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

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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 develop- ment 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 <5. # Downloaded from https://academic.oup.com/jb/article/72/5/1219/878303 by guest on 29 September 2021 s» I S. | ; i i 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.
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