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Effective dextran production from starch by dextrin dextranase with debranching enzyme
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 76, No. 5,411--413. 1993
Effective Dextran Production from Starch by Dextrin Dextranase with Debranching Enzyme KAZUYA YAMAMOTO,* KENJI YOSHIKAWA, AriD SHIGETAKA OKADA
Biochemical Research Laboratories, Ezaki Glico Co. Ltd., Utajima 4-6-5, Nishiyodogawa-ku, Osaka 555, Japan Received 9 March 1993/Accepted 15 July 1993 Dextrin dextranase (EC 2.4.1.2) is the enzyme that converts dextrins to dextran, but it is not able to synthesize dextran from unhydrolyzed starch. However, the enzyme could synthesize dextran with approx. 55-60 % yields from starch or Iow-degree-hydrolyzed starch under coexistence with pullulanase or isoamylase, when the substrate was consumed completely.
heated at 100°C for 15 min, and then analyzed. To compare the dextran production from starch with that from short-chain amylose, 1.5 ml of the mixture containing 2% short-chain amylose and 0.5 U/ml DDase was reacted. For analysis of the reaction, a 10/d aliquot was taken out to measure the reducing power by the Somogyi-Nelson method (7). Eighty microliters of 500U/ml BLA was added to 200 /~1 of the reaction mixture, incubated at 40°C for 2 h and then heated at 100°C for 15 min. Ten microliters of the BLA-treated mixture was used for measuring the reducing power, and another 150/~1 of the same mixture was added to 450 ~1 of ethanol, allowed to stand at 4°C for 2 h, and then the resulting precipitate was centrifuged and washed with 1 ml of 80% ethanol, and dried. After the precipitate was dissolved in 450/zl of distilled water, the sugar content was determined by the phenol-sulfuric acid method (8). The decrease in the reducing power after treating with BLA means the decrease in the amount of starch or amylose, and the difference between the values of reducing powers before and after BLA treatment indicates the amount of residual substrate. Also an increase in the precipitate implies the production of dextran. The yields of initial precipitates indicated the residual amounts of BLA-resistant polymer containing highly branching structures in the substrates. The structures were considered to be debranched, because it converted to dextran and disappeared during the reactions, when debranching enzymes were added to the BLAresistant polymer. The results are shown in Fig. 1. Curve A shows the value of reducing power during the reaction, and curve B the time course of reducing power after BLA treatment of the reaction mixture. The time course of dextran production from short-chain amylose is shown in Fig. la. Dextran production was almost completed in 7 h reaction, and the dextran yield was approx. 60% of the short-chain amylose initially added. The difference between the values of curves A and B had decreased to a constant value by that time. This result indicated that residual short-chain amylose was consumed almost completely, and the precipitate was entirely dextran. In the case of starch used as the substrate with isoamylase or pullulanase, similar results were obtained (Fig. lb and c). The reactions achieved a steady state after 4 and 7 h by the addition of isoamylase and pullulanase, respectively, and yields of dextrans in both reactions were approx. 55% of the amount of starch initially added. When DDase was reacted on starch without a debranching
Dextran is a glucan composed mainly of a-1,6 glucosidic linkages with minor secondary linkages such as a-l,2, a1,3, or a-l,4, and Leuconostoc mesenteroides is a wellknown dextran-producing microorganism possessing dextransucrase (EC 2.4.1.5) (1). Other dextran-producing bacteria, Acetobacter capsulatus and A. viscosus, have been reported to produce dextrin dextranase (EC 2.4.1.2; DDase), which converted dextrins to dextran (2, 3). Dextran produced by DDase was demonstrated to be similar to dextran produced by dextransucrase by immunological cross-reaction (3). However the structures of the dextrans were revealed to differ from each other by periodate oxidation and dextranase digestion (4, 5). Recently we purified DDase from A . capsulatus ATCC 11894, and examined the production of dextran by this enzyme from soluble starch and dextrins (5, 6). Though dextran was produced from soluble starch, the amount was small, and dextran was not produced from starch. However, in the case of short-chain amylose used as a substrate, a rather high amount of dextran was yielded. Here, addition of a debranching enzyme was tried as a means of improving the yield of dextran from starch and lowdegree-hydrolyzed starch, and we succeeded in obtaining dextran from the substrates effectively. DDase was partially purified by n-butanol extraction followed by Phenyl-Toyopearl HW-65S chromatography (Tosoh Co. Ltd., Tokyo) as described previously (5). The enzyme was used after dialysis against 10mM acetate buffer at pH 4.8 throughout the experiments. Isoamylase [Amano] (EC 3.2.1.68; isoamylase) and pullulanase (EC 3.2.1.41) was obtained from Amano Pharmaceuticals Co. Ltd., Nagoya and Hayashibara Biochemical Laboratories Inc., Okayama, respectively. Bacterial liquefying a-amylase (EC 3.2.1.1; BLA) was obtained from Ueda Chemicals Co. Ltd., Osaka. Starch (waxy maize) was obtained from Sanwa Denpun Kogyo Co. Ltd, Nara, and shortchain amylose was purchased from Hayashibara Biochemical Laboratories Inc. Low-degree-hydrolyzed starch (Pine-Dex #1) was obtained from Matsutani Chem. Ind. Co. Ltd., Hyogo. Dextran production from starch with the addition of isoamylase or pullulanase was examined. After a mixture (1.5ml) containing 2% starch, 0 . 5 U / m l DDase, and 59 U/ml isoamylase or 15 U/ml pullulanase was incubated at 30°C for appropriate periods, 230/d was taken out, * Corresponding author. 411
412
YAMAMOTO ET AL.
J. FERMENT.BIOENG.,
j
20 a
20
10 -O
._~ 10
_~-~o 0 i -
w
..
,
,
--
-. e-
2O
-
0-
°./ E
I
.-A I
2
4
6
FIG. 2. Effect of pullulanase addition on the production of dextran from starch. Symbols indicate the yields of precipitates from short-chain amylose (e), and from starch with 7.5, 0.8, 0.08, and 0.008 U/ml pullulanase (©, • , [], and &, respectively).
0
0
&
i
Reaction time (h)
.~ 10
~
.
;
;'
I
I
20
C
(=
10
0
t
.
0
I
10 Reaction time (h)
t
20
FIG. 1. Dextran production from amylose or starch with debranching enzyme. Symbols indicate the precipitate yielded ( • ) , and reducing power in the reaction mixture before (curve A; • ) and after (curve B; ©) BLA digestion. (a) Short-chain amylose; (b) starch with isoamylase; (c) starch with pullulanase. enzyme, the increase in precipitate and the decrease in reducing power caused by B L A treatment were not observed, and no dextran was p r o d u c e d from the starch by DDase (data not shown). In order to further examine the effect o f the a d d i t i o n o f debranching enzymes, various concentrations o f pullulanase were a d d e d to the mixture o f starch and DDase, and dextran yields were measured. A mixture (1.5 ml) containing 2 % starch, 0 . S U / m l DDase, and various concentrations o f pullulanase were incubated at 30°C; then 230 pl o f the mixture was taken out and heated at 100°C for 15 min. As shown in Fig. 2, the dextran production increased according to the increase in the pullulanase concentration, and came close to the a m o u n t o f dextran p r o d u c e d from short-chain amylose at the highest concentration o f pullulanase. A similar result was obtained when isoamylase was employed instead o f pullulanase (data not shown). To p r o d u c e a higher a m o u n t o f dextran, short-chain amylose was m o r e useful than starch with a debranching enzyme. However, as amylose retrogrades rapidly, especially at a high concentration, it is not suitable for industrial practice. Accordingly, dextran p r o d u c t i o n from lowdegree-hydrolyzed starch at a high concentration was attempted. Three milliliters o f 50% Pine-Dex #1, 2 ml o f 7.5 U / m l DDase and 8 pl o f 4 5 0 , 0 0 0 U / m l isoamylase were mixed and incubated at 40°C; then 100pl o f the reaction mixture was taken out and heated at 100°C for 15 min. The reaction mixture was diluted 10-fold with
distilled water, and then analyzed. The time course of the reaction is shown in Fig. 3. A steady state was reached in 7 h, and the yield o f dextran was approx. 60% o f the substrate used. W h e n dextran is p r o d u c e d by dextransucrase from sucrose, only glucose as the sucrose c o m p o n e n t is transformed into dextran. Fructose molecules are released in the reaction mixture but not utilized. This means that the a m o u n t o f dextran is less than a half o f the sucrose initially a d d e d under any conditions. On the other hand, dextran could be p r o d u c e d with 55 to 60% yields by DDase from starch or low-degree-hydrolyzed starch with the addition o f a debranching enzyme such as isoamylase or pullulanase. In a previous study, without a debranching enzyme, DDase could convert only one-fourth o f soluble starch to dextran, and no dextran was p r o d u c e d from starch (4). These facts suggested that DDas¢ could act on the non-reducing terminal residues o f a-l,4-1inked glucosyl linear structures, but not on structures close to the branching points in starch or soluble starch. Accordingly, though DDase can release trace glucose from starch, few glucosyl residues which can be transferred to the glucose molecules exist in starch, so that dextran cannot be produced. However in dextran p r o d u c t i o n by DDase with a debranching enzyme, starch was hydrolyzed at branching 3OO
2oo g C .O m
"~ 100 O O t-
8
o ,
0
,
i
,
,
,
,
10
,
i
,
,
20
Reaction time (h) FIG. 3. Dextran production by DDase from low-degree-hydrolyzed starch with addition of isoamylase. Symbols are the same as in Fig. 1.
VoL. 76, 1993 points by the debranching enzyme, and short-chain amyloses were released. These amyloses were then converted to dextran by DDase. T h o u g h pullulanase could not hydrolyze the inner branching points of starch, dextran was produced with the same efficiency as when isoamylase was used instead of pullulanase. DDase could act on amylose released by pullulanase, and the enzyme could transfer glucosyl residues at the external a-l,4-1inked glucosyl linear structures of the residual starch molecules. It is probable that the DDase action led to the exposure of branching points at the inner structure of the original starch, and pullulanase hydrolyzed such exposed branching points and released amylose molecules. Repetition of the reaction would result in the accumulation of dextran, and the final disappearance of the starch. Thus a debranching enzyme, isoamylase or pullulanase, could work effectively in cooperation with DDase, and DDase could produce a large a m o u n t of dextran from starch or low-degree-hydrolyzed starch in the presence of the debranching enzyme.
NOTES
413
REFERENCES
1. Sidebotham, R.L.: Dextrans. Adv. Carbohydr. Chem. Biochem., 30, 371-444 (1974). 2. Hehre, E. J. and Hamilton, D. M.: Bacterial conversion of dextrin into a polysaccharide with the serological properties of dextran. Proc. Soc. Exper. Biol. Med., 71, 336-339 (1949). 3. Hehre, E.J. and Hamilton, D.M.: The biological synthesis of dextran from dextrins. J. Biol. Chem., 192, 161-174 (1951). 4. Jeanes, A., Haynes, W. C., Wilham, C. A., Rankin, J. C., Melvin, E.H., Austin, M.J., Cluskey, J.E., Fisher, B.E., Tsuchiya, H. M., and Rist, C. E.: Characterization and classification of dextrans from ninety-six strains of bacteria. J. Am. Chem. Soc., 76, 5041-5052 (1954). 5. Yamamoto, K., Yoshikawa, K., Kitahata, S., and Okada, S.: Purification and some properties of dextrin dextranase from Acetobacter capsulatus ATCC 11894. Biosci. Biotech. Biochem., 56, 169-173 (1992). 6. Yamamoto, K., Yoshikawa, K., and Okada, S.: Detailed action mechanism of dextrin dextranase from Acetobacter capsulatus ATCC 11894. Biosci. Biotech. Biochem., 57, 47-50 (1993). 7. Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). 8. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 (1956).
Vol. 76, No. 5,411--413. 1993
Effective Dextran Production from Starch by Dextrin Dextranase with Debranching Enzyme KAZUYA YAMAMOTO,* KENJI YOSHIKAWA, AriD SHIGETAKA OKADA
Biochemical Research Laboratories, Ezaki Glico Co. Ltd., Utajima 4-6-5, Nishiyodogawa-ku, Osaka 555, Japan Received 9 March 1993/Accepted 15 July 1993 Dextrin dextranase (EC 2.4.1.2) is the enzyme that converts dextrins to dextran, but it is not able to synthesize dextran from unhydrolyzed starch. However, the enzyme could synthesize dextran with approx. 55-60 % yields from starch or Iow-degree-hydrolyzed starch under coexistence with pullulanase or isoamylase, when the substrate was consumed completely.
heated at 100°C for 15 min, and then analyzed. To compare the dextran production from starch with that from short-chain amylose, 1.5 ml of the mixture containing 2% short-chain amylose and 0.5 U/ml DDase was reacted. For analysis of the reaction, a 10/d aliquot was taken out to measure the reducing power by the Somogyi-Nelson method (7). Eighty microliters of 500U/ml BLA was added to 200 /~1 of the reaction mixture, incubated at 40°C for 2 h and then heated at 100°C for 15 min. Ten microliters of the BLA-treated mixture was used for measuring the reducing power, and another 150/~1 of the same mixture was added to 450 ~1 of ethanol, allowed to stand at 4°C for 2 h, and then the resulting precipitate was centrifuged and washed with 1 ml of 80% ethanol, and dried. After the precipitate was dissolved in 450/zl of distilled water, the sugar content was determined by the phenol-sulfuric acid method (8). The decrease in the reducing power after treating with BLA means the decrease in the amount of starch or amylose, and the difference between the values of reducing powers before and after BLA treatment indicates the amount of residual substrate. Also an increase in the precipitate implies the production of dextran. The yields of initial precipitates indicated the residual amounts of BLA-resistant polymer containing highly branching structures in the substrates. The structures were considered to be debranched, because it converted to dextran and disappeared during the reactions, when debranching enzymes were added to the BLAresistant polymer. The results are shown in Fig. 1. Curve A shows the value of reducing power during the reaction, and curve B the time course of reducing power after BLA treatment of the reaction mixture. The time course of dextran production from short-chain amylose is shown in Fig. la. Dextran production was almost completed in 7 h reaction, and the dextran yield was approx. 60% of the short-chain amylose initially added. The difference between the values of curves A and B had decreased to a constant value by that time. This result indicated that residual short-chain amylose was consumed almost completely, and the precipitate was entirely dextran. In the case of starch used as the substrate with isoamylase or pullulanase, similar results were obtained (Fig. lb and c). The reactions achieved a steady state after 4 and 7 h by the addition of isoamylase and pullulanase, respectively, and yields of dextrans in both reactions were approx. 55% of the amount of starch initially added. When DDase was reacted on starch without a debranching
Dextran is a glucan composed mainly of a-1,6 glucosidic linkages with minor secondary linkages such as a-l,2, a1,3, or a-l,4, and Leuconostoc mesenteroides is a wellknown dextran-producing microorganism possessing dextransucrase (EC 2.4.1.5) (1). Other dextran-producing bacteria, Acetobacter capsulatus and A. viscosus, have been reported to produce dextrin dextranase (EC 2.4.1.2; DDase), which converted dextrins to dextran (2, 3). Dextran produced by DDase was demonstrated to be similar to dextran produced by dextransucrase by immunological cross-reaction (3). However the structures of the dextrans were revealed to differ from each other by periodate oxidation and dextranase digestion (4, 5). Recently we purified DDase from A . capsulatus ATCC 11894, and examined the production of dextran by this enzyme from soluble starch and dextrins (5, 6). Though dextran was produced from soluble starch, the amount was small, and dextran was not produced from starch. However, in the case of short-chain amylose used as a substrate, a rather high amount of dextran was yielded. Here, addition of a debranching enzyme was tried as a means of improving the yield of dextran from starch and lowdegree-hydrolyzed starch, and we succeeded in obtaining dextran from the substrates effectively. DDase was partially purified by n-butanol extraction followed by Phenyl-Toyopearl HW-65S chromatography (Tosoh Co. Ltd., Tokyo) as described previously (5). The enzyme was used after dialysis against 10mM acetate buffer at pH 4.8 throughout the experiments. Isoamylase [Amano] (EC 3.2.1.68; isoamylase) and pullulanase (EC 3.2.1.41) was obtained from Amano Pharmaceuticals Co. Ltd., Nagoya and Hayashibara Biochemical Laboratories Inc., Okayama, respectively. Bacterial liquefying a-amylase (EC 3.2.1.1; BLA) was obtained from Ueda Chemicals Co. Ltd., Osaka. Starch (waxy maize) was obtained from Sanwa Denpun Kogyo Co. Ltd, Nara, and shortchain amylose was purchased from Hayashibara Biochemical Laboratories Inc. Low-degree-hydrolyzed starch (Pine-Dex #1) was obtained from Matsutani Chem. Ind. Co. Ltd., Hyogo. Dextran production from starch with the addition of isoamylase or pullulanase was examined. After a mixture (1.5ml) containing 2% starch, 0 . 5 U / m l DDase, and 59 U/ml isoamylase or 15 U/ml pullulanase was incubated at 30°C for appropriate periods, 230/d was taken out, * Corresponding author. 411
412
YAMAMOTO ET AL.
J. FERMENT.BIOENG.,
j
20 a
20
10 -O
._~ 10
_~-~o 0 i -
w
..
,
,
--
-. e-
2O
-
0-
°./ E
I
.-A I
2
4
6
FIG. 2. Effect of pullulanase addition on the production of dextran from starch. Symbols indicate the yields of precipitates from short-chain amylose (e), and from starch with 7.5, 0.8, 0.08, and 0.008 U/ml pullulanase (©, • , [], and &, respectively).
0
0
&
i
Reaction time (h)
.~ 10
~
.
;
;'
I
I
20
C
(=
10
0
t
.
0
I
10 Reaction time (h)
t
20
FIG. 1. Dextran production from amylose or starch with debranching enzyme. Symbols indicate the precipitate yielded ( • ) , and reducing power in the reaction mixture before (curve A; • ) and after (curve B; ©) BLA digestion. (a) Short-chain amylose; (b) starch with isoamylase; (c) starch with pullulanase. enzyme, the increase in precipitate and the decrease in reducing power caused by B L A treatment were not observed, and no dextran was p r o d u c e d from the starch by DDase (data not shown). In order to further examine the effect o f the a d d i t i o n o f debranching enzymes, various concentrations o f pullulanase were a d d e d to the mixture o f starch and DDase, and dextran yields were measured. A mixture (1.5 ml) containing 2 % starch, 0 . S U / m l DDase, and various concentrations o f pullulanase were incubated at 30°C; then 230 pl o f the mixture was taken out and heated at 100°C for 15 min. As shown in Fig. 2, the dextran production increased according to the increase in the pullulanase concentration, and came close to the a m o u n t o f dextran p r o d u c e d from short-chain amylose at the highest concentration o f pullulanase. A similar result was obtained when isoamylase was employed instead o f pullulanase (data not shown). To p r o d u c e a higher a m o u n t o f dextran, short-chain amylose was m o r e useful than starch with a debranching enzyme. However, as amylose retrogrades rapidly, especially at a high concentration, it is not suitable for industrial practice. Accordingly, dextran p r o d u c t i o n from lowdegree-hydrolyzed starch at a high concentration was attempted. Three milliliters o f 50% Pine-Dex #1, 2 ml o f 7.5 U / m l DDase and 8 pl o f 4 5 0 , 0 0 0 U / m l isoamylase were mixed and incubated at 40°C; then 100pl o f the reaction mixture was taken out and heated at 100°C for 15 min. The reaction mixture was diluted 10-fold with
distilled water, and then analyzed. The time course of the reaction is shown in Fig. 3. A steady state was reached in 7 h, and the yield o f dextran was approx. 60% o f the substrate used. W h e n dextran is p r o d u c e d by dextransucrase from sucrose, only glucose as the sucrose c o m p o n e n t is transformed into dextran. Fructose molecules are released in the reaction mixture but not utilized. This means that the a m o u n t o f dextran is less than a half o f the sucrose initially a d d e d under any conditions. On the other hand, dextran could be p r o d u c e d with 55 to 60% yields by DDase from starch or low-degree-hydrolyzed starch with the addition o f a debranching enzyme such as isoamylase or pullulanase. In a previous study, without a debranching enzyme, DDase could convert only one-fourth o f soluble starch to dextran, and no dextran was p r o d u c e d from starch (4). These facts suggested that DDas¢ could act on the non-reducing terminal residues o f a-l,4-1inked glucosyl linear structures, but not on structures close to the branching points in starch or soluble starch. Accordingly, though DDase can release trace glucose from starch, few glucosyl residues which can be transferred to the glucose molecules exist in starch, so that dextran cannot be produced. However in dextran p r o d u c t i o n by DDase with a debranching enzyme, starch was hydrolyzed at branching 3OO
2oo g C .O m
"~ 100 O O t-
8
o ,
0
,
i
,
,
,
,
10
,
i
,
,
20
Reaction time (h) FIG. 3. Dextran production by DDase from low-degree-hydrolyzed starch with addition of isoamylase. Symbols are the same as in Fig. 1.
VoL. 76, 1993 points by the debranching enzyme, and short-chain amyloses were released. These amyloses were then converted to dextran by DDase. T h o u g h pullulanase could not hydrolyze the inner branching points of starch, dextran was produced with the same efficiency as when isoamylase was used instead of pullulanase. DDase could act on amylose released by pullulanase, and the enzyme could transfer glucosyl residues at the external a-l,4-1inked glucosyl linear structures of the residual starch molecules. It is probable that the DDase action led to the exposure of branching points at the inner structure of the original starch, and pullulanase hydrolyzed such exposed branching points and released amylose molecules. Repetition of the reaction would result in the accumulation of dextran, and the final disappearance of the starch. Thus a debranching enzyme, isoamylase or pullulanase, could work effectively in cooperation with DDase, and DDase could produce a large a m o u n t of dextran from starch or low-degree-hydrolyzed starch in the presence of the debranching enzyme.
NOTES
413
REFERENCES
1. Sidebotham, R.L.: Dextrans. Adv. Carbohydr. Chem. Biochem., 30, 371-444 (1974). 2. Hehre, E. J. and Hamilton, D. M.: Bacterial conversion of dextrin into a polysaccharide with the serological properties of dextran. Proc. Soc. Exper. Biol. Med., 71, 336-339 (1949). 3. Hehre, E.J. and Hamilton, D.M.: The biological synthesis of dextran from dextrins. J. Biol. Chem., 192, 161-174 (1951). 4. Jeanes, A., Haynes, W. C., Wilham, C. A., Rankin, J. C., Melvin, E.H., Austin, M.J., Cluskey, J.E., Fisher, B.E., Tsuchiya, H. M., and Rist, C. E.: Characterization and classification of dextrans from ninety-six strains of bacteria. J. Am. Chem. Soc., 76, 5041-5052 (1954). 5. Yamamoto, K., Yoshikawa, K., Kitahata, S., and Okada, S.: Purification and some properties of dextrin dextranase from Acetobacter capsulatus ATCC 11894. Biosci. Biotech. Biochem., 56, 169-173 (1992). 6. Yamamoto, K., Yoshikawa, K., and Okada, S.: Detailed action mechanism of dextrin dextranase from Acetobacter capsulatus ATCC 11894. Biosci. Biotech. Biochem., 57, 47-50 (1993). 7. Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). 8. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 (1956).