- Email: [email protected]
ATP production by sorbitol-treated cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201
Journal Elsevier
of Biotechnology,
1 (1984)
119-127
119
JBT 00111
ATP production by sorbitol-treated cells of a methanol yeast, Candida boidinii ( Kloeckera sp.) No. 2201 Tani ‘, Tetsu Yonehara
Yoshiki ’ Research
Center for Cell and Tissue Culture, (Received
‘* , Yukio
Mitani
and 2 Department Kyoto 606, Japan
25 April
1984;
accepted
* and Hideaki
of Agricultural 22 May
Chemistry,
Yamada Kyoto
*
University,
1984)
Summary The phosphorylation system of AMP by sorbitol-treated cells of a methanolutilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201, was investigated for the production of ATP. Firstly, reaction conditions for the ATP production were optimized. Under the optimal conditions, 20-30 g 1-l of ATP were produced in the conversion rate of 60-70% from AMP. Activities of reactions involved in the ATP-producing system were compared with cells from different cultures to prepare the cells having the higher activity and to know the essential reaction limiting the rate of the system. The energy efficiency of this system was also discussed. methanol yeast, ATP production
by yeast, sorbitol-treated
yeast cells
Introduction Some attempts have been recently reported on the application of the oxidative function of methanol-utilizing yeasts as the biocatalyst (Baratti et al., 1978; Couderc and Baratti, 1980; Hou et al., 1979; Pate1 et al., 1979). In preceding papers (Tani et al., 1982, 1984a), we applied the function for a new ATP-producing system using Zymolyasee-treated cells of Candida boidinii (Kloeckera sp.) No. 2201. The phosphorylation of AMP to ADP by adenylate kinase and that of ADP to ATP by oxidative phosphorylation are also involved in the system. Catalytic activities of the * On leave
from
016%1656/84/$03.00
Toray
Industries
Inc.
0 1984 Elsevier
Science
Publishers
B.V.
120
sequential transfer of the energy yielded by the oxidation of reduced C,-compounds to ATP were retained in the Zymolyase-treated cells. Consequently, the more convenient process of the preparation of active cells was developed by the treatment of cells with sorbitol (Tani et al., 1984b). The present paper will describe results on the optimization of reaction conditions for the ATP production by the sorbitol-treated cells. The variation of activities of reactions involved in the ATP-producing system is also discussed. Materials and Methods Materials A methanol-utilizing yeast, C. boidinii (Kloeckera sp.) No. 2201 (Ogata et al., 1969; Lee and Komagata, 1980) was used. The yeast was cultivated in a medium consisting of 2% (v/v) methanol, 0.4 g NH,Cl, 0.1 g KH,PO,, 0.1 g K,HPO,, 0.05 g MgSO, - 7H,O and 0.2 g yeast extract in 100 ml of tap water, pH 6.0. A subculture of 5 ml medium with 1 g (w/v) glucose replacing the methanol was transferred to 500 ml of methanol-containing medium in a 2 1 shaking flask. The cultivation of subculture and main culture was aerobically carried out at 28OC for 24 h. Washed cells were prepared as described previously (Tam et al., 1982). Bioluminescence reagents for the Pica-Lite luminometer were purchased from Packard Instrumental Co., Ltd. Other chemicals were the same as the previous paper (Tani et al., 1984b). Preparation of sorbitol-treated cells Washed yeast cells were suspended in deionized water and were incubated for 45 min at 37OC. D-gorbitol solution was added to the suspension in a final concentration of 1.5 M. The cells were further incubated for 10 min at 37OC and used as sorbitol-treated cells. The cells could be stored at - 80 OC at least for one month. Assay methoris The standard reaction mixture for ATP production contained 50 pmol potassium phosphate buffer, pH 6.5, 5 pmol AMP. Na,, 1 pmol NAD+, 1 pmol glutathione and 50 pmol methanol. Activity measurements of ATPase, adenylate kinase, formate dehydrogenase were principally the same as described previously flani et al., 1982). The reaction mixture for each reaction contained the following compositions: for ATPase, 100 pmol potassium phosphate buffer, pH 6.5, and 5 pmol ATP . Na,; for adenylate kinase, 100 pmol potassium phosphate buffer, pH 6.5, 10 pmol ATP - Na,, 10 pmol AMP - Na, and 10 pmol Na,CO,; for for-mate dehydrogenase, 100 pmol potassium phosphate buffer, pH 6.5, 500 pmol fox-mate. Na and 5 pmol NAD+ . Reduction of NAD+ by methanol oxidation was measured in the reaction mixture containing 100 pmol potassium phosphate buffer, pH 6.5, 500 pmol methanol, 5 pmol NAD+ and 5 pm01 glutathione. All reactions were aerobically carried out at 25 ‘C in a 10 ml Erlenmeyer flask with addition of 0.2 ml cell suspension containing 10 mg dry weight of sorbitol-treated cells in 1.5 M sorbitol, and in a total volume of 0.5 ml.
121
Analysis
Amounts of ATP, ADP and NADH, and oxygen consumption in reaction mixture were determined as described previously (Tani et al., 1982, 1984a). Cell amount was estimated turbidimetrically prior to the sorbitol treatment. Endogenous ATP in cells was measured by luciferase-luciferin system with a Packard Model 6100 (Pica-Lite) luminometer, following the internal program.
Results Optimization
of reaction
conditions
for ATP
production
of methanol. Fig. 1A shows that the ATP production depends on the methanol concentration. The highest amount of ATP after 6 h incubation was obtained with 1 M methanol. The cells still showed the significant activity in the presence of 3 M methanol. The cause of ATP formation in the absence of methanol is now under investigation. At 10 h incubation, the ATP production still continued (Fig. 1B). Thus, the activity of sorbitol-treated cells is more tolerant to the high concentration of methanol and more stable during the reaction than that of Zymolyase-treated cells (Pate1 et al., 1979).
Concentration
Concentration of AMP. The effect of AMP concentration on ATP production is shown in Fig. 2. The highest amount of ATP was obtained with the AMP concentration of 80 mM and 16 h incubation. More than 60% of AMP was converted to ATP. Further increase of the AMP concentration decreased the amount of ATP and increased that of ADP. This could be explained by adenylate kinase in cells consuming the ATP, phosphorylating the excess AMP to ADP.
Methanol
cot-c. IMI
Reaction
time lh)
Fig. 1. Effect of methanol concentration on ATP production. The reaction was carried out for 6 h with different concentrations of methanol for A. For B, methanol concentrations were 0.1 M (0). 1 M (0) and 2 M (0). Other conditions were the same as described in Materials and Methods.
122
Concentration of NAD+ and glutathione. The requirement of NAD+ and glutathione in the ATP-producing system as cofactors of methanol oxidation was fulfilled with the concentration of 2 mM for 12 h incubation (Fig. 3). When the incubation was prolonged to 16 h, the optimum concentration of both cofactors increased to 10 mM with the increased production of ATP. Concentration of inorganic phosphorus. The amount of ATP produced increased with the increased addition of potassium phosphate buffer of pH 6.5 (Fig. 4). The optimum concentration was 200 mM and the higher concentration was inhibitory. A TP production under optimal conditions. Fig. 5 is the time course of ATP production with different concentrations of AMP under the reaction conditions optimized by experiments described above. Maximum amounts of ATP at 60, 80 and 100 mM of the AMP concentration were 44 mM (22 g l-l), 52 mM (26 g 1-l) and 50 mM (25 g 1-l) in conversion rates of 73, 65 and 508, respectively. A preliminary test for the feeding of substrates gave the increase of the productivity. ATP of 60 mM (30 g 1-l) was obtained from 100 mM AMP after 36 h incubation by a feeding of 1 M methanol and 50 mM potassium phosphate buffer at 16 h. Cultural conditions for preparation of sorbitol-treated cells Variation in the ATP productivity of sorbitol-treated cells was sometimes observed. To elucidate the variation and to overcome the instability of the activity, activities of the ATP-producing system in sorbitol-treated cells from different cultures were analyzed.
I
I
AMP concentration
(mM1
NAlf
GSH concentration
Fig. 2. Effect of AMP concentration on ATP production. The reaction was carried conditions except for AMP concentration: 0, ATP in 12 h incubation; 0, ATP ADP in 12 h incubation; A, ADP in 16 h incubation.
(mM I
out under the standard in 16 h incubation; A,
Fig. 3. Effect of cofactors of methanol oxidation on ATP production. The reaction was carried out under the standard conditions except for the simultaneous change of concentrations of NAD+ and glutathione (GSH). The incubation time was 12 h (0) and 16 h (0).
123
P
Growth and A TP productivity. Fig. 6 shows the activity of ATP production by cells from different cultivation time and aeration. The higher activity was obtained with the younger cells. The oxygen supply to the culture medium did not affect the activity though the ATP-producing system consisted of the oxidative sequence. Cellular ATP. Endogenous ATP might be a trigger of the system initiating the phosphorylation of AMP by adenylate kinase. Amounts of ATP in washed cells obtained from 24 h cultivation with the culture medium volumes of 100 and 500 ml
P J--&inK PB concentration
( mM I
Reaction
time (h)
Fig. 4. Effect of inorganic phosphorus concentration on ATP production. The reaction was carried out under the standard conditions except for the concentration of potassium phosphate buffer (KPB) for 12 h (0) and 16 h (0). Fig. 5. ATP production under optimal conditions. The reaction mixture contained 100 pmol potassium phosphate buffer, pH 6.5, 5 pmol NAD+, 5 pmol glutathione, 500 pmol methanol, 300 pmol sorbitol, 10 mg as dry weight of cells, and 30 pmol (0) 40 pmol (0) and 50 pmol(0) AMP in a total volume of 0.5 ml. The incubation was carried out as described in Materials and Methods.
Cultivation
time (h)
Fig. 6. ATP production by cells from different cultures. containing the medium of 100 ml (0), 250 ml (0) the growth (B) were determined as described in Materials
flask
The cultivation was carried out in 2 1 shaking and 500 ml (0). The ATP productivity (A) and and Methods.
124 TABLE ACTIVITIES
1 OF REACTION
Each activity in sorbitol-treated mixture used for ATP production
INVOLVED
IN ATP-PRODUCING
cells was measured as described was that of Fig. 5.
Reaction
Activity
of reaction
26 h cultured (nmol min7.61 15.97 1.63 1.60 287.0 1.26 1.08
0, uptake Adenylate kinase ATPase NADH production Methanol consumption ADP production ATP production
SYSTEM in Materials
and Methods.
The reaction
with
cells
' mg-' dry weight
70 h cultured
cells
cells) 6.71 8.37 1.08 2.10 373.0 0 0.08
in a 2 1 shaking flask were the same, 3.5 nmol per mg dry weight of cells. Decrease of the amount of the endogenous ATP to 2.2 nmol was observed when cells were cultured for 48 h with the culture medium of 500 ml, but not with that of 100 ml. The result indicates that the amount of endogenous ATP is not correlative with the variation of ATP productivity seen in Fig. 6. ATP in cells markedly decreased after the sorbitol treatment. Enzyme activities. Activities of reactions essentially involved in the ATP-producing system were compared with 26 h and 70 h cultured cells. As shown in Table 1, the decrease of activities of ATPase and adenylate kinase was observed in 70 h cultured cells. The marked decrease of ADP in the reaction
24
48
72
Cultivation
time
120 (h)
Fig. 7. Change of activities of adenylate kinase and ATPase by cultivation time. Activities of adenylate kinase (W), ATPase (A) and ATP production (0) in sorbitol-treated cells from different cultivation time were determined as in Table 1.
125
mixture emphasizes that the change of adenylate kinase activity could be a reason for the variation of ATP productivity. A detailed study on these activities is shown in Fig. 7. Activities of adenylate kinase, ATPase and ATP production proportionally decreased with the increased cultivation time. The reduction of NAD+ and consumption of methanol rather increased with the prolonged cultivation time (Table 1). This observation suggests that the formation of NADH is not a limiting step in the ATP-producing system. The ratio between the phosphorylation and oxygen consumption (P/O ratio) in the ATP-producing system was calculated according to the formula described in a previous paper (Tani et al., 1984a), subtracting the amount of oxygen consumed in the methanol oxidation. The values of 0.41 and 0.77 were obtained with 27 h and 120 h cultivated cells, respectively. This suggests that the activity of oxidative phosphorylation is retained in old cells. Effect of carbon source. The ATP productivity was influenced by the cultivation using methanol or glucose as the carbon source in subculture. Cells from the subculture with methanol showed the stronger activity for ATP production, but the lower growth. Table 2 shows activities of reactions involved in the ATP-producing system of these cell preparations. The subculture with methanol increased activities of ATPase, adenylate kinase and the reduction of NAD+. Based on the above-mentioned results comparing each activity in cells from different cultural conditions, the phosphorylation of AMP by adenylate kinase might be the most essential step limiting the ATP-producing system.
TABLE
2
EFFECT DUCING
OF CARBON SYSTEM
The determination Table 1. Formate
SOURCE
culture;
ACTIVITIES
OF
REACTIONS
INVOLVED
IN
ATP-PRO-
of each activity in sorbitol-treated cells grown on media indicated was the same dehydrogenase activity was determined as described in Materials and Methods.
Reaction Main
ON
Subculture: methanol:
0, uptake Adenylate kinase ATPase NADH production Formate dehydrogenase ADP production ATP production
Activity
of reaction
Glucose
1 W (w/v)
2.0% (nmol 5.32 10.57 1.17 1.14 2.09 1.25 1.24
of cells from Methanol
0.5% min-‘mg-’
dry weight 8.68 7.77 1.37 1.00 1.92 0.53 0.56
2.0% cells) 5.61 11.50 2.03 1.82 1.97 1.69 1.96
0.25%
(v/v) 0.5% 9.52 11.04 2.52 1.69 3.46 0.81 0.91
as
126
Discussion Methanol synthesized from the natural gas which is produced as oil field gas, coal field gas and biogas is now thought to be a promising substance as raw material for chemical industry and also as an energy source. The efficient bioconversion of methanol to the energy-rich compound should be useful as an energy-producing process. The present system for ATP production from AMP is characterized as not only a biotransformation system but also an energy conversion one. The calculation of energy efficiency can be discussed as follows. Table 3 shows the free energy change of each reaction involved in the ATP production by the oxidation of methanol and phosphorylation of AMP. Assuming the value of the P/O ratio in the methylotrophic growth of yeast as 2.0 (Van Dijken et al., 1981), the sequence is summarized in the Eq. (l), since 1 mol of ATP is consumed to phosphorylate AMP to ADP: AMP + 2Pi + $0, + +methanol+
ATP + ;CO, + 4H,O
0)
The theoretical value of the energy efficiency, Ke(,), in the oxidation of 1 mol methanol and reduction of 2 mol NADC by formaldehyde and formate dehydrogenases is: Ke(,, (methanol + ZNADH)
= (52.6 x 2/167.91)
x 100 = 62.7%
and that of the oxidative phosphorylation
by an electron transport from NADH
Ket,, (ZNADH
X
+ 2ATP) = (14.6
X
2/52.6
2)
X
is:
100 = 27.8%
The theoretical energy efficiency in Eq. (1) is: Keu, (methanol + ZATP) = (14.6 x 2/167.91)
x 100 = 17.4%
The practical energy efficiency, Ke(,,, was calculated using a result of ATP production where 19.46 mM ATP and 18.50 mM ADP were formed in the reaction mixture on the consumption of 906 mM methanol. Free energy changes in the reaction are: G, (methanol + CO,) = (906/1000) G’ (AMP --, ADP) = (18.5/1000) G’ (AMP -, ATP) = (19.46/1000)
TABLE FREE
X
167.91 = 152.13 kcal 1-l
x 7.3 = 0.14 kcal 1-l x 14.6 = 0.28 kcall-’
3 ENERGY
CHANGE
OF
REACTIONS
Reaction CH,OH NADH+H+ AMP+ZP,
IN
ATP
- AG + 3/20z
+ CO,
+1/20, + + ATP+ZH,O
2ADP+2Pi AMP
INVOLVED
+ ATP
+2ATP+2H,O + 2ADP
+ 2H 2O NAD+
+H,O
167.91 52.60 - 14.6 -7.3~2 0
PRODUCTION (kcal
mole’)
127
Therefore, the energy efficiency of the phosphorylation consumption of methanol is: Ke(,, = [ (0.14 + 0.28)/152.13]
x
of AMP to ATP against the
100 = 0.3%
The ratio of the practical efficiency against the theoretical efficiency is: Ke (methanol + ATP) = (Ke&Ke,,,) = [(0.14 + 0.28)/152.13/(14.6
x 2)/167.91]
x 100 = 1.3%
Thus, the energy efficiency of the present system is still low as an energy conversion system with respect to ATP accumulated in spite of the good yield from AMP.
References Baratti. J., Couderc, R., Cooney, C.L. and Wang, D.I.C. (1978) Preparation and properties of immobilized methanol oxidase. Biotechnol. Bioeng. 20, 333-348. Couderc, R. and Baratti. J. (1980) Immobilized yeast cells with methanol oxidase activity: preparation and enzymatic properties. Biotechnol. Bioeng. 22, 1155-1173. Hou, C.T., Patel. R.N., Laskin, A.I., Barnabe, N. and Marczak, 1. (1979) Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methaneand methanol-grown microbes. Appl. Environ. Microbial. 38. 135-142. Lee, J.-D. and Komagata, K. (1980) Taxonomic study of methanol-assimilating yeasts. J. Gen. Appl. Microbial. 26, 133-158. Ogata, K.. Nishikawa. H. and Ohsugi, M. (1969) A yeast capable of utilizing methanol. Agric. Biol. Chem. 33, 1519-1520. Patel. R.N., Hou, C.T., La&in, A.]., Derelanko, R. and Felix, A. (1979) Oxidation of secondary alcohols to methyl ketones by yeasts. Appl. Environ. Microbial. 38, 135-142. Tani. Y., Mitani, Y. and Yamada, H. (1982) Utilization of C,-compounds: phosphorylation of adenylate by oxidative phosphorylation in Condido boidinii (Kloeckera sp.) No. 2201. Agric. Biol. Chem. 46, 1097-1099. Tani, Y.. Mitani, Y. and Yamada, H. (1984a) ATP production by protoplasts of a methanol yeast, Condido boidinii (Kloeckera sp.) No. 2201. Agric. Biol. Chem. 48, 431-437. Tani, Y., Mitani, Y. and Yamada. H. (1984b) Preparation of ATP-producing cells of a methanol yeast, Candido boidinii (Kloeckero sp.) No. 2201. J. Ferment. Technol. 62, 99-101. Van DiJken, J.P., Harder, W. and Quayle, J.R. (1981) Energy transduction and carbon assimilation in methylotrophic yeasts. In: Microbial Growth on C, Compounds (Dalton, H., ed.), pp. 191-201. Heyden and Son Ltd., London.
of Biotechnology,
1 (1984)
119-127
119
JBT 00111
ATP production by sorbitol-treated cells of a methanol yeast, Candida boidinii ( Kloeckera sp.) No. 2201 Tani ‘, Tetsu Yonehara
Yoshiki ’ Research
Center for Cell and Tissue Culture, (Received
‘* , Yukio
Mitani
and 2 Department Kyoto 606, Japan
25 April
1984;
accepted
* and Hideaki
of Agricultural 22 May
Chemistry,
Yamada Kyoto
*
University,
1984)
Summary The phosphorylation system of AMP by sorbitol-treated cells of a methanolutilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201, was investigated for the production of ATP. Firstly, reaction conditions for the ATP production were optimized. Under the optimal conditions, 20-30 g 1-l of ATP were produced in the conversion rate of 60-70% from AMP. Activities of reactions involved in the ATP-producing system were compared with cells from different cultures to prepare the cells having the higher activity and to know the essential reaction limiting the rate of the system. The energy efficiency of this system was also discussed. methanol yeast, ATP production
by yeast, sorbitol-treated
yeast cells
Introduction Some attempts have been recently reported on the application of the oxidative function of methanol-utilizing yeasts as the biocatalyst (Baratti et al., 1978; Couderc and Baratti, 1980; Hou et al., 1979; Pate1 et al., 1979). In preceding papers (Tani et al., 1982, 1984a), we applied the function for a new ATP-producing system using Zymolyasee-treated cells of Candida boidinii (Kloeckera sp.) No. 2201. The phosphorylation of AMP to ADP by adenylate kinase and that of ADP to ATP by oxidative phosphorylation are also involved in the system. Catalytic activities of the * On leave
from
016%1656/84/$03.00
Toray
Industries
Inc.
0 1984 Elsevier
Science
Publishers
B.V.
120
sequential transfer of the energy yielded by the oxidation of reduced C,-compounds to ATP were retained in the Zymolyase-treated cells. Consequently, the more convenient process of the preparation of active cells was developed by the treatment of cells with sorbitol (Tani et al., 1984b). The present paper will describe results on the optimization of reaction conditions for the ATP production by the sorbitol-treated cells. The variation of activities of reactions involved in the ATP-producing system is also discussed. Materials and Methods Materials A methanol-utilizing yeast, C. boidinii (Kloeckera sp.) No. 2201 (Ogata et al., 1969; Lee and Komagata, 1980) was used. The yeast was cultivated in a medium consisting of 2% (v/v) methanol, 0.4 g NH,Cl, 0.1 g KH,PO,, 0.1 g K,HPO,, 0.05 g MgSO, - 7H,O and 0.2 g yeast extract in 100 ml of tap water, pH 6.0. A subculture of 5 ml medium with 1 g (w/v) glucose replacing the methanol was transferred to 500 ml of methanol-containing medium in a 2 1 shaking flask. The cultivation of subculture and main culture was aerobically carried out at 28OC for 24 h. Washed cells were prepared as described previously (Tam et al., 1982). Bioluminescence reagents for the Pica-Lite luminometer were purchased from Packard Instrumental Co., Ltd. Other chemicals were the same as the previous paper (Tani et al., 1984b). Preparation of sorbitol-treated cells Washed yeast cells were suspended in deionized water and were incubated for 45 min at 37OC. D-gorbitol solution was added to the suspension in a final concentration of 1.5 M. The cells were further incubated for 10 min at 37OC and used as sorbitol-treated cells. The cells could be stored at - 80 OC at least for one month. Assay methoris The standard reaction mixture for ATP production contained 50 pmol potassium phosphate buffer, pH 6.5, 5 pmol AMP. Na,, 1 pmol NAD+, 1 pmol glutathione and 50 pmol methanol. Activity measurements of ATPase, adenylate kinase, formate dehydrogenase were principally the same as described previously flani et al., 1982). The reaction mixture for each reaction contained the following compositions: for ATPase, 100 pmol potassium phosphate buffer, pH 6.5, and 5 pmol ATP . Na,; for adenylate kinase, 100 pmol potassium phosphate buffer, pH 6.5, 10 pmol ATP - Na,, 10 pmol AMP - Na, and 10 pmol Na,CO,; for for-mate dehydrogenase, 100 pmol potassium phosphate buffer, pH 6.5, 500 pmol fox-mate. Na and 5 pmol NAD+ . Reduction of NAD+ by methanol oxidation was measured in the reaction mixture containing 100 pmol potassium phosphate buffer, pH 6.5, 500 pmol methanol, 5 pmol NAD+ and 5 pm01 glutathione. All reactions were aerobically carried out at 25 ‘C in a 10 ml Erlenmeyer flask with addition of 0.2 ml cell suspension containing 10 mg dry weight of sorbitol-treated cells in 1.5 M sorbitol, and in a total volume of 0.5 ml.
121
Analysis
Amounts of ATP, ADP and NADH, and oxygen consumption in reaction mixture were determined as described previously (Tani et al., 1982, 1984a). Cell amount was estimated turbidimetrically prior to the sorbitol treatment. Endogenous ATP in cells was measured by luciferase-luciferin system with a Packard Model 6100 (Pica-Lite) luminometer, following the internal program.
Results Optimization
of reaction
conditions
for ATP
production
of methanol. Fig. 1A shows that the ATP production depends on the methanol concentration. The highest amount of ATP after 6 h incubation was obtained with 1 M methanol. The cells still showed the significant activity in the presence of 3 M methanol. The cause of ATP formation in the absence of methanol is now under investigation. At 10 h incubation, the ATP production still continued (Fig. 1B). Thus, the activity of sorbitol-treated cells is more tolerant to the high concentration of methanol and more stable during the reaction than that of Zymolyase-treated cells (Pate1 et al., 1979).
Concentration
Concentration of AMP. The effect of AMP concentration on ATP production is shown in Fig. 2. The highest amount of ATP was obtained with the AMP concentration of 80 mM and 16 h incubation. More than 60% of AMP was converted to ATP. Further increase of the AMP concentration decreased the amount of ATP and increased that of ADP. This could be explained by adenylate kinase in cells consuming the ATP, phosphorylating the excess AMP to ADP.
Methanol
cot-c. IMI
Reaction
time lh)
Fig. 1. Effect of methanol concentration on ATP production. The reaction was carried out for 6 h with different concentrations of methanol for A. For B, methanol concentrations were 0.1 M (0). 1 M (0) and 2 M (0). Other conditions were the same as described in Materials and Methods.
122
Concentration of NAD+ and glutathione. The requirement of NAD+ and glutathione in the ATP-producing system as cofactors of methanol oxidation was fulfilled with the concentration of 2 mM for 12 h incubation (Fig. 3). When the incubation was prolonged to 16 h, the optimum concentration of both cofactors increased to 10 mM with the increased production of ATP. Concentration of inorganic phosphorus. The amount of ATP produced increased with the increased addition of potassium phosphate buffer of pH 6.5 (Fig. 4). The optimum concentration was 200 mM and the higher concentration was inhibitory. A TP production under optimal conditions. Fig. 5 is the time course of ATP production with different concentrations of AMP under the reaction conditions optimized by experiments described above. Maximum amounts of ATP at 60, 80 and 100 mM of the AMP concentration were 44 mM (22 g l-l), 52 mM (26 g 1-l) and 50 mM (25 g 1-l) in conversion rates of 73, 65 and 508, respectively. A preliminary test for the feeding of substrates gave the increase of the productivity. ATP of 60 mM (30 g 1-l) was obtained from 100 mM AMP after 36 h incubation by a feeding of 1 M methanol and 50 mM potassium phosphate buffer at 16 h. Cultural conditions for preparation of sorbitol-treated cells Variation in the ATP productivity of sorbitol-treated cells was sometimes observed. To elucidate the variation and to overcome the instability of the activity, activities of the ATP-producing system in sorbitol-treated cells from different cultures were analyzed.
I
I
AMP concentration
(mM1
NAlf
GSH concentration
Fig. 2. Effect of AMP concentration on ATP production. The reaction was carried conditions except for AMP concentration: 0, ATP in 12 h incubation; 0, ATP ADP in 12 h incubation; A, ADP in 16 h incubation.
(mM I
out under the standard in 16 h incubation; A,
Fig. 3. Effect of cofactors of methanol oxidation on ATP production. The reaction was carried out under the standard conditions except for the simultaneous change of concentrations of NAD+ and glutathione (GSH). The incubation time was 12 h (0) and 16 h (0).
123
P
Growth and A TP productivity. Fig. 6 shows the activity of ATP production by cells from different cultivation time and aeration. The higher activity was obtained with the younger cells. The oxygen supply to the culture medium did not affect the activity though the ATP-producing system consisted of the oxidative sequence. Cellular ATP. Endogenous ATP might be a trigger of the system initiating the phosphorylation of AMP by adenylate kinase. Amounts of ATP in washed cells obtained from 24 h cultivation with the culture medium volumes of 100 and 500 ml
P J--&inK PB concentration
( mM I
Reaction
time (h)
Fig. 4. Effect of inorganic phosphorus concentration on ATP production. The reaction was carried out under the standard conditions except for the concentration of potassium phosphate buffer (KPB) for 12 h (0) and 16 h (0). Fig. 5. ATP production under optimal conditions. The reaction mixture contained 100 pmol potassium phosphate buffer, pH 6.5, 5 pmol NAD+, 5 pmol glutathione, 500 pmol methanol, 300 pmol sorbitol, 10 mg as dry weight of cells, and 30 pmol (0) 40 pmol (0) and 50 pmol(0) AMP in a total volume of 0.5 ml. The incubation was carried out as described in Materials and Methods.
Cultivation
time (h)
Fig. 6. ATP production by cells from different cultures. containing the medium of 100 ml (0), 250 ml (0) the growth (B) were determined as described in Materials
flask
The cultivation was carried out in 2 1 shaking and 500 ml (0). The ATP productivity (A) and and Methods.
124 TABLE ACTIVITIES
1 OF REACTION
Each activity in sorbitol-treated mixture used for ATP production
INVOLVED
IN ATP-PRODUCING
cells was measured as described was that of Fig. 5.
Reaction
Activity
of reaction
26 h cultured (nmol min7.61 15.97 1.63 1.60 287.0 1.26 1.08
0, uptake Adenylate kinase ATPase NADH production Methanol consumption ADP production ATP production
SYSTEM in Materials
and Methods.
The reaction
with
cells
' mg-' dry weight
70 h cultured
cells
cells) 6.71 8.37 1.08 2.10 373.0 0 0.08
in a 2 1 shaking flask were the same, 3.5 nmol per mg dry weight of cells. Decrease of the amount of the endogenous ATP to 2.2 nmol was observed when cells were cultured for 48 h with the culture medium of 500 ml, but not with that of 100 ml. The result indicates that the amount of endogenous ATP is not correlative with the variation of ATP productivity seen in Fig. 6. ATP in cells markedly decreased after the sorbitol treatment. Enzyme activities. Activities of reactions essentially involved in the ATP-producing system were compared with 26 h and 70 h cultured cells. As shown in Table 1, the decrease of activities of ATPase and adenylate kinase was observed in 70 h cultured cells. The marked decrease of ADP in the reaction
24
48
72
Cultivation
time
120 (h)
Fig. 7. Change of activities of adenylate kinase and ATPase by cultivation time. Activities of adenylate kinase (W), ATPase (A) and ATP production (0) in sorbitol-treated cells from different cultivation time were determined as in Table 1.
125
mixture emphasizes that the change of adenylate kinase activity could be a reason for the variation of ATP productivity. A detailed study on these activities is shown in Fig. 7. Activities of adenylate kinase, ATPase and ATP production proportionally decreased with the increased cultivation time. The reduction of NAD+ and consumption of methanol rather increased with the prolonged cultivation time (Table 1). This observation suggests that the formation of NADH is not a limiting step in the ATP-producing system. The ratio between the phosphorylation and oxygen consumption (P/O ratio) in the ATP-producing system was calculated according to the formula described in a previous paper (Tani et al., 1984a), subtracting the amount of oxygen consumed in the methanol oxidation. The values of 0.41 and 0.77 were obtained with 27 h and 120 h cultivated cells, respectively. This suggests that the activity of oxidative phosphorylation is retained in old cells. Effect of carbon source. The ATP productivity was influenced by the cultivation using methanol or glucose as the carbon source in subculture. Cells from the subculture with methanol showed the stronger activity for ATP production, but the lower growth. Table 2 shows activities of reactions involved in the ATP-producing system of these cell preparations. The subculture with methanol increased activities of ATPase, adenylate kinase and the reduction of NAD+. Based on the above-mentioned results comparing each activity in cells from different cultural conditions, the phosphorylation of AMP by adenylate kinase might be the most essential step limiting the ATP-producing system.
TABLE
2
EFFECT DUCING
OF CARBON SYSTEM
The determination Table 1. Formate
SOURCE
culture;
ACTIVITIES
OF
REACTIONS
INVOLVED
IN
ATP-PRO-
of each activity in sorbitol-treated cells grown on media indicated was the same dehydrogenase activity was determined as described in Materials and Methods.
Reaction Main
ON
Subculture: methanol:
0, uptake Adenylate kinase ATPase NADH production Formate dehydrogenase ADP production ATP production
Activity
of reaction
Glucose
1 W (w/v)
2.0% (nmol 5.32 10.57 1.17 1.14 2.09 1.25 1.24
of cells from Methanol
0.5% min-‘mg-’
dry weight 8.68 7.77 1.37 1.00 1.92 0.53 0.56
2.0% cells) 5.61 11.50 2.03 1.82 1.97 1.69 1.96
0.25%
(v/v) 0.5% 9.52 11.04 2.52 1.69 3.46 0.81 0.91
as
126
Discussion Methanol synthesized from the natural gas which is produced as oil field gas, coal field gas and biogas is now thought to be a promising substance as raw material for chemical industry and also as an energy source. The efficient bioconversion of methanol to the energy-rich compound should be useful as an energy-producing process. The present system for ATP production from AMP is characterized as not only a biotransformation system but also an energy conversion one. The calculation of energy efficiency can be discussed as follows. Table 3 shows the free energy change of each reaction involved in the ATP production by the oxidation of methanol and phosphorylation of AMP. Assuming the value of the P/O ratio in the methylotrophic growth of yeast as 2.0 (Van Dijken et al., 1981), the sequence is summarized in the Eq. (l), since 1 mol of ATP is consumed to phosphorylate AMP to ADP: AMP + 2Pi + $0, + +methanol+
ATP + ;CO, + 4H,O
0)
The theoretical value of the energy efficiency, Ke(,), in the oxidation of 1 mol methanol and reduction of 2 mol NADC by formaldehyde and formate dehydrogenases is: Ke(,, (methanol + ZNADH)
= (52.6 x 2/167.91)
x 100 = 62.7%
and that of the oxidative phosphorylation
by an electron transport from NADH
Ket,, (ZNADH
X
+ 2ATP) = (14.6
X
2/52.6
2)
X
is:
100 = 27.8%
The theoretical energy efficiency in Eq. (1) is: Keu, (methanol + ZATP) = (14.6 x 2/167.91)
x 100 = 17.4%
The practical energy efficiency, Ke(,,, was calculated using a result of ATP production where 19.46 mM ATP and 18.50 mM ADP were formed in the reaction mixture on the consumption of 906 mM methanol. Free energy changes in the reaction are: G, (methanol + CO,) = (906/1000) G’ (AMP --, ADP) = (18.5/1000) G’ (AMP -, ATP) = (19.46/1000)
TABLE FREE
X
167.91 = 152.13 kcal 1-l
x 7.3 = 0.14 kcal 1-l x 14.6 = 0.28 kcall-’
3 ENERGY
CHANGE
OF
REACTIONS
Reaction CH,OH NADH+H+ AMP+ZP,
IN
ATP
- AG + 3/20z
+ CO,
+1/20, + + ATP+ZH,O
2ADP+2Pi AMP
INVOLVED
+ ATP
+2ATP+2H,O + 2ADP
+ 2H 2O NAD+
+H,O
167.91 52.60 - 14.6 -7.3~2 0
PRODUCTION (kcal
mole’)
127
Therefore, the energy efficiency of the phosphorylation consumption of methanol is: Ke(,, = [ (0.14 + 0.28)/152.13]
x
of AMP to ATP against the
100 = 0.3%
The ratio of the practical efficiency against the theoretical efficiency is: Ke (methanol + ATP) = (Ke&Ke,,,) = [(0.14 + 0.28)/152.13/(14.6
x 2)/167.91]
x 100 = 1.3%
Thus, the energy efficiency of the present system is still low as an energy conversion system with respect to ATP accumulated in spite of the good yield from AMP.
References Baratti. J., Couderc, R., Cooney, C.L. and Wang, D.I.C. (1978) Preparation and properties of immobilized methanol oxidase. Biotechnol. Bioeng. 20, 333-348. Couderc, R. and Baratti. J. (1980) Immobilized yeast cells with methanol oxidase activity: preparation and enzymatic properties. Biotechnol. Bioeng. 22, 1155-1173. Hou, C.T., Patel. R.N., Laskin, A.I., Barnabe, N. and Marczak, 1. (1979) Microbial oxidation of gaseous hydrocarbons: production of methyl ketones from their corresponding secondary alcohols by methaneand methanol-grown microbes. Appl. Environ. Microbial. 38. 135-142. Lee, J.-D. and Komagata, K. (1980) Taxonomic study of methanol-assimilating yeasts. J. Gen. Appl. Microbial. 26, 133-158. Ogata, K.. Nishikawa. H. and Ohsugi, M. (1969) A yeast capable of utilizing methanol. Agric. Biol. Chem. 33, 1519-1520. Patel. R.N., Hou, C.T., La&in, A.]., Derelanko, R. and Felix, A. (1979) Oxidation of secondary alcohols to methyl ketones by yeasts. Appl. Environ. Microbial. 38, 135-142. Tani. Y., Mitani, Y. and Yamada, H. (1982) Utilization of C,-compounds: phosphorylation of adenylate by oxidative phosphorylation in Condido boidinii (Kloeckera sp.) No. 2201. Agric. Biol. Chem. 46, 1097-1099. Tani, Y.. Mitani, Y. and Yamada, H. (1984a) ATP production by protoplasts of a methanol yeast, Condido boidinii (Kloeckera sp.) No. 2201. Agric. Biol. Chem. 48, 431-437. Tani, Y., Mitani, Y. and Yamada. H. (1984b) Preparation of ATP-producing cells of a methanol yeast, Candido boidinii (Kloeckero sp.) No. 2201. J. Ferment. Technol. 62, 99-101. Van DiJken, J.P., Harder, W. and Quayle, J.R. (1981) Energy transduction and carbon assimilation in methylotrophic yeasts. In: Microbial Growth on C, Compounds (Dalton, H., ed.), pp. 191-201. Heyden and Son Ltd., London.