Sumio KITAHATA and Shigetaka OKADA

Transcription

1 Agr. Biol. Chem., 39 (11), 2185 `2191, 1975 Transfer Action of Cyclodextrin Glycosyltransferase on Starch õ Sumio KITAHATA and Shigetaka OKADA Osaka Municipal Technical Research Institute, Osaka, Japan Received May 29, 1975 The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans (BMA) were examined. No. 5 enzyme was more efficient in transglycosylation reaction than BMA in the every acceptor employed in the present study. The order of the efficient acceptors for No. 5 enzyme was maltose (G2), glucose (G1), maltotriose (G3) and sucrose (GF). On the other hand, that found for BMA was GI, G2, GF and G3. The transglycosylation products to glucose formed by the action of No. 5 enzyme on starch were G2, G3, maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) in the order of their quantities, while, in the case of BMA, they were G2, G3, G5, G7=G4 and G6. The larger transglycosylation products to sucrose formed by the action of No. 5 enzyme on starch were maltosylfructose. On the other hand, that formed by the action of BMA was maltoheptaosylfructose. It was suggested that cyclodextrin glycosyltransferase could transfer the glucosyl residues to an acceptor directly from starch, as well as through cyclodextrin. As previously reported,1) it was found that Bacillus megaterium No. 5 strain produced the cyclodextrin glycosyltransferase ( , ƒ -1,4-glucan 4-glycosyltransferase, cyclizing). The cyclodextrin glycosyltransferase (No. 5 enzyme) was highly purified and finally sepa rated into two fractions by means of Am pholine-electrophoresis (Fr. 1 and Fr. 2). Some properties and cyclization reaction of these enzyme fractions were compared with those of Bacillus macerans amylase (BMA). From the investigation,2) it was found that both fractions of No. 5 enzyme produced the cyclohexaamylose (cyclodextrin G6), cyclohep taamylose (cyclodextrin G7) and cyclooc taamylose (cyclodextrin G8) in the proportion of 1:2.4:1 from starch. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7:1:1. Another reaction attributed to this enzyme is a transglycosylation reaction whereby glycosyl moieties could be transferred from one linear oligosaccharide to another. Several studies have been so far reported on the trans glycosylation reaction of Bacillus macerans amvlase.3 `5) In those reports, however, the õ Studies on Cyclodextrin Glycosyltransferase. Part III. See References 1 and 2). authors only pointed out that the enzyme possesed transglycosylation activity which could transfer glycosyl moieties from cyclo dextrin to proper acceptors. There have been no information on the detailed reaction mode of the transglycosylation. Recently, the No. 5 enzyme was found to posses trans glycosylating activity as known in BMA, and a study was undertaken to clarify the details of transglycosylation mode using starch as well as cyclodextrin as donar. The present paper deals with the trans glycosylation reaction of the No. 5 enzyme, comparing with that of BMA, especially with respect to acceptor efficiency and quantitative distribution of the products from different donars. MATERIALS AND METHODS Material. Glucose-14C (U) (5.0mCi/mmole) and sucrose-14c (U) (376mCi/mmole) were purchased from Daiichi Pure Chemicals Co. Ltd., and used after further purification by paper chromatography. Glucose, sucrose, maltose and soluble starch were obtained from commercial sources. Maltotriose and cyclo hexaamylose were supplied by Hayashibara Co. Ltd. Enzyme preparation. The cyclodextrin glycosyl-

2 2186 S. KITAHATA and S. OKADA transferase produced by Bacillus megaterium strain No. 5 (No. 5 enzyme) and by Bacillus macerans IFO 3490 (Bacillus macerans amylase, BMA) was prepared from the culture broth, as described previously.1) The preparation of the No. 5 enzyme was homogene ous on ultracentrifugal analysis, but was separated into two fractions able to form cyclodextrins on disc electrophoresis and ampholine-electrophoresis (Fr. 1: RESULTS Structures of the oligosaccharides synthesized by transglycosylation reaction of both frac tions of No. 5 enzyme and BMA For the purpose of clarifing structures of pi 6.07 and Fr.2: pi 6.80).1) The preparation of BMA was homogeneous on both ultracentrifugal and electro phoretical analysis. Purified saccharase from yeast was purchased from the Difco Laboratories, Detroit, Michigan. The preparation of glucoamylase in crystal -line form was supplied by Ueda Chemical Co. Ltd. ƒà-amylase was prepared in the pure form free from ƒ -amylase and other ƒ -glucosidase from defatted soy bean.6) Analytical methods. The cyclodextrin glycosyl Toyo No. 50 filter paper and chromatographed transferase activity was assayed using starch as sub as described in MATERIALS AND METHODS. strate by measuring the decrease in iodine-staining The transglycosylation products were sub power as described previously.1) The amount of total sugar was measured by phenol-h2so4 method.7) jected to enzymic hydrolysis by glucoamylase, ƒà-amylase and saccharase. The transgly Determination of iodine staining power was carried out as follows. Adequate aliquots of the reaction cosylation products to glucose, maltose and mixture, were added to 2 ml of iodine solution (0.01M maltotriose gave only glucose by glucoamyl iodine in 0.25M potassium iodide) and 1ml of hydro ase action, and gave maltose, maltotriose and chloric acid. This mixture was diluted to 30ml with small amounts of other oligosaccharides by the distilled water and then the light transmission of the solution was measured at 660nm with a Hitachi ƒà- lamylase. Considering action patterns of 181 spectrophotometer. these enzymes and the hydrolytic products, Quantitative determination of cyclodextrin precipitated with trichloroethylene. To 1ml of the reaction mix ture was added 1ml of trichloroethylene (TCE), stirred vigorously for 1min and after standing at 5 Ž for 60 min, centrifuged at 3000rpm for 10min. The amount of total sugar in the supernatant was then determined. The amount of cyclodextrin precipitated with TCE was calculated by subtracting the amount of total sugar in the supernatant from that present in the original solution. Quantitative determination of transglycosylation products. The reaction mixture (20ƒÊl) were spotted on Toyo No. 50 filter paper and paper chromatographed by four ascents of the solvent system of n-butylalcohol, pyridine and water (6:4:3 by volume). Finally, the autoradiogram was prepared as described previously2) to locate the position of each maltooligosaccharides. The radioactivity of each maltooligosaccharide on the paper was measured with a Liquid Scintillation Spectro meter Packard TRI-CARB The amounts of transglycosylation products (G2-G7) were calculated from the radioactivity and specific activity. the oligosaccharide series synthesized by transglycosylation reaction of each enzyme to glucose, maltose, maltotriose and sucrose, the enzyme (40units/ml, 0.1ml) was allowed to react with 0.1ml of soluble starch (10mg) containing 10mg of various acceptors (glucose, maltose, maltotriose and sucrose) at 40 Ž for 20hr. The reaction mixture was spotted on it is quite clear that these oligosaccharides are maltooligosaccharides linked by ƒ -1,4-Dglucosidic bonds. On the other hand, trans glycosylation products to sucrose gave fructose and maltooligosaccharides by saccharase, and gave glucose and sucrose by glucoamylase. These results strongly suggest that these com pounds are the oligosaccharides terminated by sucrose at the reducing end of malto oligosaccharides. Acceptor efficiency in transglycosylation reac tion of three enzymes (Fr. 1, Fr. 2 and BMA) In an attempt to compare the transglycosyla tion reaction among both fractions of No. 5 enzyme (Fr. 1 and Fr. 2) and BMA, the efficiencies of four sugars (glucose, maltose, maltotriose and sucrose) as acceptors were examined. The rate of transglycosylation was indirectly measured by the method of decrease in iodine-staining power, on the basis of the fact that the rate of starch degra-

3 Cyclodextrin Glycosyltransferase 2187 cosylation products formed by the action of Fr. 1, Fr. 2 and BMA each other, each enzyme (4units/m1, 20ƒÊl) was reacted with soluble starch (0.4mg, 60ƒÊl) in the presence of either glucose-14c (U) (0.4mg, 0.6ƒÊCi) or sucrose-14c (U) (0.8mg, 0.6ƒÊCi) as an acceptor at 40 Ž for 10min. The amounts of transglycosyla tion products were determined by the method described in MATERIALS AND METHODS. The ratios of transglycosylation products formed from the reaction mixture were 8.7% (Fr. 2) and 6.7% (BMA) in the case of glucose as an acceptor, and in the case of sucrose as an acceptor, those were 2.2% (Fr. 2) and 2.5% FIG. 1. Degree of Acceleration of Starch Degrada tion in the Presence of Various Acceptors. Each of three enzymes (8units/ml, 0.2ml) was reacted with 0.6ml of 5% soluble starch containing different acceptors in equimolar at 40 Ž. At the stage of 20min reaction, aliquots (0.2ml) of the reaction mixture were removed to examine the decrease in iodine-staining power. *1None: absence of the acceptor. *2Relative iodine values were calculated with respect to the iodine value without acceptor. dation was accelerated in the presence of acceptor.8) Each of three enzyme (Fr. 1, Fr. 2 and BMA) (8units/ml, 0.2ml) was reacted with 0.6ml of 5% soluble starch containing different ac ceptors of same molar concentration (glucose 30mg, maltose and sucrose 57 mg and mal totriose 84 mg) at 40 Ž. At certain intervals, aliquots (0.2ml) of the reaction mixture were removed to examine the decrease in iodine staining power. The results are shown in Fig. 1. The most effective acceptor for both fractions of No. 5 enzyme (Fr. 1 and Fr. 2) was maltose, followed by glucose, maltotriose and sucrose in this order. On the other hand, the order of the efficient acceptors for BMA was glucose, maltose, sucrose and maltotriose. The distribution of the transglycosylation pro ducts formed by the action of three enzymes (Fr. 1, Fr. 2 and BMA) In order to compare the initial transgly (BMA). The distribution of the transgly cosylation products are shown in Fig. 2. The transglycosylation products to glucose formed by the action of Fr. 2 were maltose, malto triose, maltotetraose, maltopentaose, mal tohexaose and maltoheptaose in the order of their quantities, while, in the case of BMA they were maltose, maltotriose, maltopen taose, maltoheptaose=maltotetraose and mal tohexaose. The larger transglycosylation product to sucrose formed by the action of Fr. 2 was maltosylfructose, accompaning with a smaller amounts of the other oligosaccharides. On the other hand, that formed by the action of BMA was maltoheptaosylfructose, accompan ing with a smaller amounts of the other oligosaccharides. The transglycosylation pro ducts to glucose and sucrose formed by the action of Fr. 1 on starch were the same as in the case of Fr. 2. For the purpose of comparing the distribu tion of the transglycosylation products from starch as donar with those from cyclodextrin G6 as donar, the enzyme of Fr. 2 (0.4units/ml, 20ƒÊl) was reacted with either starch (2mg, 60ƒÊl) or cyclodextrin G6 (2mg, 60ƒÊl) in the presence of glucose-14c (U) (2mg, 0.6ƒÊCi) at 40 Ž for 30min. The ratios of transgly cosylation products formed from the reac tion mixture were 2.58% from starch and 1.91% from cyclodextrin G6. The distribu tion of the transglycosylation products are shown in Fig. 3. The remarkable difference

4 2188 S. KITAHATA and S. OKADA FIG. 2. The Distribution of the Transglycosylation Products to Glucose (A) and to Sucrose (B). Each of two enzymes (Fr. 2 and BMA) (4units/ml, 20ƒÊl) was incubated with soluble starch (0.4mg, 20ƒÊl) in the presence of either 40ƒÊl of glucose-14c (U) (0.4mg, 0.6ƒÊCi) or sucrose (0.8mg, 0.6ƒÊCi) as acceptors at 40 Ž for 10 min. The amounts of transglycosylation products were determined by the method described in MATERIALS AND METHODS. G2-maltose, G3-maltotriose, G4-maltote traose, G5-maltopentaose, G6-maltohexaose, G7-maltoheptaose, G8-maltooctaose, G9 -malto oligosaccharides higher than maltononaose, G2F-maltosylfructose, G3F-maltotriosylfructose, G4Fmaltotetraosylfructose, G5F-maltopentaosylfructose, G6F-maltohexaosylfructose, G7F-maltohepta osylfructose, G8F-maltooctaosylfructose, G9F5 -oligosaccharides higher than maltononaosylfruc tose. FIG. 3. The Distribution of Transglycosylation Products to Glucose from Starch and Cyclodextrin G6 as Donar. No. 5 enzyme (Fr. 2) (0.4units/ml, 20ƒÊl) was incubated with either starch (2mg, 20ƒÊl) or cyclo dextrin G6 (2mg, 20ƒÊl) in the presence of 40ƒÊl of glucose-14c (U) (2mg, 0.6ƒÊCi) at 40 Ž for 30min. The amounts of transglycosylation products were determined by the method described in MATERIALS AND METHODS. Abbreviations are the same as those in Fig. 2.

5 Cyclodextrin Glycosyltransferase 2189 between the transglycosylation products from starch and those from cyclodextrin G6 was observed in the amounts of maltooligosac charides-14c (U) higher than maltooctaose. Namely, the amounts of maltooligosaccharides higher than maltooctaose were larger when starch was used as donar than those found in the case of cyclodextrin G6 as donar. The effect of glucose concentration on the cyclization and transglycosylation reaction The enzyme of Fr. 2 (0.2units/ml, 8ml) was reacted with 8ml of 5% soluble starch containing glucose-14c (U) (0.024mg, 0.6ƒÊCi) and different amounts of glucose ((a) glucose 0mg, (b) glucose 200mg, (c) glucose 400mg) at 40 Ž. At certain intervals, aliquots (3ml) of the reaction mixture were removed to examine the iodine-staining power and the amounts of cyclodextrin and maltooligosac charides (G2-G7)-14C (U) formed by the transglycosylation reaction. As shown in Table I, at the stage of 30min reaction, the ly. Namely, the rate of starch degradation increased with increasing concentration of the acceptor, but no significant difference was found in the amounts of cyclodextrin preci pitated with TCE from the reaction mixture. On the other hand, the amounts of maltooli gosaccharides (G2-G7)-14C (U) synthesized by the transglycosylation reaction much increased with increasing concentration of the acceptor, that is, at the stage of 30min reaction, the amounts of maltooligosaccharides (G2-H7)-14C (U) were (a) 0.025mg/ml, (b) 0.98 mg/ml and (c) 1.39mg/ml. The effect of sucrose concentration on the amounts of cyclodextrin produced by the enzyme of Fr. 2 The enzyme of Fr. 2 (2units/ml, 5ml) was reacted with 5ml of 5% soluble starch con taining different amounts of sucrose ((a) 0mg, (b) 100mg and (c) 250mg) at 40 Ž. At certain intervals, aliquots (2ml) of the reac tion mixture were removed to examine the fall in iodine-staining power and the amounts TABLE I. TRANSFER ACTION AND CYCLODEXTRIN FORMATION OF CYCLODEXTRIN GLYCOSYLTRANSFERASE of cyclodextrin formed. As shown in Fig. 4 in the absence of acceptor, the amounts of cyclodextrin produced increased with the No. 5 enzyme (Fr. 2) (0.2units/ml, 8ml) was reacted with 8ml of 5% soluble starch containing glucose- 14C (U) (0.024mg, 0.6ƒÊCi) and different amounts of glucose ((a) 0mg, (b) 200mg, (c) 400mg) at 40 Ž. At the stage of 30min reaction, aliquots (3ml) of the reaction mixture were removed for analysis. progress of the reaction, resulting that at the stage of 120min reaction, the ratio of cyclo dextrin formed from the reaction mixture was 23% and at the stage of 24hr reaction, it was 26%. However, in the presence of 250 mg of sucrose as acceptor, the amounts of cyclodextrin formed increased with the pro gress of the reaction till 60min, and after a short steady state, the cyclodextrin was gradually decreased and disappeared at the stare of 24hr. DISCUSSION A number of reports3,5 have indicated that B. macerans amylase is capable to catalize coupling reaction whereby a wide variety of amounts of cyclodextrin precipitated with trichloroethylene (TCE) were (a) 1.25mg/ml, (b) 1.03mg/ml and (c) 1.00mg/ml, respective acceptors such as glucose, maltose, sucrose, etc., are added to the reducing end of the opened cyclic dextrin by ƒ -1,4-D-glucosidic bonds. But any attempt has not been done

6 2190 S. KITAHATA and S. OKADA FIG. 4. Action of Cyclodextrin Glycosyltransferase on Starch in the Presence of Various Con centration of Sucrose as acceptor. No. 5 enzyme (Fr. 2) (2units/ml, 5ml) was reacted with 5ml of 5% soluble starch containing different amounts of sucrose at 40 Ž. At certain intervals, aliquots (2ml) of the reaction mixture were removed to examine the decrease in iodine-staining power and the amounts of cyclodextrin produced. œ \ œ, decrease in iodine-staining power (O.D. at 660nm); _??_, amounts of cyclodextrin precipitated with trichloroethylene. on the transglycosylation reaction of BMA using starch as donar. In this study, the transglycosylation reac tion and the products by the enzymes of B. megaterium and BMA were examined using starch as donar. In the transglycosylation reaction of No. 5 enzyme and BMA, it was found that starch was efficient as donar and the transglycosylation products to glucose, maltose, maltotriose and sucrose from starch are the oligosaccharides terminated at the reducing end of maltooligosaccharides by a group characteristic of the acceptor used, same as found when cyclodextrin was used as donar. From the results shown in Fig. 1, it appeared that both fractions of No. 5 enzyme are more efficient in transglycosylation reaction than BMA in the every acceptors employed there. The order of the efficient acceptors for both fractions of No. 5 enzyme were similarly maltose, glucose, maltotriose and sucrose, while, that of BMA was glucose, maltose, sucrose and maltotriose. In the distribution of the initial transglycosylation products to glucose and sucrose, both fractions of No. enzyme showed the same results, but in th case of BMA the different results were obtainei from No. 5 enzyme, especially using sucros as an acceptor. As reported in the previous paper,1,2) botl fractions of No. 5 enzyme which were separate by electrofocusing procedure, showed quit same properties in the cyclization and dis proportionation reactions. Furthermore, a found in the present study, these fractions als gave same results in the transglycosylatio reaction. These fractions, therefore, were considered to be isozyme, although it is no clear whether they were originally produce as isozyme by the strain or modified durin the purification procedure. From the results shown in Table I, appeared that with increasing concentratio of the acceptor, the rate of starch degradatio and the amounts of maltooligosaccharide synthesized by the transglycosylation reaction increased, but the amounts of cyclodextri produced were not significantly varied. Froi

7 Cyclodextrin Glycosyltransferase 2191 the results obtained in the experiments of Fig. 3, it appeared that starch is more efficient as donar than cyclodextrin G6. From these facts described above, we may suggest that in the reaction system containing starch and acceptor, No. 5 enzyme can transfer the glu cosyl residues to an acceptor directly from starch, as well as through cyclodextrin. The results shown in Fig. 3 which of the transgly cosylation products from starch as donar the amounts of maltooligosaccharides higher than maltooctaose are larger than that from cyclo dextrin G6 as donar, supports the above sugges tion. Acknowledgement. The authors wish to express their hearty thanks to Dr. Y. Tsujisaka in our institute for valuable advice and encouragement and Dr. S. Emi for helpful suggestions in the preparation of this manuscript. REFERENCES 1) S. Kitahata, N. Tsuyama and S. Okada, Agr. Biol. Chem., 38, 387 (1974). 2) S. Kitahata and S. Okada, ibid., 38, 2413 (1974). 3) D. French, M. L. Levine, E. Norberg, P. Nordin, J. H. Pazur and G. Wild, J. Amer. Chem. Soc., 76, 2387 (1954). 4) E. Norberg and D. French, ibid., 72, 1202 (1950). 5) J. A. Depinto and L. L. Campbell, Arch. Biochem. Biophys., 125,253 (1968). 6) J. Fukumoto and Y. Tsujisaka, Kagaku to Kogyo, 28, 282 (1954). 7) M. Dubais, K. A. Gilles, J. K. Hamilton, P. A. Rebers and F. Smith, Anal. Chem., 28, 350 (1956). 8) S. Okada and S. Kitahata, Amylase symposium, Japan, 8, 21 (1973).