Analysis of the active center of Bacillus stearothermophilus neopullulanase

Abstract

The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanase, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopuliulanase activities toward a-(1--4)-and a-(1 >6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward a-(14)-and a-(1--6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward a-(1--*4)-and oa-(1--+6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through a-(1-36)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the a-(1-+4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanase which exhibited higher specificity toward the a-(1--36)-glucosidic linkage.

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

“…Among these is that the difference of specificities toward ␣-(134)-and ␣-(136)-glucosidic linkages is caused solely by the difference in the binding modes of substrate and enzymes. We have obtained strong evidence to prove this, because we have observed that the specificities of neopullulanase toward ␣-(134)-and ␣-(136)-glucosidic linkages can be switched by replacing amino acid residues which might be involved in substrate recognition (19).…”

“…Neopullulanase catalyzes both hydrolysis and transglycosylation at ␣-(134)-and ␣-(136)-glucosidic linkages by one active center (1,19).…”

“…Each reaction is typically catalyzed by ␣-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41)/ isoamylase (EC 3.2.1.68), cyclomaltodextrin glucanotransferase (EC 2.4.1. 19), and 1,4-␣-D-glucan branching enzyme (EC 2.4.1.18), respectively (1). These reactions and the classification of the enzymes that catalyze them have been clearly distinguished and are represented by the four individual enzymes described above.…”

Amylolytic glycoside hydrolases

“…Among these pioneering prediction in silico analyses delivered by Birte Svensson and Ann MacGregor [4][5][6][7][8][9] have received special attention. In this regard, also the discovery of neopullulanase by Takashi Kuriki [10,11], as a universal enzyme capable to catalyse all four main reactions characteristic for the family, i.e., hydrolysis and formation by transglycosylation of both a-1,4-and a-1,6-glycosidic linkages [12], deserves to be mentioned.…”

Function and Tertiary- and Quaternary-structure of Cyclodextrin-hydrolyzing Enzymes (CDase), a Group of Multisubstrate Specific Enzymes Belonging to the α-Amylase Family

“…Neopullulanase catalyzes hydrolysis and transglycosylation of 1,4 and 1,6 glycosidic linkages at a single active site. 9) The mode of action on pullulan of CDase from Flavobacterium sp. resulted in the production of a remarkably high amount of a branched tetrasaccharide due to its transglycosylation activity.…”