Structures of Singly Branched Heptaoses Produced by Bacterial Liquefying a-amylase

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1 /. Biochem., 78, (1975) Structures of Singly Branched Heptaoses Produced by Bacterial Liquefying a-amylase Kimio UMEKI and Takehiko YAMAMOTO Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka, Osaka 558 Received for publication, May 8, A singly branched heptaose produced as a limit dextrin in the digest of /3-limit dextrin with liquefying a-amylase [EC ] of Bacillus amyloliquefaciens was isolated in a paper chromatographically pure state. 2. Analysis using several enzymes revealed that the isolated branched dextrin was. a mixture of six singly branched heptaoses with different ramifying points. 3. All the branched heptaoses contained a 6 2 -a-maltosylmaltotriose moiety in their molecules, differing only in the mode of attachment of one maltose or two glucose residues by a-1,4-glucosidic bonds from this core dextrin. 4. The formation of various singly branched heptaoses (the present paper) and hexaoses (the previous paper) is discussed regarding the attack site specificity of the enzyme on /3-limit dextrin. A number of papers have been published on the mechanism underlying the specificity of a-amylase [EC ], not only for the binding mode with substrate (1), but also as regards preferential specificity (2), subsite structure (3), etc. These investigations have used amylose or linear dextrin derived therefrom as a substrate. Few studies of this kind using amylopectin, glycogen, and js-limit dextrin have been reported (4). Our previous paper (5) showed that among various branched dextrins produced on the digestion of /3-limit dextrin with liquefying a- amylase of Bacillus amyloliquefaciens Fukumoto (6~), the smallest branched dextrin was 6 2 -a-maltosylmaltotriose and the joint next smallest, three kinds of singly branched hexaose built up by joining one glucose residue to an end of the above branched pentaose. The singly branched dextrins produced by digestion of /3-limit dextrin with bacterial a- amylase were found to consist at most of nine glucose units, and the purpose of the present paper was to determine the structure of the singly branched heptaose. The study revealed that the singly branched heptaose isolated by paper chromatography was a mixture of six singly branched heptaoses. All the branched heptaoses contained the 6 2 -a-maltosylmaltotriose moiety as a core. In the present paper, the specificity of the bacterial a-amylase on j8-limit dextrin is also discussed in connection with the structure of the a-amylase limit singly branched dextrins which have so far been reported (2, 4). Vol. 78, No. 5,

K. UMEKI and T. YAMAMOTO 89 MATERIALS AND METHODS 3. BLA Limit Singly Branched Heptaose Dextrin A mixture of BLA (bacterial liquefying a-amylase) and -limit dextrin in a proportion of.

2 K. UMEKI and T. YAMAMOTO 89 MATERIALS AND METHODS 3. BLA Limit Singly Branched Heptaose Dextrin A mixture of BLA (bacterial liquefying a-amylase) and -limit dextrin in a proportion of.9 mg (5 activity units) of enzyme per gram of dextrin (final concentration, 8.%) "was incubated at 4 for 48 hr under a few drops of toluene, and the digest was subjected to preparative paper chromatography by the descending and multiple development method, using w-butanol-pyridine-water ( 6 : 4 : 3 ) as a solvent (Fig. 1). The dextrin with an Rf value between those of maltoheptaose and malto octaose was extracted as the branched dextrin -sample and used for structural investigations after examining it for possible contamination l>y other dextrins by means of chromatography Fig. 1. Progressive paper chromatograms of the digest of js-limit dextrin with liquefying a-amylase of Bacillus amyloliquefaciens (BLA). /S-Limit dextrin, 8%; BLA, 5 units per gram of /3-limit dextrin, ph 5.9, 4. Mark., markers (glucose, maltose~maltohexaose). using rc-butanol-pyridine-water (1 : 1 : 1) as a solvent. The isolated dextrin was estimated to consist of seven glucose units on the basis of the ratio of total sugar to reducing sugar (as glucose) by the method reported previously (5). 4. Methods for Structural Investigation of the Dextrin The isolated dextrin was incubated with each of the various enzymes described above, and the extent of hydrolysis and products from the dextrin were investigated. When the structures of the hydrolysis products were unknown, the products were further investigated as described in previous papers (5, 75). Total sugar content and reducing sugar as glucose were determined by the phenol-h2so4 method (16) and SomogyiNelson method (17), respectively. The hydrolysis products were isolated by the descend/. Biochem. 1. Enzymes A crystalline preparation of liquefying a-amylase of Bacillus amyloliquefaciens Fukumoto [EC ] (BLA, purchased from Seikagaku Kogyo Co.) was used for the digestion of js-limit dextrin. Crystalline preparations of saccharifying a-amylase of Bacillus subtilis [EC ] () ( 7-9 ) and glucoamylase of Rhizopus niveus [EC ] (1, 11) were also obtained from Seikagaku Kogyo Co., and used for structural investigations of dextrins, but the latter enzyme was purified further on a column of Bio-gel P-6 (12) before use. Pullulanase of a certain species of Aerobacter [pullulan 6 - glucanohydrolase, EC ] and soybean js-amylase [EC ], which were obtained and purified by the methods reported previously (5), were also used. In addition, isopullulanase [pullulan 4-glucanohydrolase, EC ] (13, 14) which hydrolyzes pullulan to produce isopanose was used. This enzyme was kindly provided by Drs. Tsujisaka and Hamada of Osaka Municipal Research Institute for Technology. The enzymes described above were assayed by methods reported previously (15). 2. p-limit Dextrin ^-Limit dextrin used as a substrate was prepared from amylopectin obtained from glutinous rice starch according to the method described in our previous paper

3 a-amylase LIMIT SINGLY BRANCHED HEPTAOSES 891 ing method of paper chromatography. Thin layer chromatography was also employed to detect the hydrolysis products from dextrins reduced with NaBH 4, prepared according to the method reported previously (5). Detection of reducing sugars on the paper and thin layer chromatograms, and that of sugar alcohols on the thin layer chromatograms were done by the silver nitrate-naoh method ( 18), tetrazolium blue method (19), and anisaldehyde-h 2 SO 4 method (2), respectively. RESULTS f t IVI a DC Fig. 2. Paper chromatogram of hydrolysates of the isolated singly branched heptaose dextrin with pullulanase and /3-amylase. M, markers (maltooligosaccharides); a, branched heptaose dextrin; b, hydrolysis with pullulanase; c, hydrolysis with /J-amylase. 1. Hydrolysis with Pullulanase A paper chromatogram obtained from the digest of branched dextrin with pullulanase (3.6 units per.1 /imole per ml, 4, 24 hr) is presented in Fig. 2, and shows that maltose, maltotriose, -tetraose, and -pentaose were produced. The branched dextrin after reduction with NaBH 4 was also digested with pullulanase under the same conditions. The thin layer chromatogram obtained from the digest is shown in Fig. 3, which indicates that maltotriitol, maltotetraitol, and maltopentaitol were present besides maltose, maltotriose, and maltotetraose. Pullulanase attacks cr-1,6-glucosidic linkages, but not ar-1,4-glucosidic bonds in branched dextrin. If the dextrin sample consisted of a single component structurally, digestion with pullulanase would produce only each one kind of maltooligosaccharide and maltooligoalditol originating from the side chain and main chain, respectively. Accordingly, Figs. 2 and 3 clearly indicate that the branched dextrin was a mixture of several branched heptaoses with main chains consisting of maltotriose, maltotetraose, and maltopentaose and side chains consisting of maltotetraose, maltotriose, and maltose. TMPIJC add Fig. 3. Thin layer chromatograms of pullulanase hydrolysate of the branched dextrin after reduction with NaBH 4. Detected with tetrazolium blue (left) and anisaldehyde-h 2 SO 4 (right); A, reduced dextrin; B, hydrolysis of the reduced dextrin with pullulanase ; M, markers; Gi, glucose; G 2, maltose; G 3, maltotriose ; Red. G 2, reduced maltose ; Red. G 3, reduced maltotriose ; etc. Vol. 78, No. 5, 1975

K. UMEKI and T. YAMAMOTO 892 O - OOO-Q OO- O- O-O- O-OO-6- Fig. 4. Hydrolysis of /3-amylase-sensitive isomers with various enzymes. /3-A, /3-amylase; Pul, pullulanase; GlcA, glucoamylase.

4 K. UMEKI and T. YAMAMOTO 892 O - OOO-Q OO- O- O-O- O-OO-6- Fig. 4. Hydrolysis of /3-amylase-sensitive isomers with various enzymes. /3-A, /3-amylase; Pul, pullulanase; GlcA, glucoamylase. O, glucose residue;, reducing end glucose residue ;, or-1, 4-glucosidic linkage ; [, o-l, 6-glucosidic linkage. Figure 5 shows that the branched heptaose sample gave maltotriose and two unknown branched tetraoses (E and F in Fig. 5) on incubation with a small amount of (.7 units of per 1 //mole of branched heptaose). On the other hand, the /S-amylase-resistant branched heptaose produced no tetraoses corresponding to E and F. The branched tetraoses E and F were found to be 62-a-glucosylmaltotriose and 62-amaltosylmaltose, respectively, because both of them were hydrolyzed by glucoamylase and formed panose as an intermediary product, but E was not hydrolyzed by pullulanase while F was hydrolyzed by pullulanase to produce maltose. The difference between the digestion products from the sample heptaose and those I MaFig. 5. Paper chromatogram of and pullulanase hydrolysates of the branched heptaose and /3-amyIaseresistant branched heptaose. A, branched heptaose+ (.7 unit/^mole); A', branched heptaose+ (7. unit///mole); B, js-amylase-resistant branched heptaose+ (.7 unit/^mole); B', js-amylaseresistant branched heptaose + (7. unit//<mole); C, /S-amylase-resistant branched heptaose -fpullulanase; Mark., markers (maltooligosaccharides). /. Biochem. 2. Hydrolysis with Isopullulanase The branched dextrin was incubated with isopullulanase (.48 units of the enzyme per 1 ^mole of dextrin, 4, 3 hr), but neither increase in reducing power nor hydrolysis products was observed. This indicates that no dextrins with a structure susceptible to isopullulanase were contained in the branched dextrins. 3. Hydrolysis with ft- Amylase The paper chromatogram obtained from the digest of the dextrin sample with /3-amylase (.7 units per 1 //mole of dextrin, 4, 24 hr) is presented in Fig. 2. The result shows that the branched dextrin consists of /?-amylase-sensitive and resistant dextrins. The sensitive dextrin was hydrolyzed by ^-amylase into maltose and the unknown pentaose, which was split into maltose and maltotriose on incubation with pullulanase. The unknown branched pentaose was also hydrolyzed by glucoamylase and produced panose as an intermediary product, indicating that the pentaose is 62-a-maltosylmaltotriose. The above results suggest that the /3amylase-sensitive branched dextrin(s) has 62-amaltotetraosylmaltotriose and/or 62-a-maltosylmaltopentaose structure(s) (Fig. 4). 4. Hydrolysis with Bacterial Saccharifying a-amylase () The paper chromatograms obtained from digests of the branched heptaose with (.7 units and 7. units of per 1 //mole of dextrin, 4, 24 hr) and that from the digest of the /3-amylase-resistant branched dextrin incubated under the same condition are shown in Fig. 5. The /3-amylase-resistant branched dextrin used in the experiment was isolated by preparative paper chromatography from a digest of the branched heptaose sample with /3-amylase under the conditions described above.

5 a-amylase LIMIT SINGLY BRANCHED HEPTAOSES 893 O-OO-O OO- OO- O O-CHZ O- not hydrolyzed 67, CA g. ( Panose ) O- O- O-g _* OO- O-O-O-& O- O-& Fig. 6. Hydrolysis of ^-amylase-sensitive isomers with several enzymes (symbols as in Fig. 4). from /3-amylase-resistant branched heptaose (Fig. 5) must be attributed to products from /S-amylase-sensitive branched heptaose. It is thus clear that the ^-amylase-sensitive branched heptaose was a mixture of two isomers, as presumed in the preceding experiment. The hydrolysis of the two - amylase-sensitive branched heptaoses by is presented schematically in Fig Hydrolysis of - Am ylase- Resistant Branched Heptaose with As can be seen from Fig. 5, /3-amylase-resistant branched heptaose was split by (7. units of per /imole of dextrin, 4, 24 hr) to produce glucose, maltose, panose and two other unknown branched dextrins which consisted of five (G) and six (H) glucose units. The branched pentaose (G) was found to be a mixture of 6 3 -o:-maltosylmaltotriose and 6 8 -a-glucosylmaltotetraose, because a part of the dextrin was split by pullulanase into maltose and maltotriose, and by isopullulanase into isopanose and maltose. The remainder of G after digestion with pullulanase and isopullulanase was hydrolyzed by glucoamylase and produced glucose and branched tetraose, which was attacked by isopullulanase to produce maltose and isomaltose. The unknown branched hexaose (H) was determined to be 6 3 -a-maltosylmaltotetraose, since it was not attacked by isopullulanase, but was hydrolyzed by pullulanase into maltose and maltotetraose. It was also attacked by glucoamylase to produce glucose and a branched tetraose as an intermediary product. The latter product was split by isopullulanase into maltose and isomaltose. The above results are presented schematically in Fig. 7. B-res B, B-res B,.7 unitv / jjmole I GlcA I MM not hydrolyzed B5 17 unit) 6 ^ j B, L B 5 (Panose) Fig. 7. Hydrolysis of /3-amylase-resistant isomers with followed by hydrolysis with various enzymes. IPul, isopullulanase ; IMM, isomaltosylmaltose (6 3 -a-glucosylmaltotriose); Gi, glucose; G 2, maltose ; G 3, maltotriose ; B 3, branched triose ; B 4, branched tetraose ; etc. Other symbols are as in Fig. 4. DISCUSSION The digestion with pullulanase of the branched heptaose before and after reduction with NaBH 4 clearly showed that the isolated branched heptaose was a mixture of several singly branched heptaoses and that their main chains were maltotriose, -tetraose, and -pentaose, with side chains of maltotetraose, -triose, and maltose, respectively. All the possible singly branched dextrins which can be built up from the main chains and side chains described above are shown in Fig. 8. However, the isolated branched heptaose contained no branched dextrins susceptibe to isopullulanase and thus the structures a, b, and c in Fig. 8 are ruled out. The branched heptaose also gave no isomaltose on incubation with glucoamylase, and the structures i, k, and 1 are therefore also ruled out. Vol. 78, No. 5, 1975

6 894 K. UMEKI and T. YAMAMOTO OQ O-O-Q - O-OOO- OOO- O-O- o-g o-o-q o-ooo O-O-O-O- O-O-O- O-O- -- O-O-OO- i* o-o-o OO-O- o-oo O-O-O- o-o-o-o O-O- Fig. 8. All possible structures of singly branched heptaose dextrins which have maltotriose, -tetraose, and -pentaose as the main chain, and maltose, maltotriose, and -tetraose as the side chain. */!-Amylasesensitive structures. Other symbols as in Fig. 4. On the other hand, the presence of branched heptaoses of structures f and j, which are sensitive to ^S-amylase, was shown by the fact that the digestion of the branched heptaose sample with js-amylase produced maltose and a branched pentaose which was split by pullulanase into maltose and maltotriose. Accordingly, the structure of /3-amylase-resistant dextrin(s) in the sample dextrin should be d, e, g, or h. On incubation with a small amount of, a portion of the /3-amylase-resistant heptaose produced maltose and a singly branched pentaose (G" in Fig. 5), a part of which in turn was split into maltose and panose on further incubation with a large amount of. Of the remaining structures, only 6 2 -amaltotriosylmaltotetraose (h) is possible as shown schematically in Fig. 9. On the other hand, the singly branched heptaoses which were hydrolyzed by to produce maltose and a - glucosylmalto - tetraose, and maltose and 6 3 -a-maltosylmaltotriose, seem to have the structures e and g (Fig. 1), respectively. Similarly, d in Fig. 8 may be taken as the branched heptaose which gave glucose and 6 3 -a-maltosylmaltotetraose on incubation with, because it seems imposo-o-o O-O-O- h CH2 Q *, OO-CHZ ooo O- O- CH3 Fig. 9. Hydrolysis of a /3-amylase-resistant isomer (h) with (symbols as in Fig. 4). O-O-Q O-O-O- e O-Q --- g o- O O-O-O- on O- O-O- PuA. not hydrolyzed O- O-O ^rw Fig. 1. Hydrolysis of a /9-amylase-resistant isomers (e and g) with various enzymes (symbols as in Fig. 4). --CM3 d O-OO- GJc/T O O-O-O Fig. 11. Hydrolysis of a /3-amylase-resistant isomer (d) with various enzymes (symbols as in Fig. 4). sible for the enzyme to produce 6 3 -a-maltosylmaltoteraose from the dextrins of structures e and g on the basis of the specificity of reported previously (5) (Fig. 11). The above discussions lead to the conclusion that the singly branched heptaose isolated from the digestion of /3-limit dextrin with BLA consists of the six isomers shown in Fig. 12. The quantitative ratio of the isomers was obtained by calculating back from the yields of products formed from the original heptaose sample, as described in the text. For instance, the maltotetraose liberated (on a molar basis) from the original branched heptaose sample on digestion with pullulanase is attributed to the branched heptaoses designated as e, f, and h (Figs. 8 and 12). On the other hand, the maltotetraose liberated from the /S-amylaseresistant dextrins by pullulanase is attributed to the dextrins shown by e and h. Therefore, subtracting the molar fractions of e and h from those of e, f, and h gives the molar fraction of the branched dextrin denoted by f. /. Biochem.

7 «-AMYLASE LIMIT SINGLY BRANCHED HEPTAOSES 895 o-o-o-o O-O- f (11?) OOOO- ( 13 % ) oo-o O-O-O- OO ( 2 % ) o-o-o - h O-OO-O- g ( 53 % ) Fig. 12. Structures of BLA-limit singly branched heptaoses and their quantitative ratio (%). O-9 - OO- o-o - OO-O-O - O-O- ( ( 4. ) o -9 OOO ooo O-OO ) Fig. 13. Structures of singly OOOO branched ' pentaose, hexaose, and heptaose produced by bacterial liquefying a-amylase. Numbers in parentheses indicates the yield from glutinous rice starch /3-limit dextrin ; BLA, 5 units per gram of dextrin, 4, 48 hr. The yields of singly branched pentaose, hexaose (5), and heptaose produced as a liquefying a-limit dextrin from /3-limit dextrin are summarized in Fig. 13. Figure 13 indicates that all the singly branched dextrins consisting of six and seven glucose units contain a 6 2 -a-maltosylmaltotriose moiety and this core branched dextrin links with glucose or maltose at the reducing or nonreducing end(s) to form the singly branched hexaoses and heptaoses. Figure 13 also shows that the yields of the singly branched dextrins were rather similar. The attack sites of bacterial liquefying a- amylase on amylose and various maltooligosaccharides have been kinetically studied by Hiromi et al. (3), and Thoma (21). They reported that the subsite structure of the enzyme binds eight to ten glucose units at a Binding mode OOOOOO-OOO-OOO OO-C>-O-CK>OO-O-O-O-O nunfcer of the subsite - Fig. 14. Complex of the subsite with branched substrate. Y////A, represents a barrier for binding with substrate; A, catalytic site. certain area of amylose, and that the catalytic site of the enzyme splits the a-1,4-glucosidic linkage between the sixth and seventh subsites counting from the non-reducing end. Their suggestions were derived from experimental results obtained at an early stage of the enzyme reaction. Our results, on the other hand, were obtained with digests which were incubated with a large excess of liquefying a- amylase. However, there should be no difference in the mechanism underlying the action specificity of the enzyme. The subsite structure of a-amylase suggested by Hiromi et al. and Thoma may be represented as shown in Fig. 14. However, the fact that all the singly branched dextrins produced by liquefying a-amylase contain a-maltosylmaltotriose as a core moiety indicates that the binding modes of d and e in Fig. 14 are non-productive. Subsites 6 and 7, both of which are adjacent to the catalytic site will not bind a glucose residue linked by an a-1, 6-glucosidic linkage at C 6 or C\ or will not give rise to straining to split the a-1,4- glucosidic linkages of the substrate. Also, the facts that the a-1,4-linked glucose chains on both sides of the ramifying point of the BLA limit dextrins are indefinite in length, and that the yield of each isomer Vol. 78, No. 5, 1975

8 896 K. UMEKI and T. YAMAMOTO - Fig. 15. Binding mode of BLA with branched substrate, a) binding with side chain most probable ; b) binding with both main and side chains improbable. of the dextrins is not significantly different, may indicate that in the binding area of the enzyme, no particular area is present which specifically binds a specific glucose residue of the substrate. Subsites 3, 4, 5, and 8 will be able to bind even a glucose residue having an a-1, 6-linkage at C 6 or C 1 with good efficiency. Liquefying a-amylase does not attack dextrins smaller than a certain size (8). This also indicates that the binding site of the enzyme covers a considerable number of glucose residues. Taking into consideration the suggestion by Hiromi et al. that the catalytic site is located nearer to the reducing end of the binding area of the enzyme, the following speculation seems reasonable; i.e., liquefying a-amylase may form an active complex with side chains as well as main chains of the substrate, but cannot form an active complex over both the main and side chains, as schematically represented in Fig. 15. REFERENCES 1. Thoma, J.A., Spradlin, J.E., & Dygert, S. (1971). in The Enzymes (Boyer, P.D., ed.) Vol. 5, pp , Academic Press, New York 2. Pazur, J.H. & Okada, S. (1966) /. Biol. Chem. 241, Iwasa, S., Aoshima, H., Hiromi, K., & Hatano, H. (1974) /. Biochem. 75, French, D. (196) Bull. Soc. Chim. Biol. 42, Umeki, K. & Yamamoto, T. (1972) /. Biochem. 72, Okada, S. & Fukumoto, J. (1963) /. Fertn. Tech. 41, Fukumoto, J., Yamamoto, T., & Ichikawa, K. (1951) Proc. Japan Acad., 27, Mantani, S., Yamamoto, T., & Fukumoto, J. (1967) Amylase Symposium (in Japanese) 2, Nishida, A., Fukumoto, J., & Yamamoto, T. (1969) Amylase Symposium (in Japanese) 4, Tsujisaka, Y., Fukumoto, J., & Yamamoto, T. (1958) Nature 181, Tsujisaka, Y. (196) Osakashiritsu Kogyokenkyusho- Hokoku (in Japanese) 23, Yamamoto, T., Ukawa, M., & Fukumoto, J. (1969) Amylase Symposium (in Japanese) 4, Tsujisaka, Y. & Hamada, N. (1971) Nihon Nogeikagakukai Taikai Koenyoshisyu (in Japanese) 46, Sakano, Y., Masuda, N., & Kobayashi, T. (1971) Agr. Biol. Chem. 35, Umeki, K. & Yamamoto, T. (1972) /. Biochem. 72, Dubois, M., Gillus, K.A., Hamilton, J.K., Rebers, P.A., & Smith, F. (1956) Anal. Chem. 28, Somogyi, M. (1952) /. Biol. Chem. 195, Trevelyan, W.E., Procter, D.P., & Harrison, J.S. (195) Nature 166, Nishikaze, O., Abraham, R., & Staudinger, HJ. (1963) /. Biochem. 54, Lisboa, B.P. (1964) /. Chromatog. 13, Thoma, J.A., Brothers, C, & Spradlin, J. (197> Biochemistry 9, 1768 /. Biochem.

Pattern of Action of Bacillus stearothermophilus Neopullulanase on Pullulan

JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 369-374 21-9193/89/1369-6$2./ Copyright 1989, American Society for Microbiology Vol. 171, No. 1 Pattern of Action of Bacillus stearothermophilus Neopullulanase on