Characteristics of transglycosylation reaction of cyclodextrin glucanotransferase in the heterogeneous enzyme reaction system using extrusion starch as a glucosyl donor

ELSEVI ER

Characteristics of transglycosylation reaction of cyclodextrin glucanotransferase in the heterogeneous enzyme reaction system using extrusion starch as a glucosyl donor Dong-Chan Park, Tae-Kwon Kim, and Yong-Hyun Lee Department of Genetic Taegu, Korea

Engineering,

College

of Natural

Sciences,

Kyungpook

National University,

Characteristics of the intermolecular transglycosylation reactions of saccharides and glucosides by cyclodextrin glucanotransferase (CGTase) in the heterogeneous enzyme reaction system using swollen extrusion starch as a glucosyl donor were analyzed. The transglycosylation yield and rate were signijicantly increased compared to the conventional reaction system using liquefied starch as the glucosyl donor. Various monosaccharides composed of the same configuration of 2-, 3-, and 4-OH with the o-glucopyranoside such as o-xylose, D-ghc0.w. L-.sorbose. and myo-inositol were found to be good acceptors for the transglycosylation reaction. Several glucosides such as stevioside and hesperidin were also identified as the suitable glucosyl acceptors. The optima/ transglycosylation reaction condition using stevioside as the glucosyl acceptor was also determined. The heterogeneous transglycosylation reaction system shows u few distinct advantages; minimized accumulation of oligosaccharides. easy removal of residual extrusion starch after reaction, and simplified purification of trunsg!ycosylated products. therefore, which will facilitate the industrial application qf the various transgl~c’osylation reactions. 0 1998 Elsevier Science Inc. Keywords: Transglycosylation; cyclodextrin system; extrusion starch as glucosyl donor

glucanotransferase;

Introduction Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) catalyzes mainly two enzyme reactions; cyclodextrin (CD) production from starch and related (Y-1,4-glucans through the intramolecular transglycosylation reaction, and the coupling and disproportionation reactions that transfer the glucosyl residue in starch or CD to the acceptor molecules through the intermolecular transglycosylation reaction. ‘.’ Various saccharides and glucosides including glucose,3 xy10se,~ sorhose,4 inositol,” sucrose,6 maltose,” lactose,7

Address reprint requests to Professor Y. H. Lee, Department of Genetic Engineering, College of Natural Sciences, Kyungpook National University, Taegu 702-70 1, South Korea Received 23 March 1996; revised 26 February 1997; accepted 22 July 1997

Enzyme and Microbial Technology 22:217-222, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

saccharides:

glucosides:

heterogeneous

enzyme reaction

stevioside,’ hesperidin,’ rutin,” and ascorbic acid,’ ’ have been identified as the acceptor compounds for an intermolecular transglycosylation reaction. The transglycosylated saccharides and glucosides that have mono- or oligoglucoside residues linked to their parent acceptor molecules usually showed significantly improved fbnctionalities such as improved sweetness, increased water solubility, stimulating growth of Bifidobacteria, and increased stability against chemicals. ” Most of the transglycosylation reactions have been performed in the homogeneous enzyme reaction system utilizing soluble starch or CDs as the glucosyl donor; however, the above homogeneous reaction systems have the following technical disadvantages for an industrial application caused by the homogeneous nature of the reaction system where both the glucosyl donor and acceptor exist in the soluble state; therefore, the separation of transglycosylated

oi4i-0229/9at$i9.00 PII SOl41-0229(97)00183-X

Papers products from the soluble state is very difficult and a significant amount of undesirable reducing sugar remains after completion of the reaction. The purification of transglycosylated products can be simplified, provided insoluble raw starch is used as the glucosyl donor, because the insoluble state residual starch can be easily removed from the reaction mixture. Also, the accumulation of undesirable oligosaccharides can be minimized because the glucosyl residues can be supplied directly from the surface of raw starch when not using oligosaccharides as the glucosyl donor. Raw starch exists in compact and crystalline structure; therefore, the rate and yield of the transglycosylation reaction were usually too low. The structural modification of raw starch is required to increase the susceptibility for the enzyme reaction. Swollen extrusion starch has been used as substrate in the production of cyclodextrin in our previous works.‘3-16 Extrusion starch suspends in water in an insoluble swollen state; therefore, the heterogeneous reaction system similar to raw starch can be maintained. According to our previous work 13-16 the direct synthesis of CD from swollen extrusion &arch can be performed effectively by CGTase without accumulation of undesirable oligosaccharides. As a consequence, the high rate and yield of CD production can be obtained. Furthermore, the reaction system facilitates the purification of CD because the residual starch can be removed by simple centrifugation or filtration. In this work, the acceptor specificities for the transglycosylation reaction of various saccharides and glucosides in the heterogeneous enzyme reaction system using extrusion starch as the glucosyl donor were analyzed. The characteristics of the transglycosylation reaction of stevioside in the heterogeneous enzyme reaction system were also studied and compared with the conventional reaction system using soluble liquefied starch. The optimal reaction conditions for the transglycosylation of stevioside, including enzyme dosage, substrate concentration, and the mixing ratio of donor and acceptor, were determined. The reaction characteristics were analyzed by following the changes in substrates, products, and intermediate compound concentrations during reaction.

Materials and methods Enzyme and determination

of activity

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19, Amano Pharmaceutical Co., Ltd., Nagoya, Japan) from Bacillus macerun~ was used. The starch-hydrolyzing activity of the CGTase was activity of 513.5 Units mg-’ protein. The starch-hydrolyzing CGTase was determined by the Kitahata methodI using 1% (w/v) soluble starch in 0.02 M Tris-malate-NaOH buffer pH 6.0 as a substrate. One unit (U) of activity was defined as the amount of CGTase corresponding to a 1% increase in transmittance at 660 nm min-‘.

Glucosyl donors The starch was extruded in a single-screw extruder, and the extrusion starch (degree of gelatinization at 70.1%) was used as the glucosyl donor. The liquefied starch was liquefied using a thermostable liquefying enzyme from Bacillus licheniformis (Ter-

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mamyl, Type LS. Novo Nordisk Co.. Bagsvaerd, Denmark) at 90°C for 20 min (DE value of 5) and was also used for comparison.

Glucosyl acceptors Various monosaccharides, disaccharides, and glucosides were used as the glucosyl acceptors. The monosaccharides included glucose, xylose, sorbose, rhamnose, inositol, ribose, and galactose and the disaccharides included maltose, cellobiose, sucrose, and lactose. Several glucosides such as stevioside, hesperidin, rutin. salicin, and naringin were also used as the glucosyl acceptors.

The transglycosylation reaction in the heterogeneous enzyme reaction system using extrusion starch as the glucosyl donor Extrusion starch (50 g) and 50 g of acceptors were suspended in 1.0 1 0.02 M Tris-malate-NaOH buffer, and then 900 U CGTase reaction was g -’ starch were added. The transglycosylation performed at 5O”C, pH 6.0, and 200 ‘pm. The amounts of extrusion starch, acceptor, and CGTase were changed accordingly. The transglycosylation yield was calculated after measuring the residual acceptor concentration.

Analytical

methods

Monosaccharides, disaccharides, tmnsglycosylated products, and cyclodextrins were analyzed by HPLC (Gilson Medical Electronics, Inc., Villiers-le-Bel, France); Cosmosil packed column 5NH, (Nacalai Tesque, Inc., Kyoto, Japan), acetonitrile/water(65/35), 1 ml min-‘, and a RI detector. The hesperidin, rutin, and naringin were also analyzed by HPLC under the same conditions but using a UV detector at a wavelength of 2.58 nm. The soluble protein concentration was determined by the Bradford method,” and the reducing sugar concentration was analyzed by the DNS method.”

Results and discussion Acceptor specificity for the transglycosylation reactions in the heterogeneous enzyme reaction system using extrusion starch as a glucosyl donor Table I shows the transglycosylation yields of various mono- and disaccharides, and glucosides using extrusion starch as the glucosyl donor. These are compared with the conventional reaction system using liquefied starch. The transglycosylation yields from extrusion starch showed much higher values compared to those obtained from liquefied starch for all acceptors. For example, the transglycosylation yields of glucose and maltose after 24 h were measured at 0.52 and 0.82 compared to 0.32 and 0.71, respectively, from liquefied starch. This corresponds to 62.5 and 15.4% increments, respectively. Extrusion starch suspends in the reaction mixture in a highly swollen state susceptible to the CGTase reaction, and an effective transfer of glucosyl residues from the surface of the swollen extrusion starch directly to the acceptor molecules has been performed. Among monosaccharides, o-glucose, o-xylose, L-sorbose, and mvo-inositol were identified as the suitable acceptors for the transglycosylation reaction of CGTase whereas o-galactose, D-ribose, o-mannose, D-arabinose, and o-fructose did not contribute as the glucosyl acceptor. The acceptor specificity from extrusion starch is generally in

Transglycosylation: Table 1 Comparison of substrate specificity for transglycosylation reaction of cyclodextrin glucanotransferase using various saccharides and glucosides as a glucosyl acceptor in the reaction system containing extrusion starch and liquefied starch

Acceptors Monosaccharides o-Glucose o-Xylose myo-lnositol L-Sorbose Disaccharides o-Maltose Sucrose Cellobiose Glucosides Stevioside Hesperidin Salicin

Extrusion Starch

Liquefied Starch

Incrementsa

0.52b 0.31 0.37 0.51

0.32 0.26 0.25 0.42

62.5 19.2 48.0 21.4

0.82 0.65 0.76

0.71 0.61 0.67

15.5 6.6 13.4

0.78 0.63 0.69

0.66 0.52 0.54

18.2 21.2 27.8

Small amounts of cyclodextrins (a-, p-, and y-CDs), the intermediates for the transglycosylation reaction, were also detected. The detailed molecular structure of the transglycosylated products and their physicochemcal properties needs to be examined after separation. Comparison stevioside

Reaction conditions: 50 g I-’ starch, 50 g I-’ each acceptor, 900 U CGTase g-r starch, pH 6.0, 5O”C, and 200 rpm a Increments of transglycosylation yield in the heterogeneous enzyme reaction system using extrusion starch compared to the conventional system using liquefied starch, % b Transglycosylation yield after 24 h

agreement with the previous report observed from soluble starchZo in which monosaccharides containing the pyranose structure (the configuration of C,-, C,-, and C,- hydroxyl groups are the same with o-glucopyranose) are good acceptors for the transglycosylation reaction of CGTase. Most of the transglycosylation yields of disaccharides showed much higher values compared to those of monosaccharides. This indicates that the CGTase has a higher affinity to disaccharides and the acceptor binding site of CGTase can recognize at least two glucopyranose molecules. Nakamura et al.” also observed that maltose and cellobiose have higher acceptor specificity than glucose for the transglycosylation reaction of CGTase. Stevioside, hesperidin, and salicin were identified as a good glucosyl acceptor, and their transglycosylation yields were 0.78, 0.63, and 0.69, respectively. These were also much higher values. On the other hand, naringin and rutin did not act as the glucosyl acceptors for both reaction systems. It has been reported that the CGTase from Bacillus macerans can catalyze the transglycosylation reaction to stevioside, but not to other glucosides, i.e., rutin.” Meanwhile, the CGTase from B. stearothermophilus or alkalophilic Bacillus sp. can induce the transglycosylation of rutin glucosides.” The transglycosylating capability of CGTase to the glucosides seems to be very dependent on the sources of CGTase, and the CGTase used in this work can contribute to the transglycosylation of stevioside, hesperidin, and salicin all together at relatively high yields. Figure 1 compares the HPLC chromatograms of acceptor compounds glucose (A), maltose (C), and stevioside (E) to those of transglycosylated products obtained after 24 h (B, D, and F, respectively). The peak height of the parent acceptor molecules were reduced, and most transglycosylated products were found to have less than one glucose molecule compared to the original acceptor molecules.

D.-C. Park et al.

of transglycosylation

in the heterogeneous

system and the conventional

yields

qf

enzyme reaction

system

Figure 2 compares the progress of three transglycosylation reactions of stevioside using extrusion starch, raw starch, and liquefied starch as the glucosyl donors. The initial rates from extrusion starch and liquefied starch were not very different from each other; however, the transglycosylation reaction using extrusion starch proceeded more rapidly. The final yield was found to be 0.78 after 24 h. This was a much higher value compared to liquefied starch at 0.68. The transglycosylation reaction from raw starch proceeded very slowly. This indicated that CGTase could not attack the crystalline structure of raw starch effectively. The amounts of reducing sugar accumulated in the reaction mixture after 24 h were also measured. Only 0.34 g 1-l for extrusion starch was observed. This was a relatively small amount compared to that of liquefied starch at 7.84 g 1-l. The catalytic reaction of CGTase on the extrusion starch seems to be performed from the nom-educing end of

I,d C)

5

10

0

5

10

Retention lime (rmn)

of glucose Figure 1 Comparison of the HPLC chromatogram (A), maltose (Cl, and stevioside (6) to the reaction mixture obtained from the transglycosylation reaction using each acceptor molecule in the heterogeneous enzyme reaction system (B, 0, and F, respectively). Peak labeled: glucose, G; maltose, M; stevioside, S; a-CD, a; P-CD, 6; y-CD, y; transglycosylated products of each acceptor molecule (numbers)

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E .g

s

‘Z

m f 8 2

0.6

0.6

0.4

f

O,OV Reactum time (h)

O 1500

Amount of CGTase (units per g of starch)

Figure 2 Comparison of the progress in the transglycosylation reaction of stevioside in the enzyme reaction system using raw (A), extrusion (01, and liquefied (0) starch as the glucosyl donor. The reaction was performed at 50 g I-’ starch, 20 g I-’ stevioside, 900 U CGTase g-’ starch, pH 6.0, 50°C, and 200 rpm for 24 h

Figure 4 Effect of the amount of CGTase on the transglycosylation yield of stevioside (0) and the accumulation of cyclodextrin (Cl) in the heterogeneous enzyme reaction system. The reaction was performed under the same conditions as Figure 2 except for the amount of CGTase

the micelle on the surface of the swollen extrusion starch as described in our previous work.22 As a consequence, the accumulation of reducing sugar representing maltooligosaccharides can be minimized. Also, the residual maltooligosaccharides can be utilized as the acceptor molecules for other transglycosylation reactions of CGTase; therefore, the high transglycosylation yield of stevioside can be achieved.

The residual reducing sugar in the reaction mixture was decreased drastically to lower than 0.3 g 1-l in the heterogeneous enzyme reaction system compared to over 8.0 g 1-l in the conventional reaction system using liquefied starch as a glucosyl donor (Figure 3). The low residual reducing sugar may be due to the characteristic reaction mode of CGTase on the extrusion starch as described in our previous work.22 The easy separation of residual extrusion starch and the low accumulation of residual reducing sugar in the reaction mixture will facilitate the purification of the various transglycosylated products.

The feasibility of separation of transglycosylated product in the heterogeneous enzyme reaction system Figure 3 compares the amounts of residual starch separable by simple centrifugation after reaction in two enzyme reaction systems during 24 h. Most of the residual starch was separated out from the reaction mixture by centrifugation at 3,000 g for 10 min because the extruded swollen starch existed in a suspended state in the reaction mixture while the residual liquefied starch in the conventional system was hardly separated (Figure 3).

0

6

12

16

24

Reaction time (h)

Figure 3 Comparison of the amounts of separable starch by simple centrifugation (0) and concentration of the residual reducing sugar in the reaction mixture (0) in enzyme reaction system using extrusion starch (closed symbols) and liquefied starch (open symbols) as the glucosyl donor. The reaction was performed under the same conditions as Figure 2

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Determination of the optimum reaction conditions for the transglycosylation reaction of stevioside Figure 4 illustrates the effect of the amount of CGTase on the transglycosylation yield of stevioside in the heterogeneous enzyme reaction system performed at different amounts of CGTase ranging from lOO--1,500 Units g-’ extrusion starch. The transglycosylation yields were increased proportionally as the amount of enzyme increased to 900 Units g-’ starch. Thereafter, they remained at the same level. Meanwhile, the accumulation of cyclodextrin in the reaction mixture was inversely decreased as the amount of enzyme increased. The excess CGTase higher than the above amount may induce unnecessary side reactions such as, the hydrolysis of cyclodextrin or a disproportionation reaction; therefore, it cannot contribute to increase the transglycosylation yield of stevioside. The optimal amount of CGTase was identified to be around 900 U g-’ extrusion starch. Figure 5 shows the effect of the mixing ratio of stevioside and extrusion starch on the transglycosylation yield and accumulation of cyclodextrin. The level of extrusion starch was fixed at 50 g 1-l and the amount of stevioside was changed from 10 to 150 g 1-l. The maximum yield was obtained at a mixing ratio of 2050 (g of stevioside:g of starch), and then decreased gradually as the amount of stevioside increased. Meanwhile, cyclodextrin decreased continuously from 21.2 to 7.4 g I-’ as

Transglycosylation:

0.0

1

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I

I

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50

75

10

00

0

100

0 Stewside

D-C. Park et al.

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12

18

24

concentration (g b’) Reaction time (h)

Figure 5 Effect of the mixing ratio of stevioside and extrusion starch on the transglycosylation yield of stevioside (0) and accumulation of cyclodextrin (0) in the heterogeneous enzyme reaction system. The reaction was performed under the same conditions as Figure 2 except for the mixing ratio of stevioside and extrusion starch

stevioside increased. The lower yields even at the increased stevioside concentration can be explained both by the limitations of glucosyl donor and CGTase. The optimum mixing ratio of stevioside and extrusion starch were identified to be in the ratio of 0.4: 1.0 (g of stevioside:g of extrusion starch). The effect of the concentration of extrusion starch (changing from 10 to 150 g 1-j) and fixing the mixing ratio of stevioside and extrusion starch to 0.4: 1.O are depicted in Figure 6. The transglycosylation yield remained constant up to an extrusion starch level of 75 g l- ‘, and then it decreased drastically at a concentration higher than 100 g 1-l. Meanwhile, cyclodextrin increased proportionally as the extrusion starch increased up to 75 g l-l, but maintained a similar level thereafter. At a high concentration, most of the water molecules are absorbed by the extrusion starch. As a consequence, the fluidity of the reaction mixture increases drastically. This prevents an effective transglycosylation reaction.

1.0 I,

30

Figure 7 The progress of the concentration changes in stevioside (V), cyclodextrin (O), transglycosylated sugar (01, and transglycosylation yield (0) during the tranqglycosylation reaction in the heterogeneous enzyme reaction system. The reaction was performed under the same conditions as Figure 2

Product profiles and mechanism of transglycosylation reaction of stevioside in the heterogeneous enzyme reaction system Figure 7 shows the progress of the transglycosylation reaction of stevioside, such as changes in stevioside as glucosyl acceptor, cyclodextrins (total CD), and transglycosylated stevioside concentrations. Cyclodextrin, known as an intermediary in the transglycosylation reaction of CGTase, accumulated noticeably at the initial stage of the reaction; however, it started to decrease slightly after 2 h. This was a different trend from our previous work,13 in which CD production was performed from extrusion starch but without the addition of stevioside. Baek et uL.,*~who studied the transglycosylation reaction of stevioside in an agitated bead reaction system using raw starch as the glucosyl donor. reported that the transglycosylation reaction occurred via two steps: firstly, the synthesis of CD from raw starch, and then secondly, the transglycosylation of glucosyl residue from CD to the acceptor molecules. Profiles of the changes of all intermediate concentrations including CD and transglycosylated saccharides showed much similarity with our previous work;” therefore, it can be postulated that the transglycosylation reaction using extrusion starch is also performed similarly. Further studies need to be conducted including kinetic and mechanistic analysis, process development, scale-up, and an economic feasibility evaluation for the practical application of the proposed reaction system.

Acknowledgments

00-o 0

50

100

150

Extrusion starch concentration (g I-‘)

Figure 6 Effect of the substrate concentration on the transglycosylation yield (0) and accumulation of cyclodextrin (0) in the heterogeneous enzyme reaction system. The reaction was performed under the same conditions as Figure 2 except for the substrate concentration, and the mixing ratio of stevioside to extrusion was fixed as 0.4:1 .O

This research was supported by a grant from the Korea Science and Engineering Foundation (No. 95-0402-05 01-3) in 1995-1996 and the 1996 research grant to Research Center for New Bio-Materials in Agriculture from the Korea Science and Engineering Foundation.

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