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Properties of sorbitol dehydrogenase from Pseudomonas sp. KS-E1806 and comparison with other sorbitol dehydrogenases
JOURNALOP FERMENTATION AND BIOENGINEERING Vol. 84, No. 3, 254-256. 1997
Properties of Sorbitol Dehydrogenase from Pseudomonas sp. KS-El806 and Comparison with Other Sorbitol Dehydrogenases TOMOYUKI
MINAMIHARA*
AND
MASARU
SUZUKI
Research & Development Division, Kikkoman Corporation, Noda, Chiba 278, Japan Received 5 November 1996/Accepted
24 June 1997
Sorbitol dehydrogenase (SDH, EC 1.1.1.14) was purified to homogeneity from a cell-free extract of Pseudomonas sp. KS-El806 by chromatographies with QAK-Sephadex A-50, QAE-Toyopearl55OC, and Bio-Gel A-1.5m. The molecular weight of the enzyme was estimated to be 64,500 and 27,400 by gel tIltration and SDS-PAGE, respectively, suggesting that it has a dimeric structure. The enzyme was stable from pH 5.5 to 10.5, and below 40°C for 30 mitt at pH 9.0. u-Sorbitol and galactitol were good substrates of the enzyme, whereas it acted only slightly on xylitol and u-mannitol. The enzyme was superior to sheep liver SDH with respect to substrate specificity and pH stability, and it was more thermostable than a previously reported Pseudomonas sp. SDH. Compared with other known SDHs, the enzyme from Pseudomonas sp. KS-El806 has several advantages for practical use in the enzymatic analysis of u-sorbitoi. [Key words:
sorbitol dehydrogenase,
Pseudomonas sp. KS-E1806, enzymatic analysis]
D-Sorbitol is a sugar alcohol naturally occurring in food. This sugar alcohol is also used as a sweetener. Recently, it was reported that the D-sorbitol levels in erythrocytes from diabetic patients have significantly increased (1). Therefore, a means of determining Dsorbitol levels that is suitable for use in clinical practice is needed. Sorbitol dehydrogenase (SDH, EC 1.1.1.14), which catalyzes the oxidation of D-sorbitol to n-fructose with NAD+ as a cofactor, has been used for the enzymatic analysis of D-sorbitol concentrations in food and in human erythrocytes (1, 2). SDHs have been found in a wide variety of animals, plants, and microorganisms (3-16), and SDHs purified from the mammalians (4-8), Bacillus subtilis (ll), and Pseudomonas sp. (12) have been investigated in detail. Although sheep liver SDH is commonly used for the determination of Dsorbitol, because of the broad substrate specificity of this enzyme it is difficult to ascertain the amount of Dsorbitol accurately when xylitol, which occurs naturally in foods, is also present. Schneider and Giffhorn (12) reported an SDH from Pseudomonas sp. that acts only slightly on xylitol, but the temperature stability of the enzyme was not sufficient for enzymatic assay because it lost 50% of its activity after treatment at 40°C for 5 min and at 20°C for 32 h. Here, we describe a new type of bacterial SDH which is stable and exhibits strict substrate specificity. The bacterium was a gram-negative, non-spore forming short rod with polar flagella, and was classified as belonging to the genus Pseudomonas according to Bergey’s Manual of Determinative Bacteriology (17). SDH activity was determined spectrophotometrically by measuring the absorbance of NADH at 340 nm (s=6.3 cm2/pmol), using a Hitachi U-2000 spectrophotometer. The standard assay for substrate oxidation was carried out at 37°C with 3 ml of a reaction mixture containing 80mM Tris-HCl buffer, pH 9.0, 183 mM D-sorbitol (Nacalai Tesque, Tokyo), 0.5 mM NAD+, and appropriately diluted enzyme solution. For substrate reduction, the assay was done with 3 ml of a reaction mixture
containing 80mM MES-NaOH buffer, pH 6.5, 167 mM D-fructose (Wako Pure Chemical Industries, Osaka), 0.22 mM NADH, and appropriately diluted enzyme solution. One unit is defined as the amount of enzyme that reduces 1 pmol of NAD+ per min under the standard assay conditions. The protein concentration was measured with a protein assay kit (Bio-Rad, USA). Bovine serum albumin was used as the standard protein. All other chemicals were of the highest grade generally available. The enzyme was purified to homogeneity from a cellfree extract of Pseudomonas sp. KS-El806 by chromatographies with QAE-Sephadex A-50, QAE-Toyopearl 55OC, and Bio-Gel A-1.5m, as shown in Table 1. In the final stage, the enzyme was purified 339-fold with a recovery of 26.5% from the crude enzyme preparation. The purified enzyme showed a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Fig. 1). The molecular weight of the native enzyme was estimated to be 64,500 by HPLC gel filtration on a TSKgel G3OOOSWxr column. From the mobility in SDS-PAGE, the molecular weight of the enzyme protein monomer was calculated to be 27,400 (Fig. 1). These results suggest that the enzyme has a dimeric structure. The enzyme showed the maximum activity at pHs of about 6.5 and 10.0 under the reduction and oxidation assay conditions, respectively, and its optimum temperature was about 50°C under the standard oxidation assay conditions (data not shown). The enzyme was stable in a pH range of 5.5-10.5 on incubation at 30°C for 4 h (Fig. 2A). To check the thermal stability, the enzyme was incubated at various temperatures for 30min in 10mM Tris-HCl buffer, pH 8.0, containing 1OOmM KCl. The remaining activity was 100% of the original level at 4O”C, 13% at 45”C, and almost zero at 50°C (Fig. 2B). In addition, the enzyme could be stored at 20°C for at least a month without any significant loss of activity in the same buffer. These results indicated that the stability of the enzyme was sufficient for its practical use in the analysis of D-sorbitol.
* Corresponding author. 254
VOL. 84, 1997 TABLE 1.
NOTES
255
Purification of Pseudomonas sp. KS-El806 sorbitol dehydrogenase
Step Crude extract 1st QAE-Sephadex A-SW (batchwise) 2nd QAE-Sephadex A-50b 1st QAE-Toyopearl 550Cc 2nd QAE-Toyopearl 550Cc Bio-Gel A-l .5md
Total activity iu)
Total protein (mg)
8000 6000
32000 1OOoO
3700 2800 2200 2120
206 70.0 35.9 25.0
Specific activity Wmg) 0.25 0.60
Yield (%)
18.0 40.0 61.3 84.7
46.3 35.0 27.5 26.5
100 75.0
a Eluted with 10 mM Tris-HCI buffer (pH 8.0) containing 200 mM KCl. b Eluted with 20 mM sodium acetate buffer (pH 6.0) containing 70 mM KCI. c Eluted with 20mM sodium acetate buffer @H 6.0) containing 60 mM KCl. d The enzyme was filtered with 10 mM Tris-HCl buffer @H 8.0) containing 100 mM KC1 and 0.02% NaN,.
In the oxidation reaction, the enzyme acted on D-sorbitol and galactitol effectively, and slightly on D-mannitol and xylitol (Table 2). No oxidation of the following substrates was observed: ribitol, D-glucose, D-fructose, D-galactose, D-mannose, D-xylose, D-arabinose, maltose, sucrose, lactose, glycerol, and ethanol. With respect to the coenzyme, it was highly specific for NAD+ and did not act on NADP+. The K,,, values for D-SOrbitol and NAD+ (in the presence of 183 mM D-sorbitol) were 14.1 mM and 58 mM, respectively. Under the reduction assay conditions, the enzyme acted on D-fructose and D-tagatose as good substrates, and did not act on D-glucose, mannose, galactose, ribose, maltose, sucrose, lactose, D-xylose, and L-sorbose. The enzyme was also highly specific for NADH and did not act on NADPH.
5
9
7
11
30
40
50
Temperature
PH
60
(“C)
FIG. 2. pH and thermal stability of Pseudomonas sp. KS-El806 sorbitol dehydrogenase. (A) Effect of pH on enzyme stability. After the enzyme solution was maintained at various pHs ( x , 0.1 M carbonate-bicarbonate; 0, 0.1 M glycine-NaOH; 0, 0.1 M Tris-HCI; A, 0.1 M potassium phosphate; A, 0.1 M sodium acetate) at 30°C for 4 h, the remaining activity was assayed. (B) Effect of temperature on enzyme stability. After the enzyme was treated in 100 mM Tris-HCl, pH9.0, at various temperatures, for 30min, the remaining activity was assayed.
The K, values for o-fructose and NADH (in the presence of 167 mM D-fructose) were 105 mM and 62 PM, respectively. We compared the properties of SDH from Pseudomona~ sp. KS-El806 with those from Pseudomonas sp. (12), sheep liver, and B. subtilis (11). As shown in Table 3, the molecular weights and subunit structure of the Pseudomonas SDHs differ from those of the sheep liver and B. subtilis enzymes. Our SDH was superior to that from sheep liver with respect to pH stability, and to the Pseudomonas sp. SDH in terns of thermal stability. The substrate specificities of Pseudomonas SDHs, differed from those of the sheep liver and B. subtilis SDHs, which showed strong activities toward D-sorbitol, L-iditol, and xylitol, but not galactitol. With regard to their kinetic parameters, the K, value for D-sorbitol of the Pseudomonas sp. KS-El806 SDH was the smallest among those of the polyols tested. On the other hand, the K,,, value for galactitol of the Pseudomonas sp. SDH previously reported was smaller than that for D-sorbitol (12). The above results show that while the Pseudomonas SDHs are clearly different from those of sheep liver and B. subtilis in several respects, our SDH also differs from PseuTABLE 2. Kinetic parameters for various substrates of Pseudomonas sp. KS-El806 sorbitol dehydrogenase Substrate
FIG. 1. SDS-PAGE of purified Pseudomonas sp. KS-El806 sorbitol dehydrogenase. SDS-PAGE was performed with Multigel (lO-20% gradient gel; Daiichi Pure Chemicals Co. Ltd., Tokyo). The following proteins were used as standards: A, Phosphorylase b (97,400); B, bovine serum albumin (66,267); C, aldolase (42,400); D, carbonic anhydrase (30,000); E, trypsin inhibitor (20,100); F, lysozyme (14,400).
(Oxidation) D-Sorbitol Galactitol L-Iditol D-Arabitol Xylitol D-Mannitol (Reduction) D-Fructose D-Tagatose D-Arabinose
14.1 42.0 149 46.3 181.8 63.8
72.5 208 29.5 12.4 2.59 0.476
5.14 4.95 0.198 0.268 0.0142 0.00746
105 85.3 187
174 837 66.8
1.66 9.81 0.357
The reactions were performed by varying the concentrations under the standard oxidation or reduction conditions described in the text. The Michaelis constants and maximal velocities for each substrate were obtained from their Lineweaver-Burk plots.
256
MINAMIHARA
AND SUZUKI
J.
TABLE 3. Property
Comparison of some properties of various sorbitol dehydrogenases
Pseudomonas sp. KS-El806
Molecular weight (subunit structure) pH stabilityb Thermal stability V,,&K,e (K,) (%) (mM) o-Sorbitol L-Iditol Xylitol Galactitol
FERMENT. BIOENG..
Pseudomonas sp. (12)
64,500 (dimer) 5.5-10.5 100%
loo 3.9 0.3 96.3
( 14.1) (149 ) (181 ) ( 42 )
65,800 (dimer) 50%d
- (9.1)
- (3.1)
Sheep 1iveIg 129,000 (tetramer) 6-8 lOO%C 100 79.6 216 0.1
( 9.1) ( 16.0) ( 4.1) (617 )
B. subtilis(11) 153,000 (tetramer)
100 78.4 79.2 5.9
( 11) ( 18) ( 14) (500)
a Data for sheep liver SDH from Sigma Chemical Company were obtained under the same conditions as described in the text. b Shown as the pH stability after treatment at 40°C for 30 min. c Shown as the remaining activity (%) after treatment at 40°C for 30 min. * Shown as the remaining activity (%) after treatment at 40°C for 5 min. e The V-/K, value for D-sorbitol was taken as 100%.
reported in its thermal sp. SDH previously stability and affinity for D-sorbitol and galactitol. The properties of the SDH from Pseudomonas sp. KS-El806 suggest that it will be applicable to the enzymatic analysis of D-sorbitol. Studies on the further development of the method are now in progress.
domonus
We would like to thank Drs. Y. Sirokane and Y. Koyama for their advice. REFERENCES 1. Kanno, K., Tozkaunaga, K., Saheki, S., Mumse, M., Takeuthi, N., Shinohara, R., and Isignro, I.: Production of active oxygen species from neutrophils and polyol pathway in hyperglycemia. Jpn. J. Clinic. Chem., 25, 109-114 (1996). 2. Beutler, H. 0.: Methods of enzymatic analysis, vol. 6, 3rd ed., p. 356-362. Verlag Chemie, Weinheim, Germany (1984). 3. Jeffery, J. and Jomvall, I-I.: Advances in enzymology and related areas of molecular biology, vol. 61, p. 47-106. John Wiley & Sons, New York (1988). 4. Smith, M.G.: Polyol dehydrogenases. Biochem. J., 83, 135144 (1962). 5. Jeffery, J., Cummins, L., Carlquist, M., and Jornvall, H.: Properties of sorbitol dehydrogenase and characterization of a reactive cysteine residue reveal unexpected similarities to alcohol dehydrogenases. Eur. J. Biochem., 120, 229-234 (1981). 6. Maret, W. and Auld, D. S.: Purification and characterization of human liver sorbitol dehydrogenase. Biochemistry, 27,
1622-1628 (1988). 7. BumeB, J. N. and Holmes, Roger, S.: Purification and properties of sorbitol dehydrogenase from mouse liver. Int. J. Biothem., 15, 507-511 (1983). 8. Heinrich, W. and Bemd, H.: Purification and characterization of sorbitol dehydrogenase from bovine brain. J. Neurochem., 52, 342-348 (1989). 9. Doehlert, D. C.: Ketose reductase activity in developing maize endosperm. Plant Physiol., 84, 830-834 (1987). 10. Horwitz, S. B. and Kaplan, N. 0.: Hexitol dehydrogenases of Bacillus subtilis. J. Biol. Chem., 239, 830-838 (1964). 11. Ng, K., Ye, R., Wu, X.-C., and Wong, S.-L.: Sorbitol dehydrogenase from Bacillus subtilis. J. Biol. Chem., 267, 2498924994 (1992). 12. Schneider, K. and GiRltom, F.: Sorbitol dehydrogenase from Pseudomonas sp.: purification, characterization and application to quantitative determination of sorbitol. Enzyme Microb. Technol., 13, 332-337 (1991). 13. Arcus, A. C. and Edson, N. L.: Polyol dehydrogenases. Biochem. J., 56, 385-394 (1956). of 14. Marcus, L. and Marr, A. G.: Polyol dehydrogenases Azotobacter agilis. J. Bacterial., 82, 224-232 (1961). 15. Scolnick, E.M. and Lin, E.C. C.: Parallel induction of Darabitol and o-sorbitol dehydrogenases. J. Bacterial., 84, 631637 (1962). 16. Drets, G. M. and Arias, A.: Metabolism of some polyols by Rhizobium meliloti. J. Bacterial., 103, 97-103 (1970). 17. Sneath, P. H. (ed.): Bergey’s manual of determinative bacteriology, 9th ed., Williams & Wilkins, Baltimore (1994).
Properties of Sorbitol Dehydrogenase from Pseudomonas sp. KS-El806 and Comparison with Other Sorbitol Dehydrogenases TOMOYUKI
MINAMIHARA*
AND
MASARU
SUZUKI
Research & Development Division, Kikkoman Corporation, Noda, Chiba 278, Japan Received 5 November 1996/Accepted
24 June 1997
Sorbitol dehydrogenase (SDH, EC 1.1.1.14) was purified to homogeneity from a cell-free extract of Pseudomonas sp. KS-El806 by chromatographies with QAK-Sephadex A-50, QAE-Toyopearl55OC, and Bio-Gel A-1.5m. The molecular weight of the enzyme was estimated to be 64,500 and 27,400 by gel tIltration and SDS-PAGE, respectively, suggesting that it has a dimeric structure. The enzyme was stable from pH 5.5 to 10.5, and below 40°C for 30 mitt at pH 9.0. u-Sorbitol and galactitol were good substrates of the enzyme, whereas it acted only slightly on xylitol and u-mannitol. The enzyme was superior to sheep liver SDH with respect to substrate specificity and pH stability, and it was more thermostable than a previously reported Pseudomonas sp. SDH. Compared with other known SDHs, the enzyme from Pseudomonas sp. KS-El806 has several advantages for practical use in the enzymatic analysis of u-sorbitoi. [Key words:
sorbitol dehydrogenase,
Pseudomonas sp. KS-E1806, enzymatic analysis]
D-Sorbitol is a sugar alcohol naturally occurring in food. This sugar alcohol is also used as a sweetener. Recently, it was reported that the D-sorbitol levels in erythrocytes from diabetic patients have significantly increased (1). Therefore, a means of determining Dsorbitol levels that is suitable for use in clinical practice is needed. Sorbitol dehydrogenase (SDH, EC 1.1.1.14), which catalyzes the oxidation of D-sorbitol to n-fructose with NAD+ as a cofactor, has been used for the enzymatic analysis of D-sorbitol concentrations in food and in human erythrocytes (1, 2). SDHs have been found in a wide variety of animals, plants, and microorganisms (3-16), and SDHs purified from the mammalians (4-8), Bacillus subtilis (ll), and Pseudomonas sp. (12) have been investigated in detail. Although sheep liver SDH is commonly used for the determination of Dsorbitol, because of the broad substrate specificity of this enzyme it is difficult to ascertain the amount of Dsorbitol accurately when xylitol, which occurs naturally in foods, is also present. Schneider and Giffhorn (12) reported an SDH from Pseudomonas sp. that acts only slightly on xylitol, but the temperature stability of the enzyme was not sufficient for enzymatic assay because it lost 50% of its activity after treatment at 40°C for 5 min and at 20°C for 32 h. Here, we describe a new type of bacterial SDH which is stable and exhibits strict substrate specificity. The bacterium was a gram-negative, non-spore forming short rod with polar flagella, and was classified as belonging to the genus Pseudomonas according to Bergey’s Manual of Determinative Bacteriology (17). SDH activity was determined spectrophotometrically by measuring the absorbance of NADH at 340 nm (s=6.3 cm2/pmol), using a Hitachi U-2000 spectrophotometer. The standard assay for substrate oxidation was carried out at 37°C with 3 ml of a reaction mixture containing 80mM Tris-HCl buffer, pH 9.0, 183 mM D-sorbitol (Nacalai Tesque, Tokyo), 0.5 mM NAD+, and appropriately diluted enzyme solution. For substrate reduction, the assay was done with 3 ml of a reaction mixture
containing 80mM MES-NaOH buffer, pH 6.5, 167 mM D-fructose (Wako Pure Chemical Industries, Osaka), 0.22 mM NADH, and appropriately diluted enzyme solution. One unit is defined as the amount of enzyme that reduces 1 pmol of NAD+ per min under the standard assay conditions. The protein concentration was measured with a protein assay kit (Bio-Rad, USA). Bovine serum albumin was used as the standard protein. All other chemicals were of the highest grade generally available. The enzyme was purified to homogeneity from a cellfree extract of Pseudomonas sp. KS-El806 by chromatographies with QAE-Sephadex A-50, QAE-Toyopearl 55OC, and Bio-Gel A-1.5m, as shown in Table 1. In the final stage, the enzyme was purified 339-fold with a recovery of 26.5% from the crude enzyme preparation. The purified enzyme showed a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Fig. 1). The molecular weight of the native enzyme was estimated to be 64,500 by HPLC gel filtration on a TSKgel G3OOOSWxr column. From the mobility in SDS-PAGE, the molecular weight of the enzyme protein monomer was calculated to be 27,400 (Fig. 1). These results suggest that the enzyme has a dimeric structure. The enzyme showed the maximum activity at pHs of about 6.5 and 10.0 under the reduction and oxidation assay conditions, respectively, and its optimum temperature was about 50°C under the standard oxidation assay conditions (data not shown). The enzyme was stable in a pH range of 5.5-10.5 on incubation at 30°C for 4 h (Fig. 2A). To check the thermal stability, the enzyme was incubated at various temperatures for 30min in 10mM Tris-HCl buffer, pH 8.0, containing 1OOmM KCl. The remaining activity was 100% of the original level at 4O”C, 13% at 45”C, and almost zero at 50°C (Fig. 2B). In addition, the enzyme could be stored at 20°C for at least a month without any significant loss of activity in the same buffer. These results indicated that the stability of the enzyme was sufficient for its practical use in the analysis of D-sorbitol.
* Corresponding author. 254
VOL. 84, 1997 TABLE 1.
NOTES
255
Purification of Pseudomonas sp. KS-El806 sorbitol dehydrogenase
Step Crude extract 1st QAE-Sephadex A-SW (batchwise) 2nd QAE-Sephadex A-50b 1st QAE-Toyopearl 550Cc 2nd QAE-Toyopearl 550Cc Bio-Gel A-l .5md
Total activity iu)
Total protein (mg)
8000 6000
32000 1OOoO
3700 2800 2200 2120
206 70.0 35.9 25.0
Specific activity Wmg) 0.25 0.60
Yield (%)
18.0 40.0 61.3 84.7
46.3 35.0 27.5 26.5
100 75.0
a Eluted with 10 mM Tris-HCI buffer (pH 8.0) containing 200 mM KCl. b Eluted with 20 mM sodium acetate buffer (pH 6.0) containing 70 mM KCI. c Eluted with 20mM sodium acetate buffer @H 6.0) containing 60 mM KCl. d The enzyme was filtered with 10 mM Tris-HCl buffer @H 8.0) containing 100 mM KC1 and 0.02% NaN,.
In the oxidation reaction, the enzyme acted on D-sorbitol and galactitol effectively, and slightly on D-mannitol and xylitol (Table 2). No oxidation of the following substrates was observed: ribitol, D-glucose, D-fructose, D-galactose, D-mannose, D-xylose, D-arabinose, maltose, sucrose, lactose, glycerol, and ethanol. With respect to the coenzyme, it was highly specific for NAD+ and did not act on NADP+. The K,,, values for D-SOrbitol and NAD+ (in the presence of 183 mM D-sorbitol) were 14.1 mM and 58 mM, respectively. Under the reduction assay conditions, the enzyme acted on D-fructose and D-tagatose as good substrates, and did not act on D-glucose, mannose, galactose, ribose, maltose, sucrose, lactose, D-xylose, and L-sorbose. The enzyme was also highly specific for NADH and did not act on NADPH.
5
9
7
11
30
40
50
Temperature
PH
60
(“C)
FIG. 2. pH and thermal stability of Pseudomonas sp. KS-El806 sorbitol dehydrogenase. (A) Effect of pH on enzyme stability. After the enzyme solution was maintained at various pHs ( x , 0.1 M carbonate-bicarbonate; 0, 0.1 M glycine-NaOH; 0, 0.1 M Tris-HCI; A, 0.1 M potassium phosphate; A, 0.1 M sodium acetate) at 30°C for 4 h, the remaining activity was assayed. (B) Effect of temperature on enzyme stability. After the enzyme was treated in 100 mM Tris-HCl, pH9.0, at various temperatures, for 30min, the remaining activity was assayed.
The K, values for o-fructose and NADH (in the presence of 167 mM D-fructose) were 105 mM and 62 PM, respectively. We compared the properties of SDH from Pseudomona~ sp. KS-El806 with those from Pseudomonas sp. (12), sheep liver, and B. subtilis (11). As shown in Table 3, the molecular weights and subunit structure of the Pseudomonas SDHs differ from those of the sheep liver and B. subtilis enzymes. Our SDH was superior to that from sheep liver with respect to pH stability, and to the Pseudomonas sp. SDH in terns of thermal stability. The substrate specificities of Pseudomonas SDHs, differed from those of the sheep liver and B. subtilis SDHs, which showed strong activities toward D-sorbitol, L-iditol, and xylitol, but not galactitol. With regard to their kinetic parameters, the K, value for D-sorbitol of the Pseudomonas sp. KS-El806 SDH was the smallest among those of the polyols tested. On the other hand, the K,,, value for galactitol of the Pseudomonas sp. SDH previously reported was smaller than that for D-sorbitol (12). The above results show that while the Pseudomonas SDHs are clearly different from those of sheep liver and B. subtilis in several respects, our SDH also differs from PseuTABLE 2. Kinetic parameters for various substrates of Pseudomonas sp. KS-El806 sorbitol dehydrogenase Substrate
FIG. 1. SDS-PAGE of purified Pseudomonas sp. KS-El806 sorbitol dehydrogenase. SDS-PAGE was performed with Multigel (lO-20% gradient gel; Daiichi Pure Chemicals Co. Ltd., Tokyo). The following proteins were used as standards: A, Phosphorylase b (97,400); B, bovine serum albumin (66,267); C, aldolase (42,400); D, carbonic anhydrase (30,000); E, trypsin inhibitor (20,100); F, lysozyme (14,400).
(Oxidation) D-Sorbitol Galactitol L-Iditol D-Arabitol Xylitol D-Mannitol (Reduction) D-Fructose D-Tagatose D-Arabinose
14.1 42.0 149 46.3 181.8 63.8
72.5 208 29.5 12.4 2.59 0.476
5.14 4.95 0.198 0.268 0.0142 0.00746
105 85.3 187
174 837 66.8
1.66 9.81 0.357
The reactions were performed by varying the concentrations under the standard oxidation or reduction conditions described in the text. The Michaelis constants and maximal velocities for each substrate were obtained from their Lineweaver-Burk plots.
256
MINAMIHARA
AND SUZUKI
J.
TABLE 3. Property
Comparison of some properties of various sorbitol dehydrogenases
Pseudomonas sp. KS-El806
Molecular weight (subunit structure) pH stabilityb Thermal stability V,,&K,e (K,) (%) (mM) o-Sorbitol L-Iditol Xylitol Galactitol
FERMENT. BIOENG..
Pseudomonas sp. (12)
64,500 (dimer) 5.5-10.5 100%
loo 3.9 0.3 96.3
( 14.1) (149 ) (181 ) ( 42 )
65,800 (dimer) 50%d
- (9.1)
- (3.1)
Sheep 1iveIg 129,000 (tetramer) 6-8 lOO%C 100 79.6 216 0.1
( 9.1) ( 16.0) ( 4.1) (617 )
B. subtilis(11) 153,000 (tetramer)
100 78.4 79.2 5.9
( 11) ( 18) ( 14) (500)
a Data for sheep liver SDH from Sigma Chemical Company were obtained under the same conditions as described in the text. b Shown as the pH stability after treatment at 40°C for 30 min. c Shown as the remaining activity (%) after treatment at 40°C for 30 min. * Shown as the remaining activity (%) after treatment at 40°C for 5 min. e The V-/K, value for D-sorbitol was taken as 100%.
reported in its thermal sp. SDH previously stability and affinity for D-sorbitol and galactitol. The properties of the SDH from Pseudomonas sp. KS-El806 suggest that it will be applicable to the enzymatic analysis of D-sorbitol. Studies on the further development of the method are now in progress.
domonus
We would like to thank Drs. Y. Sirokane and Y. Koyama for their advice. REFERENCES 1. Kanno, K., Tozkaunaga, K., Saheki, S., Mumse, M., Takeuthi, N., Shinohara, R., and Isignro, I.: Production of active oxygen species from neutrophils and polyol pathway in hyperglycemia. Jpn. J. Clinic. Chem., 25, 109-114 (1996). 2. Beutler, H. 0.: Methods of enzymatic analysis, vol. 6, 3rd ed., p. 356-362. Verlag Chemie, Weinheim, Germany (1984). 3. Jeffery, J. and Jomvall, I-I.: Advances in enzymology and related areas of molecular biology, vol. 61, p. 47-106. John Wiley & Sons, New York (1988). 4. Smith, M.G.: Polyol dehydrogenases. Biochem. J., 83, 135144 (1962). 5. Jeffery, J., Cummins, L., Carlquist, M., and Jornvall, H.: Properties of sorbitol dehydrogenase and characterization of a reactive cysteine residue reveal unexpected similarities to alcohol dehydrogenases. Eur. J. Biochem., 120, 229-234 (1981). 6. Maret, W. and Auld, D. S.: Purification and characterization of human liver sorbitol dehydrogenase. Biochemistry, 27,
1622-1628 (1988). 7. BumeB, J. N. and Holmes, Roger, S.: Purification and properties of sorbitol dehydrogenase from mouse liver. Int. J. Biothem., 15, 507-511 (1983). 8. Heinrich, W. and Bemd, H.: Purification and characterization of sorbitol dehydrogenase from bovine brain. J. Neurochem., 52, 342-348 (1989). 9. Doehlert, D. C.: Ketose reductase activity in developing maize endosperm. Plant Physiol., 84, 830-834 (1987). 10. Horwitz, S. B. and Kaplan, N. 0.: Hexitol dehydrogenases of Bacillus subtilis. J. Biol. Chem., 239, 830-838 (1964). 11. Ng, K., Ye, R., Wu, X.-C., and Wong, S.-L.: Sorbitol dehydrogenase from Bacillus subtilis. J. Biol. Chem., 267, 2498924994 (1992). 12. Schneider, K. and GiRltom, F.: Sorbitol dehydrogenase from Pseudomonas sp.: purification, characterization and application to quantitative determination of sorbitol. Enzyme Microb. Technol., 13, 332-337 (1991). 13. Arcus, A. C. and Edson, N. L.: Polyol dehydrogenases. Biochem. J., 56, 385-394 (1956). of 14. Marcus, L. and Marr, A. G.: Polyol dehydrogenases Azotobacter agilis. J. Bacterial., 82, 224-232 (1961). 15. Scolnick, E.M. and Lin, E.C. C.: Parallel induction of Darabitol and o-sorbitol dehydrogenases. J. Bacterial., 84, 631637 (1962). 16. Drets, G. M. and Arias, A.: Metabolism of some polyols by Rhizobium meliloti. J. Bacterial., 103, 97-103 (1970). 17. Sneath, P. H. (ed.): Bergey’s manual of determinative bacteriology, 9th ed., Williams & Wilkins, Baltimore (1994).