Investigation of the mechanisms of irreversible thermoinactivation of Bacillus stearothermophilus alpha-amylase.

Bacillus stearothermophilus NCIB 11412 produces a highly thermostable alpha-amylase. The enzyme displayed half-lives of irreversible thermoinactivation at 90 degrees C of 1.9 min and 12.5 min at pH 5.0 and pH 8.0, respectively. Molecular mechanisms of irreversible thermoinactivation were investigated. At both pH 5.0 and pH 8.0 irreversible thermoinactivation was due to heat-induced breakdown of non-covalent interaction within the protein molecule, resulting in unfolding and consequent formation of altered structures. Hydrophobic interactions were shown to be the most important non-covalent mechanisms involved in this phenomenon. Although not dramatically effecting the rates of irreversible thermoinactivation, electrostatic interactions, including hydrogen bonding, were also shown to have a contributory role in this process. At pH 8.0 a covalent mechanism, that of oxidation of thiols was also shown to be of secondary importance to hydrophobic interactions in the irreversible thermoinactivation of this enzyme.     

Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis alpha-amylase.

The irreversible thermal inactivation of Bacillus licheniformis alpha-amylase was studied. A two-step behaviour in the irreversible denaturation process was found. Our experimental results are consistent only with the two-step model and rule out the two-isoenzyme one. They suggest that the deactivation mechanism involves the existence of a temperature-dependent intermediate form. Therefore the enzyme could exist in a great number of active conformational states. We have shown that Ca2+ is necessary for the structural integrity of alpha-amylase. Indeed, dialysis against chelating agents leads to a reversible enzyme inactivation, though molecular sieving has no effect. Further, the key role of Ca2+ in the alpha-amylase thermostability is reported. The stabilizing effect of Ca2+ is reflected by the decrease of the denaturation constants of both the native and the intermediate forms. Below 75 degrees C, in the presence of 5 mM-CaCl2, alpha-amylase is completely thermostable. Neither other metal ions nor substrate have a positive effect on enzyme thermostability. The effect of temperature on the native enzyme and on one intermediate form was studied. Both forms exhibit the same optimum temperature. Identical activation parameters for the hydrolytic reaction catalysed by these two forms were found.     

Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133

A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.     

Site-directed mutagenesis of active site residues in Bacillus subtilis alpha-amylase.

Site-directed mutagenesis of Bacillus subtilis N7 alpha-amylase has been performed to evaluate the roles of the active site residues in catalysis and to prepare an inactive catalytic-site mutant that can form a stable complex with natural substrates. Mutation of Asp-176, Glu-208, and Asp-269 to their amide forms resulted in over a 15,000-fold reduction of its specific activity, but all the mutants retained considerable substrate-binding abilities as estimated by gel electrophoresis in the presence of soluble starch. Conversion of His-180 to Asn resulted in a 20-fold reduction of kcat with a 5-fold increase in Km for a maltopentaose derivative. The relative affinities for acarbose vs. maltopentaose were also compared between the mutants and wild-type enzyme. The results are consistent with the roles previously proposed in Taka-amylase A and porcine pancreatic alpha-amylase based on their X-ray crystallographic analyses, although different pairs had been assigned as catalytic residues for each enzyme. Analysis of the residual activity of the catalytic-site mutants by gel electrophoresis has suggested that it derived from the wild-type enzyme contaminating the mutant preparations, which could be removed by use of an acarbose affinity column; thus, these mutants are completely devoid of activity. The affinity-purified mutant proteins should be useful for elucidating the complete picture of the interaction of this enzyme with starch.     

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.     

Amino acid chemoreceptors of Bacillus subtilis.

Specificities of chemoreceptors for the 20 common amino acids, toward which Bacillus subtilis shows chemotaxis, were assessed by competition ("jamming") experiments using a modification of the traditional capillary assay, called the "sensitivity capillary assay." Many amino acids were sensed by at least two chemoreceptors. All the highest affinity chemoreceptors for the amino acids were distinct, except glutamate and aspartate, which may share one chemoreceptor, and tyrosine, for which the data could not be collected due to low solubility. The data suggest the hypothesis that each amino acid-chemoreceptor complex binds to a different signaler (from each amino acid-chemoreceptor complex binds to a different signaler (from which signals travel to the flagella to modify behavior appropriately), and that many of the signalers can also bind other attractant-chemoreceptor complexes as antagonists (no signals to flagella).     

Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus: putative role of three conserved residues.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.     

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.     

Amino acid sequence of protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25

The amino acid sequence of a protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25 (Haim II), which consists of 77 amino acid residues, including two disulfide bridges, was determined by conventional methods. One of the disulfide bridges was found to be located between Cys(6) and Cys(22), and the other between Cys(40) and Cys(67) from the results of structure analyses of the two cystine-containing peptides obtained from the thermolysin digest of the native inhibitor.     

Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus

Abstract We have cloned and expressed a novel maltogenic alpha-amylase from B. stearothermophilus on plasmid in B. subtilis . Originally the plasmid was very unstable in the absence of selection, but was stabilized due to a spontaneous, copy number reducing mutation. The promoter region and the extension of the gene have been analysed, and a provisional DNA sequence has been determined. The N-terminal of the mature amylase has been determined and shown to be in accordance with signal peptidase processing after a typical Gram-positive signal sequence of 33 amino acids.     

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.