Effects of magnesium ion and chelating agents on ... - Springer Link

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Appl Microbiol Biotechnol (1985) 21 : 143-147

Mierobiology Bioteehnology © Springer-Verlag1985

Effects of magnesium ion and chelating agents on enzymatic production of ATP from adenine Tatsuro Fujio and Akira Furuya Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Machida-shi, Tokyo 194, Japan

Summary. =As reported previously, enzymatic production of A T P from adenine by resting cells of Brevibacterium ammoniagenes (Fujio and Furuya 1983) accumulated 13.0 mg of A T P - Na 2 • 3 H20/ml , but A T P formation ceased within 6 - 8 h. Simultaneous addition of magnesium ion and phytic acid, a chelator of divalent cations, allowed A T P formation to continue longer, and 24.2mg of A T P . Na 2 • 3 H 2 0 / m l was accumulated in 10h. However, A T P formation ceased thereafter. This second cessation was found to be caused by the lack of magnesium ion active as a co-factor (Mgact). The Mg act was tentatively taken as the difference between soluble magnesium ion (Mg s°l) and the ion chelated by an equimolar amounvof A T P (MgATP), namely Mg act = Mg s°l - Mg ATP. In order to provide Mg act, sufficient phytic acid had to be added at the beginning of the reaction and magnesium ion was also added intermittently. Under these conditions A T P formation continued further, and the rate of A T P formation was increased; 37.0mg of A T P • Na 2 • 3 H 2 0 / m l was accumulated in 13 h. Since whole culture broth is preferable to frozen cells as a practical enzyme source, the conditions neccessary for use of whole culture broth of B. ammoniagenes were also investigated.

Introduction Since A T P plays an important role as an energy donor in various biochemical reactions, it has been widely used as a biochemical and therapeutic agent. Recently, A T P has attracted attention as the energy donor in a bioreactor for the production of coenzyme

Offprint request to: T. Fujio

A (Shimizu et al. 1979), glutathione, N A D P (Murata et al. 1981) and glutamine (Tachiki et al. 1981). The biochemical methods for producing A T P include: (1) extraction from animal tissues or microbes; (2) phosphorylation of the precursors such as adenosine and AMP by yeasts, using glucose as the energy donor (Tochikura et al. 1967; Asada et al. 1978); (3) phosphorylation of A M P by Escherichia coli with a hybrid-plasmid containing genes for phosphofructokinase and triose phosphate isomerase (Shimosaka et al. 1981, 1982); (4)direct ribotidation of adenine through salvage synthesis and phosphorylation by B. ammoniagenes (Tanaka et al. 1968; Fujio and Furuya 1983). In order to accumulate A T P extracellularly, yeasts or E. coli cells have to be lyophilized to alter their cytoplasmic membranes to allow A T P permeation. With B. ammoniagenes, cells grown in a medium containing a low level of manganese ion were able to excrete A T P (Nara et al. 1968). Recently we developed a method using surfactant-treated cells grown in the presence of a high level of manganese ion (Fujio and Furuya 1983). In the "surfactant-treated cell" method, the rate and maximal level of A T P accumulation was improved as compared with the "manganese-ion-limited cell" method, but the cessation of A T P formation in 6 - 8 h limited the maximum A T P accumulation at a level of approximately 13mg of A T P - Na 2 • 3 H20/ml. In this report the mechanism of this cessation was analysed in order to improve A T P production, and the significant roles of magnesium ion and chelating agents, such as phytic acid and A T P itself, were elucidated. Since whole culture broth was preferable, from practical point of view, to frozen cells of B. ammoniagenes as the enzyme source, conditions required to use whole culture broth in the conversion reaction were also investigated.

144

T. Fujio and A. Furuya: Improvement of enzymatic ATP production from adenine

Materials

and methods

Results

Microorganism. A mutant of B. ammoniagenes, KY13510 (decoyinine resistant, nucleotidase weak, and temperature sensitive) was used. This culture is practically devoid of 5'-nucleotide degrading activity.

Media and cultivation. The media composition and cultivation conditions were described in a previous report (Fujio and Furuya 1983). The whole culture broth was either used directly as the enzyme source or was initially centrifuged, the separated cells were then frozen and used after thawing at room temperature.

Reaction conditions. The enzymatic reaction was performed in a 200-ml beaker as described previously (Fujio and Furuya 1983) or in a 5-1 jar fermentor. The liquid volume in the jar fermentor was 3 1, and the aeration and agitation were 1vvm and 450 RPM, respectively. The basal reaction mixture in both beaker and jar fermentor contained: 5-10 mg of adenine, 80 mg of glucose, 15 mg of Na2HPO4, and 100-120 mg (as dry cell weight, dcw) of frozen cells/ml or, when noted, whole broth of KY13510 cultured in a fermentation medium containing 18% of glucose. The other reaction conditions were the same as those described previously ' (Fujio and Furuya 1983).

Analyses. About 0.1 ml of reaction mixture was centrifuged with a Hitachi centrifuge type 05P at 1,450 g for 10 min. The supernatant was used for assays. Soluble magnesium ion concentration was determined by an assay kit (Magnesium B-test Waka, Wake Pure Chemical Industries, Ltd.) and expressed in terms of MgSO4- 7 Ha0. Soluble phosphate ion and ATP were assayed as described previously (Fujio and Furuya 1983), and were expressed in terms of Na2HPO4 and ATP - Naz- 3 H20, respectively.

Materials. Adenine was obtained from Kyowa Hakko Kogyo Co., Ltd. Phytic acid was purchased from Mitsui Toatsu Chemicals Inc., and neutralized with sodium hydroxide before use.

E n z y m a t i c p r o d u c t i o n of A T P f r o m a d e n i n e by B. ammoniagenes cells g r o w n in a high M n 2+ m e d i u m ( a b o v e 60 ~tg/l) was described in the p r e v i o u s r e p o r t (Fujio a n d F u r u y a 1983). T h e rate of A T P f o r m a t i o n was a p p r o x i m a t e l y 3 m g / m l / h for the first 3 h, b u t t h e n it gradually decreased. A T P f o r m a t i o n s t o p p e d i n 6 - 8 h, w h e n a b o u t 13 m g / m l of A T P was accumulated.

Effect of magnesium ion and phytic acid B e c a u s e m a g n e s i u m i o n is a n essential co-factor in the A T P - p r o d u c i n g r e a c t i o n , limiting its availability should o b v i o u s l y cause the cessation of A T P form a t i o n . E x t r a a d d i t i o n of the ion, h o w e v e r , showed a n i n h i b i t o r y effect ( F u j i o a n d F u r u y a 1983). B e c a u s e the a d d i t i o n of m a g n e s i u m i o n did n o t increase soluble m a g n e s i u m i o n c o n c e n t r a t i o n (data n o t s h o w n ) , a n d b e c a u s e t h e r e was a m m o n i u m ion carried over by the cells in a d d i t i o n to p h o s p h a t e i o n in the r e a c t i o n m i x t u r e , the f o r m a t i o n of a slightly soluble salt such as m a g n e s i u m a m m o n i u m p h o s p h a t e was suggested. T h e effect of m a g n e s i u m i o n a d d i t i o n in the p r e s e n c e of a chelating agent, phytic acid, was t h e r e f o r e i n v e s t i g a t e d (Fig. 1). A s s h o w n in Fig. 1, p h o s p h a t e ion was also a d d e d i n t e r m i t t e n t l y to keep the i o n c o n c e n t r a t i o n b e t w e e n a p p r o x i m a t e l y 3 5 - 1 0 5 m M ( a p p r o x i m a t e l y 5 - 1 5 m g / m l ) , since it h a d b e e n s h o w n to b e a p r e r e q u i s i t e for A T P f o r m a t i o n ( F u j i o a n d F u r u y a 1983).

25

25

20

E

5 /t ~ l

0

i

4

l

l

l

•

10

~

5 i

I

8 12 Time ( h )

Fig. 1. Effect of magnesium ion addition on ATP production in the presence or absence of phytic acid. Reaction was carried out for 12 h in a 200-ml beaker as described in "Materials and methods" except that 5 mg/ml of MgSO4 • 7 1-120 was added at 2 h (4,) and 15 mg/ml of Na2I-IPO4 at 3,5,7, and 9 h ( t ) . (O), reaction mixture with phytic acid; ((I)) reaction mixture without phytic acid

0

f

4

i

i

i

i

8 12 Time { h )

Fig. 2. Intermittent feeding of MgSO4 • 7 H20 in the phytic acid containing reaction. The reaction was carried out as described in the legend of Fig. 1 except that 5 mg of MgSO4- 7 H20/ml was added at 2 and 7 h. Phytic acid content was 0.5 mg/ml. Na2HPOa was added intermittently (5 mg/ml at 3, 5, 8, and 11 h in this case) to keep soluble phosphate ion concentration between 35-105 mM (approximately 5-15 mg Na2HPO4/ml) (see text). (O), ATP; (O), Na2HPO4; (lq), MgSO4 • 7 H20

145


The rate and the amount of ATP formation was lower when magnesium ion was added in the absence of phytic acid. However, the initial rate of ATP formation was constant for about 7 h, and ATP reached a level of more than 20mg/ml when magnesium ion was added in the presence of phytic acid. The prevention of the precipitation of a magnesium salt by phytic acid appeared to be the main factor for continued ATP formation in the presence of additional magnesium ion.

Abrupt interruption of A TP formation and its cause Although the initial rate of ATP formation lasted longer in the presence of phytic acid, ATP formation was often abruptly terminated as shown in Fig. 2. The time course of ATP accumulation shown in Fig. 2 strongly suggested that the shortage of essential factor(s) occurred after about 10 h. Therefore, the soluble phosphate and magnesium ions present in the supernatant fraction of the reaction mixture (centrifuged at 1,450 g) were determined (Fig. 2). Phosphate ion decreased rapidly. Its addition after 5 h restored the rate of ATP formation, but showed no effect at 11 h. These results suggested that the shortage of phosphate ion was responsible for the rate reduction at 5 h, but not at 11 h. Although magnesium ion decreased less rapidly than phosphate ion, it also decreased after 2 h. It was therefore added twice to avoid a deficiency. ATP was rapidly formed during the first 2 h, even though there

was only 8-12 mM (approximately 2 - 3 mg/ml) of soluble magnesium ion, and addition of the ion at 2 h showed no stimulating effect. The addition at 7 h, however, increased ATP formation rate, and the effect lasted for a further 3 h. Thus magnesium ion concentration was rate-limiting at 7 h, even though about 30 mM (7.4 mg/ml) of soluble magnesium ion was present. These results suggested that the required concentration of magnesium ion varied with time, that is, as more ATP accumulated, more magnesium ion was required. ATP, when present in a living cell, forms a complex with magnesium ion. If this is the case in this reaction mixture, the ion should be chelated by equimolar amount of ATP. If the chelated ion (MgATP) was inactive as a co-factor, the active fraction of the ion (Mg act) should be the difference between the soluble ion (Mg s°]) and the chelated ion as shown in equation I.

(1)

M g act = M g sol _ M g A T P .

Mg aa was calculated from the data in Fig. 2, and plotted (Fig. 3). The cessation of ATP formation occurred about the time the value of Mg act changed from positive to negative, namely, at about 10 h. Just before magnesium ion addition at 7 h, when a short-term deficiency of Mg act occurred, the ATP formation rate was slightly reduced, but the rate was soon restored by the addition of the ion. These results indicated that the shortage of the ion occurred due to the formation of a complex with ATP, and this was the cause of the cessation of ATP formation at 10 h.

25 30

20 E

-~ 15 E

2o

E

~io

e2

4 =8 Time ( h )

i

o

i

4

i

i

i

i

8 12 Time ( h )

-5

Fig. 3. Time course of active magnesium ion (Mg act, see text) during ATP production shown in Fig. 2. (O), ATP; (11), MgaCt

Fig. 4. Effect of intermittent addition of MgSO4 • 7 H20 on Mg ~°l and Mg act and on ATP production. The reaction mixture contained 8 mg of phytic aciddml, and 5 mg of MgSO 4 - 7 H20/rrd was added at 2, 5, 7, and 10 h. The other reaction conditions were the same as that in Fig. 2. (O), ATP; (H), igS°l; ( 1 ) , Mg act

146


Table 1. Effect of phytic acid concentration on ATP produc-

tion

Table 2. ATP production with whole broth of B. ammoniagenes KY13510

Phytic acid (mg/ml)

ATP (mg/ml)

POESA (mg/ml)

1.5 4.0 6.0 8.0

23.0 26.0 29.2 33.9

0 4 6 8 10

Reaction was carried out as described in Fig. 2 except for addition of 5 mg of MgSO4 • 7 H20/ml at 2, 5, 7, 9, and 11 h. The indicated amounts of phytic acid were added at zero time

Maintenance of soluble magnesium ion by an increase of phytic acid In o r d e r to k e e p the M g act level p o s i t i v e , m a g n e s i u m i o n was i n t e r m i t t e n t l y a d d e d . H o w e v e r , .the a d d e d ion s o o n d i s a p p e a r e d f r o m t h e s u p e r n a t a n t , d u e p r o b a b l y to t h e f o r m a t i o n o f a slightly s o l u b l e salt with o t h e r i o n ( s ) . C o n s e q u e n t l y , A T P f o r m a t i o n s t o p p e d a l m o s t at t h e s a m e t i m e a n d at t h e s a m e level as in Fig. 3 ( d a t a n o t s h o w n ) . I n o r d e r to m a i n t a i n t h e a d d e d m a g n e s i u m i o n in a s o l u b l e f o r m in t h e s u p e r n a t a n t , p h y t i c acid c o n c e n t r a t i o n was i n c r e a s e d ( T a b l e 1). W h e n 8 m g of p h y t i c a c i d / m l was a d d e d , M g act was p o s i t i v e t h r o u g h o u t t h e r e a c t i o n t i m e , a n d A T P f o r m a t i o n c o n t i n u e d for l o n g e r t h a n 12 h. M o r e t h a n 30 m g o f A T P / m l was a c c u m u l a t e d (Fig. 4).

Xylene 0d/ml)

Time (h)

ATP (mg/rrd)

0 0 0 0 0

7

trace 0.6 4.8 5.8 5.8

0 0 0

10 20 50

8

6.1 8.8 9.9

0 2 4 8

10 10 10 10

8

6.1 14.1 17.4 13.3

The reaction was carried out for the indicated time under the same condition described in the legend of Table 1, except that 8 mg of phytic acid/ml and the indicated amounts of POESA and/or xylene were added

4O

A

3O

E

Requirements for the use of the whole broth of KY13510 as enzyme source F r o m a p r a c t i c a l p o i n t o f view, it was d e s i r a b l e to use the w h o l e c u l t u r e b r o t h o f KY13510 i n s t e a d of f r o z e n a n d t h a w e d cells as t h e e n z y m e source. T h e w h o l e b r o t h s h o w e d little activity, e v e n w h e n 4 m g of p o l y o x y e t h y l e n e s t e a r y l a m i n e ( P O E S A ) / m l was a d d e d to t h e b r o t h as in t h e case o f f r o z e n a n d t h a w e d cells. A d d i t i o n of h i g h e r c o n c e n t r a t i o n s of P O E S A r e s u l t e d in o n l y a slight a c c u m u l a t i o n of A T P ( T a b l e 2). S o m e s u r f a c t a n t s a n d o r g a n i c solvents h a v e b e e n s h o w n to b e effective for f r o z e n a n d t h a w e d cells ( F u j i o a n d F u r u y a 1983), b u t w e r e ineffective for t h e w h o l e b r o t h . C o m b i n a t i o n s of s u r f a c t a n t ( s ) and organic solvent(s) were tested, and the combination of P O E S A a n d x y l e n e was f o u n d to b e effective ( T a b l e 2).

Typical time course of A TP production from adenine T h e r e a c t i o n was c a r r i e d o u t in a 5 - 1 j a r f e r m e n t o r (Fig. 5) using w h o l e c u l t u r e b r o t h o f KY13510.

i

0

i

4

i

i

8

i

i

i

12

Time ( h ) F~. S. Typical time course of A ~

production by enzymatic

conversion from adenine. Reaction was carried out for 13 h in a 5-1 jar fermentor with whole culture broth of B. ammoniagenes KY13510 under the conditions described in "Materials and methods". Reaction mixture contained: 10 mg of adenine, 80 mg of glucose, 15 mg of Na2HPO4, 10 mg of MgSO4 • 7 H20, 11 mg of phytic acid/ml of B. ammoniagenesKY13510 broth. Five milligram of MgSO4 • 7 H20/ml was added at 1, 3, 6, 9, and 12 h. Phosphate ion concentration was kept between 75-175 mM (approximately 10-25 mg/ml as Na2HPO4) by intermittent addition of Na2HPO 4. (O), ATP; (O), Na2I-IPO4; (11), Mgact

P h o s p h a t e i o n was a d d e d i n t e r m i t t e n t l y to k e e p t h e c o n c e n t r a t i o n in t h e s u p e r n a t a n t at a b o u t 150 m M ( a p p r o x i m a t e l y 21 m g / m l ) t h r o u g h o u t t h e r e a c t i o n , w h i c h was h i g h e r t h a n in t h e b e a k e r r e a c t i o n (see discussion). M a g n e s i u m i o n was also a d d e d i n t e r m i t t e n t l y in t h e p r e s e n c e o f 11 m g of p h y t i c acid/ml and


thus Mg act was kept positive throughout the reaction. The rate of ATP formation was approximately 30% higher in the reaction containing 11 mg of phytic acid than in the reaction containing 0.5 mg/ml, that is, the average rates of ATP formation during the first 10 h were 3.2 and 2.5 mg/ml/h, respectively (Figs. 2, 5). ATP formation lasted for 13 h and 37.0 mg of A T P . Na 2 • 3 H20/ml was accumulated. The total amount of adenine in the reaction mixture was 10 mg/ml, and the conversion rate of adenine to ATP was approximately 82%.

Discussion

Because the frozen, thawed, and surfactant-treated cells of B. ammoniagenes were not viable, the ATP producing system reported here is regarded as a kind of bioreactor system. This bioreactor consists of many enzymes enclosed within the cytoplasmic membrane damaged by the surfactant. The enzymes presumed to be essential for the conversion of adenine to ATP are: (1) the enzymes synthesizing phosphoribosyl pyrophosphate (PRPP) from glucose, (2) adenine phosphoribosyltransferase, (3) AMP kinase, and (4) ADP---~ATP regenerating enzyme system. All the enzymes are shown to be stable enough to perform the conversion reaction at 32°C for 10-13 h. The AMP kinase and the ATP regenerating enzyme system are stable even at 42 ° C for more than 10 h, since the conversion of 5'-xanthylic acid to 5'-guanylic acid in a similar enzymatic reaction (in which the regeneration of ATP from AMP is essential) occurs at 42°C for 10-20 h (Fujio and Furuya 1984). The enzyme responsible for the abrupt stop of ATP formation coinciding with Mg act deficiency cannot be specified because so many enzymes are involved in the conversion reaction and most of these enzymes require magnesium ion for their activity.. However, since the effect of Mg act deficiency immediately resulted in inhibition of ATP formation, an enzyme (or enzymes) associated with the ATP regenerating system may be responsible. In the absence of phytic acid in the reaction mixture, magnesium ion was removed from the supernatant due probably to the formation of a slightly soluble precipitate. Phytic acid was shown to keep magnesium ion in soluble form in the supernatant, affording availability of the indispensable co-factor in active form. The magnesium ion complexed with ATP was suggested to be inactive as a co-factor for ATP-producing enzymes (Fig. 3). However, it is not clear as yet why the magnesium ion chelated by phytic acid is active while that chelated by ATP is inactive as co-factor.

147

The optimal concentration of phosphate ion for the ATP formation was higher in a reaction in a 5-1 jar fermentor than in a 200-ml beaker. The reason for this discrepancy is not clear but is probably related to the difference in the activity of cells, owing to the different conditions of oxygen supply. The involvement of the electron transport system in the formation of ATP has not been clarified, but is strongly suggested by the fact that aeration was indispensable for ATP production, and that a-ketoglutaric acid or latic acid could be used in place of glucose as the energy donor for the formation of ATP from adenosine (data not shown). This reaction system is expected to be a useful energy supplying system for biochemical production of various useful substances (Fujio and Furuya 1984). Acknowledgement. The helpful technical assistance of Mrs. Setsuko Endoh is greatly appreciated.

References Asada M, Nakanishi K, Matsuno R, Kariya Y, Kimura A, Kamikubo T (1978) Continuous ATP regeneration utilizing glycolysis and kinase systems of yeast. Agric Biol Chem 42 : 1533-1538 Fujio T, Furuya A (1983) Production of ATP from adenine by Brevibacterium ammoniagenes. J Ferment Technol 61 : 261-267 Fujio T, Kotani Y, Furuya A (1984) Production of 5'-guanylic acid by enzymatic conversion of 5'-xanthylic acid. J Ferment Technol 62:131-137 Murata K, Tani K, Chibata I (1981) Glycolytic pathway as an ATP regeneration system and its application to the production of glutathione and NADP. Enzyme Microbiol Technol 3 : 233-242 Nara T, Misawa M, Kinoshita S (1968) Pantothenate, thiamine and manganese in 5'-purine ribonucleotide production by Brevibacterium ammoniagenes. Agric Biol Chem 32:1153-1161 Shimizu S, Tani Y, Ogata K (1979) Synthesis of coenzyme A and its biosynthetic intermediates by microbial processes. In: McCormick DB, Wright LD (eds) Methods in enzymol, vo162. Academic Press, New York, pp 236-245 Shimosaka M, Fukuda Y, Kimura A (1981) Application of plasmid to ATP production by E. coli. Agric Biol Chem 45:1025-1027 Shimosaka M, Fukuda Y, Murata K, Kimura A (1982) Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli. J Bacteriol 152:98-103 Tachiki T, Matsumoto H, Yano T, Tochikura T (1981) Glutamine production by coupling with energy transfer employing with yeast cell-free extracts and Gluconobacter glutamine synthetase. Agric Biol Chem 45:705-710 Tanaka H, Sato Z, Nakayama K, Kinoshita S (1968) Formation of ATP, GTP and their related substances by Brevibacterium ammoniagenes. Agile Biol Chem 32:721-726 Tochikura T, Kuwahara M, Yagi S, Okamoto H, Tominaga T, Kato T, Ogata K (1967) Fermentation and metabolism of nucleic acid-related compounds in yeasts. J Ferment Technol 45:511-529 Received June 12, 1984/Revised September 4, 1984