Structures of Branched Dextrins Produced by Saccharifying A-Amylase of Bacillus Subtilis

/. Biochem., 72, 1219-1226 (1972)

Structures of Branched Dextrins Produced by Saccharifying a-Amylase of Bacillus subtilis

Kimio UMEKI and Takehiko YAMAMOTO Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 The Faculty of Science, Osaka City University, Osaka

Received for publication, May 25, 1972

The branched dextrins produced from waxy rice starch ^-limit dextrin by Bacillus subtilis' saccharifying a-amylse [EC 3.2.1.1] were isolated by the multiple development paper chromatography and their chemical structures were investigated. The analyses using £-amylase [EC 3.2.1. 2], glucoamylase [EC 3.2.1.3], pullulanase, and pullulan a-1,4-glucoside hydrolase revealed that they were doubly branched dextrins with the following structures: e'-a-^'-a-glucosylmaltosylJ-maltotriose, 68-a-, 65-a- diglucosylmaltopentaose, and 68-a-{6*-a-glucosylmaltotriosyl)-maltotriose. The results were discussed in connection with the interior structure of /3-limit dextrin.

Only a few papers have so far been published from each other (8, 9). on the action of a-amylase [EC 3.2.1.1] on The saccharifying a-amylase is character- branched substrates (1-3). French and his istic in that it hydrolyzes starch to produce coworkers have reported that the porcine reducing sugars of about twice as much as pancreatic a-amylase produces various branched those produced by the liquefying a-amylase dextrins as the limit dextrins (4). Our pre- and none of the hydrolysis products by the vious paper (5) has also shown that a-amylase saccharifying a-amylase are attacked by the of starch liquefying type from Bacillus subtilis liquefying a-amylase. Another characteristic produces various kinds of branched dextrins property of the saccharifying ar-amylase is from /3-limit dextrin, indicating that hydrolysis that in the digestion of /3-limit dextrin prepared of a-1, 4-glucosidic linkages by the enzyme is from waxy rice starch, the enzyme produces greatly affected by neighboring a-1, 6-glucosi- 68-a-glucosylmaltotriose in a yield of more dic bonds which are the ramifying points of than 20% (5). Thus, the enzyme was em- /3-limit dextrin. ployed in our previous paper to study the struc- As reported previously, there is another ture of singly branched hexaose dextrins pro- type of a-amylase in Bacillus subtilis which duced from /3-limit dextrin by the liquefying has been referred to by us as saccharifying a-amylase. a-amylase (6~). Whether an a-amylase of Recently, the structures of several branch- Bacillus subtilis is liquefying or saccharifying ed dextrins produced by the saccharifying a- type is quite dependent on the bacterial strain amylase from /3-limit dextrin were investigated which produces the enzyme (7). Also, chem- and it was found that with a sharp contrast ical and enzymatic properties of the two to those produced by other a-amylases, a cer- bacterial a-amylases are significantly different tain regularity exists among the structures of

Vol. 72, No. 5, 1972 1219 1220 K. UMEKI and T. YAMAMOTO the branched dextrins produced by the sac- conducted at 28°C by the descending method charifying a-amylase. The present paper de- on Toyo Roshi No. 50 (60x60 cm), developing scribes the experimental details for determina- multiply with a mixture of n-butanol: pyridine: tion of the structures of some of the dextrins water in the proportion described later. Loca- produced by the saccharifying a-amylase, add- tion of reducing sugars was detected by using ing some discussion on the action of the silver nitrate and NaOH (12). In the case enzyme on branched substrates as well as the of thin layer chromatography, detection of inner structure of ^-limit dextrin. sugars was made by the anisaldehyde-sulfuric acid method (13).

MATERIALS AND METHODS 3. Quantitative Determination of Sugars— Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 The Somogyi-Nelson method (14) was applied 1. fi-Limit Dextrin and Enzymes Employ- to the micro-assay of reducing sugar. Estima- ed—/S-Limit dextrin was prepared from the tion of comparatively large quantities of reduc- digests of purified waxy rice starch with /3- ing sugars was conducted by the method of amylase [EC 3. 2.1.2] from soybean, according Shaffer-Somogyi (75). Total sugar content to the method reported previously (5). of dextrins was determined by the phenol- Crystalline preparations of saccharifying sulfuric acid method (16). a-amylase of Bacillus subtilis and glucoamylase [EC 3.2.1. 3] of Rhizopus niveus were purchas- RESULTS ed from Seikagaku Kogyo Co. /3-Amylase and pullulanase were prepared from defatted 1. Hydrolysis of fi-Limit Dextrin with soybean and bacterial cells of a certain Aero- Saccharifying a-Amylase— /3-Limit dextrin (800 bacter species, respectively, by the methods mg) dissolved in 8 ml of 0.01 M acetate buffer, reported previously (5). A new enzyme, pH 5.4, was mixed with 2 ml of saccharifying which was a generous gift from Drs. Tsujisaka a-amylase solution (100 pg as protein; total and Hamada of Osaka Municipal Research activity, 40 units) and the mixture was incu- Institute for Technology and has been tenta- bated at 40°C with a few drops of toluene. At tively named by us pullulan a-l,4-glucoside certain intervals of time, 0.02 ml aliquots of hydrolase, was also used in the present work. the reaction mixture were taken and spotted Isolation of this enzyme was reported inde- on a filter paper for paper chromatography, pendently by Tsujisaka and Hamada (10) and to investigate a change in the kind of hydrolysis by Sakano et al. (11), at the same time in products as the hydrolysis proceeded. As 1971 and the enzyme was found to attack a- shown in Fig. 1, maltotriose and certain 1,4-glucosidic linkages of pullulan forming branched dextrins appeared at an early stage isopanose as the hydrolysis product. of hydrolysis, but later they gradually disap- a-Amylase, glucoamylase, and pullulanase peared. On the other hand, certain kinds of were assayed according to the methods de- branched oligosaccharides appeared (1 hr) and scribed in our previous paper (5). Pullulan increased as the hydrolysis proceeded. a-1, 4-glucoside hydrolase was assayed by the 2. Isolation of the Branched Dextrins Con- method of Tsujisaka and Hamada (10) in sisting of Six and Seven Glucose Units—jS-Limit which one unit of the enzyme activity was dextrin was hydrolyzed with saccharifying a- defined as the enzyme quantity that produced amylase under the same condition as above, 1 ^mole of reducing sugar as glucose from except that a ten-fold quantity of saccharify- pullulan per min at 40°C. ing a-amylase was employed and 48 hr later, 2. Isolation of Saccharifying a-Amylase the hydrolysate was subjected to paper chro- Branched Limit Dextrins — Separation and matography with seven times developement isolation of the dextrins produced from /9- using w-butanol: pyridine: water (6:4:3) and limit dextrin by the saccharifying a-amylase then with five times developement using a were performed by preparative scale paper mixture of an equal volume of n-butanol, py- chromatography. Paper chromatography was ridine, and water, as the solvent. The result

/. Biochem. BRANCHED DEXTRINS PRODUCED BY SACCHARIFYING a-AMYLASE 122 L

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Fig. 2. Paper chromatogram of the digests of /3- limit dextrin with saccharifying a-amylase of Bacillus subtilis (BSA). 500 units BSA per g of /3-limit dextrin, 40°C, 48 hr; as to the procedure of paper chromatography, see the text. Fig. 1. Paper chromatogram of the progressive hydrolysis products of /3-limit dextrin with sac- sis products were identified by paper chroma- charifying a-amylase of Bacillus subtilis. a-Amylase ; tography and by enzymatic method. 50 units per g of ^-limit dextrin, pH 5.4, 40°C. 7) Dextrin No. 3: Dextrin No. 3 (0.3 mg) was incubated at 40°C with pullulanase (12 is presented in Fig. 2, showing that the oli- units) in 5 ml of water under a few drops of gosaccharides of No. 3, 4-1, and 4-2 in the toluene. After reaction for 24 hr, the mixture paper chromatograms obtained after develop- was heated in boiling water for 15 min to in- ment with the former solvent were further activate pullulanase, and then concentrated to distinctly separated by multiple development a small volume under reduced pressure. The with the latter solvent. Dextrin No. 3, 4-1, paper chromatography of the concentrate and 4-2 were recovered from the chromato- showed that No. 3 had been hydrolyzed into grams in approximate yield of 2, 6, and 4%, maltotriose and a branched trisaccharide with respectively, based on the amount of /3-limit an Rf value corresponding to panose, as shown dextrin used. The polymerization degree of in Fig. 3. The branched trisaccharide formed the isolated branched dextrins was determined there was extracted from the paper, and then according to the' method reported previously incubated with glucoamylase. The periodical (5). Dextrin No. 3, 4-1, and 4-2 were esti- analyses of the hydrolysate by paper chroma- mated to be composed of 6, 7, and 7 glucose tography revealed that the saccharide was units, respectively. split into glucose and maltose, and no isomal- 3. Structures of Dextrin No. 3, 4-1, and tose was formed, indicating that the branched 4-2—The isolated branched dextrins No. 3, 4- trisaccharide was panose. 1, and 4-2 were separately hydrolyzed by fi- Dextrin No. 3 (1.2 mg) was, on the other amylase, pullulanase, pullulan o-l, 4-glucoside hand, incubated with pullulan o-l, 4-glucoside hydrolase, and glucoamylase, and the hydroly- hydrolase (0.053 units; the total volume of the

Vol. 72, No. 5, 1972 1222 K. UMEKI and T. YAMAMOTO

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M. A B CM. Fig. 4. Paper chromatogram of hydrolysates of dextrin No. 3 with pullulan a-l, 4-glucoside hydrolase. Fig. 3. Paper chromatogram of the hydrolysates of Symbols are as in Fig. 3. dextrin No. 3 with pullulanase. M., markers; Gi- G6, a series of maltooligodextrins; IM, isomaltose; P, panose; IP, isopanose; IMM, isomaltosylmaltose. A, hydrolysate of /3-limit dextrin with BSA; B, BSA- 8-0 limit dextrin No. 3; C, hydrolysate of BSA-limit ?\>\ 0-0-0 dextrin No. 3 with pullulanase. reaction mixture, 0.6 ml) at 40°C. At appro- 0-0-0'®/ priate intervals of time, 0.15 ml aliquots of the reaction mixture were taken out and subjected to paper chromatography after heating at 100°C for a few minutes to inactivate the enzyme. not hydrolyzed In Fig. 4 is presented the result, showing that Fig. 5. Schematic presentation of hydrolysis of at an early stage of the incubation, maltose dextrin No. 3 with several enzymes. /9-A, j3- and an unknown oligosaccharide whose i?/was amylase; Pul., pullulanase; PGH, pullulan ar-1,4- between maltopentaose and maltohexaose were glucoside hydrolase; O, glucose residue; 0, reduc- observed. However, at a later stage of the ing end glucose residue; —, a-l, 4-glucosidic linkage; enzyme reaction, the unknown oligosaccharide 1, a-l, 6-glucosidic linkage. described above appeared to be hydrolyzed to isomaltose. Dextrin No. 3 was not attacked units) in 5 ml of water at 40°C for 24 hr. The by /9-amylase. These results are schematically reaction mixture was then treated as described shown in Fig. 5, clearly indicate that dextrin 3 2 in "RESULTS" 3-1), and an aliquot of the con- No. 3 is 6 - a - (6 - a - glucosylmaltosyl) - malto - centrate was analyzed. However, no hydrol- triose. ysis of No. 4-1 was observed with pullulanase 2) Dextrin No. 4-1: Dextrin No. 4-1 (Fig. 6). On the other hand, No. 4-1 was at- (0.35 mg) was incubated with pullulanase (12 tacked by pullulan a-l, 4-glucoside hydrolase.

/. Biochem. BRANCHED DEXTRINS PRODUCED BY SACCHARIFYING a-AMYLASE 1223

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Fig. 6. Thin layer chromatogram of the hydrolysates of dextrin No. 4-1 and 4-2 with pullulanase. H. 4-1 M. Symbols are as in Fig. 3. PGH . P8H Fig. 7. Paper chromatogram of the hydrolysates of The experiment was conducted under the same dextrin No. 4-1 and 4-2 with pullulan a-1,4-glucoside condition as in the case of dextrin No. 3, hydrolase. Symbols are as in Fig. 3. except that 0.35 mg of the dextrin was incubated with 0.013 units of the enzyme. Figure not hydrolyzed 7 shows the result indicating that dextrin No. 4-1 was split by the enzyme to isomaltose and an unknown branched pentasaccharide. This 6-O-6-O0 branched saccharide was hydrolyzed by glucoamylase into glucose. But isomaltosylmaltose was formed as an intermediary product. Dex- trin No. 4-1 was not attacked by /3-amylase. not hydrolyzed The enzymatic hydrolysis of dextrin No. Fig. 8. Schematic presentation of hydrolysis of dextrin No. 4-1 with several enzymes. GlcA., gluco- 4-1 described above is presented schematically amylase ; other symbols are as in Fig. 5. in Fig. 8, indicating that the structure of the dextrin is 63-a-, 65-a-diglucosylmaltopentaose. 3) Dextrin No. 4-2: Dextrin No. 4-2 was was then split into glucose. This is an indica- incubated with pullulanase under the same tion that the branched tetrasaccharide was conditions as described in "RESULTS" 3-2). isomaltosylmaltose. The thin layer chromatogram of the hydrol- On the other hand, the hydrolysis of No. ysate is presented in Fig. 6, showing that 4-2 with pullulan a-1, 4-glucoside hydrolase the dextrin was hydrolyzed by pullulanase to which was conducted under the same condition maltotriose and an unknown branched tetrasac- as described above, yielded maltose, isomaltose, charide. The branched saccharide formed, and isopanose (Fig. 7). however, was hydrolyzed by glucoamylase The hydrolysis of No. 4-2 with pullulanase into maltotriose and glucose, and the former and pullulan a-1, 4-glucoside hydrolase is

Vol. 72, No. 5, 1972 1224 K. UMEKI and T. YAMAMOTO

6-0-0 O-O-0 Isopanose

pullulan

X 0O-0 6/0-0 0 O-0 not hydrolyzed Isomaltosylmaltose Isomaltose + maltose Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 Fig. 9. Schematic presentation of hydrolysis of dextrin No. 4-2 with several enzymes. Symbols are 0 0 as in Fig. 8. panose Isomaltose + glucose schematically presented in Fig. 9. Thus, No. Fig. 10. Specificity of pullulan a-1,4-glucoside hy- 4-2 is concluded to be 68-a-(68-a-glucosylmal- drolase. totriosyl)-maltotriose. linkage, as shown in Fig. 10. The enzyme DISCUSSION did not attack the a-1,4-glucosidic linkages due to the glucose residue which was also That the structure of BSA-limit branched linked to another two glucose residue, one at J dextrin No. 3 is 6'-a-(6 -a-glucosylmaltosyl)- C« and the other at Ct* We have confirmed maltotriose, is understandable from the fact this characteristic specificity of the enzyme that the dextrin was split by pullulanase to and thus structures of several branched dex- panose ^-a-glucosylmaltose) and maltotriose. trins were elucidated in the present paper. No other structures than that described above The chemical structure of branched dex- will be assigned for the dextrin from the spe- trin No. 4-2, 6'-a-(68-a-glucosylmaltotriosyl)- cificity of pullulanase and saccharifying a- maltotriose, is supported by the fact that the amylase. doubly branched dextrin with the same prop- The fact that the branched dextrin No. erties on the enzymatic hydrolyses could be 4-1 was hydrolyzed by a new enzyme "pullulan obtained from the partial hydrolysate of pul- a-1, 4-glucoside hydrolase" to form isomaltose lulan with pullulanase followed by digestion and 63-a-glucosylmaltotetraose is sufficient to with the saccharifying a-amylase, as shown prove that the structure of the dextrin is 6*-a-, in Fig. 11. 5 6 -a-digIucosylmaltopentaose, because the en- The saccharifying a-amylase limit dextrins zyme has been found to attack only the a-1, 4-glucosidic bond between two glucose residues • Y. Tsujisaka and N. Hamada, Presented at the in which the glucose at the non-reducing end Meeting of Agricultural Chemical Society of Japan, side is linked with another glucose by a-1, 6- Osaka, December 1971.

-O-0

Pullulan

Fig. 11. Preparation of 6>-or-{6'-ar-glucosylmaltotriosyl)-maltotriose by hydrolysis of pullulan with pullulanase followed by digestion with saccharifying a-amylase. Pul., pullulanase; BSA, saccharifying a-amylase of Bacillus subtilis.

J. Biochem. BRANCHED DEXTRINS PRODUCED BY SACCHARIFYING a-AMYLASE 1225

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00000 Fig. 12. Action patterns of saccharifying (BSA) and liquefying (BLA) a-amylases from Bacillus subtilis on brancheetc. d substrate. Within a certain region around the branching point, the hydrolysis of any one of the ar-1,4-glucosidic linkage results in inertness of the remaining ff-1,4-linkages toward BLA.

investigated in the present work were only branching point of dextrins produced by the those which were consisting of six and seven first attack, finally to form isomaltosylmaltose glucose units. However, it is of particular (6'-a-glucosylmaltotriose), as shown in Fig. interest that they were all doubly branched 12. and, regarded as the condensation products As has been pointed out by Kainuma and of 63 - a - glucosylmaltotriose. However, the French (4), our present study also provides above fact may be quite reasonable from the evidence that the amylopectin of waxy rice specificity of the saccharifying a-amylase that starch is ramified sometimes next by next the enzyme never attack the two o-l, 4- or every two glucose residues by the following glucosidic linkages existing in 6'-a-glucosyl- two ways: one, the Staudinger type as rep- maltotriose. This will be understandable in resented by branched dextrin No. 4-1, and connection with the fact that the amylase the other, the Haworth type as dextrin No. forms the limit dextrin in a yield of more than 3 and No. 4-2 in the present paper. 20% from /3-limit dextrin. Our previous papers (5, 17) reported that REFERENCES liquefying a-amylase from Bacillus subtilis acting on amylopectin produces by the first 1. D.H. Brown, B. Illingworth, and R. Kornfeld, attack various branched dextrins with a certain Biochemistry, 4, 486 (1965). regularity in the structure, and that the en- 2. K. Kainuma and D. French, FEBS letter, 5, 257 zyme shows no more action towards the (1969). 3. K. Kainuma and D. French, FEBS letter, 6, 182 branched dextrin produced there, thus leaving (1970). them as the a-amylase limit dextrins, as shown 4. K. Kainuma, Proceeding of the Symposium on in Fig. 12. Amylase, Osaka, October, p. 35 (1970) (in Contrarily, the present paper has revealed Japanese). that saccharifying a-amylase hydrolyzes the 5. K. Umeki and T. Yamamoto, /. Biochem., 72, or-1,4-glucosidic linkages neighboring the 101 (1972).

Vol. 72, No. 5, 1972 1226 K. UMEKI and T. YAMAMOTO

6. J. Fukumoto, K. Ichikawa, and T. Yamamoto, abstracts of papers, 42nd National Meeting of Proceedings of the Japan Academy, 27, 352 (1951). Agricultural Chemical Society of Japan, Tokyo, 7. S. Okada and J. Fukumoto, /. Fertn. Tech., 41, p. 278 (1971) (in Japanese); Agr. Biol. Chem., 427 (1963). 35, 971 (1971). 8. T. Yamamoto, A. Nishida, S. Mantani, and J. 12. W.E. Trevelyan, D.P. Procter, and J.S. Harrison, Fukumoto, Proceeding of the 7th International Nature, 166, 444 (1950). Congress of Biochemistry, Tokyo, August 19, p. 13. B.P. Lisboa, /. Chromatog., 16, 136 (1964). 766 (1967). 14. M. Somogyi, /. Biol. Chem., 195, 19 (1952). 9. J. Fukumoto, T. Yamamoto, and S. Mantani, 15. P.A. Shaffer and M. Somogyi, /. Bid. Chem., Koso Kagaku Symposium, 18, 223 (1966) (in 100, 695 (1933).

Japanese). 16. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 10. Y. Tsujisaka and N. Hamada, The abstracts of Rebers, and F. Smith, Anal. Chem., 28, 350 papers, 42nd National Meeting of Agricultural (1956). Chemical Society of Japan, Tokyo, p. 214 (1971) 17. T. Yamamoto and K. Umeki, Proceeding of the (in Japanese). Symposium on Amylase, Osaka, October, p. 49 11. Y. Sakano, N. Masuda, and T. Kobayashi, The (1971) (in Japanese).

/. Biochem.