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 acti- vation, 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 hig- her 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 con- stant 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.