Industrial Production of L-Alanine from Ammonium Fumarate Using Immobilized Microbial Cells of Two Kinds

INDUSTRIAL PRODUCTION OF L-ALANINE FROM AMMONIUM FUMARATE USING IMMOBILIZED MICROBIAL CELLS OF TWO KINDS

Satoru TAKAMATSU,Tetsuya TOSA and Ichiro CHIBATA Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Osaka 532

Key Words: Biochemical Engineering, Immobilized Microbial Cell, L-Alanine Production, Sequential EnzymeReaction, Immobilized Escherichia coli, Immobilized Pseudomonas dacunhae Rate equations were derived for a two-step enzymereaction whichproducedL-alanine from ammoniumfumarate via L-aspartic acid by aspartase of immobilized Escherichia coli cells and L-aspartate /?-decarboxylase of immobilized Pseudomonas dacunhae cells. To establish an efficient system for industrial production of L-alanine, we investigated the design of adequate bioreactors on the basis of the simulation of this sequential reaction by solving the simultaneous equations derived. As a result, L-alanine was found to be produced efficiently by use of two sequential column reactors: a conventional column reactor containing immobilized E. coli cells and a closed column reactor containing immobilized P. dacunhae cells.

To develop the most suitable production system for Introduction L-alanine, rate equations of aspartase and L-aspartate L-Alanine had been produced from L-aspartic acid /?-decarboxylase reactions should be obtained, and a by a batch enzymatic method, using the activity of l- strategy for operation should be mathematically de- aspartate /?-decarboxylase of intact Pseudomonas dac- termined using these equations. In the previous unhae cells.1} Since 1982, however, Tanabe Seiyaku papers,1143'14) rate equations were derived for as- Co., Ltd. has been producing L-alanine from am- partase reaction of E. coli cells immobilized within moniumfumarate via L-aspartic acid by the con- polyacrylamide gel and the strategy for operation tinuous column reaction system, using the aspartase was proposed using the rate equations. activity of immobilized Escherichia coli cells and l- In the present work, the rate equation was modified aspartate /?-decarboxylase activity of immobilized P. for aspartase in the sequential two-step reaction, and dacunhae cells.9) a rate equation was derived for L-aspartate /?-de- These reactions proceed as follows: carboxylase of immobilized P. dacunhae cells in the (aspartase) two-step reaction. Furthermore, we simulated this

Fumaric acid+NH3<( åºl-Aspartic acid L-aspartate sequential reaction of L-alanine production from am- #-decarboxylase moniumfumarate by means of numerical analysis, and designed an industrial production system for l- åºl-Alanine + CO2 alanine. In the previous paper,10) we studied the conditions for production of L-alanine from ammonium fu- 1. Experimental marate via L-aspartic acid in a single-batch reactor 1.1 Materials using a mixture of immobilized E. coli cells and K>Carrageenanwas purchased from Sansho Co., immobilized P. dacunhae cells, and coimmobilized E. Ltd., Osaka. Pyridoxal-5'-phosphate (PLP) was pur- coli-P. dacunhae cells. chased from Kyowa Hakko Co., Ltd., Tokyo, and As a result, the productivity of L-alanine by the other reagents were purchased from Katayama mixture of the two kinds of immobilized microbial Kagaku Co., Ltd., Osaka. cells was found to be superior to that by the coim- 1.2 Preparation of immobilized microbial cells mobilized microbial cells. Further, L-alanine was 1) Immobilized E. coli cells As described in the found to be most efficiently produced when the previous paper,10) E. coli EAPc-77) having higher reaction started at pHaround 8. aspartase activity, a kind of mutant of E. coli ATCC Received April 1 1, 1985. Correspondence concerning this article should be addressed 1 1 303, was cultivated, pH-treated12) and immobilized to S. Takamatsu. with K-carrageenan. The resulting gels were suspen-

VOL 19 NO. 1 1986 31 ded in 0.2 kmol-m 3 acetate buffer (pH 5.0) contain- dStV^UK'.+ l^ -So} (1) ing 10 mol-m"3 L-aspartic acid and 2% KC1 at 37°C dt (K'e+\)Sx-S0+KmlK'e for 24h for activation, and washed thoroughly with 2% KC1 solution. ^i = ^*iSr7exp(-3140/r+ 10.13) (2) 2) Immobilized P. dacunhae cells As described in the previous paper,10) P. dacunhae IAM 1152 was *:ml = o.68sr4 (3) cultivated, pH-treated, glutaraldehyde-treatedn) and K'e= -(3.603^0-0.284)7+1200S0-78 (4) immobilized with /c-carrageenan. The resulting gels On the basis of the assumption that these equations were suspended in 0.2 kmol-m~3 acetate buffer (pH could be adapted to aspartase reaction of E. coli cells 5.0) containing 10 mol*m~3 L-aspartic acid, 1 mol- immobilized with K-carrageenan, Eq. (1) was ex- m~3 PLP and 2% KC1 at 37°C for 24h for activation, and washed thoroughly with 2% KC1 tended as follows, considering the shift of the equilib- solution. rium of aspartase towards L-aspartic acid formation 1.3 Measurement of kinetic constants of aspartase caused by the coexistent L-aspartate /?-decarboxylase: reaction dS, V^K'e+DSt-So+P} (5) Kml, Vml and Ke of aspartase reaction were de- At (K;+l)S1-S0+KmlK' termined at pH 6-9 using the methods described in the previous paper.13) As Eq. (1) was derived at pH 8.5, effects of pH on 1.4 Assay of L-aspartate /?-decarboxylase activity K*l9 Kmland K'e were examined to extend Eqs. (2), Ten milliliters of various substrate solutions (pH (3) and (4) to the two-step reaction. K*x and Kml were 5.5-9.0) consisting of 0.01-2kmol-m~3 ammonium found to be represented as an exponential function of L-aspartate, 0-2kmol-m~3 L-alanine, 0-0.5 kmol- pH by the following equations. m~3ammoniumfumarate, l mol-m~3 pyruvic acid K*1=exp(10.271npH-21.98+lnF*1*) (6) and 0.1 mol-m~3 PLP was added to 1g of immobi- J£ml =exp(0.816pH -7.322) (7) lized P. dacunhae cells, and the mixture was shaken in a 100-ml Erlenmeyer flask at 30-45°C for 0.5-1 h. Further, Ke value was found to increase linearly After removal of the immobilized cells by filtration, against pH, and its coefficient of correlation was the amount of L-alanine formed was determined by independent of So and temperature. For example, K'e bioassay with Leuconostoc citrovorum ATCC8801.8) was represented as Eq. (8) at 1 kmol-m~3 of So and 1.5 Batch reaction using two kinds of immobilized 37°C. microbial cells Twenty milliliters of 1.2 kmol-m""3 ammonium K'e=7.52pH+29.2 (8) fumarate (pH 8) containing 1 mol-m"3 Mg2+, 1 In addition, effects of L-alanine, pyruvic acid and mol-m~3 pyruvic acid and 0.1 mol-m~3 PLP was PLPon aspartase activity were tested, because these added to a mixture of 0.5 or l.Og of immobilized E. materials were not involved in a substrate solution for coli cells and 4.5 or 4.0g of immobilized P. dacunhae conventional aspartase reaction but were involved in cells, and the mixture was incubated in a 100-ml reaction mixture of L-aspartate /?-decarboxylase. The Erlenmeyer flask at 37°C for 8h with shaking. aspartase activity was found not to be affected by Samples were removedat appropriate intervals from these materials. Consequently, the aspartase activity the reaction mixture for analysis. Remaining fumaric of immobilized E. coli cells in the two-step reaction is acid was spectrophotometrically determined2) and l- represented by Eqs. (5), (9), (10) and (ll). aspartic acid was determined by bioassay with Fml =^-77 exp(-3140/r+ 10.13) Leuconostoc mesenteroides P-60.4) xexp(10.271npH-21.98+ln K*f) (9) 2. Results and Discussion iCml = S'S-O4exp(0.816pH -7.322) (10) 2.1 Kinetics of aspartase reaction of immobilized E. coli cells ^=7.52pH-(3.603S0-0.284)r The simplified equation of the two-step enzyme +1200^0-141.9 (ll) reaction described in this paper was represented as follows: where Eqs. (9), (10) and (ll) were obtained by L-aspartate combination ofEqs. (2) and (6), Eqs. (3) and (7), and aspartase ^-decarboxylase Eqs. (4) and (8), respectively. S\< >$2 *P 2.2 Kinetics of L-aspartate /f-decarboxylase reaction Aspartase activity of E. coli cells immobilized with of immobilized P. dacunhae cells polyacrylamide was represented by the following To obtain the rate equation of L-aspartate /?- equations13): decarboxylase activity of immobilized P. dacunhae

32 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN cells, effects of L-aspartic acid, L-alanine and fumaric acid on its activity were examined at various pH. The L-aspartate j8-decarboxylase activity was found to be inhibited by L-aspartic acid (Fig. 1). The inhibition constant was calculated to be 8.9 kmol-m"3. The inhibition constant was further examined in the higher pHregion, and was found to be represented as follows: A:/2 =exp(-0.7535pH+6.721) (12) Fig. 1. Effect of substrate concentration on L-aspartate /?- Lineweaver-Burk plots of L-aspartate /?-decarbox- decarboxylase activity of immobilized P. dacunhae cells (pH ylase activity of immobilized P. dacunhae cells were 6). obtained at pH 6.0 (Fig. 2), and the Michaelis constant was calculated to be 0.055 kmol- m"3. However, Michaelis constants in the higher pH region could not be obtained because of concave Lineweaver-Burk plots. Therefore, the Michaelis constant obtained at pH6.0 was used for further simulation. Km2 =0.055 (13) Furthermore, L-aspartate /?-decarboxylase activity of immobilized P. dacunhae cells was found to be competitively inhibited by fumaric acid, and the inhibition constant was calculated to be 1 kmol-m~3 at pH 6-9 (Fig. 3). Therefore, Eq. (14) was used for further simulation. Kn=l (14) Fig. 2. Lineweaver-Burk plots of L-aspartate ^-decarboxylase immobilized P. dacunhae cells (pH 6). On the basis of these results, the rate equation of the L-aspartate /?-decarboxylase of immobilized P. dacunhae cells was derived by the steady-state ap- proach of King and Altmann5) as follows. _^2 = Vm2S2 /15j dt Km2(l+S1/KI1)+S2(l+S2/KI2) Generally, Vm2is varied with temperature and pH. Therefore, effects of temperature and pH on Vm2were examined, and the following equation was obtained. Vm2= VU-0.2661pH+2.6) Fig. 3. Effect of fumaric acid on L-aspartate /?-decarbo- x exp(-5380/7+ 17.35) (16) xylase activity of immobilized P. dacunhae cells (pH 6). 2.3 Simulation of L-alanine production from ammoniumfumarate in two-step enzymereaction ii) F**:O.03 mol-h^-g-ger1 In the previous paper,10) L-alanine was found to be iii) F*2: 0.003 molà"h"xà"g-gel"x most efficiently produced from ammoniumfumarate iv) reaction temperature: 37°C using immobilized E. coli cells and immobilized P. v) pH: 7.8 for the first half and 8.0 for the latter dacunhae cells when initial pH of substrate solution half was adjusted to around 8. Therefore, simulation of l- The results of simulation are shown in Fig. 4. alanine production from ammoniumfumarate was carried out at such pH condition by solving the Calculated curves for concentrations of L-alanine, l- simultaneous differential equations (5) and (15). aspartic acid and fumaric acid were found to be in Detailed conditions used for the simulation were as good agreement with those from batch experiments, follows: which were carried out using 10% and 20% ratios of i) concentration of substrate solution: 1.2 immobilized E. coli cells to total immobilized cells. kmol-m~3 Consequently, Eqs. (5) and (15) are considered to be applicable to aspartase and L-aspartate jS-decar-

VOL 19 NO. 1 1986 33 Fig. 5. Effect of pH on L-alanine production from ammonium fumarate using two immobilized microbial cells. Fig. 4. Simulation of sequential enzyme reaction for l- Conditions for calculation: total gel weight= 100 g; substrate alanine production from ammoniumfumarate using two solution= 1 /; reaction temperature= 37°C. immobilized microbial cells.

boxylase respectively in the two-step enzyme reac- section. tion. Figure 6 shows various types of bioreactors con- 2.4 Industrial production of L-alanine with column sidered for L-alanine production from ammonium reactor fumarate using immobilized E. coli cells and immobi- To design an efficient system for industrial pro- lized P. dacunhae cells. In method A, after attainment duction of L-alanine, the effect of operation con- of the desired conversion of aspartase reaction in one ditions, ratio of the two kinds of immobilized cells reactor, L-aspartate /?-decarboxylase reaction was ini- and method of combination of columns on the time tiated in the other reactor. MethodB is the method required for desired conversion was considered by described in previous section, using a single reactor. solving the simultaneous differential equations (5) Method C is an improved method with two reactors. and (15) on the assumption of plug-flow throughout That is, a second reactor involving immobilized P. the columns. Then, the optimum condition at which dacunhae cells was employedafter attainment of the the time required for desired conversion was mi- desired conversion of aspartase reaction in the first nimumwas determined. reactor involving two kinds of immobilized cells. 1) Single reactor For conventional column reac- Method D is a further improved method with three tion, simultaneous differential equations (5) and (15) reactors. That is, only aspartase reaction was run in were solved at pH 8.0, because the highest pro- the first reactor because L-aspartate /?-decarboxylase ductivity of L-alanine in batch reaction was obtained hardly acts at low concentration of L-aspartic acid. at this pH value.10) On the other hand, the closed- Then the second and third reactors were operated column reactor (operated at higher pressure) for l- similarly as in Method C. In these four methods, the aspartate /5-decarboxylase reaction was developed as reactions were run at pH 7.0 without pH adjustment. an advantageous reactor.3) By using this reactor, In MethodE, however, aspartase reaction was run at liberated CO2gas is dissolved into the reaction mix- optimum pH 8.5 in the first reactor and L-aspartate /?- ture, and the pH of reaction mixture is fairly stable decarboxylase reaction was initiated at optimum pH at around 7. Therefore, for the closed-column reac- 6.2 in the second reactor after pH adjustment. In this tion, simultaneous differential equations (5) and (15) case no increase in volumeof the substrate solution in were solved at pH 7.0. Figure 5 shows the rela- the pH-adjustment process was assumed in the fol- tionship between the time required for 0.999 conver- lowing simulation. Furthermore, Eq. (15) was solved sion and the ratio of the two kinds of immobilized stepwise for each 0.05-unit rise in pH value using the microbial cells in these two columns as a parame- relationship between pH and conversion obtained ter of initial substrate concentration. The optimum previously. ratio of immobilized E. coli cells at which the The time required for desired conversion was ob- time required for 0.999 conversion was minimum tained by solving Eqs. (5) and (15) for each ratio of was found to be dependent on initial substrate con- two kinds of immobilized microbial cells, and then the centration for each column. Furthermore, the closed minimumtime and the optimumratio of kinds of cells column was found to be superior to a conventional were determined for methods A-E. Figure 7 shows one regardless of the initial substrate concentration. relative productivities of L-alanine using methods 2) Multi-reactor L-Alanine may be more efficiently A-E, under respective optimum conditions. Method produced when multi-reactor systems are used. E was found to be most efficient; that is, the time Productivities of L-alanine with various types of required for desired conversion was about 30%short- bioreactors were investigated and are described in this er than that by Method A. Method B was expected to 34 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN Table 1. Comparison of productivity of L-alanine from ammoniumfumarate among several combination methods of bioreactors (S0= 1.5kmol-m-3, 37°C)

Relative time required Desired conversion [-] for reaction [%]

AAspartase, L-AspartateF, Method of combination p-decarboxylase of bioreactors A B C D E

0.97 0.99 100* 113 100 99.0 73.2 0.999 100 113 100 99.1 74.4 Fig. 6. Various bioreactors for continuous production ofl- 0.98 0.99 100 110 100 98.9 72.6 alanine from ammoniumfumarate using two immobilized 0.999 100 115 100 98.9 73.9 microbial cells. #, immobilized E. coli cells; O, immobilized 0.99 0.99 100 104 99.9 99.0 70.5 P. dacunhae cells. 0.999 100 107 99.8 97.9 71.7

a These methods are illustrated in Fig. 6. b Reaction time of method A was taken as 100%.

Fig. 7. Comparison of L-alanine productivity from ammoniumfumarate in various bioreactors using two immobilized microbial cells. Conditions for calculation: desired conversion of aspartase=0.99; desired conversion of l- aspartate /?-decarboxylase=0.999; So = 1.5kmol-m~3; reac- Fig. 8. Flow sheet of industrial system for L-alanine tion temperature = 37°C. 1, first column; 2, second column; 3, production. third column; E, immobilized E. coli cells; P, immobilized P. dacunhaecells; these values showthe ratio of columnvol- umeor the ratio of gel volume. reaction by two enzymes in a single reactor was scarcely observed in this L-alanine production system. be superior to MethodA because of simultaneous This reason is considered to be as follows: optimum reaction by two enzymes.. The time required for pH values of the two enzymes differ from each other, desired conversion by Method B, however, was found and fumaric acid inhibits L-aspartate /?-decarboxylase to about 7% be longer than that by Method A. The reaction. reason is considered to be that the desired conversion 3) Industrial production system Although the ad- of L-aspartate /?-decarboxylase reaction is hardly ac- ditional process for pH adjustment in Method E is complished because of gradual production of l- disadvantageous for industrialization, this disadvan- aspartic acid by immobilized E. coli cells located near tage would be outweighed by good efficiency. So, the reactor outlet. Therefore, the final conversion of Method E was chosen as an industrializable method aspartase reaction by Method B became higher than for production of L-alanine from ammonium fu- the desired conversion. marate. Figure 8 shows the flow sheet of a production Few merits of Methods C and D were observed. system for L-alanine industrialized in 1982. Using this Table 1 shows relative productivities of L-alanine for system, L-alanine is being produced at the expected several desired conversions. The relative time required lower cost, compared with the conventional batch for the desired conversion was found to be little reactor. This is considered to be the first industrial changed by the desired conversion. application of sequential enzyme reactions using two As described above, the merit of simultaneous kinds of immobilized microbial cells.

VOL 19 NO. 1 1986 35 Nomenclature 638 (1965). 2) Bock, R. M. and R. A. Alberty: /. Am. Chem. Soc, 75, 1921 modified equilibrium constant for aspartase [-] (1953). competitive inhibition constant by fumaric acid 3) Furui, M. and K. Yamashita: /. Ferment. Technol., 61, 587 for L-aspartate /?-decarboxylase [kmol - m~3] (1983). substrate inhibition constant for L-aspartate 4) Henderson, L. M. and E. E. Snell: /. Biol. Chem., Ill, 15 /?-decarboxylase [kmol - m ~ 3] (1948). Michaelis constant for aspartase [kmol - m~3] Michaelis constant for L-aspartate /?-decarbo- 5) King, E. L. and C. Altmann: J. Phys. Chem., 60, 1375 (1956). 6) Nishida, Y., T. Sato, T. Tosa and I. Chibata: Enzyme

p xylase [kmol - m"3] concentration of L-alanine [kmol à" m~3] Microbial Technol., 1, 95 (1979). So 7) Nishimura, N. and M. Kisumi: Appl. Environ. Microbiol., 48, initial concentration of fumaric acid [kmol-m"3] 1072 (1984). concentration of fumaric acid [kmol - m~3] 8) Snell, E. E.: "Methods in Enzymology," Vol. 3, p. 477, s2 concentration of L-aspartic acid [kmol - m~3] t reaction time [h] Academic Press, New York (1957). 9) Takamatsu, S., T. Tosa and I. Chibata: J. Chem. Soc. Jpn., T absolute temperature [k] No. 9, 1369 (1983). maximumaspartase reaction rate [mol-h^ -g-ger1] 10) Takamatsu, S., T. Tosa and I. Chibata: /. Chem. Eng. Japan, Vml in the case of 1kmol-m"3 So at 37°C 18, 66 (1985). [mol-h^ -g-gel"1] ll) Takamatsu, S., M. Ueba and K. Yamashita: J. Chem. Eng. Japan, 17, 647 (1984). F*i at pH 8.5 [mol-h-^g-gel"1] maximumL-aspartate /?-decarboxylase reaction 12) Takamatsu, S., I. Umemura, K. Yamamoto, T. Sato, T. Tosa rate [mol-h"1 -g-gel"1] and I. Chibata: Eur. J. Appl. Microbiol. Biotechnol., 15, 147 (1982). vt, Vm2 at pH 6.0 and 37°C [mol-h"1-g-gel"1] 13) Takamatsu, S. and K. Yamashita: J. Chem. Eng. Japan, 17, 406 (1984). Literature Cited 14) Takamatsu, S., K. Yamashita and A. Sumi: J. Ferment. 1) Chibata, I., T. Kakimoto and J. Kato: J. Appl. Microbiol., 13, Technol., 58, 129 (1980).

MULTIVARIABLE CONTROL OF CSTR WITH TIME- DELAYS VIA A DECOUPLING STRATEGY

Em NAKANISHI AND Seiji OHTANI Department of Chemical Engineering, Kobe University, Kobe 657

KeyWords: Multivariable Process Control, Chemical Reactor Control, Nonlinear Decoupler Design, Time- Delay Decoupler, Smith Compensator Decoupling control of nonisothermal CSTRwith dead times in control variables was investigated on the basis of a linear time-varying time-delay model by which both nonlinearity and time-delay characteristics involved in the CSTRdynamics are simultaneously considered. A pair of nonlinear time-delay decouplers were theoretically derived and the feasibility of designing a decoupling control system which requires the estimation of future values of the state variables wasensured by using a predictive schemein combination with the control system. For the purpose of improving the control performance of the decoupling control system in which incorrect design of the decouplers is caused by identification error of uncertain parameters present in the CSTRdynamics, a feedback control system with the Smith compensator was designed for incompletely decoupled CSTRand the robustness of such feedback control system to disturbance was shown by simulations.

on the linearized model of CSTR. Strong non- Introduction linearity of temperature is involved in the reaction Manystudies have been carried out for investigat- term of mass and heat balance equations of CSTR ing control of CSTR,but in most of these inves- while the dead times present in the control variables tigations1 ~3'5'10) control system designs were based due to actuation lags of final control elements cannot be neglected in the operation of commercial-scale

Received May20, 1985. Correspondence concerning this article should be addressed CSTR.Thus the traditional procedure on the basis of to E. Nakanishi. the linear time-invariant delay-free model of reactor

36 JOURNAL OF CHEMICAL ENGINEERING OF JAPAN