K. and Nakajima, M. 2009. Production of monodis-perse water-in-oil emulsions consisting of highly uniform droplets using asymmetric straight-through microchannel arrays. Microfluid. Nanofluid. 7: 107-119.

24. Vladisavljevic, G.T., Kobayashi, I., and Nakajima, M. 2008. Generation of highly uniform droplets using asymmetric microchannels fabricated on a

single crystal silicon plate: Effect of emulsifier and oil types. Powder Technol., 183: 37-45.

25. Yin, L. J., Kobayashi, I., Nakajima, M. 2008. Effect of polyglycerol esters of fatty acids on the physicochemical properties and stability of β-Carotene emulsions during digestion in simulated gastric fluid. Food Biophys. 3: 213-218.

Biotechnological Modification of Bioactive Natural Compounds for Food Industry

KWAN HWA PARK*

Department of Food Service Management and Nutrition, Sangmyung University, South Korea

ABSTRACT

Enzymatic transglycosylation takes place between an oligosaccharide donor and a relatively large number of structurally diverse acceptor molecules such as bioactive natural compounds. The enzymes including maltogenic amylase (MAase), α-glucanotransferase (α-GTase), and cyclodextrin glucanotransferase (CGTase) are involved in this transglycosylational bioconversion. We have investigated the biotechnological modification of natural compounds such as isoflavones by transglycosylation activity of MAase. MAases catalyze not only hydrolysis of substrate, but also transglycosylation reaction in the presence of various acceptor molecules by preferentially forming α-(1,6)-glycosidic linkage. The mechanism of transglycosylation was elucidated on the basis of three-dimensional structure and site-directed mutagenesis. The results suggested that Glu-332, which is located in a pocket, plays an important role on the formation and accumulation of transfer products by aligning the acceptor molecule in the nucleophilic attack of the glycosyl-enzyme intermediate. In vitro MAase is capable of transferring glucosyl moiety of donor molecules to acceptor such as genistin and puerarin to give an α-(1,6)-glycosidic linkage. We found that transglycosylation reactions have successfully increased the water solubility and the water-soluble products fully maintained the biological activities. In this presentation, some potential applications of the transglycosylated bioactive products in food industry will be discussed.

Key words: transglycosylation, bioactive natural compound, maltogenic amylase, isoflavone, acarbose

                                                       *Author for correspondence. Tel: +82-32-835-8249, 4620; Fax: +82-32-835-0763; E-mail: parkkh@incheon.ac.kr

 

INTRODUCTION

The transglycosylation reaction has been known to modify certain characteristics of substances including glycosides. By the use of transfer reactions using distinct enzymes, a wide variety of transfer products have been obtained between a segment of a donor (R1- glycoside, Figure 1) and various kinds of acceptors (R2- glycoside, Figure 1). In general, the transglycosylation reaction takes place from a specific donor molecule to a relatively large number of different acceptors in structure. The specific-ity of the transfer seems to depend on the type of the en-zymes, determining the configuration of the glycosidic bond formed between donor and acceptor molecule.

Figure 1. Enzymatic transglycosylation between a segment of a donor and acceptor.

Enzymatic methods of glycosylation have many

advantages over chemical methods of synthesizing oligo-saccharides and their derivatives. Complicated proce-dures of the specific protection and deprotection of hydroxyl groups are not required and the enzymes often specifically transfer to one hydroxyl group of the acceptor molecule. This leads to fewer reaction steps, simpler purification procedures, and higher yields.

A new type of amylase named Bacillus stearother-mophilus maltogenic amylase (BSMA) was obtained from B. stearothermophilus in the group, which has transglycosylation activity as well as hydrolytic activity(2). The BSMA showed that it hydrolyzed cyclomaltohep-taose, pullulan, starch, and acarbose and exhibited trans-glycosylation activity, forming both α-(1,6)- and α- (1,4)-glucosidic linkages. When acarbose was incubated with α-amylase, the α-(1,4)-glycosidic linkages were not cleaved and transglycosylation can not occur. The mech-anism of transglycosylation was first illustrated by ob-servation of an extra sugar binding space in the crystal structure of the enzyme(3).

This paper describes maltogenic amylases (MAases), which possess properties distinct from those of other am-ylolytic enzymes. The enzymes hydrolyze both α-(1,4)- and α-(1,6)-glycosidic linkages of starch to yield maltose as the major product. In addition to the hydrolysis activ-

 

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ity, MAases catalyze the transglycosylation reaction in the presence of various acceptor molecules and accumu-late branched glycosidic natural compounds.

The insolubility of bioactive natural compound like isoflavones often limits its biological availability. Therefore, transglycosylation reaction of the enzymes has been used to increase the solubility of the compounds using the transglycosylation process. In addition to increase of the solubility the glycosylated compounds maintained biological activities(3).

I. Transglycosylation Reaction of Maltogenic Amylases from Bacillus Stearothermophilus (BSMA) and Thermus sp. (ThMA)

BSMA was obtained from recombinant Escherichia coli DH5 α that harbors plasmid pSG18. ThMA was also cloned using the similar way like BSMA. It catalyzes cleavage of α-(1,4)-glucosidic linkage and can transfer the products to the sugar moiety of various acceptor mol-ecules. Park and colleagues(2) demonstrated that acarbose was cleaved by BSMA and ThMA and the products were transferred to acceptor molecules by formation of α-(1,3)-, α-(1,4)-, and α-1,6-glycosidic linkages.

The crystal structure of ThMA was determined at 2.8 Ǻ(8). The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (α/β)8 barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical α-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transgly-cosylation and to allow binding of a branched oligosac-charide. In this model the C4-OH group of a maltose at the nonreducing end is 3.6 Ǻ away from Glu-357. The space may be responsible for the transglycosylation activ-ity of ThMA. A mono- or disaccharide occupying the space could serve as an acceptor molecule to compete with a water molecule for attacking the enzyme-substrate intermediate as shown in Figure 2. In the extra sug-ar-binding space, an acceptor sugar molecule may be able to position either the C3-, C4-, or C6-OH group in a proper orientation for nucleophilic attack of the glycosyl enzyme intermediate.

The role of Glu-332 in the hydrolysis and the trans-glycosylation activity of ThMA was studied by site-directed mutagenesis. Replacing Glu-332 with his-tidine reduced transglycosylation activity signifycantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transgly-cosylation activity. These results suggest that an acidic

side chain at position 332 of MAase located in the pocket plays an important role in the formation and accumulation of the transfer products.

Figure 2. Mechanism for transglycosylation reaction of maltogenic amylase.

The role of Glu-332 in the hydrolysis and the trans-

glycosylation activity of ThMA was studied by site-directed mutagenesis. Replacing Glu-332 with his-tidine reduced transglycosylation activity signifycantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transgly-cosylation activity. These results suggest that an acidic side chain at position 332 of MAase located in the pocket plays an important role in the formation and accumulation of the transfer products.

II. Synthesis of Acarbose Transfer Products by BSMA with Various Acceptors

In the presence of the acceptor molecule, BSMA could transfer pseudotrisaccharide (PTS) from acarbose to the acceptor molecule by forming α-(1,3)-, α-(1,4)-, or α-(1,6)-glycosidic linkages (Park et al., 1998).

Figure 3. Schematic diagram of procedures for synthesis and analysis acarbose transglycosylation products.

Acarbose, a pseudotetrasaccharide that has a pseudo

sugar ring at the nonreducing end [4,5,6-trihydroxy-3- (hydroxymethyl)-2-cyclohexen-1-yl] linked to the nitro-gen of 4-amino-4,6-dideoxy-D-glucopyranose (4-amino- 4-deoxy-D-quinovopyranose), which is linked α-(1,4)- to

maltose, is widely recognized as a potent inhibitor of several carbohydrases such as α-glucosidase, glucoamy-lase, α-amylase, and cyclodextrin glucanosyltransferase (CGTase).

Various transfer products including simmondsin were modified with acarbose using the transglycosylation ac-tivity of BSMA (Figures 3 and 4).

Simmondsin, a material related to food intake inhibi-tion from jojoba (Simmondsia chinensis), was transgly-cosylated by reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55oC. The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorp-tion ionization with time of flight (MALDI-TOF)/mass spectrometry (MS) and 13 C-NMR. The major transgly-cosylation product was (PTS)-simmondsin, in which PTS was attached by an α-(1,6)-glycosidic linkage to sim-mondsin (Figure 4). The administration of transglycosy-lated simmondsin with acarbose (200 mg/kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob/ob mice. The transfer product (10 mg/kg) significantly suppressed the postprandial blood glucose response to starch (2 g/kg) by 68%, comparable to 60% of acarbose in Zucker fa/fa rats. Thus, the transfer products would be effective agents in lowering both food intake and blood glucose.

III. Transglycosylation of Bioactive Natural Products: Isoflavones

The isoflavones are often referred to as phytoestro-

gens because of their estrogenic activity, which results from their interactions with estrogen receptors in cells (11,

12). Dietary intake of isoflavones can reduce the risks of hormone-dependent and -independent cancers and car-diovascular diseases. MTase(11) has a unique transfer ac-tivity confined to the transfer of maltosyl units and dis-proportionate maltooligosaccharides to form a set of maltosyl transfer products (e.g., maltose, maltotetraose, maltohexaose). The maltosyl-transfer activity of MTase from T. maritima was used to synthesize new highly soluble isoflavone derivatives. The transglycosylation reaction was successfully conducted with daidzin and maltotriose as the acceptor and donor, respectively.

Incubating maltosyltransferase with starch led to the formation of products with repeated maltose units in-cluding maltosyl-daidzin as major and maltotetrao-syl-daidzin as second product.

Transglycosylation of puerarin (daidzin 8 - C gluco-side) was carried out using various enzymes to increase the water-solubility of puerarin. BSMA was found to be the most effective enzyme among transferases such as MTase, 4-α-glucanotransferase (4-α-GTase) and other transferases used in the reaction. The transglycosylation indicated that MTase and 4-α-GTase did not have acceptor specificity for puerarin which lacks O-glucoside linkage between D-glucose and 7-OH-daidzein.

Figure 4. Transfer products synthesized by transglycosylation reaction of maltogenic amylase (MAase).

(1)

(9)

(3)

(4)

(1)

(10)

References

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ity, MAases catalyze the transglycosylation reaction in the presence of various acceptor molecules and accumu-late branched glycosidic natural compounds.

The insolubility of bioactive natural compound like isoflavones often limits its biological availability. Therefore, transglycosylation reaction of the enzymes has been used to increase the solubility of the compounds using the transglycosylation process. In addition to increase of the solubility the glycosylated compounds maintained biological activities(3).

I. Transglycosylation Reaction of Maltogenic Amylases from Bacillus Stearothermophilus (BSMA) and Thermus sp. (ThMA)

BSMA was obtained from recombinant Escherichia coli DH5 α that harbors plasmid pSG18. ThMA was also cloned using the similar way like BSMA. It catalyzes cleavage of α-(1,4)-glucosidic linkage and can transfer the products to the sugar moiety of various acceptor mol-ecules. Park and colleagues(2) demonstrated that acarbose was cleaved by BSMA and ThMA and the products were transferred to acceptor molecules by formation of α-(1,3)-, α-(1,4)-, and α-1,6-glycosidic linkages.

The crystal structure of ThMA was determined at 2.8 Ǻ(8). The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (α/β)8 barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical α-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transgly-cosylation and to allow binding of a branched oligosac-charide. In this model the C4-OH group of a maltose at the nonreducing end is 3.6 Ǻ away from Glu-357. The space may be responsible for the transglycosylation activ-ity of ThMA. A mono- or disaccharide occupying the space could serve as an acceptor molecule to compete with a water molecule for attacking the enzyme-substrate intermediate as shown in Figure 2. In the extra sug-ar-binding space, an acceptor sugar molecule may be able to position either the C3-, C4-, or C6-OH group in a proper orientation for nucleophilic attack of the glycosyl enzyme intermediate.

The role of Glu-332 in the hydrolysis and the trans-glycosylation activity of ThMA was studied by site-directed mutagenesis. Replacing Glu-332 with his-tidine reduced transglycosylation activity signifycantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transgly-cosylation activity. These results suggest that an acidic

side chain at position 332 of MAase located in the pocket plays an important role in the formation and accumulation of the transfer products.

Figure 2. Mechanism for transglycosylation reaction of maltogenic amylase.

The role of Glu-332 in the hydrolysis and the trans-

glycosylation activity of ThMA was studied by site-directed mutagenesis. Replacing Glu-332 with his-tidine reduced transglycosylation activity signifycantly, but enhanced hydrolysis activity on α-(1,3)-, α-(1,4)-, and α-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transgly-cosylation activity. These results suggest that an acidic side chain at position 332 of MAase located in the pocket plays an important role in the formation and accumulation of the transfer products.

II. Synthesis of Acarbose Transfer Products by BSMA with Various Acceptors

In the presence of the acceptor molecule, BSMA could transfer pseudotrisaccharide (PTS) from acarbose to the acceptor molecule by forming α-(1,3)-, α-(1,4)-, or α-(1,6)-glycosidic linkages (Park et al., 1998).

Figure 3. Schematic diagram of procedures for synthesis and analysis acarbose transglycosylation products.

Acarbose, a pseudotetrasaccharide that has a pseudo

sugar ring at the nonreducing end [4,5,6-trihydroxy-3- (hydroxymethyl)-2-cyclohexen-1-yl] linked to the nitro-gen of 4-amino-4,6-dideoxy-D-glucopyranose (4-amino- 4-deoxy-D-quinovopyranose), which is linked α-(1,4)- to

maltose, is widely recognized as a potent inhibitor of several carbohydrases such as α-glucosidase, glucoamy-lase, α-amylase, and cyclodextrin glucanosyltransferase (CGTase).

Various transfer products including simmondsin were modified with acarbose using the transglycosylation ac-tivity of BSMA (Figures 3 and 4).

Simmondsin, a material related to food intake inhibi-tion from jojoba (Simmondsia chinensis), was transgly-cosylated by reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55oC. The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorp-tion ionization with time of flight (MALDI-TOF)/mass spectrometry (MS) and 13 C-NMR. The major transgly-cosylation product was (PTS)-simmondsin, in which PTS was attached by an α-(1,6)-glycosidic linkage to sim-mondsin (Figure 4). The administration of transglycosy-lated simmondsin with acarbose (200 mg/kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob/ob mice. The transfer product (10 mg/kg) significantly suppressed the postprandial blood glucose response to starch (2 g/kg) by 68%, comparable to 60% of acarbose in Zucker fa/fa rats. Thus, the transfer products would be effective agents in lowering both food intake and blood glucose.

III. Transglycosylation of Bioactive Natural Products: Isoflavones

The isoflavones are often referred to as phytoestro-

gens because of their estrogenic activity, which results from their interactions with estrogen receptors in cells (11,

12). Dietary intake of isoflavones can reduce the risks of hormone-dependent and -independent cancers and car-diovascular diseases. MTase(11) has a unique transfer ac-tivity confined to the transfer of maltosyl units and dis-proportionate maltooligosaccharides to form a set of maltosyl transfer products (e.g., maltose, maltotetraose, maltohexaose). The maltosyl-transfer activity of MTase from T. maritima was used to synthesize new highly soluble isoflavone derivatives. The transglycosylation reaction was successfully conducted with daidzin and maltotriose as the acceptor and donor, respectively.

Incubating maltosyltransferase with starch led to the formation of products with repeated maltose units in-cluding maltosyl-daidzin as major and maltotetrao-syl-daidzin as second product.

Transglycosylation of puerarin (daidzin 8 - C gluco-side) was carried out using various enzymes to increase the water-solubility of puerarin. BSMA was found to be the most effective enzyme among transferases such as MTase, 4-α-glucanotransferase (4-α-GTase) and other transferases used in the reaction. The transglycosylation indicated that MTase and 4-α-GTase did not have acceptor specificity for puerarin which lacks O-glucoside linkage between D-glucose and 7-OH-daidzein.

Figure 4. Transfer products synthesized by transglycosylation reaction of maltogenic amylase (MAase).

(1)

(9)

(3)

(4)

(1)

(10)

References

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Two major products were identified as gluco-syl-α-(1,6)-puerarin and maltosyl-α-(1,6)-puerarin. The solubilities of the transfer products were 14 and 200 times higher than that of puerarin, respectively. In Fig.4 various transfer products synthesized by MAase were listed.

IV. Transglycosylation Reaction with Cyclodextrin Di-O-α-maltosyl-β-cyclodextrin [(G2)2-β-CD] was syn-thesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. The synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (7)

was identified to be isomers of 61,63- and 61,64-dimaltosyl-β-CD.

CONCLUSIONS A variety of modified bioactive compounds

have been synthesized by enzymatic transfer reactions involving maltogenic amylase, α-glucanotransferase, cyclodextrin glucanotransferase and some debranching enzyme. The major advantage of the glycosylation of isoflvones is that its glycosides greatly improve the water solubility while maintaining the antioxidant activities. The newly formed α-(1,6)-glycosidic linkage of the transglycosylation products was easily hydrolysable in the human body by intestinal microorganisms that use glycosyl hydrolases and digestive enzymes, implying that a high concentration of isoflavone glycosides may be metabolized in the same way as natural isoflavone. In conclusion, the glycosylated bioactive natural compounds have similar antioxidant activity levels. Considering their excellent water solubility, transfer products may have potential as a healthy nutraceutical and a functional food ingredient.

ACKNOWLEDGMENTS

This work was supported in part by Basic Science

Research Program (2009-0087146) through the National Research Foundation and in part by the Technology Development Program of Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea (109153032WT111).

REFERENCES

1. Baek, J. S, Kim, H. Y., Yoo, S. S, Cheong, T. K.,

Kim, M. J., Lee, S. B., Abbott, T. P., Song, H. J., Rhyu, M. R., Oh, B. H. and Park, K. H. 2000. Synthesis of acarbose transfer products by Bacillus stearothermophilus maltogenic amylase with simmondsin. Ind. Crops Prod. 12: 173-182.

2. Cha, H. J., Yoon, H. G., Kim, Y. W., Lee, H. S., Kim,

J. W., Kweon, K. S., Oh, B. H. and Park, K. H. 1998. Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur. J. Biochem. 253: 251-262.

3. Chung, M. J., Kang, A. Y., Lee, K. M., Oh, E., Jun, H. J., Kim, S. Y., Auh, J. H., Moon, T. H., Lee, S. J. and Park, K. H. 2006. Water-soluble genistin glycoside isoflavones up-regulate antoxidant metallothionein expression and scavenge free radicals. J. Agric. Food Chem. 54: 3819-3826.

4. Chung, M. J., Sung, N. J., Park, C. S., Kweon, D. K., Mantovani, A., Moon, T. W., Lee, S. J. and Park, K. H. 2008. Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57 BL/6J mice. Eur. J. Pharmacol. 578: 159-170.

5. Fiechter, G., Opacak, I., Raba, B. and Mayer, H. K. 2011. A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: application to analyze isoflavone levels in soybean cultivars. Food Res. Int. Doi: 10.1016/j.foodres. 2011.03.038.

6. Kang, H. K., Cha, H., Yang, T. J., Park, J.T., Lee, S., Kim, Y. W., Auh, J. H., Okada, Y., Kim, J. W., Cha, J., Kim, C. H., Park, K. H. 2007. Enzymatic synthesis of dimaltosyl-β-cyclodextrin via a transglycosylation reaction using TreX, a sulfolobus solfataricus P2 debranching enzyme. Biochem. Biophys. Res. Commun. 366: 98-103.

7. Kim, M. J., Lee, H. S., Cho, J. S., Kim, T. J., Moon, T. H., Oh, S. T., Kim, J. W., Oh, B. H. and Park, K. H. 2002. Preparation and characterization of α-D-Glucopyranosyl-α-acarviosinyl-D-glucopyranose, a novel inhibitor specific for maltose-producing amylase. Biochem. 41: 9099-9108.

8. Kim, J. S., Cha, S. S., Kim, H. J., Kim, T. J., Ha, N. C., Oh, S. T., Cho, H. S., Kim, M. J., Kim, J. W., Choi, K. Y., Park, K. H. and Oh, B. H. 1999. Crystal structure of a maltogenic amylase: provides insights into a catalytic versatility. J. Biol. Chem. 274: 26279-26286.

9. Lee, S. J, Kim, J. C., Kim, M. J., Kitaoka, M., Park, C. S., Lee, S. Y., Ra, M. J., Moo, T. H., Robyt, J. F. and Park, K. H. 1999. Transglycosylation of naringin by Bacillus stearothermophilus maltogenic amylase to give glycosylated naringin. J. Agric. Food Chem. 47: 3669-3674.

10. Lee, Y. S., Lee, M. H., Lee, H. S., Lee, S. J., Kim, Y. W., Zhang, R., Withers, S. G., Kim, K. S., Lee, S. J. and Park, K. H. 2008. Enzymatic synthesis of a selective inhibitor for α-glucosidases: α-acarviosinyl-(1→9)-3- α-D-glucopyranosylpropen. J. Agric. Food Chem. 56: 5324-5330.

11. Meissner, H. L. and Leibl, W. 1998. Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. Eur. J. Biochem. 250:

1050-1058. 12. Schwarts, H. and Sontag, G. 2009. Comparison of

sample preparation methods for analysis of isoflavones in foodstuffs. Anal. Chim. Acta. 633: 204-215.

 

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Two major products were identified as gluco-syl-α-(1,6)-puerarin and maltosyl-α-(1,6)-puerarin. The solubilities of the transfer products were 14 and 200 times higher than that of puerarin, respectively. In Fig.4 various transfer products synthesized by MAase were listed.

IV. Transglycosylation Reaction with Cyclodextrin Di-O-α-maltosyl-β-cyclodextrin [(G2)2-β-CD] was syn-thesized from 6-O-α-maltosyl-β-cyclodextrin (G2-β-CD) via a transglycosylation reaction catalyzed by TreX, a debranching enzyme from Sulfolobus solfataricus P2. The synthesis of dimaltosyl-β-CD occurred exclusively via transglycosylation of an α-1,6-glucosidic linkage. Based on the HPLC elution profile, the transfer product (7)

was identified to be isomers of 61,63- and 61,64-dimaltosyl-β-CD.

CONCLUSIONS A variety of modified bioactive compounds

have been synthesized by enzymatic transfer reactions involving maltogenic amylase, α-glucanotransferase, cyclodextrin glucanotransferase and some debranching enzyme. The major advantage of the glycosylation of isoflvones is that its glycosides greatly improve the water solubility while maintaining the antioxidant activities. The newly formed α-(1,6)-glycosidic linkage of the transglycosylation products was easily hydrolysable in the human body by intestinal microorganisms that use glycosyl hydrolases and digestive enzymes, implying that a high concentration of isoflavone glycosides may be metabolized in the same way as natural isoflavone. In conclusion, the glycosylated bioactive natural compounds have similar antioxidant activity levels. Considering their excellent water solubility, transfer products may have potential as a healthy nutraceutical and a functional food ingredient.

ACKNOWLEDGMENTS

This work was supported in part by Basic Science

Research Program (2009-0087146) through the National Research Foundation and in part by the Technology Development Program of Ministry for Food, Agriculture, Forestry, and Fisheries, Republic of Korea (109153032WT111).

REFERENCES

1. Baek, J. S, Kim, H. Y., Yoo, S. S, Cheong, T. K.,

Kim, M. J., Lee, S. B., Abbott, T. P., Song, H. J., Rhyu, M. R., Oh, B. H. and Park, K. H. 2000. Synthesis of acarbose transfer products by Bacillus stearothermophilus maltogenic amylase with simmondsin. Ind. Crops Prod. 12: 173-182.

2. Cha, H. J., Yoon, H. G., Kim, Y. W., Lee, H. S., Kim,

J. W., Kweon, K. S., Oh, B. H. and Park, K. H. 1998. Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose. Eur. J. Biochem. 253: 251-262.

3. Chung, M. J., Kang, A. Y., Lee, K. M., Oh, E., Jun, H. J., Kim, S. Y., Auh, J. H., Moon, T. H., Lee, S. J. and Park, K. H. 2006. Water-soluble genistin glycoside isoflavones up-regulate antoxidant metallothionein expression and scavenge free radicals. J. Agric. Food Chem. 54: 3819-3826.

4. Chung, M. J., Sung, N. J., Park, C. S., Kweon, D. K., Mantovani, A., Moon, T. W., Lee, S. J. and Park, K. H. 2008. Antioxidative and hypocholesterolemic activities of water-soluble puerarin glycosides in HepG2 cells and in C57 BL/6J mice. Eur. J. Pharmacol. 578: 159-170.

5. Fiechter, G., Opacak, I., Raba, B. and Mayer, H. K. 2011. A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: application to analyze isoflavone levels in soybean cultivars. Food Res. Int. Doi: 10.1016/j.foodres. 2011.03.038.

6. Kang, H. K., Cha, H., Yang, T. J., Park, J.T., Lee, S., Kim, Y. W., Auh, J. H., Okada, Y., Kim, J. W., Cha, J., Kim, C. H., Park, K. H. 2007. Enzymatic synthesis of dimaltosyl-β-cyclodextrin via a transglycosylation reaction using TreX, a sulfolobus solfataricus P2 debranching enzyme. Biochem. Biophys. Res. Commun. 366: 98-103.

7. Kim, M. J., Lee, H. S., Cho, J. S., Kim, T. J., Moon, T. H., Oh, S. T., Kim, J. W., Oh, B. H. and Park, K. H. 2002. Preparation and characterization of α-D-Glucopyranosyl-α-acarviosinyl-D-glucopyranose, a novel inhibitor specific for maltose-producing amylase. Biochem. 41: 9099-9108.

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