Purification and some properties of a novel .ALPHA.-amylase produced by a strain of Thermoactinomyces vulgaris.

Shimizu, Mizuho; Kanno, Mutsuo; Tamura, Masaki; Suekane, Mikio

doi:10.1271/bbb1961.42.1681

Cited by 66 publications

(28 citation statements)

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“…The pullulanase II examined in this study had properties similar to those of three previously characterized types of puilulan-degrading enzymes: an isopullulanase from A. niger, an a-amylase of Thermoactinomyces vulgaris (13,17), and a neopullulanase of Bacillus stearothermophilus (7). The Bacteroides pullulanase II, Thermoactinomyces a-amylase, and Bacillus neopullulanase produce panose from pullulan hydrolysis.…”

Section: G3supporting

confidence: 60%

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1

Smith

Salyers

1991

J Bacteriol

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Previously, we constructed a gene disruption in the pullulanase I gene of Bacteroides thetaiotaomicron 5482A. This mutant, designated B. thetaiotaomicron 95-1, had a lower level of pullulanase specific activity than did wild-type B. thetaiotaomicron but still exhibited a substantial amount of pullulanase activity. Characterization of the remaining pullulanase activity present in B. thetaiotaomicron 95-1 has identified an a(1->4)-D-glucosidic bond cleaving pullulanase which has been tentatively designated a neopullulanase. The neopullulanase (pullulanase II) is a 70-kDa soluble protein which cleaves a(1-4)-D-glucosidic bonds in pullulan to produce panose. The neopullulanase also cleaved a(1-4) bonds in amylose and in oligosaccharides of maltotriose through maltoheptaose in chain length. An a-glucosidase from B. thetaiotaomicron 95-1 was characterized. The a-glucosidase was partially purified to a preparation containing three proteins of 80, 57, and 50 kDa. Puliulan and amylose were not hydrolyzed by the a-glucosidase. a(1->4)-D-Glucosidic oligosaccharides from maltose to maltoheptaose were hydrolyzed to glucose by the a-glucosidase. The a-glucosidase also hydrolyzed a(1-6)-linked oligosaccharides such as panose (the product of the pullulanase II action on pullulan) and isomaltotriose.Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe that is found in the human colon in high numbers (6), can ferment a wide variety of polysaccharides (16). Among these polysaccharides are the starch amylose and the starchlike polysaccharide pullulan (Fig. 1). Previously, we reported the cloning and characterization of pullulanase from B. thetaiotaomicron. The cloned pullulanase (puilulanase I) was active as a monomer of approximately 77 kDa and cleaved ac(1-6)-D-glucosidic linkages of pullulan to produce maltotriose (18). Directed insertional mutagenesis was used to construct a pullulanase I-minus mutant of B. thetaiotaomicron so that the physiological significance of the cloned pullulanase could be investigated. Subsequent examination of B. thetaiotaomicron 95-1 (the pullulanase I-minus mutant) revealed that although the cloned pullulanase comprised approximately 30 to 50% of the total pullulanase activity in B. thetaiotaomicron, this enzyme was not essential for growth of B. thetaiotaomicron on pullulan. In fact, the 95-1 mutant was able to grow on pullulan at a rate similar to that of wild-type B. thetaiotaomicron. Therefore, there must be a second pullulan-degrading enzyme in B. thetaiotaomicron.Four types of pullulan-hydrolyzing enzymes have been described: (i) a glucoamylase (EC 3.2.1.3) (11,19) which hydrolyzes pullulan from the nonreducing ends to produce glucose, (ii) a pullulanase (EC 3.2.1.41) (1) from Klebsiella pneumoniae which hydrolyzes a(1-*6)-D-glucosidic linkages of puilulan to produce maltotriose, (iii) an isopullulanase (EC 3.2.1.57) (14) from Aspergillus niger which hydrolyzes a(1-*4)-D-glucosidic linkages of pullulan to produce isopanose, and (iv) a neopullulanase from Bacillus stearothermophilus w...

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Section: G3supporting

confidence: 60%

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1

Smith

Salyers

1991

J Bacteriol

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“…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)(14)(15) and should be considered as a new enzyme (8).…”

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confidence: 94%

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

Imanaka

Kuriki

1989

J Bacteriol

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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 62-0-a-(63-0-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 63-0-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 3.2.1.3) (15), which hydrolyzes pullulan from nonreducing ends to produce glucose; (ii) pullulanase (ax-dextrin endo-1,6-a-glucosidase; EC 3.2.1.41) (2), from Klebsiella pneumoniae, which hydrolyzes a-(1-+6)-glucosidic linkages of pullulan to produce maltotriose; (iii) isopullulanase (EC 3.2.1.57) (13), from Aspergillus niger, which hydrolyzes ot-(1-4)-glucosidic linkages of pullulan to produce isc panose (6-0-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 TRS40 in our previous paper (8).Neopullulanase from B. stearothermophilus TRS40 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 ne...

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“…The enzyme activity has been reported for Thermoactinomyces vulgaris a-amylase (19). These action patterns are diagrammatically shown in Fig.…”

mentioning

confidence: 95%

“…In contrast, Thermoactinomyces a-amylase can hydrolyze not only starch but also pullulan. However, the enzyme can mainly function as a starch-saccharyfying a-amylase, and the pullulan-hydrolyzing activity is known to be a side effect (16,17,19).…”

mentioning

confidence: 99%

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis

1988

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A new type of pullulanase which mainly produced panose from puliulan was found in Bacilus stearothermophilus and purified. The enzyme can hydrolyze puliulan efficiently and only hydrolyzes a small amount of starch. When puliulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacilus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new puliulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65°C, and about 90% of the enzyme activity was retained even after treatment at 60°C for 60 min. The optimum pH for the enzyme was 6.0.

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Purification and some properties of a novel .ALPHA.-amylase produced by a strain of Thermoactinomyces vulgaris.

Cited by 66 publications

References 1 publication

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1

Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis

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