Received 22 January 1998/Accepted 29 May 1998

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1998, p Vol. 64, No /98/$ Copyright 1998, American Society for Microbiology. All Rights Reserved. Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker s Yeast SHINJI WAKISAKA, 1 YOSHIFUMI OHSHIMA, 2 MASAHIRO OGAWA, 2 TATSUROKURO TOCHIKURA, 3 AND TAKASHI TACHIKI 4 * Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Rithumeikan University, Kusatsu, 4 Wakunaga Pharmaceutical Co., Ltd., Hiroshima, 1 Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto, 2 and Faculty of Home Economics, Kobe Women s University, Kobe, 3 Japan Received 22 January 1998/Accepted 29 May 1998 Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain. Glutamine is one of the most important compounds in nitrogen metabolism; it is not only a constituent of proteins but is also a donor of the amino (amido) moiety in the biosynthesis of other amino acids, purines, pyrimidines, pyridine coenzymes, and complex carbohydrates. Glutamine is also used in the treatment of gastric ulcers and has been produced commercially by direct fermentation with certain bacteria (6 10). In recent years, enzymatic synthesis has come to rival direct fermentation as a means of producing amino acids. In the case of glutamine, however, the need for a stoichiometric supply of ATP for the endoergonic reaction of glutamine synthetase (GS) precludes the development of an economically valuable method, unless ATP can be regenerated and recycled. Processes for the production of various substances using dried yeast cells as an enzyme source were established by Tochikura and colleagues (2, 4, 16, 18 20). The processes are driven by the chemical energy of ATP released by the alcoholic fermentation by the yeast, which has been wasted in alcoholic brewing (17). Tochikura and colleagues also designed a process in which the yeast fermentation of sugar is combined with an endoergonic reaction catalyzed by an enzyme from a different microorganism (3). The results suggest that the process offers the possibility of producing many compounds at a high yield by using various biosynthetic reactions and high concentrations of substrates. Tochikura et al. introduced the general * Corresponding author. Mailing address: Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Rithumeikan University, Kusatsu, Japan. Phone: Fax: tachiki@se.ritsumei.ac.jp. idea of coupled fermentation with energy transfer for the process; its principle is indicated in Fig. 1, with glutamine production as an example. In the process of coupled fermentation with energy transfer, a catalytic amount of ATP is regenerated with the energy of sugar fermented by yeast, in the form of baker s yeast (4, 16, 18, 19, 23). The energy-utilizing system for the synthesis can involve the enzyme(s) of yeast itself or those of other organisms. It should be noted that, from another point of view, the use of the energy-utilizing system results in ADP regeneration to complete the fermentation of glucose, and that, if there is no ADP regeneration, the yeast fermentation of sugar can proceed only as follows, in the presence of inorganic phosphate (the Harden-Young effect of inorganic phosphate [1]), 2 glucose 2 inorganic phosphate 3 fructose 1,6-bisphosphate (FBP) 2 C 2 H 5 OH 2 CO 2 (Harden-Young equation), where ADP regeneration for the fermentation of 1 mol of glucose is carried out by the phosphorylation of another mole of glucose to FBP. We previously reported glutamine production, obtained by employing a combination of baker s yeast cells and GS from Gluconobacter suboxydans, as the first application of the coupled fermentation with energy transfer method for the production of a nonphosphorylated compound (12, 13). In addition, we achieved high-yield glutamine production by using the Corynebacterium glutamicum (Micrococcus glutamicus) enzyme and larger amounts of the substrates (15). The maximum amounts of glutamine formed (23 to 25 g/liter) and the yield based on glutamate (50 to 100%) were to some extent satisfactory, but the yield based on the energy source (glucose) for 2952

2 VOL. 64, 1998 GLUTAMINE PRODUCTION BY YEAST AND BACTERIAL GS 2953 of glucose regenerates 2 mol of ATP, 1 mol of glucose [or fructose] monophosphate regenerates 3 mol of ATP, and 1 mol of FBP regenerates 4 mol of ATP): glucose as an energy source, hexose monophosphate as an energy source, glutamine formed (mm) 100 (%); glucose consumed (mm) 2 or FBP as an energy source, glutamine formed (mm) 100 (%); hexose monophosphate (mm) 3 glutamine formed (mm) 100 (%). FBP consumed (mm) 4 FIG. 1. Scheme of glutamine production by the coupled fermentation with energy transfer method. 1, glycolytic pathway is abridged. 2, inorganic phosphate (P i ) is recycled. ATP regeneration was not satisfactory (about 40% of the theoretical value; 2 mol of glutamine can be formed when 1 mol of glucose is consumed). In the present study, we examined the characteristics of glutamine production regarding product yield based on the energy source for ATP regeneration and regarding the reactivity of GS during glutamine production, which is closely related to the product yield. The results of preliminary attempts to improve glutamine production are also described. In these experiments, a yeast mutant which has a low assimilating ability for glycerol and/or ethanol was used. MATERIALS AND METHODS Enzyme preparation. Two yeast preparations were used. (i) Dried yeast cells. Pressed baker s yeast supplied by Oriental Yeast Co. (Tokyo, Japan) was dried as described previously (18). Saccharomyces cerevisiae (haploid, mating type a) and its mutant strain O-21 were grown in 2-liter Sakaguchi flasks at 28 C for 24 h on 500 ml of a medium (preparation medium) containing 4% glucose, 1% peptone, 0.5% yeast extract, 0.5% K 2 HPO 4, 0.5% KH 2 PO 4, and 0.3% MgSO 4 7H 2 O at ph 7. The cells were collected, washed with water, and dried as described previously (18). (ii) Cell extract. Washed cells of S. cerevisiae wild type or O-21 suspended in 0.01 M potassium phosphate buffer (ph 7.0) were disrupted by ultrasonication (Kaijo Denki Co., Tokyo, Japan; 20 kc, 20 min, below 10 C). After centrifugation (10,000 g, 20 min), the supernatant was dialyzed overnight at 5 C against 0.05 M Tris-HCl buffer (ph 7.4) containing 2 mm 2-mercaptoethanol and 2 mm EDTA and was used for the enzyme assays. GS. Two preparations from C. glutamicum (ATCC 13032; American Type Culture Collection, Manassas, Va.) or from Brevibacterium flavum (ATCC 14062) were used. (i) Partially purified enzyme. A cell extract of each bacterium was treated up to the step of DEAE-cellulose column chromatography, as outlined previously (11, 14), and was used in the glutamine production experiments. (ii) Purified enzyme. GS was isolated as described previously (11, 14) and used in the spectrophotometric experiments. One unit of GS was defined as the enzyme amount forming 1 mol of -glutamylhydroxamate per min at 37 C in the transfer reaction (11, 14) and producing 0.2 mol of glutamine at 30 C in the standard GS assay mixture consisting of 50 mm sodium glutamate, 25 mm ammonium chloride, 7.5 mm ATP, 30 mm MgCl 2, and 100 mm imidazole buffer (ph 7.0) (11, 14). Reaction conditions. (i) Mixture for glutamine production. The mixture contained 100 to 400 mm glucose or sugar phosphate(s); 100 to 400 mm potassium phosphate buffer (ph 7.0); 100 to 400 mm sodium glutamate; 100 to 400 mm ammonium chloride; 0 to 10 mm adenosine, AMP, and/or ATP; 0 to 50 mm MgCl 2 ; 0 to 600 U of GS per ml; and 0 to 60 mg of dried baker s yeast cells per ml in a total volume of 2 ml (detailed concentrations are described for each experiment). Reactions were carried out at 28 to 30 C with shaking and were terminated by immersing the reaction tubes in boiling water for 3 min. The supernatant obtained by centrifugation at 1,500 g for 10 min was submitted to the assay. The yield of glutamine obtained with an energy source for ATP regeneration at the time of glutamine production was calculated as the amount of ATP regenerated (which is expected to be the same as the amount of glutamine formed) by the amount of energy source for ATP regeneration consumed (1 mol (ii) GS assay mixtures with a regenerating system for a small amount of ATP. The spectrophotometric assay mixture (SA mixture) was designed to examine the reactivity of GS with high sensitivity. The GS reaction mixture described by Wakisaka et al. (22) was modified to contain 50 mm sodium glutamate, 25 mm ammonium chloride, to 0.8 mm ATP, 50 mm MgCl 2, 100 mm imidazole buffer (ph 7.0), 90 mm potassium chloride, 0.15 mm NADH, 1 mm phosphoenolpyruvate, 25 U of pyruvate kinase per ml, and 30 U of lactate dehydrogenase per ml, and purified GS. Reactions were started at 30 C by the addition of GS or one of the substrates to each assay mixture. The GS activity was estimated spectrophotometrically by determining the rate of NADH oxidation at 340 nm (ε ). It was confirmed that pyruvate kinase and lactate dehydrogenase were not rate limiting in the complete reaction. The second mixture used was SG mixture (modified SA mixture that simulates the glutamine-producing conditions). The SA mixture was modified to simulate the glutamine-producing conditions as follows: (i) 200 mm glucose was added, (ii) imidazole buffer was replaced by 100 mm potassium phosphate buffer (ph 7.0), and (iii) the concentrations of sodium glutamate, ammonium chloride, and MgCl 2 were changed to 400, 400, and 15 mm, respectively. Isolation of yeast mutant. In an attempt to improve the glutamine yield obtained with glucose as an energy source for ATP regeneration, we used a yeast mutant that grew well on glucose as a carbon source but did not grow well on glycerol and/or ethanol as did the wild-type strain. We expected, in light of the enzymatic features of glycolysis and gluconeogenesis, that such a mutant might be deficient in, or decrease, the specific phosphatase(s) that consumes sugar phosphate(s). Two culture media were used for the mutagenesis and investigation of the growth characteristics of the yeast strains. GE medium contained 3% glycerol, 3% ethanol, 2% peptone, 1% yeast extract, 30 mg of K 2 HPO 4 per ml, 0.2% MgSO 4 7H 2 O, and 0.15% KCl (ph 7). For D medium, the composition was the same as that of the GE medium except that 3% glycerol and 3% ethanol were replaced by 3% glucose as a carbon source. S. cerevisiae (mating type a) was cultured statistically in 10 ml of the preparation medium in a 16.5-mm test tube at 30 C for 24 h; the culture was then diluted 30-fold with sterilized water (about 10 6 cells/ml). A 30-ml portion was transferred to a sterilized petri dish (9 cm in diameter) and was irradiated with a UV lamp (18-W Toshiba germicidal lamp) for 7 min at a distance of 30 cm, while being stirred by a magnetic stirrer. Under these conditions, about 0.01% of the cells survived. The treated cell suspension was spread on an agar plate containing GE medium and was incubated at 30 C for 4 to 5 days. Tiny colonies were selected and replicated onto agar plates containing D medium. After incubation at 30 C for 24 h, large colonies were isolated. Small colonies were excluded as respiratory-deficient mutants. Dried cells of the isolates were prepared by the procedures outlined above, and their ATP regeneration activity was examined on the basis of glutamine yield obtained with glucose as an energy source for ATP regeneration. The isolate (strain O-21) used in the present study showed the following growth characteristics on carbon sources (indicated as turbidity at 610 nm after shaking of culture at 30 C for 72 h on 3 ml of medium): O-21, 17.7 on glucose, 2.4 on glycerol, and 7.0 on ethanol; wild type, 25.9 on glucose, 7.5 on glycerol, and 26.8 on ethanol. Assays. The activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphoglucomutase, fructose 1,6-bisphosphatase (FBPase), phosphofructokinase, aldolase, and triose phosphate isomerase in the yeast cell extracts were estimated enzymatically according to the methods of Maitra and Lobo (5). Glucose 6-phosphatase activity was measured by determining glucose with glucose oxidase. Glutamine, glutamate, protein, glucose, and FBP were determined as described in previous reports (12, 13). The amounts of ADP-ATP excreted from yeast cells into the GP mixture were determined spectrophotometrically with the ammonia assay mixture described by Wakisaka et al. (21), slightly modified. Reagents. Phosphoglucoisomerase (yeast), aldolase (yeast), triose phosphate isomerase (rabbit muscle), and glycerol phosphate dehydrogenase (rabbit muscle) were supplied by Boehringer Mannheim (Mannheim, Germany); ATP, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase were obtained from Sigma Chemical Co. (St. Louis, Mo.); NADH and glucose 6-phosphate dehydrogenase were provided by Oriental Yeast Co.; and glucose oxidase (glucose C test; Wako) was supplied by Wako Chemical Co. (Osaka, Japan).

3 2954 WAKISAKA ET AL. APPL. ENVIRON. MICROBIOL. FIG. 2. Glutamine production in mixtures containing varying amounts of GS. The reaction mixture contained 100 mm glucose, 100 mm potassium phosphate buffer (ph 7.0), 200 mm glutamate, 220 mm ammonium chloride, 15 mm MgCl 2, and 30 mg of dried yeast cells per ml. The amounts of partially purified GS of C. glutamicum added to the reaction mixtures were 0 (A), 20 (B), 60 (C), and 100 (D) U/ml. The glutamine formed in each mixture is indicated in panels A-1, B-1, C-1, and D-1, and the sugar components are shown in panels A-2, B-2, C-2, and D-2. Symbols: F, glutamine;, glucose; Œ, FBP. RESULTS AND DISCUSSION Characteristics of the reaction of glutamine production with dried yeast cells and partially purified GS. Figure 2 shows that glutamine formation was supported by the addition of GS and increased as the enzyme increased. The reactions proceeded without the addition of ATP or other adenosine derivatives, indicating that the endogenous amounts of these substances in yeast cells were effective for energy transfer between the yeast fermentation of sugar and the GS reaction (300 to 400 M ADP-ATP was found in the supernatant of the mixture). The supplement of ATP at 5 to 10 mm (an effective level for the Harden-Young equation [1]) was found to have an inhibitory effect on the initial rate of glutamine formation (20). In addition, FBP accumulated temporarily prior to glutamine formation, and the amount of accumulated FBP decreased as GS increased, although the rate of glucose consumption was not variable (Fig. 2). This accumulation of FBP means that the yeast fermentation of sugar proceeded basically according to the Harden-Young equation. However, the 60% yield of FBP compared to the theoretical value in Fig. 2A (maximum about 30 mm, compared to the theoretical value, 50 mm) and the disappearance of FBP after the consumption of glucose suggested that some ADP-forming (i.e., ATP-consuming) reaction(s), not shown in Fig. 1, was still active in the yeast preparation. The decrease of the maximum amount of FBP, corresponding to the amounts of GS, implies that the sugar-phosphorylating enzymes, which function in ADP regeneration to enhance sugar fermentation according to the Harden-Young equation, might be partly replaced by GS, and eventually the chemical change of sugars in the reaction mixture would be restored to that in Neuberg s first equation: 2 glucose 3 4 C 2 H 5 OH 4 CO 2. The yield of glutamine obtained with glucose as an energy source for ATP regeneration in the mixture containing sufficient GS (100 U/ml) was about 55% (Fig. 2D), and the amount of ATP utilized for glutamine production was almost equal to that expected based on the accumulated FBP in the control mixture lacking GS (Fig. 2A). It could therefore be expected that the glutamine yield would be improved if the reaction were to be carried out under conditions in which glucose, if GS is absent, is converted more efficiently to FBP (i.e., where the Harden-Young effect on sugar fermentation is more clearly observed). Figure 3 compares the glutamine production levels obtained with various reaction mixtures, together with the FBP accumulations in the corresponding control mixtures lacking GS. The concentrations of inorganic phosphate, MgCl 2, and adenosine were decided by taking into account the potentiality for FBP formation by toruol-treated yeast cells. In another investigation (16), 75 to 100 g of FBP per liter was produced with a 60 to 80% yield based on glucose, probably being achieved due to the inhibitory effect of inorganic phosphate on phosphatase(s) and due to the partial reactivation of phosphate-repressed glycolysis by adenosine nucleotides and Mg 2. The rates of glucose consumption in the mixtures A-1, B-1, C-1, and D-1 were almost the same as those in the control mixtures lacking GS (A-2, B-2, C-2, and D-2). This result indicates that the final yields of glutamine were related to the amounts of FBP accumulated in the control mixtures. Table 1 summarizes similar experiments with higher concentrations of GS and substrates. The yields of glutamine obtained with glucose as an energy source for ATP regeneration in experiments 1, 2, and 3 (about 70%) were much higher than those in a previous report (maximum, 40%) (18) and in other examples of coupled fermentation with energy transfer (5 to 15%) (2, 3, 12, 13, 16, 18 20). However, the glutamine yields obtained with glucose as an energy source were still less than FIG. 3. Relationship between glutamine production in the mixtures used for glutamine production and FBP accumulation in the control mixtures lacking GS. Each reaction mixture contained, in common, 100 mm glucose, 160 mm glutamate, 200 mm ammonium chloride, and 5 mm CoCl 2 (15). Other compounds were varied as follows. (A-1) A total of 100 mm potassium phosphate buffer (ph 7.0), 15 mm MgCl 2, 100 U of partially purified GS of C. glutamicum per ml, and 30 mg of dried yeast cells per ml were added. (B-1) Adenosine (3 mm) was added to the mixture from panel A-1. (C-1) A total of 300 mm potassium phosphate buffer (ph 7.0), 30 mm MgCl 2, 3 mm adenosine, 100 U of partially purified GS of C. glutamicum per ml, and 30 mg of dried yeast cells per ml were added. (D-1) The amount of dried yeast cells in the mixture for panel C-1 was changed to 40 mg/ml. Mixtures shown for panels A-2, B-2, C-2, and D-2 were controls, in which GS was omitted from the mixtures for panels A-1, B-1, C-1, and D-1, respectively, to determine the FBP accumulation. Symbols: F, glutamine;, glucose; Œ, FBP.

4 VOL. 64, 1998 GLUTAMINE PRODUCTION BY YEAST AND BACTERIAL GS 2955 TABLE 1. Glutamine production in improved reaction mixtures, obtained with glucose or FBP as the energy source Expt a Glutamine formed (mm, g/liter) Yield based on glutamate (%) Yield based on energy source b (glucose or FBP) (%) Maximum c rate of glutamine formation (mm/h) Expected d maximum rate of glutamine formation (mm/h) Maximum rate/expected maximum rate (100) (%) 1 135, (glucose) , (glucose) 32 3, , (glucose) 22 3, , (FBP) 55 7, , (FBP) 53 5, a For experiment 1, the results are also shown in Fig. 2D (7-h incubation). In experiment 2, the mixture contained 180 mm glucose, 400 mm potassium phosphate buffer (ph 7.0), 370 mm glutamate, 450 mm ammonium chloride, 30 mm MgCl 2, 5 mm CoCl 2, 3 mm adenosine, 400 U of partially purified GS of C. glutamicum per ml, and 30 mg of dried yeast cells per ml (12-h incubation). In experiment 3, the mixture contained 270 mm glucose, 400 mm potassium phosphate buffer (ph 7.0), 560 mm glutamate, 680 mm ammonium chloride, 30 mm MgCl 2, 5 mm CoCl 2, 3 mm adenosine, 3 mm ATP, 500 U of partially purified GS of C. glutamicum per ml, and 50 mg of dried yeast cells per ml (24-h incubation). In experiment 4, the mixture contained 85 mm FBP, 250 mm potassium phosphate buffer (ph 7.0), 400 mm glutamate, 450 mm ammonium chloride, 15 mm MgCl 2, 5 mm CoCl 2, 900 U of partially purified GS of C. glutamicum per ml and 50 mg of dried yeast cells per ml (9-h incubation). In experiment 5, the mixture contained 80 mm FBP, 250 mm potassium phosphate buffer (ph 7.0), 390 mm glutamate, 450 mm ammonium chloride, 15 mm MgCl 2, 5 mm CoCl 2, 700 U of partially purified GS of C. glutamicum per ml, and 50 mg of dried yeast cells per ml (12-h incubation). b See Materials and Methods. c Calculated graphically. d Calculated from the enzyme unit (i.e., reactivity in the standard reaction mixture). those achieved with FBP as an energy source, in experiments 4 and 5 of Table 1 (90 to 100%). Experiment A in Table 2 compares the glutamine yields obtained with several sugar phosphates as energy sources. The data indicates that the yield was increased in the following order of energy sources: glucose, glucose monophosphate, fructose 6-phosphate, and FBP. This finding suggested that some unidentified factor(s) reduces the efficiency of energy utilization at the early stage of glycolysis (from glucose to FBP). In experiment B, the addition of a higher concentration of inorganic phosphate improved the yield of glutamine obtained with each energy source for ATP regeneration, suggesting that phosphatase(s) might be involved in the above reduction (because a phosphatase[s] that can consume ATP is usually inhibited by inorganic phosphate) and that the addition of inorganic phosphate could be effective in increasing glutamine yields. Reactivity of GS during glutamine production. Table 1 also compares the maximum rates of glutamine production (i.e., the reactivities of GS in the mixtures used for glutamine production) with the calculated rates of glutamine formation in the standard GS assay mixture (see definition of GS unit in Materials and Methods); the former rates were very much lower than the latter. Figure 4 illustrates the relationship between ATP concentration and GS activity in the SA and SG mixtures, which contained ATP-regenerating systems. The enzyme activity in the SA mixture increased linearly with increasing ATP concentrations, whereas the activity in the SG mixture (the mixture that simulates the glutamine-producing conditions) increased only slightly. The SA and SG mixtures were distinguishable from each other in regard to the concentration of inorganic phosphate (SA mixture, nil; SG mixture, 100 mm), and the SG mixture also had high concentrations of glutamate, ammonia, and MgCl 2. The velocity of the GS reaction in the SG mixture did not exhibit first-order kinetics in relation to the enzyme amounts (data not shown). In these experiments, the GS activity found in the SG mixture was approximately as low as that found in the mixture for glutamine production (0.5 to 10% of that expected in the standard GS assay mixture) and variable (corresponding to the concentrations of ATP and the enzyme itself). To determine the unusual behavior of GS in the SG mixture (glutamine-producing conditions), variation in the GS activity in SA mixtures supplemented with glutamate, ammonium chloride, glucose, and/or inorganic phosphate was demonstrated. As shown in Table 3, inorganic phosphate markedly inhibited the enzyme activity. The other supplements showed slight inhibition at higher concentrations, but ammonium chloride (400 mm) caused a reduction of GS activity when added to an SA mixture containing 200 mm inorganic phosphate and 0.4 to 0.8 mm ATP (data not shown). The inhibition of inorganic phosphate was noncompetitive in regard to ATP (k i 80 mm) and was of a mixed type in regard to glutamate and ammonia (k i 70 and 99 mm, respectively). Glutamine production with the yeast mutant O-21. The results described above indicated that the low reactivity of GS in the glutamine-producing conditions might be caused mainly by the high concentration of inorganic phosphate added to mixtures with a system for generating a small amount of ATP. Since inorganic phosphate is not involved with the stoichiometry of glutamine production (Fig. 1), it would be expected that a decrease in, or the removal of, inorganic phosphate from the GP mixture would bring about higher glutamine yields due to the increased reactivity of GS. However, the data shown in Table 1 indicated that the presence of inorganic phosphate at a high concentration is necessary for the effective operation of Energy source TABLE 2. Glutamine production obtained with various energy sources a Yield obtained with energy source for ATP regeneration (%) Expt A Expt B Glucose Glucose 1-phosphate Glucose 6-phosphate Fructose 6-phosphate FBP a The yield of glutamine obtained with an energy source for ATP regeneration at the time of glutamine production was calculated as the amount of ATP regenerated (which is expected to be same as the amount of glutamine formed) by the amount of energy source for ATP regeneration consumed. One mole of glucose could regenerate 2 mol of ATP. One mole of hexose monophosphate could regenerate 3 mol of ATP. One mole of FBP could regenerate 4 mol of ATP (Fig. 1). In experiment A, the reaction mixture contained 75 to 150 mm energy source (FBP, 75 mm; hexose monophosphate, 100 mm; glucose, 150 mm), 100 mm potassium phosphate buffer (ph 7.0), 400 mm glutamate, 450 mm ammonium chloride, 15 mm MgCl 2, 5 mm CoCl 2, 525 U of partially purified GS of B. flavum per ml, and 50 mg of dried yeast cells per ml. In experiment B, the concentration of potassium phosphate buffer in the mixture was increased to 200 mm, and 5 mm adenosine was added. Reactions were carried out for 6 h.

5 2956 WAKISAKA ET AL. APPL. ENVIRON. MICROBIOL. FIG. 4. Response of GS activity to ATP concentration in SA and SG mixtures. Reactions in mixtures containing varying concentrations of ATP were started by the addition of 2Uofpurified GS of B. flavum per ml. Other conditions are described in Materials and Methods. Symbols: E, GS activity in SA mixture; F, GS activity in SG mixture. yeast glycolysis for glutamine production. Such contradictory requirements regarding the inorganic phosphate concentration of both enzyme systems (low for GS reaction and high for yeast glycolysis) might be resolved (i) by defining new reaction conditions in which yeast glycolysis proceeds effectively for the GS reaction (i.e., the activity of phosphatase[s] is reduced at a certain level) without the addition of inorganic phosphate at a high concentration, and/or (ii) by using yeast strains with low phosphatase activity which easily show the Harden-Young effect in sugar fermentation even at a low inorganic phosphate concentration. Figure 5 compares the glutamine production obtained with the wild-type yeast strain and that with the mutant O-21 strain, which is expected to have reduced phosphatase activity, under conditions of differing inorganic phosphate concentrations (400 and 250 mm). The maximum amount of glutamine formed in the mixture containing the O-21 strain was not changed (or was slightly increased) by decreasing the phosphate concentration (i.e., the glutamine yield obtained with glucose as an energy source for ATP regeneration did not vary), whereas that in the mixture TABLE 3. Effects of glutamate, ammonium chloride, glucose, and/ or potassium phosphate buffer on GS activity in SA mixture a Expt no. and additive(s) mm additive Relative activity (%) 1, glutamate and ammonium chloride b 50, , , , , , , , glucose , phosphate buffer a Various concentrations of glutamate, ammonium chloride, and/or potassium phosphate buffer (ph 7.0) were added to SP mixture. GS activity is indicated relative to activity without the additive. b The two values are respective. FIG. 5. Effect of phosphate concentration on glutamine production with wild-type and mutant strains of S. cerevisiae. The reaction mixtures contained 200 mm glucose, 400 or 250 mm potassium phosphate buffer (ph 7.0), 350 mm glutamate, 400 mm ammonium chloride, 15 mm MgCl 2, 5 mm CoCl 2,3mM AMP, 200 U of partially purified GS of B. flavum per ml, and 40 mg of dried cells of the wild-type or the mutant (O-21) strain per ml. The concentration of potassium phosphate buffer and the yeast strain used were 400 mm and wild type in the A-1 mixture, 250 mm and wild type in the A-2 mixture, 400 mm and O-21 in the B-1 mixture, and 250 mm and O-21 in the C-2 mixture, respectively. The glucose in each mixture was completely consumed in a 6- to 10-h incubation (data not shown). Symbols: F, glutamate; E, glutamine. containing the wild-type strain decreased (the yield was lowered). The data in Table 4, summarizing the enzyme activities in cell extracts of the yeasts, indicates that, in O-21, the activity of the enzymes involved in gluconeogenesis, especially the activity of FBPase, was lower than that in the wild-type strain. Furthermore, the enzyme activity for glycolysis was almost the same in the two strains. FBPase in the O-21 strain was strikingly inhibited by inorganic phosphate (45% inhibition was produced by the addition of 10 mm inorganic phosphate, and 80% was produced by the addition of 30 mm), whereas FBPase in the wild-type strain was not strikingly inhibited (27% inhibition by 30 mm inorganic phosphate). The phosphofructokinase in both strains was not greatly affected by inorganic phosphate (2 to 5% inhibition by 30 mm inorganic phosphate). These results suggested that the consumption of ATP by a futile cycle in the glycolytic pathway (apparent hydrolysis of ATP caused by the combined action of sugar-phosphorylating enzyme and phosphatase) might be a cause of the reduced glutamine yield obtained with glucose as an energy source for ATP regeneration and, further, that higher concentrations of inorganic phosphate repressed the ATP hydrolysis by inhibit- TABLE 4. Activity of enzymes involved in glycolysis and gluconeogenesis in cell yeast strain extracts a Enzyme Enzyme activity ( mol of product/min/mg of protein) Wild type O-21 Hexokinase (glucose) 1.55 (100) 1.64 (106) Hexokinase (fructose) 1.31 (100) 1.41 (108) Glucose 6-phosphatase (100) (80) Glucose 6-phosphate dehydrogenase (100) (95) Phosphoglucomutase (100) (57) Phosphoglucoisomerase 2.56 (100) 3.17 (123) Phosphofructokinase (100) (115) FBPase (100) (19) a The activity of hexokinase was determined with glucose or fructose as a substrate. The values in parentheses indicate the activities relative to the activities in the cell extract of the wild-type strain.

6 VOL. 64, 1998 GLUTAMINE PRODUCTION BY YEAST AND BACTERIAL GS 2957 ing phosphatase (in particular, sugar phosphate-specific phosphatase). This concept should be tested by detailed biochemical examinations of the organisms, which would be valuable for the propagation of yeast strains effective in energy-generating systems for coupled fermentation with energy transfer. REFERENCES 1. Harden, A., and W. Young The alcoholic ferment of yeast-juice. Proc. R. Soc. Lond. Ser. B 77: Kariya, Y., K. Aisaka, A., Kimura, and T. Tochikura Fermentative production of CDP-chlorine-related compounds (I). Amino Acid Nucleic Acid 29: (In Japanese.) 3. Kariya, Y., Y. Owada, A. Kimura, and T. Tochikura Fermentative phosphorylation of galactose by yeast. Agric. Biol. Chem. 43: Kawaguchi, K., H. Kawai, and T. Tochikura Production of sugar nucleotides by fermentation. Methods Carbohydr. Chem. 3: Maitra, P. K., and Z. Lobo Control of enzyme synthesis in yeast by products of the hexokinase reaction. J. Biol. Chem. 246: Nabe, K., T. Ujimaru, N. Izuo, S. Yamada, and I. Chibata Production of L-glutamine by a penicillin-resistant mutant of Flavobacterium rigense. Appl. Environ. Microbiol. 40: Nabe, K., T. Ujimaru, S. Yamada, and I. Chibata Influence of nutritional conditions on production of L-glutamine by Flavobacterium rigense. Appl. Environ. Microbiol. 41: Nakanishi, T Roles of ammonium and chloride in the conversion of L-glutamic acid fermentation to L-glutamine and N-acetyl-L-glutamine fermentation by Corynebacterium glutamicum. J. Ferment. Technol. 56: Nakanishi, T Enzymes concerned in the conversion of L-glutamic acid fermentation to L-glutamine and N-acetyl-L-glutamine fermentation by Corynebacterium glutamicum. J. Ferment. Technol. 56: Nakanishi, T Studies on L-glutamine and N-acetyl-L-glutamine fermentation and its metabolic regulations. Hakko Kogaku Kaishi 58: Sung, H. C., T. Tachiki, H. Kumagai, and T. Tochikura Production and preparation of glutamine synthetase from Brevibacterium flavum. J. Ferment. Technol. 62: Tachiki, T., H. Matsumoto, T. Yano, and T. Tochikura Glutamine production by coupling with energy transfer: employing yeast cell-free extract and Gluconobacter glutamine synthetase. Agric. Biol. Chem. 45: Tachiki, T., H. Matsumoto, T. Yano, and T. Tochikura Glutamine production by coupling with energy transfer: employing yeast cells and Gluconobacter glutamine synthetase. Agric. Biol. Chem. 45: Tachiki, T., S. Wakisaka, H. Kumagai, and T. Tochikura Crystallization of glutamine synthetase from Micrococcus glutamicus. Agric. Biol. Chem. 45: Tachiki, T., H. Suzuki, S. Wakisaka, T. Yano, and T. Tochikura Glutamine production in high concentrations with energy transfer employing glutamine synthetase from Micrococcus glutamicus. J. Gen. Appl. Microbiol. 29: Tochikura, T Production of nucleotide-related substances by yeast and utilization of fermentation energy to biosynthetic processes. Hakko Kogaku Kaishi 56: (In Japanese.) 17. Tochikura, T Production of industrial enzymes from waste materials and utilization of waste ATP energy for synthesis, p In A. M. Martin (ed.), Bioconversion of waste materials to industrial products. Elsevier Science Publishers Ltd., London, England. 18. Tochikura, T., M. Kuwahara, S. Yagi, H. Okamoto, Y. Tominaga, T. Kano, and K. Ogata Fermentation and metabolism of nucleic acid-related compounds in yeast. J. Ferment. Technol. 45: Tochikura, T., Y. Kariya, T. Yano, T. Tachiki, and A. Kimura Studies on utilization of fermentation and respiratory energy to biosynthetic processes. I. Fermentative production of phosphoric acid anhydrides and phosphate esters. Amino Acid Nucleic Acid 29: (In Japanese.) 20. Tochikura, T., T. Tachiki, T. Yano, and S. Kadowaki Biochemical reactors using ATP energy of yeast fermentation. Yuki Gosei Kagaku Kyokaishi 39: (In Japanese.) 21. Wakisaka, S., H. C. Sung, T. Tachiki, H. Kumagai, T. Tochikura, and S. Matsui A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria. Anal. Biochem. 163: Wakisaka, S., T. Tachiki, T. Aikawa, and T. Tochikura Properties of Brevibacterium flavum glutamine synthetase in an in vivo-like system. J. Ferment. Bioeng. 67: Yoshinaga, F., and S. Nakamori Production of amino acids, p In K. M. Herrmann and R. Somerville (ed.), Amino acids. Biosynthesis and genetic regulation. Addison-Wesley Publishing Co., Inc., Reading, Mass. Downloaded from on April 24, 2019 by guest