Carbohydrate Research 387 (2014) 1–3
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Glucansucrase acceptor reactions with D-mannose Gregory L. Côté a,⇑, Ryan S. Cormier a, Karl E. Vermillion b a Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA b Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Glucansucrase Acceptor reactions Dextransucrase Mannose
a b s t r a c t The main acceptor product of glucansucrases with D-mannose has not previously been identified. We used glucansucrases that form water-insoluble a-D-glucans to produce increased yields of acceptor products from D-mannose, and identified the major product as 6-O-a-D-glucopyranosyl-D-mannose. Glucansucrases that synthesize insoluble a-D-glucans produced higher yields of the disaccharide compared to typical dextransucrases. Published by Elsevier Ltd.
Glucansucrases such as dextransucrase (EC 2.4.1.5) are wellknown not only for their ability to synthesize glucans directly from sucrose, but also to transfer D-glucopyranosyl units from sucrose to acceptor sugars to form novel glucosyl saccharides. In our research on various glucansucrases, we have noted a gap in the literature regarding acceptor reactions with D-mannose. Initially, it was reported that D-mannose was not an acceptor for dextransucrase from Leuconostoc mesenteroides NRRL B-512F.1 However, Iriki and Hehre later isolated three acceptor products from Leuconostoc citreum NRRL B-742 as well as L. mesenteroides NRRL B-512F reactions with D-mannose.2 Of the three, two were not characterized, but a minor product was shown to be a-D-glucopyranosyl (1M1) b-D-mannopyranoside. Robyt and Eklund studied the relative effects of various acceptors with the B-512F dextransucrase, and noted two products from D-mannose.3 Although neither was isolated and structurally characterized, they cited Iriki and Hehre’s structure2 for one of the products, and stated the other to be an unknown.3 We have described the acceptor reaction products of alternansucrase (EC 2.4.1.140) with a number of carbohydrates, including 1-O-methyl a- and b-D-mannopyranoside,4 and discovered the major product to be the corresponding 6-O-glucosylated saccharide. More recently, we have isolated and described glucansucrases that produce water-insoluble D-glucans containing large proportions of a-(1?3)-linkages.5,6 Therefore, it was of interest to compare their ⇑ Corresponding author. Tel.: +1 309 681 6319; fax: +1 309 681 6040. E-mail address: [email protected] (G.L. Côté). http://dx.doi.org/10.1016/j.carres.2014.01.013 0008-6215/Published by Elsevier Ltd.
acceptor reaction products with those of alternansucrase and dextransucrases. 1. Experimental Culture fluids from sucrose-grown bacteria were used as the source of glucansucrases, as previously described.1–3 The following strains were used: Lactobacillus satsumensis NRRL B-59839, L. mesenteroides NRRL B-512F and B-523, and L. citreum NRRL B-742. Cloned DsrI from L. mesenteroides NRRL B-1118 was expressed in Escherichia coli and prepared as previously described.5 Acceptor reactions were performed at room temperature in 20 mM pH 5.6 sodium acetate buffer containing 2 mM CaCl2 and 0.01% NaN3. For determination of yields, 70 mmol of sucrose (24 g) and 167 mmol of D-mannose (30 g) were dissolved in 300 mL of buffer containing either 250 units of B-512F dextransucrase or 125 units of cloned B-1118 DsrI (U = lmol glc transferred min 1). Thin-layer chromatography was carried out on 20 cm silica gel 60 plates, developed for two ascents in acetonitrile–water (4:1, v:v), and compounds were made visible with acidic N-(1-naphthyl)ethylenediamine dihydrochloride.7 Reactions were carried to completion as judged by disappearance of sucrose on tlc analysis. Oligosaccharide products were separated according to degree of polymerization (dp) over a 5 120 cm column of Bio-Gel P-2, eluted with water. Structural analysis was carried out by 1H, 13C, COSY, HSQC, HSQC-TOCSY, and HMBC NMR experiments recorded on a Bruker 500 MHz instrument in D2O at 27 °C and methylation analysis as previously described.4,5
2
G. L. Côté et al. / Carbohydrate Research 387 (2014) 1–3 Table 1 H NMR peak assignments for 6-O-a-D-glucopyranosyl-D-mannopyranose
2. Results and discussion
1
All glucansucrases tested gave the same pattern of products when analyzed by tlc. Although the main products were chromatographically identical, we chose to focus on those from strains B-512F and B-1118 (DsrI) because those two enzymes are the most well-characterized. It was evident from the size and intensity of tlc spots corresponding to the major disaccharide product that those enzymes which synthesized insoluble glucans (B-523, B-59839, and B-1118 DsrI) produced higher yields of the disaccharide than those which produced soluble glucans (B-512F and B-742). A quantitative indication of this difference is shown in Figure 1, a densitometric scan of the tlc lanes for B-1118 DsrI and B-512F dextransucrase. In that instance, the B-512F dextransucrase gave a 14% yield of the main disaccharide after isolation, based on sucrose, whereas the B-1118 DsrI gave a 34% yield. By way of contrast, under identical conditions of sucrose and acceptor concentrations, Iriki and Hehre2 recovered a 0.4% yield of a-D-glucopyranosyl (1M1) b-D-mannopyranoside. In our experiments, the higher yield of product from DsrI seemed to be accompanied by lower amounts of leucrose, leading to easier purification of the glucosylated mannose. We noted the appearance of traces of a disaccharide migrating slightly ahead of leucrose on tlc, but this was not isolated. It could be either isomaltulose or a-D-glucopyranosyl (1M1) b-D-mannopyranoside, or a mixture thereof, but at best represented only a trace fraction relative to the major disaccharide product. There is little doubt that the yields could be further optimized by varying the absolute and relative concentrations of sucrose and mannose; however, such studies are beyond the scope of this brief note. Numerous studies have been published and it is well-established that the single most important factor in determining such yields is the [sucrose]/[acceptor] ratio.8,9 Complicating the issue is the fact that each oligosaccharide acceptor product may also act as an acceptor for further glucosylation, thus yielding a series of oligosaccharide products. Isolation of the main disaccharide product yielded a disaccharide which was further analyzed by NMR and methylation. Methylation analysis yielded 2,3,4,6-tetra-O-methyl glucose and 2,3,4-triO-methyl mannose derivatives, in approximately equal amounts. 1 H NMR data for the anomeric protons appear in Table 1 with 13 C NMR data in Table 2 as assigned using the data obtained by the 2D NMR experiments. These shifts are just as predicted for 6O-a-D-glucopyranosyl-D-mannose by comparison to the corresponding methyl glycoside4 as well as by computer simulation
C1
a-D-Glcp
4.89 4.88 5.10 4.84
b-D-Manp a-D-Manp a b
(d, (d, (d, (d,
J = 3.47 Hz)a J = 3.78 Hz)b J = 1.89 Hz) J = 1.26 Hz)
6-O-a-D-Glucopyranosyl-a-D-mannopyranose. 6-O-a-D-Glucopyranosyl-b-D-mannopyranose.
Table 2 C NMR peak assignments for 6-O-a-D-glucopyranosyl-D-mannopyranose
13
a-D-Glcp b-D-Manp a-D-Manp a b
C1
C2
C3
C4
C5
C6
97.95a 97.92b 93.93 94.27
71.50a 71.46b 71.79 70.65
73.10ab
69.54ab
60.50ab
73.26 70.51
66.30 66.56
71.74a 71.72b 74.37 70.75
65.78 65.81
6-O-a-D-Glucopyranosyl-a-D-mannopyranose. 6-O-a-D-Glucopyranosyl-b-D-mannopyranose.
using CASPER.10 Glycosidic linkages were confirmed via the HMBC NMR experiments. The fact that the major disaccharide product from glucansucrase acceptor reactions with D-mannose arises from glucosylation at position 6 is not surprising, as this is the main position of glucosylation with a variety of acceptors. What is surprising is that this had not previously been reported. Iriki and Hehre2 isolated a minor product, a-D-glucopyranosyl (1M1) b-D-mannopyranoside, but neglected to report the structure of their major product. We hope our current work corrects this historical oversight. Of equal significance is our finding that glucansucrases which produce insoluble glucans may be useful for the synthesis of increased yields of acceptor products when compared to ‘typical’ dextransucrases such as the commercial NRRL B-512F strain. Preliminary results using cellobiose and lactose as acceptors support this generalization, and are currently under study in our laboratory. Acknowledgments We would like to thank Angie Purlee and Suzanne Unser for technical assistance in the preparation and analysis of the products, and Dr. Christopher Skory for the molecular biology aspects
mannose & fructose
leucrose
DP2 product
fru
400
600
800
1000
1200
1400
1600
1800
2000
distance from bottom of plate (pixels) Figure 1. Densitogram of thin-layer chromatography lanes for mannose acceptor reactions. Solid line is for cloned B-1118 DsrI, dashed line is for L. mesenteroides NRRL B512F dextransucrase, and dotted line shows elution profile for leucrose and fructose standards.
G. L. Côté et al. / Carbohydrate Research 387 (2014) 1–3
of the enzyme preparation. This work was supported by the United States Department of Agriculture. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer. References 1. Koepsell, H. J.; Tsuchiya, H. M.; Hellman, N. N.; Kazenko, A.; Hoffman, C. A.; Sharpe, E. S.; Jackson, R. W. J. Biol. Chem. 1953, 200, 793–801.
2. 3. 4. 5. 6. 7. 8. 9. 10.
3
Iriki, Y.; Hehre, E. J. Arch. Biochem. Biophys. 1969, 134, 130–138. Robyt, J. F.; Eklund, S. H. Carbohydr. Res. 1983, 121, 279–286. Côté, G. L.; Dunlap, C. A. Carbohydr. Res. 2003, 338, 1961–1967. Côté, G. L.; Skory, C. D. Appl. Microbiol. Biotechnol. 2012, 93, 2387–2394. Côté, G. L.; Skory, C. D.; Unser, S. M.; Rich, J. O. Appl. Microbiol. Biotechnol. 2013, 97, 7265–7273. Bounias, M. Anal. Biochem. 1980, 106, 291–295. Paul, F.; Oriol, E.; Auriol, D.; Monsan, P. Carbohydr. Res. 1986, 149, 433–441. Heincke, K.; Demuth, B.; Jördening, H.-J.; Buchholz, K. Enzyme Microb. Technol. 1999, 24, 523–534. Ronnols, J.; Pendrill, R.; Fontana, C.; Hamark, C.; d’Ortoli, T. A.; Engstrom, O.; Stahle, J.; Zaccheus, M. V.; Sawen, E.; Hahn, L. E.; Iqbal, S.; Widmalm, G. Carbohydr. Res. 2013, 380, 156–166.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Glucansucrase acceptor reactions with D-mannose Gregory L. Côté a,⇑, Ryan S. Cormier a, Karl E. Vermillion b a Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA b Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA
a r t i c l e
i n f o
Article history: Received 16 December 2013 Received in revised form 10 January 2014 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Glucansucrase Acceptor reactions Dextransucrase Mannose
a b s t r a c t The main acceptor product of glucansucrases with D-mannose has not previously been identified. We used glucansucrases that form water-insoluble a-D-glucans to produce increased yields of acceptor products from D-mannose, and identified the major product as 6-O-a-D-glucopyranosyl-D-mannose. Glucansucrases that synthesize insoluble a-D-glucans produced higher yields of the disaccharide compared to typical dextransucrases. Published by Elsevier Ltd.
Glucansucrases such as dextransucrase (EC 2.4.1.5) are wellknown not only for their ability to synthesize glucans directly from sucrose, but also to transfer D-glucopyranosyl units from sucrose to acceptor sugars to form novel glucosyl saccharides. In our research on various glucansucrases, we have noted a gap in the literature regarding acceptor reactions with D-mannose. Initially, it was reported that D-mannose was not an acceptor for dextransucrase from Leuconostoc mesenteroides NRRL B-512F.1 However, Iriki and Hehre later isolated three acceptor products from Leuconostoc citreum NRRL B-742 as well as L. mesenteroides NRRL B-512F reactions with D-mannose.2 Of the three, two were not characterized, but a minor product was shown to be a-D-glucopyranosyl (1M1) b-D-mannopyranoside. Robyt and Eklund studied the relative effects of various acceptors with the B-512F dextransucrase, and noted two products from D-mannose.3 Although neither was isolated and structurally characterized, they cited Iriki and Hehre’s structure2 for one of the products, and stated the other to be an unknown.3 We have described the acceptor reaction products of alternansucrase (EC 2.4.1.140) with a number of carbohydrates, including 1-O-methyl a- and b-D-mannopyranoside,4 and discovered the major product to be the corresponding 6-O-glucosylated saccharide. More recently, we have isolated and described glucansucrases that produce water-insoluble D-glucans containing large proportions of a-(1?3)-linkages.5,6 Therefore, it was of interest to compare their ⇑ Corresponding author. Tel.: +1 309 681 6319; fax: +1 309 681 6040. E-mail address: [email protected] (G.L. Côté). http://dx.doi.org/10.1016/j.carres.2014.01.013 0008-6215/Published by Elsevier Ltd.
acceptor reaction products with those of alternansucrase and dextransucrases. 1. Experimental Culture fluids from sucrose-grown bacteria were used as the source of glucansucrases, as previously described.1–3 The following strains were used: Lactobacillus satsumensis NRRL B-59839, L. mesenteroides NRRL B-512F and B-523, and L. citreum NRRL B-742. Cloned DsrI from L. mesenteroides NRRL B-1118 was expressed in Escherichia coli and prepared as previously described.5 Acceptor reactions were performed at room temperature in 20 mM pH 5.6 sodium acetate buffer containing 2 mM CaCl2 and 0.01% NaN3. For determination of yields, 70 mmol of sucrose (24 g) and 167 mmol of D-mannose (30 g) were dissolved in 300 mL of buffer containing either 250 units of B-512F dextransucrase or 125 units of cloned B-1118 DsrI (U = lmol glc transferred min 1). Thin-layer chromatography was carried out on 20 cm silica gel 60 plates, developed for two ascents in acetonitrile–water (4:1, v:v), and compounds were made visible with acidic N-(1-naphthyl)ethylenediamine dihydrochloride.7 Reactions were carried to completion as judged by disappearance of sucrose on tlc analysis. Oligosaccharide products were separated according to degree of polymerization (dp) over a 5 120 cm column of Bio-Gel P-2, eluted with water. Structural analysis was carried out by 1H, 13C, COSY, HSQC, HSQC-TOCSY, and HMBC NMR experiments recorded on a Bruker 500 MHz instrument in D2O at 27 °C and methylation analysis as previously described.4,5
2
G. L. Côté et al. / Carbohydrate Research 387 (2014) 1–3 Table 1 H NMR peak assignments for 6-O-a-D-glucopyranosyl-D-mannopyranose
2. Results and discussion
1
All glucansucrases tested gave the same pattern of products when analyzed by tlc. Although the main products were chromatographically identical, we chose to focus on those from strains B-512F and B-1118 (DsrI) because those two enzymes are the most well-characterized. It was evident from the size and intensity of tlc spots corresponding to the major disaccharide product that those enzymes which synthesized insoluble glucans (B-523, B-59839, and B-1118 DsrI) produced higher yields of the disaccharide than those which produced soluble glucans (B-512F and B-742). A quantitative indication of this difference is shown in Figure 1, a densitometric scan of the tlc lanes for B-1118 DsrI and B-512F dextransucrase. In that instance, the B-512F dextransucrase gave a 14% yield of the main disaccharide after isolation, based on sucrose, whereas the B-1118 DsrI gave a 34% yield. By way of contrast, under identical conditions of sucrose and acceptor concentrations, Iriki and Hehre2 recovered a 0.4% yield of a-D-glucopyranosyl (1M1) b-D-mannopyranoside. In our experiments, the higher yield of product from DsrI seemed to be accompanied by lower amounts of leucrose, leading to easier purification of the glucosylated mannose. We noted the appearance of traces of a disaccharide migrating slightly ahead of leucrose on tlc, but this was not isolated. It could be either isomaltulose or a-D-glucopyranosyl (1M1) b-D-mannopyranoside, or a mixture thereof, but at best represented only a trace fraction relative to the major disaccharide product. There is little doubt that the yields could be further optimized by varying the absolute and relative concentrations of sucrose and mannose; however, such studies are beyond the scope of this brief note. Numerous studies have been published and it is well-established that the single most important factor in determining such yields is the [sucrose]/[acceptor] ratio.8,9 Complicating the issue is the fact that each oligosaccharide acceptor product may also act as an acceptor for further glucosylation, thus yielding a series of oligosaccharide products. Isolation of the main disaccharide product yielded a disaccharide which was further analyzed by NMR and methylation. Methylation analysis yielded 2,3,4,6-tetra-O-methyl glucose and 2,3,4-triO-methyl mannose derivatives, in approximately equal amounts. 1 H NMR data for the anomeric protons appear in Table 1 with 13 C NMR data in Table 2 as assigned using the data obtained by the 2D NMR experiments. These shifts are just as predicted for 6O-a-D-glucopyranosyl-D-mannose by comparison to the corresponding methyl glycoside4 as well as by computer simulation
C1
a-D-Glcp
4.89 4.88 5.10 4.84
b-D-Manp a-D-Manp a b
(d, (d, (d, (d,
J = 3.47 Hz)a J = 3.78 Hz)b J = 1.89 Hz) J = 1.26 Hz)
6-O-a-D-Glucopyranosyl-a-D-mannopyranose. 6-O-a-D-Glucopyranosyl-b-D-mannopyranose.
Table 2 C NMR peak assignments for 6-O-a-D-glucopyranosyl-D-mannopyranose
13
a-D-Glcp b-D-Manp a-D-Manp a b
C1
C2
C3
C4
C5
C6
97.95a 97.92b 93.93 94.27
71.50a 71.46b 71.79 70.65
73.10ab
69.54ab
60.50ab
73.26 70.51
66.30 66.56
71.74a 71.72b 74.37 70.75
65.78 65.81
6-O-a-D-Glucopyranosyl-a-D-mannopyranose. 6-O-a-D-Glucopyranosyl-b-D-mannopyranose.
using CASPER.10 Glycosidic linkages were confirmed via the HMBC NMR experiments. The fact that the major disaccharide product from glucansucrase acceptor reactions with D-mannose arises from glucosylation at position 6 is not surprising, as this is the main position of glucosylation with a variety of acceptors. What is surprising is that this had not previously been reported. Iriki and Hehre2 isolated a minor product, a-D-glucopyranosyl (1M1) b-D-mannopyranoside, but neglected to report the structure of their major product. We hope our current work corrects this historical oversight. Of equal significance is our finding that glucansucrases which produce insoluble glucans may be useful for the synthesis of increased yields of acceptor products when compared to ‘typical’ dextransucrases such as the commercial NRRL B-512F strain. Preliminary results using cellobiose and lactose as acceptors support this generalization, and are currently under study in our laboratory. Acknowledgments We would like to thank Angie Purlee and Suzanne Unser for technical assistance in the preparation and analysis of the products, and Dr. Christopher Skory for the molecular biology aspects
mannose & fructose
leucrose
DP2 product
fru
400
600
800
1000
1200
1400
1600
1800
2000
distance from bottom of plate (pixels) Figure 1. Densitogram of thin-layer chromatography lanes for mannose acceptor reactions. Solid line is for cloned B-1118 DsrI, dashed line is for L. mesenteroides NRRL B512F dextransucrase, and dotted line shows elution profile for leucrose and fructose standards.
G. L. Côté et al. / Carbohydrate Research 387 (2014) 1–3
of the enzyme preparation. This work was supported by the United States Department of Agriculture. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer. References 1. Koepsell, H. J.; Tsuchiya, H. M.; Hellman, N. N.; Kazenko, A.; Hoffman, C. A.; Sharpe, E. S.; Jackson, R. W. J. Biol. Chem. 1953, 200, 793–801.
2. 3. 4. 5. 6. 7. 8. 9. 10.
3
Iriki, Y.; Hehre, E. J. Arch. Biochem. Biophys. 1969, 134, 130–138. Robyt, J. F.; Eklund, S. H. Carbohydr. Res. 1983, 121, 279–286. Côté, G. L.; Dunlap, C. A. Carbohydr. Res. 2003, 338, 1961–1967. Côté, G. L.; Skory, C. D. Appl. Microbiol. Biotechnol. 2012, 93, 2387–2394. Côté, G. L.; Skory, C. D.; Unser, S. M.; Rich, J. O. Appl. Microbiol. Biotechnol. 2013, 97, 7265–7273. Bounias, M. Anal. Biochem. 1980, 106, 291–295. Paul, F.; Oriol, E.; Auriol, D.; Monsan, P. Carbohydr. Res. 1986, 149, 433–441. Heincke, K.; Demuth, B.; Jördening, H.-J.; Buchholz, K. Enzyme Microb. Technol. 1999, 24, 523–534. Ronnols, J.; Pendrill, R.; Fontana, C.; Hamark, C.; d’Ortoli, T. A.; Engstrom, O.; Stahle, J.; Zaccheus, M. V.; Sawen, E.; Hahn, L. E.; Iqbal, S.; Widmalm, G. Carbohydr. Res. 2013, 380, 156–166.
