Denitrification enzymes of Bacillus stearothermophilus

Abstract A hybrid trpPO:lacO regulatory sequence was cloned upstream of a promoterless lacZ gene and recombined onto a Λ bacteriophage. Escherichia coli lysogens representing the four possible phenotypes for lacI and trpR were constructed and the synthesis of β -galactosidase was assayed under various growth conditions. The results illustrated that both control elements could be efficiently and independently regulated by the addition or omission of appropriate accessory molecules.     

Effect of calcium and anaerobiosis on the thermostability of Bacillus stearothermophilus.

Bacillus stearothermophilus NCA 2184 lost viability and subsequently released cytoplasmic components when suspended in 0.1 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.2) and incubated at 60 degrees C. Cell lysis was prevented by the addition of 10 mM CaCl2 to the Tris-buffer suspension. Cells which were incubated under anaerobic conditions for 20 min in the growth medium before they were collected were stable in the Tris-buffer suspension without added calcium. Anaerobic incubation effected an increase in membrane cardiolipin which appeared to be related to the increase in the thermostability of the cells.     

Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.     

Crystal structure of Bacillus stearothermophilus alpha-amylase: possible factors determining the thermostability

The crystal structure of a thermostable alpha-amylase from Bacillus stearothermophilus (BSTA) has been determined at 2.0 A resolution. The main-chain fold is almost identical to that of the known crystal structure of Bacillus licheniformis alpha-amylase (BLA). BLA is known to be more stable than BSTA. A structural comparison between the crystal structures of BSTA and BLA showed significant differences that may account for the difference in their thermostabilities, as follows. (i) The two-residue insertion in BSTA, Ile181-Gly182, pushes away the spatially contacting region including Asp207, which corresponds to Ca(2+)-coordinating Asp204 in BLA. As a result, Asp207 cannot coordinate the Ca(2+). (ii) BSTA contains nine fewer hydrogen bonds than BLA, which costs about 12 kcal/mol. This tendency is prominent in the (beta/alpha)(8)-barrel, where 10 fewer hydrogen bonds were observed in BSTA. BLA forms a denser hydrogen bond network in the inter-helical region, which may stabilize alpha-helices in the barrel. (iii) A few small voids observed in the alpha-helical region of the (beta/alpha)(8)-barrel in BSTA decrease inter-helical compactness and hydrophobic interactions. (iv) The solvent-accessible surface area of charged residues in BLA is about two times larger than that in BSTA.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin

Summary An extracellular neutral protease, of Bacillus stearothermophilus KP 1236 (a soil isolate) able to grow at 39°–71°C was purified to homogeneity. The molecular weight, sedimentation coefficient in water at 20°C, and isoelectric point were estimated as 33,000, 3.46 S and 7.5, respectively. The enzyme was most active at 80°C and pH 7.0. The activity was stable for 10 min up to 80°C at pH 7.5 and for 18 h at 60°C over pH 6.0–8.8. The enzyme and thermolysin (microbial metalloproteinase, EC 3.4.24.4) shared their antigenic determinants in part.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan on Sapporo, July 30, 1985 (Abstracts, p. 333)     

Cloning and Expression of Thermostable alpha-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

Role of cysteine residues in esterase from Bacillus stearothermophilus and increasing its thermostability by the replacement of cysteines

Bacillus stearothermophilus esterase contains two free cysteine residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70°C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes. Correspondence to: T. Yamane     

Thermostable peroxidase from Bacillus stearothermophilus

A peroxidase from Bacillus stearothermophilus was purified to homogeneity. The enzyme (Mr 175,000) was composed of two subunits of equal size, and showed a Soret band at 406 nm. On reduction with sodium dithionite, absorption at 434 nm and 558 nm was observed. The spectrum of reduced pyridine haemochrome showed peaks at 418, 526 and 557 nm; the reduced minus oxidized spectrum of pyridine haemochrome showed peaks of 418, 524 and 556 nm with a trough at 452 nm. These results indicate that the enzyme contained protohaem IX as a prosthetic group. The optimum pH was about 6 and the apparent optimum temperature was 70 degrees C. The enzyme was relatively stable up to 70 degrees C; at 30 degrees C it was stable for a month. The enzyme had peroxidase activity toward a mixture of 2,4-dichlorophenol and 4-aminoantipyrine with a Km for H2O2 of 1.3 mM. It also acted as a catalase with a Km for H2O2 of 7.5 mM.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease.

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.     

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.     

Isolation of a Bacillus stearothermophilus mutant exhibiting increased thermostability in its restriction endonuclease.

A procedure was developed for the selection of spontaneous mutants of Bacillus stearothermophilus NUB31 that are more efficient than the wild type in the restriction of phage at elevated temperatures. Inactivation studies revealed that two mutants contained a more thermostable restriction enzyme and one mutant contained three times more enzyme than the wild type. The restriction endonucleases from the wild type and one of the mutants were purified to apparent homogeneity. The mutant enzyme was more thermostable than the wild-type enzyme. The subunit molecular weight, amino acid composition, N-terminal and C-terminal amino acid residues, tryptic peptide map, and catalytic properties of the two enzymes were determined. The two enzymes have similar catalytic properties, but the molecular size of the mutant enzyme is approximately 6 to 7 kilodaltons larger than that of the wild-type enzyme. The mutant enzyme contains 54 additional amino acid residues, of which 26 to 28 are aspartate/asparagine, 8 to 15 are glutamate/glutamine, and 8 to 9 are tyrosine residues. The two enzymes contained similar amounts of the other amino acids, identical N-terminal residues, and different C-terminal residues. Tryptic peptide analyses revealed a high degree of homology between the two enzymes. The increased thermostability observed in the mutant enzyme appears to have been achieved by a mutation that resulted in the addition of amino acid residues to the wild-type enzyme. A number of mechanisms are discussed that could account for the observed difference between the mutant and wild-type enzymes.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis

Genome features of the Bacillus cereus group genomes (representative strains of Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis sub spp. israelensis) were analyzed and compared with the Bacillus subtilis genome. A core set of 1381 protein families among the four Bacillus genomes, with an additional set of 933 families common to the B. cereus group, was identified. Differences in signal transduction pathways, membrane transporters, cell surface structures, cell wall, and S-layer proteins suggesting differences in their phenotype were identified. The B. cereus group has signal transduction systems including a tyrosine kinase related to two-component system histidine kinases from B. subtilis. A model for regulation of the stress responsive sigma factor sigmaB in the B. cereus group different from the well studied regulation in B. subtilis has been proposed. Despite a high degree of chromosomal synteny among these genomes, significant differences in cell wall and spore coat proteins that contribute to the survival and adaptation in specific hosts has been identified.     

Extracellular esterase activity from Bacillus stearothermophilus

Bacillus stearothermophilus has been reported to produce an extracellular esterase with molecular weight of 42–47 kDa. Extracellular esterase activity in Bacillus stearothermophilus (NCIB 13335) was found to reside in protein with a molecular weight less than 10 kDa. This small esterase was responsible for all the esterase activity observed in this strain under the conditions studied.