Cyclodextrins Are Not the Major Cyclic alpha -1,4-Glucans Produced by the Initial Action of Cyclodextrin Glucanotransferase on Amylose*

(Received for publication, January 14, 1997, and in revised form, March 27, 1997)

Yoshinobu Terada Dagger , Michiyo Yanase , Hiroki Takata , Takeshi Takaha and Shigetaka Okada

From the Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Utajima 4-6-5, Nishiyodogawa-ku, Osaka 555, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilic Bacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic alpha -1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known alpha -, beta -, and gamma -cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic alpha -1,4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic alpha -1,4-glucans and into the final major product, beta -CD. CGTase from Bacillus macerans also produced large cyclic alpha -1,4-glucans except that the final major product was alpha -CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.


INTRODUCTION

Cyclodextrin glucanotransferase (CGTase,1 EC 2.4.1.19), found in several bacterial species, catalyzes the inter- and intramolecular transglycosylation of alpha -1,4-glucan. Such activity of CGTase on inter- and intramolecular transglycosylation of alpha -1,4-glucan is called the disproportionation reaction and the cyclization reaction, respectively. It is also known that CGTase catalyzes the transglycosidic linearization (coupling reaction) of cyclic alpha -1,4-glucan in the presence of a suitable acceptor molecule to produce linear alpha -1,4-glucan. The cyclization reaction of CGTase has been of great interest since this is the only enzyme that can produce alpha -, beta - and gamma -cyclodextrin (CD), which are generally known as the cyclic alpha -1,4-glucan with DP of 6, 7, or 8. These CDs all have a hydrophobic central cavity, incorporate various inorganic or organic compounds, and form inclusion complexes (1). Therefore, these CDs are widely used in the pharmaceutical, food, agricultural, and cosmetic industries (2).

Extensive analyses on various CGTases indicated that all CGTases convert amylose or amylopectin into a mixture of alpha -, beta -, and gamma -CD and remaining dextrins; differences, however, were found in their product specificities (alpha -, beta -, and gamma -CD ratios). Thus, CGTase is sometimes classified into three types (alpha -, beta -, and gamma -CGTase), depending on the major CD produced. Since alpha -, beta -, and gamma -CD all have a dimensionally distinct central cavity and different specificity for guest molecules, recent studies on CGTase have focused on trying to understand the mechanism of the cyclization reaction and to find or engineer a CGTase that produces a specific type of CD.

Several approaches have been carried out to obtain the structural explanation of the cyclization reaction of CGTase. Analyses of the three-dimensional structure of CGTase have been carried out using several types of CGTases (3-6). Additionally, the structures of CGTases with substrates (5, 7-9) and with inhibitor molecules (9, 10) were also analyzed. From these studies, models of CGTase activity cleaving the target alpha -1,4-linkage were proposed. In the case of CGTase from Bacillus circulans strain 251, three active site residues, Asp-229, Glu-257, and Asp-328, which are conserved in all CGTase primary sequences, play important roles for this step of reaction (8, 10). A similar catalytic mechanism has been reported from structural studies on amylases (11-14) that hydrolyze alpha -1,4-glucan. Several amino acid residues involved in substrate binding or in the determination of product specificity have also been proposed by three-dimensional structure analysis (5, 6, 8, 9) or by protein engineering approaches (15-17). However, it is less clearly understood how CGTase catalyzes the following intramolecular transfer reaction to produce cyclodextrins.

Although alpha -, beta -, and gamma -CD are the major products of CGTase, it has been known that trace amounts of larger cyclic glucans (delta -, epsilon -, zeta -, eta -, and theta -CD) were also present in the reaction mixture of CGTase on starch (18, 19). The structures of these larger cyclic glucans are still not well understood, because they seem to be a mixture of cyclic alpha -1,4-glucans, outer-branched cyclic alpha -1,4-glucans, and inner-branched cyclic glucans (18). Kobayashi and Yamasaki (20) carried out further structural analyses on putative delta -, epsilon -, zeta -, eta -, and theta -CD fractions and reported that the delta -CD fraction contained a large amount of unbranched cyclic alpha -1,4-glucan with DP 9. However, the proportion of unbranched cyclic alpha -1,4-glucan in the following fractions decreased dramatically (50% in epsilon -CD, 25% in zeta -CD, and almost 0% in eta - and theta -CD fractions). From this study, it is thought that the presence of cyclic alpha -1,4-glucan with DP larger than 12 in the CGTase reaction products is unlikely. Recently, however, we found that potato D-enzyme (disproportionating enzyme or 4-alpha -glucanotransferase, EC 2.4.1.25) catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic alpha -1,4 glucans with DP range from 17 to several hundred (21). The time-course analysis of D-enzyme action on amylose revealed that large cyclic alpha -1,4-glucans were preferentially produced in the initial stage of the cyclization reaction, and subsequently converted into small cyclic alpha -1,4-glucans, although alpha -, beta -, and gamma -CD were never produced (21). D-enzyme also catalyzes disproportionating reactions on malto-oligosaccharides (22) and transglycosidic linearization of cyclic alpha -1,4-glucans in the presence of a suitable acceptor (21). In all these respects, D-enzyme and CGTase both seem to catalyze the same reaction, with the major difference being the DP of the cyclic alpha -1,4-glucan produced. Furthermore, we also reported that the glycogen-branching enzyme (EC 2.4.1.18) from Bacillus stearothermophilus also catalyzes the intramolecular transglycosylation of amylose and amylopectin to produce branched cyclic glucans with DP larger than 18 (23, 24). From these studies on glycosyltransferases other than CGTase, we are interested in the action of CGTase in producing cyclic glucans larger than CDs. In this paper, we investigated the initial action of CGTase from an alkalophilic Bacillus sp. A2-5a (25) on synthetic amylose and found that the CGTase also produced cyclic alpha -1,4-glucans with DP ranging from 9 to more than 60, in addition to alpha -, beta -, and gamma -CD, from synthetic amylose in the very early stage of the reaction.


EXPERIMENTAL PROCEDURES

Chemicals and Enzymes

Synthetic amylose with an average molecular mass of 30 kDa (amylose AS-30) and soluble starch were purchased from Nakano Vinegar Co., Ltd. (Aichi, Japan) and E. Merck AG (Darmstadt, Germany), respectively. Glucoamylase from Rhizopus sp. was purchased from Toyobo Co., Ltd. (Osaka, Japan). CGTase from an alkalophilic Bacillus sp. A2-5a was purified to a homogeneous state (25). CGTases from B. macerans was purchased from Amano Pharmaceutical Co., Ltd. (Aichi, Japan) and was used without further purification. The activity of CGTase was assayed using soluble starch as the substrate by measuring the decrease in iodine-staining power as described previously (25).

Preparation of Amylose Solution

Amylose AS-30 (8 mg) was dissolved in 200 µl of 1 N NaOH solution then neutralized by adding 200 µl of 1 M sodium acetate buffer (pH 5.5), 200 µl of 1 N HCl, and 400 µl of distilled water. The solution was used immediately after neutralization.

Analysis of Reaction Products of CGTase on Amylose

CGTase (0.75 unit/ml) was incubated at 40 °C with amylose AS-30 (0.4% (w/v)) in 0.2 M sodium acetate buffer, pH 5.5, and reactions were terminated by boiling the solutions for 10 min. The reaction mixture containing 20 µg of glucan was incubated with glucoamylase (0.2 units) in 20 mM sodium acetate buffer (pH 5.5) for 16 h at 40 °C and then boiled for 5 min. The products in the reaction mixture were determined with high performance anion exchange chromatography (HPAEC, see below). The amounts of alpha -, beta -, and gamma -CD were measured with high performance liquid chromatography (HPLC, see below). The amount of glucoamylase-resistant molecules was calculated by subtracting the amount of glucose released by glucoamylase from that of total glucan in the reaction mixture. The amount of glucose was measured by the glucose oxidase method (26).

HPAEC

HPAEC was carried out based on the DX-300 system (Dionex) with a pulsed amperometric detector (model PAD-II, Dionex) using a Carbopac PA-100 column (4 mm × 250 mm). A sample (25 µl) containing 40 µg of glucan was injected and eluted with a gradient of sodium acetate (0-2 min, 50 mM; 2-37 min, increasing from 50 mM to 350 mM with the installed gradient program 3; 37-45 min, increasing from 350 mM to 850 mM with the installed gradient program 7; 45-47 min, 850 mM) in 150 mM NaOH with a flow rate of 1 ml min-1.

HPLC

HPLC was carried out based on the DX-300 system (Dionex) using an Aminex HPX-42A column. To remove glucose from the reaction mixtures, a sample (50 µl) containing 80 µg of glucan was charged on a Waters Sep-Pak C18 cartridge (Millipore), washed with 10 ml of H2O and eluted with 1.5 ml of 50% methanol. The eluate was dried up in vacuo, and dissolved in 50 µl of water. The sample was then injected and eluted with water with a flow rate of 0.6 ml min-1 at 80 °C. The eluate from the column was mixed with 0.3 M LiOH using an anion micromembrane suppressor (model AMMS-II, Dionex), after which the carbohydrate in the eluate was detected with a pulsed amperometric detector (mode PAD-II, Dionex).

"Time of Flight" Mass Spectrometry (TOF-MS)

A reaction mixture (5 ml) containing 10 mg of amylose AS-30 and CGTase (0.7 unit) was incubated in 0.2 M sodium acetate buffer, pH 5.5, at 40 °C for 1 h, and then boiled for 10 min. The reaction mixture was incubated with 10 units of glucoamylase at 40 °C for 16 h, and then boiled for 5 min. After removing glucose with the Waters Sep-Pak C18 cartridge, the molecular masses of glucoamylase-resistant glucans were determined with a Kompact Maldi I TOF-MS system (Shimadzu, Kyoto, Japan).


RESULTS

Analysis of Initial Glucoamylase-resistant Molecules of CGTase on Amylose

Synthetic amylose AS-30 was incubated with CGTase from alkalophilic Bacillus sp. A2-5a. The enzyme reaction was terminated at the early stage of the reaction (10 min), and then the reaction mixture was incubated with glucoamylase to digest the linear amylose into glucose. When the glucoamylase-resistant molecules thus obtained were analyzed with HPAEC, many peaks were detected (Fig. 1A). Note that these peaks were not found in a control experiment with heat-inactivated CGTase (result not shown). Most of these peaks were eluted in the region where cyclic alpha -1,4-glucans with DPs of over 17, produced by potato D-enzyme on synthetic amylose (Fig. 1F), were eluted. This result suggests that CGTase produced such cyclic alpha -1,4-glucans. The large peak around 42 min may indicate the presence of glucoamylase-resistant molecules with DP more than 60, since these molecules were not resolved in this HPAEC condition and eluted together.


Fig. 1. HPAEC analysis of glucoamylase-resistant molecules produced by the action of CGTase from alkalophilic Bacillus sp. A2-5a on amylose AS-30. A-E, amylose AS-30 (0.4% (w/v)) was incubated with purified CGTase from alkalophilic Bacillus sp. A2-5a (0.75 unit/ml) at 40 °C for 10 min (A), 20 min (B), 30 min (C), 60 min (D), and 360 min (E). The products in each reaction mixture were analyzed after glucoamylase treatment by HPAEC. F, elution profile of the cyclic alpha -1,4-glucans produced by potato D-enzyme from amylose AS-30. alpha , beta , and gamma  indicate the positions where alpha -, beta -, and gamma -CD were eluted. DPs of cyclic alpha -1,4-glucans are indicated above each peak (cG17, cG20, etc.).
[View Larger Version of this Image (15K GIF file)]

Then to prove the cyclic structure of glucoamylase-resistant molecules produced by CGTase, their molecular masses were determined with TOF-MS (Fig. 2). A non-cyclic glucan with DP of n has a molecular mass of 162.1436n + 18.01534 Da, whereas a cyclic glucan should have a molecular mass of 162.1436n Da. The molecular mass of each glucoamylase-resistant molecule was compared with the theoretical value for non-cyclic and cyclic glucans (Table I). As shown in Table I, experimental values of glucoamylase-resistant molecules were consistent with theoretical values for cyclic glucan but not with those for non-cyclic glucan.


Fig. 2. TOF-MS analysis of glucoamylase-resistant molecules. Glucoamylase-resistant molecules were purified as described under "Experimental Procedures," and subjected to TOF-MS analysis. Numbers above each peak indicate the molecular mass (in daltons) of the molecule plus the mass of Na+ (23 Da).
[View Larger Version of this Image (9K GIF file)]

Table I. Experimental and theoretical masses of glucans

Experimental masses of glucans were determined by TOF-MS. Theoretical masses of cyclic and noncyclic glucans were calculated as 162.1436n + 22.9898 and 162.1436n + 22.9898 + 18.01534, respectively. Values are rounded and presented as whole numbers. 162.1436, the mass of glucosyl residue; n, DP; 22.9898, the mass of sodium ion; 18.01534, the mass of H2O.

DP Experimental Theoretical
Cyclic glucan Noncyclic glucan

6 995 996 1014
7 1157 1158 1176
8 1319 1320 1338
9 1483 1482 1500
10 1643 1644 1662
11 1807 1807 1825
12 1969 1969 1987
13 2131 2131 2149
14 2292 2293 2311
15 2456 2455 2473
16 2617 2617 2635
17 2778 2779 2797
18 2942 2942 2960
19 3104 3104 3122
20 3267 3266 3284

To confirm that the glucoamylase-resistant molecules produced by CGTase are alpha -1,4-glucans, the structure of these glucans were further examined by treatment with several enzymes. alpha -Amylase from Bacillus subtilis, an endo-type amylase, completely degraded these molecules to glucose and maltose, and isoamylase and pullulanase, which degrade alpha -1,6-linkage of glucans, did not (data not shown). These results indicate that the glucoamylase-resistant molecules produced by CGTase were alpha -1,4-glucans.

Based on all the results mentioned above, we concluded that the molecules produced by the CGTase reaction on amylose, as shown in Fig. 1A, were cyclic alpha -1,4-glucans with DPs ranging from 6 to more than 60.

Time Course of Reaction of CGTase on Amylose

It is known that the glucoamylase-resistant products of the CGTase reaction on amylose, after a prolonged reaction time, are alpha -, beta -, gamma -CD and negligible amounts of other glucoamylase-resistant glucans. Our result apparently contradicts this widely held view since the major cyclic alpha -1,4-glucans produced in the initial stage of the CGTase reaction were not alpha -, beta -, and gamma -CD but were those with high DPs. To investigate how large cyclic alpha -1,4-glucans, which were found in the initial stage of CGTase reaction, were replaced by alpha -, beta -, and gamma -CD, the time course of the reaction of CGTase were monitored. Amylose AS-30 was incubated with CGTase from alkalophilic Bacillus sp. A2-5a for up to 360 min. Each sample was treated with glucoamylase and was analyzed by HPAEC (Fig. 1). As shown in Fig. 1 (A-E), cyclic alpha -1,4-glucans with DPs of over 60 were most prevalent after a reaction time of 10 min (Fig. 1A). However, with prolonged reaction, cyclic alpha -1,4-glucans with high DPs gradually decreased, and those with low DPs increased. At the end of the reaction, the main product of CGTase was beta -CD (Fig. 1E).

Fig. 3 shows the time course of the amount of cyclic alpha -1,4-glucans. The amount of total cyclic alpha -1,4-glucans increased rapidly from the beginning of the reaction, and their yield reached about 80% at 120 min. However, the amounts of alpha -, beta -, and gamma -CD increased at a more slower rate than those of total cyclic alpha -1,4-glucans. As a result, cyclic alpha -1,4-glucans, apart from alpha -, beta -, and gamma -CD, increased at the early stage of the reaction and reached a yield of about 52% at 50 min, but decreased thereafter.


Fig. 3. Time course of the amount of cyclic alpha -1,4-glucans produced by CGTase on amylose AS-30. The amount of total cyclic alpha -1,4-glucan (open circle ), and total CD (the sum of alpha -, beta -, and gamma -CD, square ) were determined as described under "Experimental Procedures." Cyclic alpha -1,4-glucans apart from alpha -, beta -, and gamma -CD (bullet ) were calculated by subtracting the amount of total CD from that of total cyclic alpha -1,4-glucan.
[View Larger Version of this Image (17K GIF file)]

Analysis of the Initial Action of Other CGTases

CGTases found in many bacterial species are classified into three types, alpha -, beta -, or gamma -CGTase, depending on the major product of the cyclization reaction. The CGTase employed above is beta -CGTase because it mainly produces beta -CD (25). To examine whether the production of a large cyclic alpha -1,4-glucan is the specific feature only found in this CGTase or is the common feature for others, similar experiments were carried out by using CGTases from B. macerans, which is classified as alpha -CGTase. This enzyme also produced large cyclic alpha -1,4-glucans in the initial stage of reaction (Fig. 4, A and B), which were subsequently converted into small cyclic alpha -1,4-glucans. However, the final major cyclic product was alpha -CD (Fig. 4E).


Fig. 4. HPAEC analysis of cyclic alpha -1,4-glucans produced by CGTase from B. macerans. Amylose AS-30 (0.4% (w/v)) was incubated with CGTase from B. macerans (0.75 unit/ml) at 40 °C for 0.5 h (A), 1 h (B), 2 h (C), 4 h (D), and 24 h (E). The products in each reaction mixture were analyzed after glucoamylase treatment by HPAEC. alpha , beta , and gamma  indicate the positions where alpha -, beta -, and gamma -CD were eluted.
[View Larger Version of this Image (15K GIF file)]


DISCUSSION

It is generally believed that the cyclization reaction of CGTase on amylose is an exo-type attack (2), where the enzyme recognizes the 6-8 glucose units from the non-reducing end, attacks the adjacent alpha -1,4-linkage, and transfers it to the C-4 position of the non-reducing end to produce alpha -, beta -, or gamma -CD (Fig. 5A). This view was only confirmed from the analysis of CGTase action on 14C-labeled linear alpha -1,4-glucans with DP 7-12 (27), but not investigated in high molecular weight glucans. If this view can be applied to the CGTase action on high molecular weight glucans, cyclic products throughout the reaction on amylose are expected to be only alpha -, beta -, and gamma -CD. However, the results presented in this paper clearly demonstrate that the cyclic glucans produced in the initial stage of cyclization reaction of CGTase are not only alpha -, beta -, and gamma -CD, but are cyclic alpha -1,4-glucans with various DP ranging from 6 to more than 60. Large cyclic alpha -1,4-glucans were preferentially produced in the initial stage of cyclization reaction, which were subsequently converted into small cyclic alpha -1,4-glucans and into the final major products, alpha -CD or beta -CD. Thus these findings apparently contradict the widely held view of the action model of CGTase, and so we propose a new model for the action of CGTase as shown in Fig. 5B. CGTase probably attacks any alpha -1,4-linkage within the amylose molecule, and then transfers the newly formed reducing end of the substrate either to the non-reducing end of a separate linear acceptor molecule or glucose (the intermolecular transglycosylation or disproportionation reaction), or to its own non-reducing end (the intramolecular transglycosylation or cyclization reaction, Fig. 5B). This random cyclization reaction produces wide ranges of cyclic alpha -1,4-glucans with DP 6 to more than 60. The reversibility of these reactions allows large cyclic molecules to be linearized again by transglycosylation, and smaller cyclic molecules to be subsequently produced. The equilibrium of the whole reaction tends toward the formation of alpha - or beta -CD as the final major products.


Fig. 5. Models of cyclization reaction of CGTase on amylose. A is a conventional model for the action of CGTase. B is a proposed model. Solid lines and circles indicate alpha -1,4-glucan chains, where the relative length represents their relative DP. R, glucosyl residue with reducing end; down-triangle, alpha -1,4 linkage attacked by CGTase; CD, alpha -, beta -, or gamma -CD.
[View Larger Version of this Image (9K GIF file)]

Both CGTase and D-enzyme catalyze the cyclization and disproportionation of alpha -1,4-glucan and transglycosidic linearization of cyclic alpha -1,4-glucan in the presence of a suitable acceptor molecule. During the cyclization reaction, large cyclic alpha -1,4-glucans were preferentially produced in the initial stage, but were subsequently converted into smaller cyclic alpha -1,4-glucans in both cases. Thus both enzymes seem to catalyze the same reaction, with the major difference being in the smallest size of the cyclic alpha -1,4-glucans produced. The DP of the smallest cyclic alpha -1,4-glucan produced by CGTase is 6. On the other hand, D-enzyme never produced alpha -, beta -, and gamma -CD and the smallest cyclic alpha -1,4-glucan has DP of 17. It is very interesting to know how the specificities of these products are determined differently between D-enzyme and CGTase. In our previous paper (23), we discussed that cyclic alpha -1,4-glucan with DP 6-8 (alpha -, beta -, and gamma -CD) and those with DP more than 17 may have different structures. The structures of cyclic alpha -1,4-glucan with DP 6-8 (alpha -, beta -, and gamma -CD) may fit well to the active site of CGTase, and those with DP more than 17 may fit well to D-enzyme. However, this idea seems to be unlikely because it is now understood that CGTase can also produce cyclic alpha -1,4-glucans with DP more than 17. One possible explanation for the different product specificity is as follows. The equilibrium of the reaction catalyzed by both enzymes tends toward the formation of smaller cyclic alpha -1,4-glucans; however, the smallest cyclic alpha -1,4-glucan molecule to be produced by D-enzyme has DP of 17, whereas that of CGTase is 6. At the moment, we do not know the mechanism to determine the smallest cyclic product of each enzyme, although we speculate that the difference in the smallest products of each enzyme is attributable to the active site structure. Despite the similarity found in their activities, D-enzyme and CGTase show no similarity in their primary sequences (22). The tertiary structure of CGTase has already been obtained by x-ray crystallographic studies (3-6). Determination of the structure of D-enzyme and its comparison with CGTase will be necessary to answer this question.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 81-6-477-8425; Fax: 81-6-477-8271; E-mail: biochem-lab{at}glico.co.jp.
1   The abbreviations used are: CGTase, cyclodextrin glucanotransferase; CD, cyclodextrin; DP, degree of polymerization; D-enzyme; disproportionating enzyme; HPAEC, high performance anion exchange chromatography; HPLC, high performance liquid chromatography; TOF-MS, time of flight mass spectrometry.

ACKNOWLEDGEMENT

We especially thank Shimadzu Co. for TOF-MS analyses.


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