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Enzymatic production of d -mannose from d -fructose by mannose isomerase
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 76, No. 3, 237-239. 1993
Enzymatic Production of D-Mannose from D-Fructose by Mannose Isomerase* YOSHIYUK1 T A K A S A K I , * K O W A S H I H I N O K I , Y O S H I O K A T A O K A , SE1JIRO F U K U Y A M A , N O B U H I R O N I S H I M U R A , S A C H I O H A Y A S H I , AND K I Y O H I S A I M A D A
Department of Materials Science, Faculty of Engineering, Miyazaki University, 1-1, Nishi, Gakuen-kibanadai, Miyazaki 889-21, Japan Received 20 January 1993/Accepted 29 June 1993
An enzymatic process for the production of t)-mannose from D-fructose was investigated by using a thermostable mannose isomerase from Pseudomonas sp. MI which was isolated from soil. The optimum temperature was around 60°C. The isomerization reaction progressed effectively at pH 8.0-8.5 within the tested pH range of 7.0 to 8.5 and with increasing concentrations of D-fructose between 5 and 40%, and about 25% of t~fructose was converted to t~-mannose. The conversion of o-glucose to D-mannose was also investigated under 30% D-glucose concentration with both mannose isomerase and glucose isomerase. At equilibrium, a mixture containing D-glucose (about 50%), D-fructose (about 38%) and a-mannnse (about 12%) was obtained.
o - M a n n o s e has recently been used for variety o f purp o s e s - a s a material for the syntheses o f mannitol, drugs and sugar chains in glycoproteins, and as a constituent o f chemically defined media for cell culture. Also, it has recently been shown that o - m a n n o s e inhibits the growth o f Salmonella typhimurium and may be used as a feed additive for fowl such as chickens to prevent c o n t a m i n a t i o n by the bacterium (I-3). However, o - m a n n o s e is very expensive because it is prepared by the acid-hydrolysis o f plant m a n n a n or by molybdate-catalyzed epimerization o f glucose. In order to develop a new technique for the production o f D-mannose, the authors have studied the enzymatic conversion o f D-fructose to o - m a n n o s e by mannose isomerase. This enzyme, which converts between Dmannose and o-fructose reversibly, was found by Palleroni and D o u d o r o f f as the first enzyme isomerizing free hexose in Pseudomonas saccharophila (4) and by the first author in Xanthomonas rubrilineans (5-7) and Streptomyces aerocolorigenes (8). However, these enzymes have an optimum temperature o f 35--40°C and are thus unsuitable for industrial application. It was found that a bacterium newly isolated from soil and identified as Pseudomonas sp. MI produced a thermostable m a n n o s e isomerase. The strain was cultivated in a medium (40ml, p H 7.0) containing: fish meal extract 2.5o/o, o-glucose (or D-galactose) 1.0%, K2HPO4 0.25°/0, MgSO4-7H20 0 . 3 % , and MnSO4 6 × 10 4M in a 200-ml Erlenmeyer flask. The cultivation was carried out for 24 h at 30°C on a rotary shaker at 150rpm. The cells were harvested by centrifugation (10,000rpm, 3 rain), washed with distilled water and then sonicated for 20 s at 20KC or autolyzed in the presence o f 0.1% cetylpyridinium chloride at pH 8.0 and at 30°C. The cell debris were removed by centrifugation (10,000rpm, 5 min). To the resultant supernatant (100ml), a m m o n i u m sulfate was a d d e d to 60% saturation. The precipitate formed was collected, dis-
solved in distilled water, and then dialyzed against 2.5 x 10 3M Tris-HCl buffer (pH 7.0). The dialysate was put on a D E A E - S e p h a r o s e column (2.1 × 27.5 cm) equilibrated with 2.5 x 10 3 M Tris-HCl buffer (pH 7.0) containing 1 × 10 3 M L-cysteine. The column was washed with the same buffer and then the enzyme was eluted linearly, increasing the concentration o f KC1 in the buffer from 0 to 1.0 M. The enzyme fractions were collected, concentrated with a m e m b r a n e filter (Amicon YM 10 membrane), and then dialyzed against distilled water. The fraction thus obtained (2.05 units/Azs0,m) was used for this study. The assay o f the mannose isomerase was done in a mixture (0.2 ml, pH 7.0) containing 0.1 M o-mannose, 5 × 10 2 M p h o s p h a t e buffer and the enzyme solution. The
60 >,
"~ 50 % ~ 40
c~
20
I
I
30
L,O
I
~
I
I
6C
7C
r e i n 2 e r a t ure(*C~
FIG. 1. Effect of temperature on activity and stability of the mannose isomerase. Symbols: ,;, optimum temperature [The reaction was done in a mixture (0.4ml) containing 5 x 10 2 M phosphate buffer (pH7.0), 0.1M D-mannose and 0.13 units of mannose isomerase, which was incubated for 30mini; O, heat stability [A mixture (0.2ml) containing 5 x 10 ZM phosphate buffer (pH 7.0), a 0.2 units of mannose isomerase was incubated for 10 min at various temperatures as indicated and the residual enzyme activity was measured].
* Corresponding author. t A part of this paper was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, March 30-April 2, 1990 (Abstracts of Papers, p. 578). 237
238
TAKASAKI ET AI,.
J. t"ERMI:NI. BIoh?,,'(i.,
:00
80
-~ 20
6O 0 u
o L~
"o---------o_
o c- i 0 c
uO
5 2O
i
i
i
I
l
10
20
30
40
50
I
React ton t irne(h)
10
FIG. 2. Effect of pH on the mannose isomerase reaction. The reaction was done in a mixture (I.0 ml) containing 20(1 mg (1t D-fructose, 4 " 10 ' M Tris buffer at various pHs as indicated, and 0.2 units of m a n n o s e i s o m e r a s e at 50°C. Symbols: , , pH 7.0: • , pH 7.5; ,., pHS. 0; .:, p118.5.
I
I
20 30 40 R e a c t i o n t line(h)
1
50
FIG. 4. Time course of conversion of D-glucose to i)-mannose with the mannose isomerase and glucose isomerase. The reaction was done in a mixture ( I m l ) containing 300 mg D-glucose, 5 × 10 -"M Tris butl'er (pH 7.0), 1 , 10 : M MgSO4, 0.3 units of mannose isomerase and 0.15 units of glucose isomerase at 50~C. Symbols: , D-ghtcose; D, D-fructose: O, t)-mannose.
100
merase reactions at various pHs ranging from 7.0 to 8.5. The reaction was done under 20%o D-fructose for 2 d at 50°C. The most suitable pH of the reaction seemed to be 8.0-8.5 under these conditions. Figure 3 shows the effect of the concentration of D-fructose on the mannose reaction. The reaction progressed steadily without significant inhibition at increasing substrate concentrations, and about 25°~ of D-fructose was
o 60
~ 1-0
converted - -
-0--
•
2O
I
1O
I
I
20 30 React ~on tlrne(t')
I
i
z.o
5C,
FIG. 3. I.ffcct (1t concentration of l)-fructose on tile manuose isomerase reaction. The reaction ~ a s done in a mixture 11.0 ml) containiug 50-404) mg of D-fructose, 4 • 10 -' M Tris buffer (pH 7.0), and 0.4 units of man nose isomerase at 5 0 ' ( '. Symbols lot substratc concentrations: @, 5!~.; , i()0;~. A 20o,i~: .., 30of~; i , 40oi,.
mixture was incubated for 30 min at 50°C. The reaction was stopped by adding 0.8 ml of 0.5 M HC104 and Dfructose formed was measured by the cysteine-carbazole method (10, 11). One unit of the enzyme was defined as the amount of enzyme producing o n e / t m o l of D-fructose from D-mannose in one rain under these assay conditions. The sugar constituent of the enzyme reaction mixture was analyzed by high-performance liquid chromatography using a column of Wakobeads T-131-E with distilled water as the eluent. Figure 1 shows the effects of temperature on the activity and stability of the enzyme. The o p t i m u m temperature was around 60°C (30 min-reaction under 0.1 M o-mannose). The enzyme was unstable above 50°C. A m o n g sugars tested, only D-mannose and D-lyxose were isomerized to corresponding ketoses. Fugure 2 shows the effect of pH on the mannose iso-
to D-mannose.
Figure 4 shows the time courses of conversion of Dglucose to o-mannose with the mannose isomerase and glucose isomerase (9). At the equilibrium state, a mixture containing o-glucose (about 5 0 ~ ) , D-fructose (about 38%o) and D-mannose (about 12°o/) was obtained. From the results it appears that this enzymatic process for the production of D-mannose using mannose isomerase is feasible. REFERENCES
1. Oyofo, B. A., Droleskey, R . E . , Norman, J . O . , Mollenhauer, H. H., Ziprin, R. L., Collier, 1). E., and I)eLoach, J. R.: Inhibition by mannose of in vitro colonization of chicken small intestine by Salmonella typhimurium. Poultry Science, 68, 1351 - 1356 (1989). 2. Oyofo, B . A . , Deloach, J. R., Corrier, D . E . , Norman, J . O . , Ziprin, R. 1.., and Mollenhauer, H . H . : Prevention of Sahnonella typhimurium colonization of broilers with D-mannose. Poultry Science, 68, 1357-1360 (1989).
3 Izat, A. 1.., Hierholzer, R . E . , Koper, J . M . , Adams, M . H . , Reiber, M . A . , and Mcginnis, J . P . : Research note: effects of D-mannose on incidence and levels of Salmonella ill ceca and carcass samples of market age broilers. Poultry Science, 69. 2244-2247 (1990). 4. Palleroni, N . J . and Doudoroff, M.; Mannose isomcrase o l Pseudomonas saccharophila. J. Biol. Chem., 218, 535-548 (1959). 5. Ta ka s a ki , Y. and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VI. On the o-mannose isomerase of strain S-48. J. Agr. (?hem. Soc. Japan, 37, 524-528 (1963). 6. Takasaki, Y. and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VII. Constitutive production of mannose
VoL. 76, 1993 isomerase by Xanthomonas species. Agric. Biol. Chem., 28, 601604 (1964). 7. Takasaki, ¥,, Takano, S., and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VIII. Purification and some properties of mannose isomerase from Xanthomonas rubrilinearts S-48. Agric. Biol. Chem., 28, 605-609 (1964). 8. Takasaki, Y.: Kinetic and equilibrium studies on D-mannose-D-
NOTES
239
fructose isomerization catalyzed by mannose isomerase from Streptono,ces aerocolorigenes. Agric. Biol. Chem., 31,435-440 (1967). 9. Takasaki, ¥.: Studies on sugar-isomerizing enzymes. Production and utilization of glucose isomerase from Streptom.vces sp. Agric. Biol. Chem., 30, 1247-1253 (1966).
Vol. 76, No. 3, 237-239. 1993
Enzymatic Production of D-Mannose from D-Fructose by Mannose Isomerase* YOSHIYUK1 T A K A S A K I , * K O W A S H I H I N O K I , Y O S H I O K A T A O K A , SE1JIRO F U K U Y A M A , N O B U H I R O N I S H I M U R A , S A C H I O H A Y A S H I , AND K I Y O H I S A I M A D A
Department of Materials Science, Faculty of Engineering, Miyazaki University, 1-1, Nishi, Gakuen-kibanadai, Miyazaki 889-21, Japan Received 20 January 1993/Accepted 29 June 1993
An enzymatic process for the production of t)-mannose from D-fructose was investigated by using a thermostable mannose isomerase from Pseudomonas sp. MI which was isolated from soil. The optimum temperature was around 60°C. The isomerization reaction progressed effectively at pH 8.0-8.5 within the tested pH range of 7.0 to 8.5 and with increasing concentrations of D-fructose between 5 and 40%, and about 25% of t~fructose was converted to t~-mannose. The conversion of o-glucose to D-mannose was also investigated under 30% D-glucose concentration with both mannose isomerase and glucose isomerase. At equilibrium, a mixture containing D-glucose (about 50%), D-fructose (about 38%) and a-mannnse (about 12%) was obtained.
o - M a n n o s e has recently been used for variety o f purp o s e s - a s a material for the syntheses o f mannitol, drugs and sugar chains in glycoproteins, and as a constituent o f chemically defined media for cell culture. Also, it has recently been shown that o - m a n n o s e inhibits the growth o f Salmonella typhimurium and may be used as a feed additive for fowl such as chickens to prevent c o n t a m i n a t i o n by the bacterium (I-3). However, o - m a n n o s e is very expensive because it is prepared by the acid-hydrolysis o f plant m a n n a n or by molybdate-catalyzed epimerization o f glucose. In order to develop a new technique for the production o f D-mannose, the authors have studied the enzymatic conversion o f D-fructose to o - m a n n o s e by mannose isomerase. This enzyme, which converts between Dmannose and o-fructose reversibly, was found by Palleroni and D o u d o r o f f as the first enzyme isomerizing free hexose in Pseudomonas saccharophila (4) and by the first author in Xanthomonas rubrilineans (5-7) and Streptomyces aerocolorigenes (8). However, these enzymes have an optimum temperature o f 35--40°C and are thus unsuitable for industrial application. It was found that a bacterium newly isolated from soil and identified as Pseudomonas sp. MI produced a thermostable m a n n o s e isomerase. The strain was cultivated in a medium (40ml, p H 7.0) containing: fish meal extract 2.5o/o, o-glucose (or D-galactose) 1.0%, K2HPO4 0.25°/0, MgSO4-7H20 0 . 3 % , and MnSO4 6 × 10 4M in a 200-ml Erlenmeyer flask. The cultivation was carried out for 24 h at 30°C on a rotary shaker at 150rpm. The cells were harvested by centrifugation (10,000rpm, 3 rain), washed with distilled water and then sonicated for 20 s at 20KC or autolyzed in the presence o f 0.1% cetylpyridinium chloride at pH 8.0 and at 30°C. The cell debris were removed by centrifugation (10,000rpm, 5 min). To the resultant supernatant (100ml), a m m o n i u m sulfate was a d d e d to 60% saturation. The precipitate formed was collected, dis-
solved in distilled water, and then dialyzed against 2.5 x 10 3M Tris-HCl buffer (pH 7.0). The dialysate was put on a D E A E - S e p h a r o s e column (2.1 × 27.5 cm) equilibrated with 2.5 x 10 3 M Tris-HCl buffer (pH 7.0) containing 1 × 10 3 M L-cysteine. The column was washed with the same buffer and then the enzyme was eluted linearly, increasing the concentration o f KC1 in the buffer from 0 to 1.0 M. The enzyme fractions were collected, concentrated with a m e m b r a n e filter (Amicon YM 10 membrane), and then dialyzed against distilled water. The fraction thus obtained (2.05 units/Azs0,m) was used for this study. The assay o f the mannose isomerase was done in a mixture (0.2 ml, pH 7.0) containing 0.1 M o-mannose, 5 × 10 2 M p h o s p h a t e buffer and the enzyme solution. The
60 >,
"~ 50 % ~ 40
c~
20
I
I
30
L,O
I
~
I
I
6C
7C
r e i n 2 e r a t ure(*C~
FIG. 1. Effect of temperature on activity and stability of the mannose isomerase. Symbols: ,;, optimum temperature [The reaction was done in a mixture (0.4ml) containing 5 x 10 2 M phosphate buffer (pH7.0), 0.1M D-mannose and 0.13 units of mannose isomerase, which was incubated for 30mini; O, heat stability [A mixture (0.2ml) containing 5 x 10 ZM phosphate buffer (pH 7.0), a 0.2 units of mannose isomerase was incubated for 10 min at various temperatures as indicated and the residual enzyme activity was measured].
* Corresponding author. t A part of this paper was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Kyoto, March 30-April 2, 1990 (Abstracts of Papers, p. 578). 237
238
TAKASAKI ET AI,.
J. t"ERMI:NI. BIoh?,,'(i.,
:00
80
-~ 20
6O 0 u
o L~
"o---------o_
o c- i 0 c
uO
5 2O
i
i
i
I
l
10
20
30
40
50
I
React ton t irne(h)
10
FIG. 2. Effect of pH on the mannose isomerase reaction. The reaction was done in a mixture (I.0 ml) containing 20(1 mg (1t D-fructose, 4 " 10 ' M Tris buffer at various pHs as indicated, and 0.2 units of m a n n o s e i s o m e r a s e at 50°C. Symbols: , , pH 7.0: • , pH 7.5; ,., pHS. 0; .:, p118.5.
I
I
20 30 40 R e a c t i o n t line(h)
1
50
FIG. 4. Time course of conversion of D-glucose to i)-mannose with the mannose isomerase and glucose isomerase. The reaction was done in a mixture ( I m l ) containing 300 mg D-glucose, 5 × 10 -"M Tris butl'er (pH 7.0), 1 , 10 : M MgSO4, 0.3 units of mannose isomerase and 0.15 units of glucose isomerase at 50~C. Symbols: , D-ghtcose; D, D-fructose: O, t)-mannose.
100
merase reactions at various pHs ranging from 7.0 to 8.5. The reaction was done under 20%o D-fructose for 2 d at 50°C. The most suitable pH of the reaction seemed to be 8.0-8.5 under these conditions. Figure 3 shows the effect of the concentration of D-fructose on the mannose reaction. The reaction progressed steadily without significant inhibition at increasing substrate concentrations, and about 25°~ of D-fructose was
o 60
~ 1-0
converted - -
-0--
•
2O
I
1O
I
I
20 30 React ~on tlrne(t')
I
i
z.o
5C,
FIG. 3. I.ffcct (1t concentration of l)-fructose on tile manuose isomerase reaction. The reaction ~ a s done in a mixture 11.0 ml) containiug 50-404) mg of D-fructose, 4 • 10 -' M Tris buffer (pH 7.0), and 0.4 units of man nose isomerase at 5 0 ' ( '. Symbols lot substratc concentrations: @, 5!~.; , i()0;~. A 20o,i~: .., 30of~; i , 40oi,.
mixture was incubated for 30 min at 50°C. The reaction was stopped by adding 0.8 ml of 0.5 M HC104 and Dfructose formed was measured by the cysteine-carbazole method (10, 11). One unit of the enzyme was defined as the amount of enzyme producing o n e / t m o l of D-fructose from D-mannose in one rain under these assay conditions. The sugar constituent of the enzyme reaction mixture was analyzed by high-performance liquid chromatography using a column of Wakobeads T-131-E with distilled water as the eluent. Figure 1 shows the effects of temperature on the activity and stability of the enzyme. The o p t i m u m temperature was around 60°C (30 min-reaction under 0.1 M o-mannose). The enzyme was unstable above 50°C. A m o n g sugars tested, only D-mannose and D-lyxose were isomerized to corresponding ketoses. Fugure 2 shows the effect of pH on the mannose iso-
to D-mannose.
Figure 4 shows the time courses of conversion of Dglucose to o-mannose with the mannose isomerase and glucose isomerase (9). At the equilibrium state, a mixture containing o-glucose (about 5 0 ~ ) , D-fructose (about 38%o) and D-mannose (about 12°o/) was obtained. From the results it appears that this enzymatic process for the production of D-mannose using mannose isomerase is feasible. REFERENCES
1. Oyofo, B. A., Droleskey, R . E . , Norman, J . O . , Mollenhauer, H. H., Ziprin, R. L., Collier, 1). E., and I)eLoach, J. R.: Inhibition by mannose of in vitro colonization of chicken small intestine by Salmonella typhimurium. Poultry Science, 68, 1351 - 1356 (1989). 2. Oyofo, B . A . , Deloach, J. R., Corrier, D . E . , Norman, J . O . , Ziprin, R. 1.., and Mollenhauer, H . H . : Prevention of Sahnonella typhimurium colonization of broilers with D-mannose. Poultry Science, 68, 1357-1360 (1989).
3 Izat, A. 1.., Hierholzer, R . E . , Koper, J . M . , Adams, M . H . , Reiber, M . A . , and Mcginnis, J . P . : Research note: effects of D-mannose on incidence and levels of Salmonella ill ceca and carcass samples of market age broilers. Poultry Science, 69. 2244-2247 (1990). 4. Palleroni, N . J . and Doudoroff, M.; Mannose isomcrase o l Pseudomonas saccharophila. J. Biol. Chem., 218, 535-548 (1959). 5. Ta ka s a ki , Y. and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VI. On the o-mannose isomerase of strain S-48. J. Agr. (?hem. Soc. Japan, 37, 524-528 (1963). 6. Takasaki, Y. and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VII. Constitutive production of mannose
VoL. 76, 1993 isomerase by Xanthomonas species. Agric. Biol. Chem., 28, 601604 (1964). 7. Takasaki, ¥,, Takano, S., and Tanabe, O.: Studies on the isomerization of sugars by bacteria. VIII. Purification and some properties of mannose isomerase from Xanthomonas rubrilinearts S-48. Agric. Biol. Chem., 28, 605-609 (1964). 8. Takasaki, Y.: Kinetic and equilibrium studies on D-mannose-D-
NOTES
239
fructose isomerization catalyzed by mannose isomerase from Streptono,ces aerocolorigenes. Agric. Biol. Chem., 31,435-440 (1967). 9. Takasaki, ¥.: Studies on sugar-isomerizing enzymes. Production and utilization of glucose isomerase from Streptom.vces sp. Agric. Biol. Chem., 30, 1247-1253 (1966).