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Production of l -leucine from α-ketoisocaproic acid by cell-free extract of Euglena gracilis Z
JOURNAL OF FERMENTATIONAND BIOENGINEERING VOI. 70, NO. 6, 427-428. 1990
NOTES Production of L-Leucine from a-Ketoisocaproic Acid by Cell-Free Extract of Euglena gracilis Z TOHRU YOSHIMURA,* NORIO KOIKE, YOSHIMITSU KIMURA, RYOHEI YAMAOKA, AND KEIZO HAYASHIYA
Laboratory of Applied Biochemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received 5 July 1990/Accepted 28 September 1990
L-Leucine was produced from a-ketoisocaproic acid at about 100% conversion with L-glutamate as an amino donor using cell-free extracts of Euglena gracilis Z. a-Ketoglutarate decarboxylase in Euglena drives the conversion to completion by removal of a-ketoglularate formed during the transamination.
One of the methods for conversion of keto acids to amino acids is to use a transaminase which catalyzes the transfer of an amino group between amino acids and keto acids. Transaminase reaction are reversible, and proceeds through a ping-pong-bi-bi mechanism (1). Therefore, the conversion of keto acids to amino acids is usually not completed, unless the elimination of produced amino acids or keto acids occurs. Calton et al. (2) have attained a maximum conversion of 98%/00 in the production of phenylalanine from phenylpyruvic acid with z-aspartate as an amino donor. In this case, the conversion was driven to completion by the elimination of oxaloacetate which was formed from t-aspartate through decarboxylation to pyruvate. Recently, a novel enzyme, a-ketoglutarate decarboxylase, has been found in Euglena gracilis Z. The enzyme catalyzes the decarboxylation of a-ketoglutarate to produce succinate semialdehyde in the presence of thiamine pyrophosphate, MgC12, and NADP +, the latter of which is not converted to NADPH during the reaction (3). The discovery of the enzyme led us to investigate the production of amino acids from keto acids and Lglutamate using cell-free extracts of E. gracilis. It was expected that ~-ketoglutarate decarboxylase eliminated (~ketoglutarate formed from the amino donor z-glutamate during the transamination to complete the conversion of keto acids to amino acids. E. gracilis Z was kindly donated from Professor S. Kitaoka of Osaka Prefectural University. Cells were cultured in Oda medium (pH 3.3) which contained 1 g of (NH4)2SO4, 19 g of glucose, 5 g of L-glutamate, 0.4 g of KH2PO4, 0.5 g of MgSO4-7H20, 0.2 g of CaCO3, 25 mg of ZnSO4.7H20, 20 mg of MnC12- 4H20, 1 mg of Fe2(SO4)3. xHzO, 1 mg of NaMoO4.2H20, 1 mg of C u S O 4 - 5 H 2 0 , l mg of C O S O 4 - 7 H 2 0 , 1 mg of H3BO3, 0.5 mg of NiSO4. 6 H 2 0 , 2.5 mg of thiamine HC1, and 10 ¢tg of cyanocobalamine in 1 / of medium. The culture was carried out at 25°C under illumination (1500 lx) with shaking for 96 h. Cultured cells (about 4 g wet weight) were suspended in a small amount of 50 mM potassium phosphate buffer (pH 7.5) containing 30%o ethylene glycol, 0.02% 2-mercaptoethanol, and 0.1% Triton X-100, and homogenized with
a l u m i n i u m oxide and sea sand at 4°C, followed by centrifugation at 12,000 rpm for 30 min. The supernatant solution dialyzed against 20 mM potassium phosphate buffer (pH 7.5) containing 300/00 ethylene glycol and 0.02% 2mercaptoethanol at 4°C was designated as the cell-free extract. The activity of a-ketoglutarate decarboxylase was assayed by measuring the succinate semialdehyde formed from a-ketoglutarate by the o-aminobenzaldehyde method as described previously (3). One unit of enzyme was defined as the amount that increased one absorbance unit at 440 nm per min. In the presence of MgC12, thiamine pyrophosphate, and NADP *, the prepared cell-free extract exhibited decarboxylase activity (0.011 units/mg protein), whereas the activity was not detected without the cofactors. The standard reaction mixture for amino acid produc-
* Corresponding author. Present address: Institute for Chemical Research, Kyoto University, Uji, Kyoto-fu 611, Japan.
FIG. 1. Time course of L-leucine production in the presence ( o ) or absence ( • ) of the cofactors of (,-ketoglutarate decarboxylase.
20
15 c
G
Ji
10
..3
5
v
427
I
I
&
8
I
I
I
12 16 Time, h
I
20
24
428
J. FERMENT.BIOENG.,
YOSHIMURA ET AL. TABLE 1. L-Leucineproduction from a-ketoisocaproic acid and L-glutamate with E, gracilis Cofactors a
+ + +
3-Mercaptopropionic acid (mM)
L-Leu (mM)
L-Gluremained (mM)
y-Aminobutyrate (mM)
Conversion ratio b (%)
0 0 1.0 5.0
6.27 7.58 8.30 9.60
23.1 18.6 19.0 18.5
0.58 3.42 2.74 1.43
62.7 75.8 83.0 96.0
a MgCI2, Thiamine pyrophosphate, and NADP ÷. b Final concentration of L-leucine/initial concentration of ~-ketoisocaproic acid. tion (0.5ml) contained 100mM potassium phosphate buffer (pH 7.5), 1 m M MgC12, 0.2 mM thiamine pyrophosphate, 1 m M N A D P ~, 1 m M 2-mercaptoethanol, 0.02 m M pyridoxal 5'-phosphate, 0.25 mg of NAN3, various concentrations of sodium L-glutamate and each keto acid, as well as 0.71 mg protein of cell-free extract. The reaction was performed at 30°C for 12 h, then terminated by the addition of 0.5 ml of 2 N HC1. After centrifugation, the a m i n o acid concentration of the supernatant was determined with a Shimadzu LC-6A amino acid analysis system. Protein was assayed by the method of Lowry et al. (4), with bovine serum a l b u m i n as the standard. To examine the effect of a-ketoglutarate decarboxylase on amino acid production, the conversion of a-ketoisocaproic acid to z-leucine with or without the cofactors of decarboxylase (substrate concentration: ~-ketoisocaproic acid, 10mM; z-glutamate, 30raM) was performed. As shown in Table 1, the conversion ratio was slightly enhanced from 62.7%0 to 75.8% by the presence of the cofactors, although the complete conversion could not be attained. A m i n o acid analysis revealed the newly formed amino acid which was identified as y-aminobutyric acid due to its retention time. The results suggest that the cellfree extract contained L-glutamate decarboxylase which decreased leucine production through the c o n s u m p t i o n of Lglutamate. To eliminate the effect of z-glutamate decarboxylase, the reaction was carried out in the presence of 3mercaptopropionic acid, a potent inhibitor of z-glutamate decarboxylase (5). In the presence of 5 mM 3-mercaptopropionic acid, a conversion of 96% was attained for Lleucine from ~-ketoisocaproic acid (Table 1). Figure 1 shows the time course of z-leucine production with 2 0 m M cr-ketoisocaproic acid, 3 0 m M z-glutamate, and 5 m M 3-mercaptopropionic acid. In the presence of aketoglutarate decarboxylase cofactors, complete conversion of a-ketoisocaproic acid to z-leucine was attained in 18h. Without cofactors, the reaction reached an equilibrium at which about only 50%0 of the initial a m o u n t
of a-ketoisocaproic acid had been converted to z-leucine. These results confirm that ~r-ketoglutarate decarboxylase drove the reaction to completion. Under the same conditions, a-ketoisovaleric acid, phenylpyruvic acid, and pyruvic acid were converted to the corresponding amino acids by 18-h incubation with conversion ratios of 100, 70, and 15%0, respectively. The results may reflect the different activities of transaminases in E. gracilis Z. The chirality of the products, leucine and valine, were determined to be Lform by enantioselective H P L C analysis (data not shown). L-Glutamate is one of the least expensive amino acids, and acts as an amino donor for many transaminases. Thus, the results obtained here may contribute to the production of amino acids from keto acids. The authors wish to thank Professor S. Kitaoka and Associate Professor Y. Nakano of Osaka Prefectural University, and Professor K. Soda of Kyoto University for their helpful advice. REFERENCES
1. Braunstein, A. E.: Amino group transfer, p. 379-481. In Boyer, P. D. (ed.), The enzymes, 3rd ed., 9. Academic Press, New York (1973). 2. Calton, G.J., Wood, L. L., Upflike, M. H., Lantz, L. II., and Hamman, J.P.: The production of L-phenylalanine by polyazetidine immobilized microbes. Bio/Technology, 4, 317320 (1986). 3. Shigeoka, S., Onishi, T., Maeda, K., Nakano, Y., and Kitaoka, S.: Occurrence of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in mitochondria of Euglena gracilis. FEBS Lett., 195, 43-47 (1986). 4. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). 5. Taberner, P. V., Pearce, M.J., and Watkins, J. C.: The inhibition of mouse brain glutamate decarboxylase by some structural analog of L-glutamic acid. Biochem. Pharmacol., 26, 345-349 (1977).
NOTES Production of L-Leucine from a-Ketoisocaproic Acid by Cell-Free Extract of Euglena gracilis Z TOHRU YOSHIMURA,* NORIO KOIKE, YOSHIMITSU KIMURA, RYOHEI YAMAOKA, AND KEIZO HAYASHIYA
Laboratory of Applied Biochemistry, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606, Japan Received 5 July 1990/Accepted 28 September 1990
L-Leucine was produced from a-ketoisocaproic acid at about 100% conversion with L-glutamate as an amino donor using cell-free extracts of Euglena gracilis Z. a-Ketoglutarate decarboxylase in Euglena drives the conversion to completion by removal of a-ketoglularate formed during the transamination.
One of the methods for conversion of keto acids to amino acids is to use a transaminase which catalyzes the transfer of an amino group between amino acids and keto acids. Transaminase reaction are reversible, and proceeds through a ping-pong-bi-bi mechanism (1). Therefore, the conversion of keto acids to amino acids is usually not completed, unless the elimination of produced amino acids or keto acids occurs. Calton et al. (2) have attained a maximum conversion of 98%/00 in the production of phenylalanine from phenylpyruvic acid with z-aspartate as an amino donor. In this case, the conversion was driven to completion by the elimination of oxaloacetate which was formed from t-aspartate through decarboxylation to pyruvate. Recently, a novel enzyme, a-ketoglutarate decarboxylase, has been found in Euglena gracilis Z. The enzyme catalyzes the decarboxylation of a-ketoglutarate to produce succinate semialdehyde in the presence of thiamine pyrophosphate, MgC12, and NADP +, the latter of which is not converted to NADPH during the reaction (3). The discovery of the enzyme led us to investigate the production of amino acids from keto acids and Lglutamate using cell-free extracts of E. gracilis. It was expected that ~-ketoglutarate decarboxylase eliminated (~ketoglutarate formed from the amino donor z-glutamate during the transamination to complete the conversion of keto acids to amino acids. E. gracilis Z was kindly donated from Professor S. Kitaoka of Osaka Prefectural University. Cells were cultured in Oda medium (pH 3.3) which contained 1 g of (NH4)2SO4, 19 g of glucose, 5 g of L-glutamate, 0.4 g of KH2PO4, 0.5 g of MgSO4-7H20, 0.2 g of CaCO3, 25 mg of ZnSO4.7H20, 20 mg of MnC12- 4H20, 1 mg of Fe2(SO4)3. xHzO, 1 mg of NaMoO4.2H20, 1 mg of C u S O 4 - 5 H 2 0 , l mg of C O S O 4 - 7 H 2 0 , 1 mg of H3BO3, 0.5 mg of NiSO4. 6 H 2 0 , 2.5 mg of thiamine HC1, and 10 ¢tg of cyanocobalamine in 1 / of medium. The culture was carried out at 25°C under illumination (1500 lx) with shaking for 96 h. Cultured cells (about 4 g wet weight) were suspended in a small amount of 50 mM potassium phosphate buffer (pH 7.5) containing 30%o ethylene glycol, 0.02% 2-mercaptoethanol, and 0.1% Triton X-100, and homogenized with
a l u m i n i u m oxide and sea sand at 4°C, followed by centrifugation at 12,000 rpm for 30 min. The supernatant solution dialyzed against 20 mM potassium phosphate buffer (pH 7.5) containing 300/00 ethylene glycol and 0.02% 2mercaptoethanol at 4°C was designated as the cell-free extract. The activity of a-ketoglutarate decarboxylase was assayed by measuring the succinate semialdehyde formed from a-ketoglutarate by the o-aminobenzaldehyde method as described previously (3). One unit of enzyme was defined as the amount that increased one absorbance unit at 440 nm per min. In the presence of MgC12, thiamine pyrophosphate, and NADP *, the prepared cell-free extract exhibited decarboxylase activity (0.011 units/mg protein), whereas the activity was not detected without the cofactors. The standard reaction mixture for amino acid produc-
* Corresponding author. Present address: Institute for Chemical Research, Kyoto University, Uji, Kyoto-fu 611, Japan.
FIG. 1. Time course of L-leucine production in the presence ( o ) or absence ( • ) of the cofactors of (,-ketoglutarate decarboxylase.
20
15 c
G
Ji
10
..3
5
v
427
I
I
&
8
I
I
I
12 16 Time, h
I
20
24
428
J. FERMENT.BIOENG.,
YOSHIMURA ET AL. TABLE 1. L-Leucineproduction from a-ketoisocaproic acid and L-glutamate with E, gracilis Cofactors a
+ + +
3-Mercaptopropionic acid (mM)
L-Leu (mM)
L-Gluremained (mM)
y-Aminobutyrate (mM)
Conversion ratio b (%)
0 0 1.0 5.0
6.27 7.58 8.30 9.60
23.1 18.6 19.0 18.5
0.58 3.42 2.74 1.43
62.7 75.8 83.0 96.0
a MgCI2, Thiamine pyrophosphate, and NADP ÷. b Final concentration of L-leucine/initial concentration of ~-ketoisocaproic acid. tion (0.5ml) contained 100mM potassium phosphate buffer (pH 7.5), 1 m M MgC12, 0.2 mM thiamine pyrophosphate, 1 m M N A D P ~, 1 m M 2-mercaptoethanol, 0.02 m M pyridoxal 5'-phosphate, 0.25 mg of NAN3, various concentrations of sodium L-glutamate and each keto acid, as well as 0.71 mg protein of cell-free extract. The reaction was performed at 30°C for 12 h, then terminated by the addition of 0.5 ml of 2 N HC1. After centrifugation, the a m i n o acid concentration of the supernatant was determined with a Shimadzu LC-6A amino acid analysis system. Protein was assayed by the method of Lowry et al. (4), with bovine serum a l b u m i n as the standard. To examine the effect of a-ketoglutarate decarboxylase on amino acid production, the conversion of a-ketoisocaproic acid to z-leucine with or without the cofactors of decarboxylase (substrate concentration: ~-ketoisocaproic acid, 10mM; z-glutamate, 30raM) was performed. As shown in Table 1, the conversion ratio was slightly enhanced from 62.7%0 to 75.8% by the presence of the cofactors, although the complete conversion could not be attained. A m i n o acid analysis revealed the newly formed amino acid which was identified as y-aminobutyric acid due to its retention time. The results suggest that the cellfree extract contained L-glutamate decarboxylase which decreased leucine production through the c o n s u m p t i o n of Lglutamate. To eliminate the effect of z-glutamate decarboxylase, the reaction was carried out in the presence of 3mercaptopropionic acid, a potent inhibitor of z-glutamate decarboxylase (5). In the presence of 5 mM 3-mercaptopropionic acid, a conversion of 96% was attained for Lleucine from ~-ketoisocaproic acid (Table 1). Figure 1 shows the time course of z-leucine production with 2 0 m M cr-ketoisocaproic acid, 3 0 m M z-glutamate, and 5 m M 3-mercaptopropionic acid. In the presence of aketoglutarate decarboxylase cofactors, complete conversion of a-ketoisocaproic acid to z-leucine was attained in 18h. Without cofactors, the reaction reached an equilibrium at which about only 50%0 of the initial a m o u n t
of a-ketoisocaproic acid had been converted to z-leucine. These results confirm that ~r-ketoglutarate decarboxylase drove the reaction to completion. Under the same conditions, a-ketoisovaleric acid, phenylpyruvic acid, and pyruvic acid were converted to the corresponding amino acids by 18-h incubation with conversion ratios of 100, 70, and 15%0, respectively. The results may reflect the different activities of transaminases in E. gracilis Z. The chirality of the products, leucine and valine, were determined to be Lform by enantioselective H P L C analysis (data not shown). L-Glutamate is one of the least expensive amino acids, and acts as an amino donor for many transaminases. Thus, the results obtained here may contribute to the production of amino acids from keto acids. The authors wish to thank Professor S. Kitaoka and Associate Professor Y. Nakano of Osaka Prefectural University, and Professor K. Soda of Kyoto University for their helpful advice. REFERENCES
1. Braunstein, A. E.: Amino group transfer, p. 379-481. In Boyer, P. D. (ed.), The enzymes, 3rd ed., 9. Academic Press, New York (1973). 2. Calton, G.J., Wood, L. L., Upflike, M. H., Lantz, L. II., and Hamman, J.P.: The production of L-phenylalanine by polyazetidine immobilized microbes. Bio/Technology, 4, 317320 (1986). 3. Shigeoka, S., Onishi, T., Maeda, K., Nakano, Y., and Kitaoka, S.: Occurrence of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in mitochondria of Euglena gracilis. FEBS Lett., 195, 43-47 (1986). 4. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). 5. Taberner, P. V., Pearce, M.J., and Watkins, J. C.: The inhibition of mouse brain glutamate decarboxylase by some structural analog of L-glutamic acid. Biochem. Pharmacol., 26, 345-349 (1977).