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A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47Ϫ70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an A-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β-cyclodextrinϪhydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.Keywords : Bacillus stearothermophilus; maltogenic amylase ; acarbose ; transglycosylation; site-directed mutagenesis.Many types of amylases with unique properties have been megaterium [7], and A-amylase of Thermoactinomyces vulgaris [8] have been reported to hydrolyze the A-1,4 linkages of pulluisolated and characterized for various applications in the starch industry [1,2]. These proteins share many structural and mecha-lan to produce panose. Amylolytic enzymes, such as cyclodextrin glucanotransferases (CGTase) and CDase exhibit their nistic characteristics. However, amylases can be divided into several groups according to substrate specificities, patterns of highest levels of activity on cyclomaltodextrins [9Ϫ11]. Some starch cleavage, transglycosylation or cyclization activities, and amylolytic enzymes, including debranching enzymes and structural features. Classical A-amylases (e.g. 1,4-A-D-glucan CGTase, catalyze transglycosylation by forming A-1,4 or A-1,6 glucanohydrolase) catalyze hydrolysis of A-1,4-glucosidic link-linkages. ages in starch, and different amylases give rise to oligosacchaJespersen et al.[12] used sequence alignments and structurerides with specific lengths of glucose as major product [2]. De-prediction models to predict the presence of A-amylase-type branching enzymes are capable of hydrolyzing A-1,6-glucosidic (β/A) 8 -barrel domains and the positions of the β-strands and Alinkages in starch and/or pullulan [1, 3Ϫ5] to produce maltotri-helices found in 47 amy...

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ⌬malP (encoding maltodextrin phosphorylase), ⌬malQ (encoding amylomaltase), ⌬glgA (encoding glycogen synthase), and ⌬glgA ⌬malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ⌬malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ⌬malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ⌬malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ⌬glgA and ⌬glgA ⌬malP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.The synthesis of glycogen in bacteria occurs when they are grown with limited nutrients but an abundance of a carbon source (33,34). Escherichia coli accumulates glycogen at levels of more than half of its cell mass under optimal conditions. The glycogen gene cluster in E. coli consists of two operons oriented in tandem, glgBX and glgCAP, encoding enzymes that synthesize and degrade glycogen (12). The encoded enzymes are a branching enzyme (glgB), a debranching enzyme (glgX), an ADP-glucose pyrophosphorylase (glgC), a glycogen synthase (glgA), and a glycogen phosphorylase (glgP). The polymerization of the ␣-1,4-linked glucosyl chain is mediated via the transfer of glucose from ADP-glucose by GlgA, the glycogen synthase, onto the nonreducing ends of linear dextrins that are subsequently branched (formation of ␣-1,6-glycosyl linkage) by GlgB, the branching enzyme. The expression of the glg gene cluster is complicated. It involves the global carbon storage regulator CsrA (2, 53), the cyclic AMP (cAMP)/catabolite gene activator protein (CAP) system (39), and the stringent response (38). In addition, the two-component regulatory system PhoP-PhoQ (29) connects the system to Mg 2ϩ levels, and even the phosphotransferase system appears to affect the glycogen phosphorylase involved in the degradation of glycogen (42,43). glgS, an additional gene involved in glycogen synthesis, is not part of the glg gene cluster. It is not essential for ...

A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium,Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60°C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed β-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar tomethyl-α-d-glucopyranoside by forming an α-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.