evaporated again to dryness, and the residue was dissolved in 1mL of MeOH for analysis. The values in the semilog plot are shown in Figure 3. Racemization of (R)-Phe-O-iPr*HCl.A solution of 1.0 g (4.1mmol) of (R)-Phe-O-iPr-HCl[ [ c Y I ~ ~=D-36.7" (c, 2.0; EtOH), mp 223-224 "C] in 100 mL of saturated NaHC03 was extracted with toluene (3 X 50 mL). The
combined extract was dried over anhydrous Nap904 and concentrated to 25 mL in vacuo, and to the solution was added 800 mg (weight ratio of amino acid ester to catalyst = 1) of the resin-immobilized salicylaldehyde catalyst (Mirviss, 1987). After being refluxed for 3 h, the catalyst was removed by filtration, the toluene was evaporated, and the residual amino acid ester was converted into the hydrochloride by crystallization from 10mL of 2-propanol/ HC1, to yield white needles. Yield: 960 mg (96%). mp: 199-200 "C. [cUIz5D = 0.0" (c, 2; EtOH). The lH NMR spectrum was identical to that of the starting material. Results and Discussion Figure 1compares the time course of the hydrolysis of the phenylalanine methyl and isopropyl esters by subtilisin, in 0.2 M bicarbonate buffer, pH 8.5, substrate to enzyme ratio = 10, at 25 "C. From the data, it is clear that the improvement in enzyme enantioselectivity resulting from the change of methyl to isopropylreflects the inability of the (R)-isopropylester to enter into productive binding at the active site of subtilisin. Hydrolysis of the (R)-isopropyl ester was not apparent up to 65 h of reaction time, under conditions in which the (&)-methylester was fully hydrolyzed at 10 h. This refractoriness of the (R)-isopropyl ester was seen as a valuable asset that would allow for more flexible processing conditions, in the sense that its residence time in the reaction could be varied without undue concern for enzyme overexposure. Conditions for enzymatic continuous reactions can be modeled conveniently in the laboratory with hollow fiber supported liquid membrane (SLM) reactors (Matson and Quinn, 1986; Matson, 1989; Araki and Tsukube, 1990).In a preferred configuration, an SLM hollow fiber module is made of microporous polypropylene fibers, having the porous walls filled with water-immiscible hydrophobic organic liquids such as isohexadecanol,undecanone, dodecane, etc. The organic liquid is held in place by capillary forces, and it provides a functional SLM of high interfacial surface. In the module, the SLM interfaces with two aqueous phases that can be circulated countercurrently, one phase through the lumen of the fibers (tube phase) and the other along the interfibrillar space (shell phase). Uncharged compounds present in solution in either of the aqueous phases can cross the SLM according to their osmotic gradients, but ionized species are fully rejected. As indicated in Scheme I, the hydrolysis of (R,S)-Phe0-iPr at pH 7.5 results in a mixture of permeable (R)Phe-0-iPr and nonpermeable (S)-Phe-COz-. Circulation of the mixture through the shell side of an SLM module against water, pH 3.5, in the tube side will result in the selective transport of the free (R)-amino acid ester across the SLM and the trapping of its protonated form in the tube phase, while leaving behind the nonpermeable (S)-Phe-COz-and the enzyme. Resolution of the substrate results from the combined effects of the enzyme and SLM selectivities when operation is at the appropriate pH in each phase. These are kept constant during runs through the use of pH-stats. A schematic view of the experimental apparatus is shown in Figure 2.
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Blotechnol. Prog., lW2, Vol. 8, No. 3
Table 11. Continuous Resolutions of (R,S)-Phe-O-iPr.HClat a 2 mM/h Feed Rate, using Various Concentrations of Subtilisin, pH 7.5, 25 OC
(R)-Phe-O-iPr-HCl(permeate phase)
(S)-Phe-OH (reaction phase) subtilisin concentration (g/L) 0.70 (oDtimal) . _ 0.35 0.17
0
Vm (mM/h) 0.93 1.12 0.36
re1 rate ( % )
[alf (deg)
eea (%)
93 112 36
-32.2 -34.8 -34.5
95 99 96
V,
(mM/h) re1 rate (%) 1.02 1.20 1.30
[a]: (deg)
een (%)
-29.0 -22.0 -13.2
81 54 36
102 120 130
By chiral HPLC analysis (cf. Experimental Procedures).
Although the experiments done here represented 4-h runs, the physical stability of the SLM in the hollow fiber is far greater. For the organic liquid and conditions of pH and temperature used here, lifetimes of about 3 months have been observed. However, the SLM can be regenerated repeatedly by reloading the pores of the microporous support with fresh organic liquid. The choice of operational pH is a compromise between subtilisin activity (optimal pH = 8.5) and the partition of the substrate into the water phase, as both influence reaction rates and respond oppositely to pH changes. The variation of K = Corg/Cwakr with pH, measured for phenylalanine isopropyl ester between toluene and water at 25 "C, is shown in Figure 3. The steep distribution of the substrate into the organic phase at alkaline pH suggested pH 7.5 as an acceptable standoff between the kinetics of (S)-esterhydrolysis and the rate of permeation of the (R)ester. The results shown in Figure 4 and Table I illustrate a typical 4-h run conducted in water, pH 7.5, at a substrate feed rate of 2 mM/h and 0.7 g/L subtilisin, in which a steady-state condition was achieved. This is shown by the low level of substrate maintained in the reaction mixture, the theoretical rates of enantiomer formation observed, and the high enantiomeric excessof the resulting products. On the other hand, Figure 5 shows the course of events when the rate of addition of (R,S)-Phe-0-iPr.HC1 is doubled (4 mM/h). The unbalance created between feed and permeation rates has caused an accumulation of (R)ester, which depressed the rate of (S)-Phe-OH formation to 68% of the theoretical value (Table I). The inhibitory effect of the @)-ester resulted from nonproductive binding at the active site (Fersht, 1985), in competition with the normal substrate. This inhibitory effect of the (R)-isopropyl ester was not evident in the case of the (R)-methyl ester (Table I), because its binding to subtilisin led to hydrolysis, resulting in high phenylalanine yields of poor enantiomeric purity. The optimal concentration found for subtilisin (0.7 g/ L) is required to secure complete resolution in less than 12 min, which is the retention time imposed to the contents of vessel A before vessel B is reached (Figure 2). The interdependence between chemical kinetics, circulation rates, and membrane area was further evidenced by reducing the enzyme level. As shown in Table 11, the decrease of subtilisin concentration below the optimal 0.7 g/L slowed down the resolution rate, thus forcing the accumulation of @)-ester in the reaction mixture. The consequence of this is seen (a) in the lowering of the (5')Phe-OH resolution rate and (b) in the low values of the ee percentage found for the permeated (R)-ester due to the co-permeation of unreacted @)-ester (Table 11). Maintenance of a constant resolution rate VRSwill require the continuous removal of the inhibitory (R)-ester as it is fed, or VRS = V,,,, = l/2Vfeed (mM/h). To accomplish this, it is necessary to adjust the mechanics of the system accordingly, as to provide the proper retention time for
Table 111. Effects of Enzyme Binding upon the Velocity of Permeation of (R)-Phe-0-iPr,Measured in an SLM Hollow Fiber Module, at 25 'Ca
expt no.
control
plus enzyme
1 2 3
27.0 28.2 26.4 27.2
21.7 21.0 19.2 20.6
av
A% 20 25 27 24
Conditions: SLM, 900 cm2,Nfl-diethyldodecanamide (33%) in dodecane; [R]= 10 mM (R)-Phe-O-iPr.HCl, in 0.1 M phosphate buffer,pH7.5; [El = 0.04mMsubtilisinCarlsberg, in0.1Mphosphate buffer, pH 7.5. Table IV. Kinetic Parameters for the Hydrolysis of (S)-Phe-O-iPr.HClwith Subtilisin Carlsberg, in 0.1 M Phosphate Buffer, pH 7.5, at 25 OC, Given by the Reaction Ks
E + S e ES Darameter [SI (mM) [El (mM) VS"(mM-min-l)
-+ ka
E
product
value
Darameter value kz (min-l) 140.9 KS (mM) 4.65b kz/Ks (mM-l-min-l) 30.3 "Initial velocity, measured during the first 5 min. bCalculated as Ks = Vs/2. 100 6.6 X 9.3
Scheme I1 (Sbph.OH
t
rl
"lr
u the total hydrolysis of the substrate and enough SLM surface to achieve the rate of permeation needed. The independent determination of the individual dissociation constants K s = 4.65 mM and K R = 24.1 mM has allowed the calculation of K M . For the case of the racemate, K Mtakes the meaning of the apparent dissociation constant of the enzyme/substrate complex when [Rl = [SI,which is a function of K s and K R (Fersht, 1985):
KM = [1/[1 + ( K s / K R ) ] ] K=s 0.84Ks = 3.9 mM (1) where the factor [1/ [1+ ( K s / K ~ ) l lfd = (fs +f R ) represents the fraction of sites occupied by the productive (S)-enantiomer over the total number of sites (Bender and KBzdy, 1965). Thus, the equilibration of subtilisin Carlsberg with the racemic Phe-0-iPr results in 84% of the binding sites taken by the (5')-enantiomer, as its affinity for the enzyme is 5 times higher than that of the (R)-enantiomer. The
202
Biofechnol. Rag., 1992, Vol. 8, No. 3
Table V. Kinetic Parameters for the Hydrolysis of (R,S)-Phe-O-iPr.HClwith Subtilisin Carlsberg, at pH 7.5,25 "C, Given by the Reaction
E + (R,S)
A. batch reaction, in 0.1 M phosphate buffer, pH 7.5 B. continuous reactions in water pH 7.5 (i) steady state (ii) under inhibition
- -
KM
kcat
ER-ES
E + product
100
3.1 X
4.0
2.30 X 1.33 X
2.30 X 0.57 x
1.16 X 0.62 x
121.2
3.91b
31.0 30.2c 81.8c
Initial velocity, measured during the first 5 min. Calculated as KM = 11/11+ (Ks/K~)llKs. Calculated as (kcaJK~)app = VW/[E][R,SI. Table VI. Constancy of the Specificity Constant for the Hydrolysis of (S)-Phe-O-iPr by Subtilisin Carlsberg, at pH 7.5, 25 O C kJK. =
(R,S)-Phe-O-iPr.HCl (batch) (R,S)-Phe-O-iPr.HCI (continuous, steady state) (R,S)-Phe-O-iPr.HCl (continuous, under inhibition)
0.9 0.9
31.0 30.2
27.9 27.1
0.36*
81.8
29.4
Calculated as fd(fs + f ~ =) kaJkz (Tables IV and V). Calculated as fs/(fs + f ~ )[(VRS)inhid(VRS)Ststatel X 0.9 (Table V). Table VII. Continuous Resolution of (R,S)-Phe-O-iPr.HCl with a-Chymotrypsin,pH 7.5,25 O c a
(S)-Phe-OH (reaction phase) (R)-Phe-O-iPr.HCl (permeate phase) V- (mM/h) 1.23 V ,, (mM/h) 0.46 relrate (%) 123 re1 rate (%) 46 [a]; (deg) -23.5 [a]: (deg) -35.2 ee (96) 67 ee (%) 96 0 Conditions: feed rate, 2 mM/h; enzyme concentration, 0.6 g/L.
weaker binding of the (R)-ester to subtilisin, as measured by KR,facilitates the permeation process and the acquisition of the steady-state condition:
where [E] is the concentration of the free enzyme, and [R,S] is the concentration of racemic substrate. The complex set of equilibria operating at the steady state are represented in Scheme 11. As shown in Tables IV and V, the specificity constant for the hydrolysis of (S)-Phe-O-iPr by subtilisin Carlsberg remained invariant during the resolution of the racemate, when it was performed either in a batch mode or in a continuous steady-state fashion. However, when the system was inhibited by an excess of (R)-ester, a much larger value for the apparent constant was observed. This can be reconciled by accepting that in all cases the enzyme concentration is given by the expression [El [fd(fs+ fR)], that is, the enzyme activity results from the molar concentration of active sites times the relative distribution of those sites between the productive and nonproductive enantiomers, a distribution that depends on the relative magnitude of their dissociation constants KR and Ks (Bender and KBzdy, 1965). Thus, the rate equation (eq 2) can be written
or (4) (kcat/KM)app[fS/(fS + fR)] = k2/KS As shown in Table VI, the relationship given by eq 4 holds ) during well for the various values offs/(fs + f ~experienced this work. The racemization of (R)-Phe-O-iPr is a necessary step for a process of this type. It was done successfully for this ester by adopting the conditions described in the patent literature (Mirviss,1987),refluxing it in anhydrous toluene in the presence of an immobilized salicylaldehydecatalyst, with quantitative recovery of the (R,S)-Phe-O-iPrand no observable formation of the DKP by HPLC. The rate of racemization increased with the increase in the proportion of catalyst to substrate; equal weights of substrate and catalyst were needed for reaction times of about 3 h in refluxing anhydrous toluene. The catalyst was recovered and reused ten times without any decrease of the racemization rate. Because of the mechanistic similarities of the two serine proteases subtilisin and a-chymotrypsin (Polgar, 1987), and the prior assertion (Purdie and Benoiton, 1970)that (R)-Phe-O-iPr was an inhibitor of the hydrolysis of the (&-ester by the latter, it was of interest to examine the enantioselectivity of a-chymotrypsin in a continuous resolution. The experiment of Table VI1 was done on (R,S)-Phe-O-iPr under the optimal conditions found for subtilisin, and the results indicated that a substantial hydrolysis of (R)-Phe-O-iPr has occurred. This finding emphasized the uniqueness of the absolute selectivity shown by subtilisin and reinforced the importance of binding characteristics (strength, orientation of bound form) to the manifestation of enzyme specificity.
Notation initial velocity of hydrolysis for the substrate (S)-Phe-O-iPr,mM.h-' initial velocity of hydrolysis for the substrate (R,S)-Phe-O-iPr,mM-h-l velocity of permeation of (R)-Phe-O-iPracross the SLM, in mM-h-l velocity of addition of reactant (R,S)-Phe-OiPr to the reaction mixture, mM.h-' concentration of free enzyme, mM concentration of (R,S)-Phe-O-iPr,mM fraction of enzyme active sites occupied by (S)-Phe-O-iPr fraction of enzyme active sites occupied by (R)-Phe-O-iPr fractional distribution of (S)-sites over total occupied sites dissociation constantof the Michaelis-Menten complex E-@),mM
Bbtechnol. Prog., 1992, Vol. 8, No. 3 KR KM
kz kcat
kdKS
209
dissociation constant of the Michaelis-Menten complex E G ) , mM dissociation constant of the Michaelis-Menten complex E.(R,S),m M turnover number, or first-order rate constant, for the reaction E@) -E product, min-l turnover number for the reaction E.(R,S) E product, min-1 specificity constant (second-order rate constant) for the reaction E.(R,S) E product [ = ( k e a t / K M ) a p p [ f S ( f S + fR)11, mM-1.min-1
+
+
-
+
Acknowledgment We thank Drs. James G. Sweeny and George A. King I11 of these laboratories for helpful discussions and Mr. Daniel Brose and Dr.Paul van Eikeren of Bend Research Inc. (Bend, OR) for advice and guidance in the use of SLM modules. Literature Cited Aida, K.; Chibata, I.; Nakayama, K.; Takinami, K.; Yamada, H., Eds. Biotechnology of Amino Acid Production, Progress in Industrial Microbiology; Kodansha: Tokyo, 1986;Vol. 24,pp 188-206. Araki, T.; Tsukube, H., Eds. Liquid Membranes: Chemical Applications; CRC Press: Boca Raton, 1990. Bender, M. L.; KBzdy, F. J. Mechanism of Action of Proteolytic Enzymes. Annu. Rev. Biochem. 1965,34,49-76. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. Quantitative Analyses of Biochemical Kinetic Resolutions of Enantiomers. J. Am. Chem. SOC.1982,104,7294-7299. Chen, S.-T.; Wang, K.-T.; Wong, C.-H. Chirally Selective Hydrolysis of DL-Amino Acid Esters by Alkaline Protease. J. Chem. SOC.,Chem. Commun. 1986,1514-1516.
Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman and Co.: New York, 1985;pp 109-111. Klibanov, A. M. Asymmetric Transformations Catalyzed by Enzymes in Organic Solvents. Acc. Chem. Res. 1990,23,114120. Laane, C.; Tramper, J.; Lilly, M. D., Eds. Biocatalysis in Organic Media; Elsevier Science Publishers B.V.: Amsterdam, 1987. Mataon, S. L. Method for Resolution of Stereoisomers in Multiphase and Extractive Membrane Reactors. U.S.Patent 4,800,162,January 24, 1989. Mataon, S.L.; Quinn, J. A. Membrane Reactors in Bioprocessing. Ann. N.Y. Acad. Sci. 1986,469,152-165. Meienhofer, J. Large Scale Peptide Synthesis: A Review. In Peptides; Marsson, U. R., Ed.; Almquist and Wiksell International: Stockholm, 1984,pp 19-34. Mirviss, S. B. Racemization of Amino Acids. US. Patent 4,713,470,December 15,1987. Mirviss, S. B.; Dahod, S. K.; Empie, M. W. Synthesis of L-Phenylalanine Methyl Ester. Ind. Eng. Chem. Res. 1990,29,651659. Polgar, L. Structure and Function of Serine Proteases. In Hydrolytic Enzymes; Neuberger, A.; Brocklehurst, K., Eds.; Elsevier Science Publishers B.V.: Amsterdam, 1987;pp 159200. Purdie, J. E.; Benoiton, N. L. The Interaction of a-Chymotrypsin with Phenylalanine Derivatives Containing a Free aAmino Group. Can. J. Biochem. 1970,48,1058-1065. Sakurai, T.; Margolin,A. L.; Ruesell,A. J.;Klibanov, A. M. Control of Enzyme Enantioseledivity by the Reaction Medium. J. Am. Chem. SOC.1988,110,7236-1237. Wretlind, K. A. J. Resolution of Racemic Phenylalanine. J.Biol. Chem. 1950,186,221-224. Accepted February 12,1992. Registry No. (R,S)-Phe-0-iPr, 81084-82-4; (S)-Phe-OH,63a-chy91-2;(R,S)-Phe-OMe, 15028-44-1;subtilisin, 9014-01-1; motrypsin, 9004-07-3.
