Pattern of Action of Bacillus stearothermophilus Neopullulanase on Pullulan

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JOURNAL OF BACTERIOLOGY, Jan. 1989, p. 369-374 21-9193/89/1369-6$2./ Copyright 1989, American Society for Microbiology Vol. 171, No.

1 JOURNAL OF BACTERIOLOGY, Jan. 1989, p /89/1369-6$2./ Copyright 1989, American Society for Microbiology Vol. 171, No. 1 Pattern of Action of Bacillus stearothermophilus Neopullulanase on Pullulan TADAYUKI IMANAKA* AND TAKASHI KURIKI Department of Fermentation Technology, Faculty of Engineering, Osaka University, Yamadaoka, Suita-shi, Osaka 565, Japan Received 18 May 1988/Accepted 18 October 1988 The action of neopullulanase from Bacillus stearothermophilus on many oligosaccharides was tested. The enzyme hydrolyzed not only a-(1-+4)-glucosidic linkages but also specific a-(1-+6)-glucosidic linkages of several branched oligosaccharides. When pullulan was used as a substrate, panose, maltose, and glucose, in that order, were produced as final products at a final molar ratio of 3:1:1. According to these results, we proposed a model for the pattern of action of neopullulanase on puliulan as follows. In the first step, the enzyme hydrolyzes only a-(1-)4)-glucosidic linkages on the nonreducing side of a-(1->6) linkages of puliulan and produces panose and several intermediate products composed of some panose units. In the second step, taking 62--a-(63--a-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, the enzyme hydrolyzes either a-(1-,4) (the same position as that described above) or a-(1-6) linkages and produces panose or 63-_-a-glucosyl-maltotriose plus maltose, respectively. In the third step, the a-(1-4) linkage of 63--aL-glucosyl-maltotriose is hydrolyzed by the enzyme, and glucose and another panose are produced. To confirm the model of the pattern of action, we extracted intermediate products produced from pullulan by neopullulanase and analyzed the structures by glucoamylase, pullulanase, and neopullulanase analyses. The experimental results supported the above-mentioned model of the pattern of action of neopullulanase on pullulan. Four types of pullulan-hydrolyzing enzymes have been reported: (i) glucoamylase (glucan 1,4-a-glucosidase; EC ) (15), which hydrolyzes pullulan from nonreducing ends to produce glucose; (ii) pullulanase (ax-dextrin endo-1,6- a-glucosidase; EC ) (2), from Klebsiella pneumoniae, which hydrolyzes a-(1-+6)-glucosidic linkages of pullulan to produce maltotriose; (iii) isopullulanase (EC ) (13), from Aspergillus niger, which hydrolyzes ot-(1-4)- glucosidic linkages of pullulan to produce isc panose (6--amaltosyl-glucose); and (iv) neopullulanase, which hydrolyzes a-(1-+4)-glucosidic linkages of pullulan to produce panose (62-O-a-glucosyl-maltose). The last enzyme was reported to be a new type of pullulanase from Bacillus stearothermophilus TRS4 in our previous paper (8). Neopullulanase from B. stearothermophilus TRS4 could hydrolyze pullulan efficiently and only hydrolyzed a small amount of starch. When pullulan was used as a substrate, the main product was panose, and small amounts of glucose and maltose were simultaneously produced (8). Therefore, neopullulanase was obviously different from the pullulan-hydrolyzing enzymes previously reported (2, 13-15) and should be considered as a new enzyme (8). To produce glucose and maltose in addition to panose from pullulan, neopullulanase has to hydrolyze a-(1-+6)- glucosidic linkages as well as a-(1--+4)-glucosidic linkages. The present investigation was conducted to reveal the attack point of glucosidic linkages of pullulan by neopullulanase. This paper describes the action of neopullulanase on many oligosaccharides and pullulan. According to these observations, a model of the pattern of action of the enzyme on pullulan is proposed and discussed. * Corresponding author. 369 MATERIALS AND METHODS Substrates and reagents. Maltotriose, maltotriitol, panose, and pullulan were purchased from Hayashibara Co., Ltd., Okayama, Japan. Isomaltose and maltotetraose were obtained from Sigma Chemical Co., St. Louis, Mo. Unless otherwise specified, glucose, maltose, 3-cyclodextrin, and all other reagents were purchased from Wako Pure Chemical Industries, Osaka, Japan. Enzymes. Neopullulanase from B. stearothermophilus TRS4 was purified as described previously (8). Saccharifying a-amylase (EC ) was prepared from B. subtilis as described by Fukumoto and Okada (5). Cyclodextrin glucanotransferase (cyclomaltodextrin glucanotransferase; EC ) from B. macerans was purchased from Amano Pharmaceutical Co., Ltd., Nagoya, Japan. Pullulanase from Klebsiella (Aerobacter) aerogenes and glucoamylase from Aspergillus niger were obtained from Sigma. Assay of neopullulanase activity. Neopullulanase activity was assayed as described previously (8). The reaction mixture (5,ul) consisted of 1% pullulan in.2 M sodium acetate buffer (ph 6.) and the enzyme. The reaction was stopped after an appropriate incubation period at 6 C by the addition of 3,5-dinitrosalicylic acid reagent (1 ml). The reagent was prepared by mixing.5 g of 3,5-dinitrosalicylic acid dissolved in 2 ml of 2 N NaOH and 3 g of sodium potassium tartrate dissolved in 5 ml of deionized water and adjusting the volume to 1 ml with deionized water. One unit of neopullulanase activity was defined as the amount of enzyme which released 1,xmol of reducing sugar as glucose per min under the assay conditions described above. Analysis of hydrolysis products. Paper chromatography was carried out in the ascending mode on Toyo no. 5 filterpaper with a solvent mixture of n-butanol-pyridine-water (6: 4:3 or 6:4:4, by volume). Preparative-scale (4- by 4-cm) and analytical-scale (18- by 2-cm) paper chromatograms

2 37 IMANAKA AND KURIKI were developed seven and four times, respectively. Sugars on the paper were detected by the silver nitrate dip method (11). High-performance liquid chromatography (LC-6A; Shimazu, Kyoto, Japan; ZORBAX-NH2 column; Du Pont Co., Wilmington, Del.) was also used for the analysis of reaction products. Paper chromatography gave superior discrimination between a-(1---4)- and a-(1--36)-linked oligosaccharides, as compared with high-performance liquid chromatography or thin-layer chromatography. The retardation of an a-(1--+6) linkage was about one-half to two-thirds of that of an additional a-(1->4)-linked D-glucose unit (1). Quantitative analysis of reducing sugars in the hydrolysate was performed as described above for the neopullulanase assay. Total carbohydrate was measured by the phenol-sulfuric acid method (4). Extraction of oligosaccharides from paper chromatograms. After the preparative-scale paper chromatograms were developed and dried, the oligosaccharide fraction was cut out and dipped into boiling water (1 ml) for 5 min. The eluted oligosaccharide solution was collected by filtration (Whatman no. 1 filter paper). These elution steps were repeated twice, and the solution was concentrated by lyophilization. The yield of oligosaccharide was about 1%, with no contamination by other oligosaccharides. RESULTS AND DISCUSSION Preparation of branched oligosaccharides. Isomaltose was incubated with starch and cyclodextrin glucanotransferase to generate various branched oligosaccharides terminated by isomaltose at the reducing end (7). The oligosaccharides in the mixture obtained by the coupling reaction with cyclodextrin glucanotransferase were fractionated by preparativescale paper chromatography. As a branched fraction with a degree of polymerization (DP) of 3, isopanose and 61-O-aglucosyl-maltose were produced simultaneously from isomaltose, so it was not feasible to isolate them as individual compounds (1). In the same manner, panose was incubated with starch and cyclodextrin glucanotransferase to generate various branched oligosaccharides terminated by panose at the reducing end, and these oligosaccharides were fractionated. As a branched DP 4 fraction, 62-O-ot-maltosyl-maltose and 62-O-a-glucosyl-maltotriose were produced (7) and could be separated by paper chromatography. The former migrated more slowly than did the latter (16). The structures of these branched oligosaccharides were confirmed by pullulanase and glucoamylase analyses. The mixture of isopanose and 61-O-a-glucosyl-maltose was not hydrolyzed by pullulanase (1) but was hydrolyzed to produce glucose and isomaltose by glucoamylase (9). 62-O-a- Maltosyl-maltose was hydrolyzed by pullulanase, and maltose was produced (1). It was also hydrolyzed by glucoamylase to produce glucose and to accumulate panose as an intermediate product (9). On the other hand, waxy maize amylopectin was digested completely by saccharifying a-amylase. The products were analyzed by preparative-scale paper chromatography, and the smallest limit dextrin was extracted as 63-O-a-glucosylmaltotriose (16). Action of neopullulanase on DP 2 and DP 3 oligosaccharides. Maltose, isomaltose, panose, maltotriose, maltotriitol, and the mixture of isopanose and 61-O-a-glucosyl-maltose were digested with neopullulanase, and the time course of the products was analyzed by paper chromatography. Each reaction mixture (5 RI) consisted of.5% substrate in 5 mm acetate buffer (ph 6.) and.5 U of neopullulanase. After an appropriate incubation period at 5 C, 5-,ul samples were collected, and the reaction was stopped by heat treatment at 1 C for 5 min. A 1-,Il amount of each sample was applied to chromatography paper. Neopullulanase did not hydrolyze maltose, isomaltose, and panose (data not shown). Maltotriose and maltotriitol could be hydrolyzed, and both maltose and glucose were produced, indicating that neopullulanase hydrolyzed a-(1-+4) linkages next to both nonreducing and reducing ends (Fig. la and b). It must be noted that maltotriitol does not produce a spot on a paper chromatogram with silver nitrate reagent because of the reduction of a reducing end. The mixture of isopanose and 61-O-a-glucosyl-maltose could be also hydrolyzed, and both maltose and glucose were produced (Fig. lc). These facts indicated that neopullulanase hydrolyzed not only oa-(1-4) linkages but also a-(1--*6) linkages. Action of neopullulanase on DP 4 oligosaccharides. Using the same procedures as those described above, we analyzed the patterns of action of neopullulanase on maltotetraose, 63-O-a-glucosyl-maltotriose, and 62-O-a-maltosyl-maltose. Neopullulanase hydrolyzed maltotetraose and produced maltose and a smaller amount of glucose (Fig. 2a). These results showed that the middle position of the three ot-(1--4) linkages was mainly cleaved. 63-O-a-Glucosyl-maltotriose was hydrolyzed, and panose and glucose were produced (Fig. 2b). On the other hand, neopullulanase attacked the a-(1--*6) linkage of 62-O-a-maltosyl-maltose and produced maltose (Fig. 2c). A very small amount of glucose was visible on the paper chromatogram; the reason is discussed below. Time course of pullulan hydrolysis by neopullulanase. To investigate the pattern of action of neopullulanase more precisely, we quantitatively analyzed the time course of the sugars making up the pullulan hydrolysate by high-performance liquid J. BACTERIOL. chromatography (data not shown). Only panose was detected in the early stage of the reaction ( to 3 min); maltose production followed after 3 min. Glucose could not be detected in the early stage at all but could be detected after 6 min. The final molar ratio of panose, maltase, and glucose (after 36 min) was 3:1:1. Model of action of puilulan hydrolysis by neopuflulanase. According to the results mentioned above, a model of the action of neopullulanase on pullulan is proposed (Fig. 3). In the first step, neopullulanase attacks only a-(1-4)-glucosidic linkages on the nonreducing side of a-(1-*6)-glucosidic linkages of pullulan at random and produces panose and several intermediate products composed of some panose units. The endo-cleaving fashion of neopullulanase was confirmed by the fact that the enzyme could hydrolyze P-cyclodextrin (data not shown). In the second step, taking 62-O-a-(63-O-a-glucosyl-maltotriosyl)-maltose as an example of one of the intermediate products, neopullulanase attacks either a-(1--4) or a-(1-*6) linkages and produces panose or 63-O-a-glucosyl-maltotriose plus maltose, respectively. In other words, the enzyme can hydrolyze not only a-(1--4) linkages but also a-(1-36) linkages of partial hydrolysates of pullulan (intermediate products), recognizing the structure terminated by 62-O-a-maltosyl-maltose at the reducing end (Fig. 2c). In the third step, 63-O-a-glucosylmaltotriose is hydrolyzed by the enzyme, and glucose is produced together with panose (Fig. 2b). This model is basically consistent with the fact that when pullulan was hydrolyzed by neopullulanase, panose, maltose, and glucose appeared, in that order, with some intervals, that the molar ratio of maltose and glucose was 1:1, and that the amount of panose was greater than those of maltose and glucose.

a G1 2-9G G3-9s* 4- V G5- *, * * 9 9 * 9 a v....b...s I ooqr (G - oks Ma l totriose (3 3 Std. b GI -_ G2-4 G3- I G4-1 G5- A 1 2 3 6 Time (min) _eon 12 24 Std. * -OH - Mlal tot riito.

3 a G1 2-9G G3-9s* 4- V G5- *, * * 9 9 * 9 a v....b...s I ooqr (G - oks Ma l totriose (3 3 Std. b GI -_ G2-4 G3- I G4-1 G5- A Time (min) _eon Std. * -OH - Mlal tot riito.1 {3 OH Downloaded from Std. C GI - G2-* G3- C) Ti me (min) Std..* qi 46 4 do - I sopanose on December 25, 218 by guest G4- W G5- g * (3 Std Std. 61-O--a-Glucosylmaltose Time (min) FIG. 1. Action of neopullulanase on DP 3 oligosaccharides. Paper chromatograms and the schematic representation of the results are shown. Glucose, maltose, maltotriose, maltotetraose, and maltopentaose are indicated by Gi, G2, G3, G4, and G5, respectively. Std. represents the standard mixture of oligosaccharides. Symbols:, glucose; (, glucose with a reducing end; OH, sorbitol; -, at-(l-*4) linkage; I, oa-(1-3-6) linkage. Thick and thin horizontal arrows indicate relatively fast and slow enzyme reactions in the system, respectively. 371

372 IMANAKA AND KURIKI J. BACTERIOL. *1,, * * * Q-3 13 i 4) ;. 1. E I 2 (. A3 to )2 24 SLd. *R 4 to A* a 9 6 * / Std. 1 2 3 6 12 24 Stid. Time (min) c1 -. C _~~ cg2-- 14t.

4 372 IMANAKA AND KURIKI J. BACTERIOL. *1,, * * * Q-3 13 i 4) ;. 1. E I 2 (. A3 to )2 24 SLd. *R 4 to A* a 9 6 * / Std Stid. Time (min) c1 -. C _~~ cg2-- 14t..( be11i 11 m I b1 * ---*o41 4) 1 G12-- Ci. (Gl GJ I..) (C13 (,l..~t. G(;4I.) * 1 3a 4 4 p - 1l II ;RI "I t ) I 1- B1 I 2.V1zt t:)-- ii a1 ', 3~ -(~ -3: '' s o-c(: Std Std. Time (miii) FIG. 2. Action of neopullulanase on DP 4 oligosaccharides. PA, Panose. Other abbreviations and symbols are as in Fig. 1.

VOL. 171, 1989 PATTERN OF ACTION OF NEOPULLULANASE 373 1 st step 2nd step 3rd step 62 O a -(63-O- a -GlucosYI - aaltotriosyl) -maltose ----- 1st step FIG. 3. Model of pullulan hydrolysis by neopullulanase.

5 VOL. 171, 1989 PATTERN OF ACTION OF NEOPULLULANASE st step 2nd step 3rd step 62 O a -(63-O- a -GlucosYI - aaltotriosyl) -maltose st step FIG. 3. Model of pullulan hydrolysis by neopullulanase. Symbols are as in Fig. 1. The oligosaccharides enclosed by rectangles are the final products..1. Gi- 4kl -A 3 G;2- I& G3-* Confirmation of the model of action. To examine whether the action model is correct, we analyzed intermediate products produced from pullulan by neopullulanase. A 1% pullulan solution (4.5 ml) and 5 U of neopullulanase were incubated at 5 C for 4 h, and the reaction was stopped by heat treatment at 1 C for 5 min. The reaction mixture was analyzed by preparative-scale paper chromatography, and a part of the reaction mixture was visualized by paper chromatography after treatment by the glucoamylase dip method (6) to convert weakly reducing oligosaccharides into glucose (Fig. 4). Spots A, B, and C corresponded to glucose, maltotriose, and panose, respectively, as final products. Spot D seemed to be a branched DP 4 oligosaccharide, and the fraction was extracted from the paper chromatogram. The sample was hydrolyzed by neopullulanase, and glucose and a branched DP 3 oligosaccharide were produced as final products. The extracted fraction was hydrolyzed by glucoamylase, and only glucose was produced as the final product. These results indicated that spot D was 63-O-a-glucosylmaltotriose. Spot E corresponded to a branched DP 6 oligosaccharide, and the fraction was extracted. The sample was hydrolyzed by pullulanase to produce maltose and a branched DP 4 oligosaccharide and was also hydrolyzed by glucoamylase to produce glucose and panose. These results suggested that spot E was 62-O-a-(63-O-a-glucosyl-maltotriosyl)-maltose. In this context, the extracted sample from the spot E fraction was hydrolyzed by neopullulanase, and panose, maltose, and glucose were produced. From the data mentioned above, the model of action of neopullulanase G4- G5- G6- G7-1 -B Final Q products -C i 9-D ( -b 1 A -E I Std. FIG. 4. Paper chromatography of the partial hydrolysis products produced from pullulan by neopullulanase. Lane 1 contains the sample. Maltohexaose and maltoheptaose are indicated by G6 and G7, respectively. Other abbreviations and symbols are as in Fig. 1.

374 IMANAKA AND KURIKI a b - - - -.. n O ----- 41. n 6-- a -(63--a -Clucosylmaltotriosyl) -glucose FIG. 5.

6 374 IMANAKA AND KURIKI a b n O n 6-- a -(63--a -Clucosylmaltotriosyl) -glucose FIG. 5. Schematic representation of a side reaction of neopullulanase on 62-O-an-maltosyl-maltose (a) and 62-O-a-(63-O-_C-glucosylmaltotriosyl)-maltose (b). Broken arrows indicate the very weak side reaction of neopullulanase. Symbols are as in Fig. 1. (Fig. 3) must be reliable enough to explain the action of neopullulanase on pulllan. In addition to these main reactions, a slight side reaction of neopullulanase was found. Neopullulanase attacked mainly the a-(1-*6) linkage of 62-O-a-maltosyl-maltose and produced maltose. However, a very small amount ofglucose was found (Fig. 2c), Therefore, it was most likely that neopuliulanase hydrolyzed the a-(1->4) linkage of 62-O-amaltosyl maltose very slightly and produced glucose and maltose (Fig. 5a). A similar reaction might occur with other substrates. Taking 62-OD-a-(63-O-a-glucosyl-maltotriosyl)- maltose as an example of an intermediate product, a very small amount of isomaltose might be produced as one of the final products from pullulan after neopuilulanase treatment (Fig. 5b). In fact, a faint spot corresponding to isomaltose was found on the paper chromatogram in Fig. 4, spot a. Spot b in -Fig, 4 was thought to be the intermediate product 6-O-oc-(63-O-cz-glucosyl-maltotriosyl)-glucose (Fig. Sb). Recently, some enzymes that demonstrate dual specificity towards a-(1-.4)- and a-q-1-6)-glucosidic linkages have been reported (3, 1, 12). Thermoanaerobium sp. strain Tok6-B1 puliulanase (1) and Thermoanaerobium brockii debranching enzyme (3) can cleave the a-(i--*6) linkages [and none of the a-(l-*4) bonds] of pullulan and the a-(1-4) linkages of starch. On the other hand, Thermoactinomyces vulgaris a-amylase attacks some of the a-(1-.6) linkages in partial hydrolysates ofpullulan as well as a-(1-34) linkages in starch and puliulan (12). From the viewpoint ofthe pattern of action on pullulan and partial hydrolysates of pullulan, B. stearothermophilus neopullulanase has something in common with T. vulgaris a-amylase. When compared on the same basis of a final hydrolysis percentage [(reducing sugar/total carbohydrate) x 1], T. vulgaris a-amnylase was shown to hydrolyze pullulan and starch 35 and 51%, respectively (14), whereas.(3 B. stearothermophilus neopuliulanase hydrolyzed pullulan efficiently (45%) but hydrolyzed starch less efficiently (13%) (8). Hence, B. stearothermophilus neopuliulanase should be (3 3 clearly distinguished from a-amylase. Further investigations are now in progress to better understand the substrate specificity of the enzyme by using several polysaccharides, such as amylose, amylopectin, glycogen, and a-limit dextrin. LITERATURE CITED J. BACTERIOL. 1. Abdullah, M., and D. French Substrate specificity of puilulanase. Arch. Biochem. Biophys. 137: Bender, H., and K. Wallefels Untersuchungen an Pullulan. II. Spezifisher Abbau durch tin Bacterielles Enzyme. Biocheip. Z. 334: Coleman, R. D., S. S. Yang, and M. P. McAlhter Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis. J. Bacteriol. 169: Dubois, M., K. A. Gilles, J. K., P. A. Rebers, and F. Smith Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: Fukumoto, J., and S. Okada Studies on bacterial amylktse. Amylase types ofbacillus subtilis species. J. Ferment. Technol. 41: Kninuma, K., and D. French Action of pancreatic amylase on starch ofigosaccharides containing single glucose side chains. FEBS Lett. 5: Kanuma, K., S. Kobayashi, and T. Harada Action of Pseudomonas isoamylase on various branched oligo- and polysaccharides. Carbohydr. Res. 61: Kurili, T., S. Okada, and T. Imanaka New type of pullulanase from Bacillus stearot4ermophilus and moleoular cloning and expression of the gene in Bacillus subtilis. J. Bacteriol. 17: Pazur, J. H., and T. Ando The hydrolysis of glucosyl oligosaccharides with a-d-(1-4) and a-d-(1--*6) tonds by fungal amyloglucosidase. J. Biol. Chemn. 23S: Plant, A. R., R. M. Clemens, H. W. Morgan, d R. M. Daniel, Active-site- and substrate-spepificity of Thermpanaerobium Tok6-B1 puliulanase. Biochem. J. 2; Robyt, J., and D. French Action pattem and specificity of an amylase from Bacillus subtilis. Arch. Biochem. Biophys. 1: Sakano, Y., S. Hiralwa, J. Fukhm, and T. Kobayashi Enzymatic properties and action patterns of Thermoactinomyces vulgaris oa-amylase. Agric. Biol. Chem. 46: Sakano, Y., N. M da, d T. Kobaynhl Hydrolysis of pullulan by a novel enzyme from Aspergillus niger. Agric. Biol. Chem. 35: Shinizu, M., M. Kanno, M. Tanura, ad M. S Purification and some properties of a novel oa-amylase produced by a strain of Thermoactinomyces vulgaris. Agric. Biol. Chem. 42: Ueda, S., 1K. Fujita, Komatu, N. Nakshhn Polysaccharide produced by the genus Pullularia. I. Production of polysaccharide by growing cells. Appl. Microbiol. 11: Umeki, K., and T. Yanamoo Enzymatic determination of structure of singly branched hexaose dextrins formed by liquefying a-amylase of Bacillus subtilis. J. Biochem. (Tokyo) 72:11-19.

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

/. Biochem., 78, 889-896 (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,