Active-site- and substrate-specificity of <i>Thermoanaerobium</i> Tok6-B1 pullulanase

Plant, Adrian R.; Clemens, Robyn M.; Morgan, Hugh W.; Daniel, Roy M.

doi:10.1042/bj2460537

Cited by 58 publications

(23 citation statements)

References 18 publications

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“…Recently, some enzymes that demonstrate dual specificity towards a-(1-.4)-and a-Q-1-6)-glucosidic linkages have been reported (3,10,12). Thermoanaerobium sp.…”

Section: -O-a-(63-o-a-glucosyl-maltotriosyl)-maltose As An Exam-mentioning

confidence: 99%

“…Thermoanaerobium sp. strain Tok6-B1 puliulanase (10) 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) (3 3 clearly distinguished from a-amylase.…”

Section: -O-a-(63-o-a-glucosyl-maltotriosyl)-maltose As An Exam-mentioning

confidence: 99%

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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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“…Recently, some enzymes that demonstrate dual specificity towards a-(1-.4)-and a-Q-1-6)-glucosidic linkages have been reported (3,10,12). Thermoanaerobium sp.…”

Section: -O-a-(63-o-a-glucosyl-maltotriosyl)-maltose As An Exam-mentioning

confidence: 99%

Section: -O-a-(63-o-a-glucosyl-maltotriosyl)-maltose As An Exam-mentioning

confidence: 99%

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

Imanaka

Kuriki

1989

J Bacteriol

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“…Recently, other pullulan-hydrolyzing enzymes that hydrolyze both a-(1->4)-and a-(1--+6)-glucosidic linkages have been reported (2,15,16). Melasniemi et al (11) reported that the primary structure of the a-amylase-pullulanase from Clostridium thermohydrosulfuricum closely resembled that of neopullulanase.…”

mentioning

confidence: 99%

“…On the other hand, Plant et al (15) suggested that the same active center participated in the dual activity toward a-(1-->4)-and a-(1-*6)-glucosidic linkages, as indicated by * Corresponding author. studies of chemical modification and enzymatic kinetics of Thermoanaerobium sp.…”

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confidence: 99%

Analysis of the active center of Bacillus stearothermophilus neopullulanase

et al. 1991

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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.

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“…The protein encoded by the pul gene has putative N-terminal pullulanase and C-terminal alpha-amylase domains, potentially able to act on pullulan, amylopectin, or glycogen (20,35). The pul gene lies directly upstream from the glg operon, suggesting that the encoded protein might be involved in determining IPS branch structure and/or IPS metabolism.…”

Section: Discussionmentioning

confidence: 99%

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

et al. 2009

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Intracellular polysaccharide (IPS) is accumulated byStreptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.Streptococcus mutans is a facultative colonizer of the human dental plaque, the microbial pellicle that covers the surface of the teeth. It is the major etiological agent of dental caries (17). Sugar metabolism is central to the behavior of S. mutans (4, 7). It can use a variety of sugars. The sugars are fermented by glycolysis with production of organic acids, particularly lactic acid (4, 7). In addition to providing energy, sucrose is used to produce extracellular polysaccharides to form the biofilm matrix that aids in the association of S. mutans with the dental plaque. Once the S. mutans biofilm becomes part of the dental plaque, the acidic by-products of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries (17). The presence of sugars in the dental plaque is periodic and reflects the intake of dietary sugars. If there is excess sugar available, in addition to producing organic acids and matrix, intracellular (iodophilic) polysaccharide (IPS; glycogen) is formed.The IPS of S. mutans is a polymer of the glycogen-amylopectin type, with ␣-(1, 4)-and ␣-(1, 6)-linked glucose, and is stored as intracellular granules (10). Intracellular glycogen storage reserves in various bacterial species are synthesized from glucose-1-P via ADP-glucose (1). The synthesis involves at least three enzymes: glycogen synthase, glucose-1-phosphate pyrophosphorylase, and branching enzyme. The genes encoding these enzymes are commonly found in a glg operon, although the order of genes differs between species. In two gram-positive species, Bacillus subtilis and Bacillus stearothermophilus, the gene order is glgB-glgC-glgD-glgA-glgP (15, 29): glgA encodes glycogen synthase, glgB encodes glucan branching enzyme, and glgC and glgD encode subunits of glucose-1-phosphate pyrophosphorylase. The glgP gene encodes glycogen phosphorylase, which is unlikely to be involved in glycogen synthesis (29). Genes putatively encoding similar enzymes are present in the same order in the genome of S. mutans (29); they are thought also to form an operon.The IPS can be used as a source of carbohydrate for fermentation upon nutrient depletion (11, 13). In planktonic cultures, IPS reserves are largely consumed within 12 h of the imposition of sugar starv...

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Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase

Cited by 58 publications

References 18 publications

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan

Analysis of the active center of Bacillus stearothermophilus neopullulanase

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

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