Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

Abstract

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...

Controlling Substrate Preference and Transglycosylation Activity of Neopullulanase by Manipulating Steric Constraint and Hydrophobicity in Active Center

“…Transglycosylation Activity of Wild-type and Mutated Neopullulanase-Maltotriose is the most simple substrate for neopullulanase (1,18). Neopullulanase hydrolyzes maltotriose to produce glucose and maltose.…”

“…-O-␣-Maltosyl-maltose was obtained from panose and starch by the coupling reaction with cyclomaltodextrin glucanotransferase (18). The oligosaccharide was purified by preparative paper chromatography (18).…”

“…The oligosaccharide was purified by preparative paper chromatography (18). 6 3 -O-␣-Glucosyl-maltotriose was purchased from Sigma.…”

“…We have been studying a new type of pullulan-hydrolyzing enzyme, neopullulanase from Bacillus stearothermophilus (11,17,18). Neopullulanase catalyzes both hydrolysis and transglycosylation at ␣-(134)-and ␣-(136)-glucosidic linkages by one active center (1,19).…”

Introduction of Raw Starch-Binding Domains into Bacillus subtilis α-Amylase by Fusion with the Starch-Binding Domain of Bacillus Cyclomaltodextrin Glucanotransferase

“…␣-Amylase activity was assayed based on the 3,5-dinitrosalicylic acid method, as described previously (11). The reaction mixture (200 l) consisted of 0.5% soluble starch (Merck, Darmstadt, Germany) in 20 mM sodium acetate buffer (pH 5.5) and the enzyme.…”