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Production of poly-β-hydroxybutyrate and measurement of related enzyme activities in Alcaligenes eutrophus
JOURNAl. Or FERMENTATION A N D BIOENGINEERING Vol. 76, No. 5, 416---418. 1993
Production of Poly-/3-Hydroxybutyrate and Measurement of Related Enzyme Activities in Alcaligenes eutrophus IN YOUNG LEE, 1 SOO WAN NAM, 1 EUN SOO CHOI, l H O NAM CHANG, 2 AND YOUNG HOON PARK 1. Genetic Engineering Research Institute, KIST, P.O. Box 17, Taedok Science Town, Taejon 305-606, ~ and Department of Chemical Engineering, Korea Advanced Institute of Science and Technology, Taedok Science Town, Taejon 305-701, 2 Korea Received 10 May 1993/Accepted 27 July 1993
Activities of ~-ketothiolase (KT) and glucose-6-phosphate dehydrogenase (GPDH) were determined during the fermentation of Alcaligenes eutrophus NCIMB 11599 at three different values of C / N ratio. As the C / N ratio increased the cell growth decreased due to the limitation of nitrogen, while the cellular poly-~-hydroxybutyrate (PHB) content increased. At an initial C / N ratio of 32.8, the cellular PHB content reached 90.0Yo after 32 h cultivation. The specific activity of ~-ketothiolase in each fermentation was the highest immediately after exhaustion of the nitrogen source. Specific activities of ~-ketothiolase and glucose-6-phosphate dehydrogenase were maintained stably within the tested range of C / N ratio even after the N-source was completely exhausted.
the fermentation medium. The air flow rate and agitation speed were 0.5-1.0vvm and 300-600rpm, respectively. The pH was controlled automatically at 7.0 with 4 N NaOH. Cell growth was monitored by measuring the optical density of culture broth at 600 nm. The cell concentration was also determined by measuring the dry cell weight. Glucose concentration was determined by a glucose analyzer (YSI, Ohio, USA) and ammonia was measured by the indophenol method (5). Protein was determined by a modification of Lowry's method. The PHB concentration was determined by gas chromatography (Hewlett Packard, Avondale, USA) with benzoic acid as an internal standard (6). Cells harvested from the fermentation broth were resuspended with 50mM Tris-HC1 (pH8.1) containing 1 mM EDTA and 1 mM PMSF as protease inhibitor and then disrupted with a sonicator (Sonic and Materials Inc., USA). The lysate was centrifuged at 5,000 × g for 10 rain, and the supernatant was used for the determination of enzyme activity. The enzyme activity of glucose-6-phosphate dehydrogenase (GPDH) was determined by measuring the N A D P H produced (7). 3-Ketothiolase (KT) was assayed by using the thiolysis reaction (8) and citrate synthase (CS) was determined by measuring the appearance of the free SH group of the released CoASH by use of 5,5'-dithio bis-(2-nitrobenzoate) (9). Cell growth and PHB accumulation were monitored at three different values of C / N ratio (4.1, 7.1, and 32.8). The ratios were calculated on a molar basis by varying the ammonium sulfate concentration at a fixed glucose concentration, 20 g/l. The effects of the C / N ratio on cell growth and PHB accumulation in batch fermentations of A. eutrophus NCIMB 11599 are summarized in Fig. 1. The highest cell growth was achieved at a C / N ratio of 4.1, while high PHB accumulations, with concentrations of up to 6.0 g/l, were obtained at C / N ratios of both 4.1 and 7.1. At the C / N ratio of 4.1, PHB accumulation stopped after 31 h of cultivation due to glucose exhaustion. However, a higher PHB accumulation would have been achieved by additional feeding of glucose since the G P D H and KT activities were stably maintained. As the C / N ratio increased, so did the PHB content (see Fig. 2a).
In most bacteria synthesizing poly-/3-hydroxybutyrate (PHB), the accumulation of the polymer is reported to be triggered by limitation of nutrients such as nitrogen or phosphate (1, 2). Mass production of PHB has therefore been achieved by controlled carbon/nitrogen (C/N) feeding (3). At low C / N ratios cell growth is favored, while PHB accumulation increases at higher C / N values. This indicates that the enzyme activities related to PHB synthesis are directly affected by C / N ratio. However, very little information is available on the enzymes involved in PHB synthesis during fermentation. It is well known that glucose-6-phosphate dehydrogenase (GPDH) and /3ketothiolase (KT) (4) are the key enzymes in glucose assimilation and PHB synthesis, respectively. The enzyme activities of both G P D H and KT need therefore to be controlled optimally for the fermentative production of PHB. In addition, since acetyl-CoA, a precursor metabolite of PHB, is oxidized by citrate synthase (CS), the activity of CS is also of considerable importance in PHB synthesis. In this study, we investigated the effects of the C / N ratio on cell growth and PHB accumulation during the cultivation of Alcaligenes eutrophus NCIMB 11599, a glucose-utilizing mutant, by using glucose and ammonium sulfate as the carbon and nitrogen sources, respectively. The enzyme activities of G P D H , KT, and CS were also determined during the fermentation, and their relationships with PHB accumulation analyzed. The fermentation medium contained (per liter): 20g glucose, 3.32 g Na2HPO4.12H20, 0.83 g KH2PO4, 0.2 g MgSO4-7HzO, 20 mg FeSO4.7H20, 10 mg CaC12.2H20, 1 ml of trace element solution (0.3 g H3BO3, 0.2 g COC126H20, 0.1g ZnSO4.7H20, 30mg MnCI2.4H20, 30mg NaMoO4- 2H20, 20 mg NiCI2.6H20, 10 mg CuSO4.5H20 per liter of 0.1 N HCI) and a predetermined amount of (NH4)2SO4. Fermentation was carried out in a 5-1 jar fermentor (Korea Fermentor Co., Inchon, Korea) equipped with a DO analyzer and a pH controller. Two hundred ml of the seed culture cultivated at 30°C for 24 h in shake flasks was transferred to the fermentor containing 2.8 1 of * Corresponding author. 416
Vot. 76, 1993
NOTES
(b)
(a) ,
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,
417
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....... ,
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7
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.v. -V. ~5.o )
m
~2.5
0.0 1.8
•
~
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1.2
009 0.6
0.06
v
0.03 0.00
0.0 10
20
30
40
10
20
30
10
40
20
30
40
50
Culture time (h) FIG. 1. Effect of initial molar ratio of carbon to nitrogen (C/N ratio) on substrate consumption, cell growth, PHB accumulation, and specific activities of enzymes. C/N ratios are: (a) 4.1, (b) 7.1, and (c) 32.8. Specific activities of enzymes were determined by enzyme activity per unit intracellular protein concentration. Details are described in the text. In case o f the highest C / N ratio o f 32.8, the cellular P H B content rose to 90.00//oo after 3 2 h cultivation, while as shown in Fig. 2b, the yield o f P H B from glucose reached 43%. Considering that the theoretical m a x i m u m yield is calculated to be 4 8 ~ ( w / w ) (10), this value reveals that glucose was almost completely converted to P H B synthesis at the high C / N ratio. Specific activities o f G P D H , KT, and CS during the ferm e n t a t i o n were measured for the three tested values o f C / N ratio (Fig. 1). C o m p a r e d with CS and G P D H , the specific activity o f KT was relatively high, reaching its maxim u m immediately after the exhaustion o f the nitrogen source for all cases. This m a y suggest that when nitrogen source is exhausted a c e t y l - C o A is preferably converted to acetoacetyl-CoA, the m a j o r precursor for P H B synthesis, due to higher KT activity. Utilization o f acetyl-CoA for cellular energy metabolism (i.e. the T C A cycle) is even further diminished under nitrogen-limited conditions since the CS activity is significantly inhibited by a high intracellular concentration o f N A D H or N A D P H , as reported by A n d e r s o n et al. (11). The flow o f acetyl-CoA to aceto- acetyl-CoA can thus be even m o r e facilitated. This also supports the previous observation that P H B synthesis is commenced after the exhaustion o f the nitrogen source (1, 2). It was also noted that the specific activity o f G P D H was stably maintained, and the specific glucose c o n s u m p t i o n
rate was kept within a range o f 0.11-0.27 (g-glucose/gD C W / h ) during the P H B accumulation phase. It was interesting that the specific G P D H activity was not significantly affected by the C / N ratio o f the culture medium. However, since a greater glucose uptake is expected to increase the intracellular availability o f acetyl-CoA, it is believed i m p o r t a n t to achieve a stable and higher level o f G P D H for P H B accumulation. In this connection, a m o r e elaborate study to enhance the enzyme activity is considered necessary. In conclusion, employing a suitable C / N ratio in the cul-
(a) ~ I 0 0
.~
,
,
(b) A'4
80
.~ 60
5O
,
-
~'~ 4 0
-
.E~3o
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i
t
t
i
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i
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I
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0 I0 20 30 40 50 Culture time (h)
o
o I0 20 30 40 50 Culture time (h)
FIG. 2. Cellular PHB content (a) and PHB yield from glucose (b). Symbols: C/N ratio=4.1 (©); 7.1 (o); 32.8 (4).
418
J. FERMENT. BIOENG.,
LEE ET AL.
ture m e d i u m is o n e o f the i m p o r t a n t requisites for achieving high p r o d u c t i v i t y in P H B synthesis, in that it can p r o vide a m o r e efficient o r b e t t e r - b a l a n c e d e n z y m e system for P H B a c c u m u l a t i o n . Efforts to i m p r o v e the yield o f the ferm e n t a t i o n system, i n c l u d i n g genetic m a n u p u l a t i o n o f the strain, are c u r r e n t l y being m a d e t a k i n g this o b s e r v a t i o n into a c c o u n t . REFERENCES 1. Oeing, V. and Schlegel, H.G.: /%Ketothiolase from Hydrogenomonas eutropha HI6 and its significance in the regulation of poly-/3-hydroxybutyrate metabolism. Biochem. J., 134, 239-248 (1973). 2. Schlegel, H. G., Gottschalk, G., and Bartha, R. V.: Formation and utilization of poly-t3-hydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature, 29, 463-465 (1961). 3. Suzuki, T., Yamane, Y., and Shimizu, S.: Mass production of poly-t3-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding. Appl. Microbiol. Biotechnol., 24, 370374 (1986). 4. Haywood, G. H., Anderson, A. J., Chu, L., and Dawes, E. A.: Characterization of two 3-ketothiolase in the polyhydroxyal-
5.
6.
7. 8. 9. 10. 11.
kanoates synthesizing organism Alcaligenes eutrophus. FEMS Microbiol. Lett., 52, 91-96 (1988). Srienc, F., Arnold, B., and Bailey, J . E . : Characterization of intracellular accumulation of poly-13-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 flow cytometry. Biotechnol. Bioeng., 26, 982-987 (1984). Braunegg, G., Sonnleitner, B., and Lafferty, R. M.: A rapid gas chromatographic method for the determination of poly-/3-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol., 6, 29-37 (1978). Olive, C. and Levy, H. R.: Methods in enzymology, vol. 41, p. 196-201. Academic Press, New York (1975). Nishimura, T., Saito, T., and Tomita, K.: Purification and properties of /3-ketothiolase from Zoogloea ramigera. Arch. Microbiol., 116, 21-27 (1978). Srere, P. A.: Methods in enzymology, vol. 13, p. 3-11. Academic Press, New York (1969). Yamane, T.: Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study. Biotechnol. Bioeng., 41, 165-170 (1992). Anderson, A . J . and Dawes, E.A.: Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev., 54, 450-472 (1990).
Production of Poly-/3-Hydroxybutyrate and Measurement of Related Enzyme Activities in Alcaligenes eutrophus IN YOUNG LEE, 1 SOO WAN NAM, 1 EUN SOO CHOI, l H O NAM CHANG, 2 AND YOUNG HOON PARK 1. Genetic Engineering Research Institute, KIST, P.O. Box 17, Taedok Science Town, Taejon 305-606, ~ and Department of Chemical Engineering, Korea Advanced Institute of Science and Technology, Taedok Science Town, Taejon 305-701, 2 Korea Received 10 May 1993/Accepted 27 July 1993
Activities of ~-ketothiolase (KT) and glucose-6-phosphate dehydrogenase (GPDH) were determined during the fermentation of Alcaligenes eutrophus NCIMB 11599 at three different values of C / N ratio. As the C / N ratio increased the cell growth decreased due to the limitation of nitrogen, while the cellular poly-~-hydroxybutyrate (PHB) content increased. At an initial C / N ratio of 32.8, the cellular PHB content reached 90.0Yo after 32 h cultivation. The specific activity of ~-ketothiolase in each fermentation was the highest immediately after exhaustion of the nitrogen source. Specific activities of ~-ketothiolase and glucose-6-phosphate dehydrogenase were maintained stably within the tested range of C / N ratio even after the N-source was completely exhausted.
the fermentation medium. The air flow rate and agitation speed were 0.5-1.0vvm and 300-600rpm, respectively. The pH was controlled automatically at 7.0 with 4 N NaOH. Cell growth was monitored by measuring the optical density of culture broth at 600 nm. The cell concentration was also determined by measuring the dry cell weight. Glucose concentration was determined by a glucose analyzer (YSI, Ohio, USA) and ammonia was measured by the indophenol method (5). Protein was determined by a modification of Lowry's method. The PHB concentration was determined by gas chromatography (Hewlett Packard, Avondale, USA) with benzoic acid as an internal standard (6). Cells harvested from the fermentation broth were resuspended with 50mM Tris-HC1 (pH8.1) containing 1 mM EDTA and 1 mM PMSF as protease inhibitor and then disrupted with a sonicator (Sonic and Materials Inc., USA). The lysate was centrifuged at 5,000 × g for 10 rain, and the supernatant was used for the determination of enzyme activity. The enzyme activity of glucose-6-phosphate dehydrogenase (GPDH) was determined by measuring the N A D P H produced (7). 3-Ketothiolase (KT) was assayed by using the thiolysis reaction (8) and citrate synthase (CS) was determined by measuring the appearance of the free SH group of the released CoASH by use of 5,5'-dithio bis-(2-nitrobenzoate) (9). Cell growth and PHB accumulation were monitored at three different values of C / N ratio (4.1, 7.1, and 32.8). The ratios were calculated on a molar basis by varying the ammonium sulfate concentration at a fixed glucose concentration, 20 g/l. The effects of the C / N ratio on cell growth and PHB accumulation in batch fermentations of A. eutrophus NCIMB 11599 are summarized in Fig. 1. The highest cell growth was achieved at a C / N ratio of 4.1, while high PHB accumulations, with concentrations of up to 6.0 g/l, were obtained at C / N ratios of both 4.1 and 7.1. At the C / N ratio of 4.1, PHB accumulation stopped after 31 h of cultivation due to glucose exhaustion. However, a higher PHB accumulation would have been achieved by additional feeding of glucose since the G P D H and KT activities were stably maintained. As the C / N ratio increased, so did the PHB content (see Fig. 2a).
In most bacteria synthesizing poly-/3-hydroxybutyrate (PHB), the accumulation of the polymer is reported to be triggered by limitation of nutrients such as nitrogen or phosphate (1, 2). Mass production of PHB has therefore been achieved by controlled carbon/nitrogen (C/N) feeding (3). At low C / N ratios cell growth is favored, while PHB accumulation increases at higher C / N values. This indicates that the enzyme activities related to PHB synthesis are directly affected by C / N ratio. However, very little information is available on the enzymes involved in PHB synthesis during fermentation. It is well known that glucose-6-phosphate dehydrogenase (GPDH) and /3ketothiolase (KT) (4) are the key enzymes in glucose assimilation and PHB synthesis, respectively. The enzyme activities of both G P D H and KT need therefore to be controlled optimally for the fermentative production of PHB. In addition, since acetyl-CoA, a precursor metabolite of PHB, is oxidized by citrate synthase (CS), the activity of CS is also of considerable importance in PHB synthesis. In this study, we investigated the effects of the C / N ratio on cell growth and PHB accumulation during the cultivation of Alcaligenes eutrophus NCIMB 11599, a glucose-utilizing mutant, by using glucose and ammonium sulfate as the carbon and nitrogen sources, respectively. The enzyme activities of G P D H , KT, and CS were also determined during the fermentation, and their relationships with PHB accumulation analyzed. The fermentation medium contained (per liter): 20g glucose, 3.32 g Na2HPO4.12H20, 0.83 g KH2PO4, 0.2 g MgSO4-7HzO, 20 mg FeSO4.7H20, 10 mg CaC12.2H20, 1 ml of trace element solution (0.3 g H3BO3, 0.2 g COC126H20, 0.1g ZnSO4.7H20, 30mg MnCI2.4H20, 30mg NaMoO4- 2H20, 20 mg NiCI2.6H20, 10 mg CuSO4.5H20 per liter of 0.1 N HCI) and a predetermined amount of (NH4)2SO4. Fermentation was carried out in a 5-1 jar fermentor (Korea Fermentor Co., Inchon, Korea) equipped with a DO analyzer and a pH controller. Two hundred ml of the seed culture cultivated at 30°C for 24 h in shake flasks was transferred to the fermentor containing 2.8 1 of * Corresponding author. 416
Vot. 76, 1993
NOTES
(b)
(a) ,
~
,
417
(C)
E
2O
"-I ~
15
O 0.45
10(
0.30
....... ,
5
0
0.15 0.00
7
I0.0
•
~>
'7.5
G
.v. -V. ~5.o )
m
~2.5
0.0 1.8
•
~
~>
1.2
009 0.6
0.06
v
0.03 0.00
0.0 10
20
30
40
10
20
30
10
40
20
30
40
50
Culture time (h) FIG. 1. Effect of initial molar ratio of carbon to nitrogen (C/N ratio) on substrate consumption, cell growth, PHB accumulation, and specific activities of enzymes. C/N ratios are: (a) 4.1, (b) 7.1, and (c) 32.8. Specific activities of enzymes were determined by enzyme activity per unit intracellular protein concentration. Details are described in the text. In case o f the highest C / N ratio o f 32.8, the cellular P H B content rose to 90.00//oo after 3 2 h cultivation, while as shown in Fig. 2b, the yield o f P H B from glucose reached 43%. Considering that the theoretical m a x i m u m yield is calculated to be 4 8 ~ ( w / w ) (10), this value reveals that glucose was almost completely converted to P H B synthesis at the high C / N ratio. Specific activities o f G P D H , KT, and CS during the ferm e n t a t i o n were measured for the three tested values o f C / N ratio (Fig. 1). C o m p a r e d with CS and G P D H , the specific activity o f KT was relatively high, reaching its maxim u m immediately after the exhaustion o f the nitrogen source for all cases. This m a y suggest that when nitrogen source is exhausted a c e t y l - C o A is preferably converted to acetoacetyl-CoA, the m a j o r precursor for P H B synthesis, due to higher KT activity. Utilization o f acetyl-CoA for cellular energy metabolism (i.e. the T C A cycle) is even further diminished under nitrogen-limited conditions since the CS activity is significantly inhibited by a high intracellular concentration o f N A D H or N A D P H , as reported by A n d e r s o n et al. (11). The flow o f acetyl-CoA to aceto- acetyl-CoA can thus be even m o r e facilitated. This also supports the previous observation that P H B synthesis is commenced after the exhaustion o f the nitrogen source (1, 2). It was also noted that the specific activity o f G P D H was stably maintained, and the specific glucose c o n s u m p t i o n
rate was kept within a range o f 0.11-0.27 (g-glucose/gD C W / h ) during the P H B accumulation phase. It was interesting that the specific G P D H activity was not significantly affected by the C / N ratio o f the culture medium. However, since a greater glucose uptake is expected to increase the intracellular availability o f acetyl-CoA, it is believed i m p o r t a n t to achieve a stable and higher level o f G P D H for P H B accumulation. In this connection, a m o r e elaborate study to enhance the enzyme activity is considered necessary. In conclusion, employing a suitable C / N ratio in the cul-
(a) ~ I 0 0
.~
,
,
(b) A'4
80
.~ 60
5O
,
-
~'~ 4 0
-
.E~3o
~
i
t
t
i
P
i
k
I
2o
~ eO e~
0 I0 20 30 40 50 Culture time (h)
o
o I0 20 30 40 50 Culture time (h)
FIG. 2. Cellular PHB content (a) and PHB yield from glucose (b). Symbols: C/N ratio=4.1 (©); 7.1 (o); 32.8 (4).
418
J. FERMENT. BIOENG.,
LEE ET AL.
ture m e d i u m is o n e o f the i m p o r t a n t requisites for achieving high p r o d u c t i v i t y in P H B synthesis, in that it can p r o vide a m o r e efficient o r b e t t e r - b a l a n c e d e n z y m e system for P H B a c c u m u l a t i o n . Efforts to i m p r o v e the yield o f the ferm e n t a t i o n system, i n c l u d i n g genetic m a n u p u l a t i o n o f the strain, are c u r r e n t l y being m a d e t a k i n g this o b s e r v a t i o n into a c c o u n t . REFERENCES 1. Oeing, V. and Schlegel, H.G.: /%Ketothiolase from Hydrogenomonas eutropha HI6 and its significance in the regulation of poly-/3-hydroxybutyrate metabolism. Biochem. J., 134, 239-248 (1973). 2. Schlegel, H. G., Gottschalk, G., and Bartha, R. V.: Formation and utilization of poly-t3-hydroxybutyric acid by Knallgas bacteria (Hydrogenomonas). Nature, 29, 463-465 (1961). 3. Suzuki, T., Yamane, Y., and Shimizu, S.: Mass production of poly-t3-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding. Appl. Microbiol. Biotechnol., 24, 370374 (1986). 4. Haywood, G. H., Anderson, A. J., Chu, L., and Dawes, E. A.: Characterization of two 3-ketothiolase in the polyhydroxyal-
5.
6.
7. 8. 9. 10. 11.
kanoates synthesizing organism Alcaligenes eutrophus. FEMS Microbiol. Lett., 52, 91-96 (1988). Srienc, F., Arnold, B., and Bailey, J . E . : Characterization of intracellular accumulation of poly-13-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 flow cytometry. Biotechnol. Bioeng., 26, 982-987 (1984). Braunegg, G., Sonnleitner, B., and Lafferty, R. M.: A rapid gas chromatographic method for the determination of poly-/3-hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol., 6, 29-37 (1978). Olive, C. and Levy, H. R.: Methods in enzymology, vol. 41, p. 196-201. Academic Press, New York (1975). Nishimura, T., Saito, T., and Tomita, K.: Purification and properties of /3-ketothiolase from Zoogloea ramigera. Arch. Microbiol., 116, 21-27 (1978). Srere, P. A.: Methods in enzymology, vol. 13, p. 3-11. Academic Press, New York (1969). Yamane, T.: Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study. Biotechnol. Bioeng., 41, 165-170 (1992). Anderson, A . J . and Dawes, E.A.: Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev., 54, 450-472 (1990).