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...
A gene (ssg) encoding a putative glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was cloned and expressed in Escherichia coli, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90°C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated -D-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from S. solfataricus produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from S. solfataricus has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.
Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both ␣-amylase and cyclodextrin-hydrolyzing enzyme. Like typical ␣-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and -cyclodextrin, which are resistant to ␣-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent ␣-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both ␣-amylase and cyclodextrin-hydrolyzing enzyme.The recent advent of genomic research has produced vast amounts of sequence information for many different taxa of Bacteria, Eukarya, and Archaea, now collected in databases such as GenBank; the full genome sequences of more than 140 different microorganisms have been completed. With a generally applicable combination of conventional genetic engineering and genomic research techniques, many extremely thermostable enzymes are being developed for biotechnological purposes. The genome sequences of some hyperthermophilic microorganisms, such as Thermotoga maritima, Pyrococcus furiosus, and Sulfolobus solfataricus, are of considerable biotechnological interest because they encode many highly heat-stable enzymes that are active under conditions previously regarded as incompatible with biological materials (20,25). Amylolytic enzymes are of great significance in many industrial processes, including those for foods, textiles, and detergents. Many hyperthermophilic microorganisms possess starch-hydrolyzing enzymes, such as ␣-amylase, ␣-glucosidase, pullulanase, and cyclodextrinase, in their genomes even though they live in environments where starch is rare (23).Analysis of the full genome of P. furiosus, a hyperthermophilic archaeon, has revealed that this microorganism has several amylolytic enzymes. An amylopullulanase and two distinct ␣-amylase genes of P. furiosus were identified and expressed in E. coli. These enzymes can hydrolyze a wide variety of substrates, such as soluble starch, amylose, amylopectin, glycogen, and oligosaccharides. However, ␣-amylase does not hydrolyze pullulan and cyclodextrin, whereas amylopullulanase can degrade pullulan (7,8).The multiple sequence alignment of amylolytic enzymes from P. furiosus revealed some interesting features of the gene (PF1939) homologous with those for various cyclodextrin-hydrolyzing enzymes such as cyclodextrinase, maltogenic amylase, and neopullulanase in glycoside hydrolase family 1...
The relation between the quaternary structure and the substrate specificity of Thermus maltogenic amylase (ThMA) has been investigated. Sedimentation diffusion equilibrium ultracentrifugation and gel filtration analyses, in combination with the crystal structure determined recently, have demonstrated that ThMA existed in a monomer/dimer equilibrium. The truncation of ThMA by removing the N-terminal domain, which is composed of 124 amino acid residues, resulted in the complete monomerization of the enzyme (ThMADelta124) accompanied by a drastic decrease in the activity for beta-cyclodextrin (beta-CD) and a relatively smaller reduction of the activity for starch. Despite the overall low activity of ThMADelta124, the activity was higher toward starch than beta-CD, and the ratio of the specific activities toward these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KCl to wild-type ThMA shifted the monomer/dimer equilibrium toward the monomer. In the presence of 1.0 M KCl, the relative activity of ThMA toward beta-CD decreased to 74%, while that for soluble starch increased to 194% compared to the activities in the absence of KCl. Thus, the ThMA monomer and dimer are both inferred to be enzymatically active but with a somewhat different substrate preference. Kinetic parameters of the wild-type and truncated enzymes also are in accordance with the changes in their specific activities. We thus provide evidence in support of a model, which shows that the relative multisubstrate specificity of ThMA is influenced by the monomer/dimer equilibrium of the enzyme.
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