Bacillus Stearothermophilus Strain Research Articles

Novel Phospholipase C with High Catalytic Activity from a Bacillus stearothermophilus Strain: An Ideal Choice for the Oil Degumming Process

A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLCBs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLCBs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn2+ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLCBs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn2+. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLCBs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLCBs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLCBs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLCBs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.

Sequence, genome organization, annotation and proteomics of the thermophilic, 47.7-kb Geobacillus stearothermophilus bacteriophage TP-84 and its classification in the new Tp84virus genus.

Bacteriophage TP-84 is a well-characterized bacteriophage of historical interest. It is a member of the Siphoviridae, and infects a number of thermophilic Geobacillus (Bacillus) stearothermophilus strains. Its’ 47.7-kbp double-stranded DNA genome revealed the presence of 81 coding sequences (CDSs) coding for polypeptides of 4 kDa or larger. Interestingly, all CDSs are oriented in the same direction, pointing to a dominant transcription direction of one DNA strand. Based on a homology search, a hypothetical function could be assigned to 31 CDSs. No RNA or DNA polymerase-coding genes were found on the bacteriophage genome indicating that TP-84 relies on the host’s transcriptional and replication enzymes. The TP84 genome is tightly packed with CDSs, typically spaced by several-to-tens of bp or often overlapping. The genome contains five putative promoter-like sequences showing similarity to the host promoter consensus sequence and allowing for a 2-bp mismatch. In addition, ten putative rho-independent terminators were detected. Because the genome sequence shows essentially no similarity to any previously characterised bacteriophage, TP-84 should be considered a new species in an undefined genus within the Siphoviridae family. Thus a taxonomic proposal of a new Tp84virus genus has been accepted by the International Committee on Taxonomy of Viruses. The bioinformatics genome analysis was verified by confirmation of 33 TP-84 proteins, which included: a) cloning of a selected CDS in Escherichia coli, coding for a DNA single-stranded binding protein (SSB; gene TP84_63), b) purification and functional assays of the recombinant TP-84 SSB, which has been shown to improve PCR reactions, c) mass spectrometric (MS) analysis of TP-84 bacteriophage capsid proteins, d) purification of TP-84 endolysin activity, e) MS analysis of the host cells from infection time course.

Kinetics of Deoxy-CTP Incorporation Opposite a dG-C8- N-2-Aminofluorene Adduct by a High-Fidelity DNA Polymerase

The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2′-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2′-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry.

Bacillus stearothermophilus YC4194에 의한 Pythium 모잘록병의 생물학적 방제

Bacillus stearothermophilus YC4l94는 in vitro 와 in vivo 에서 병원균 Pythium aphanidermatum에 대한 억제 효과를 보였다 in vitro 실험에서 P. aphanidermatum의 유주포자낭 형성과 피낭포자 발아를 억제하였을 뿐 아니라 식물체에 적접 길항균 제제 처리 시 50% 이상의 방제가를 보였다. 그리고 다른 화학 살균제와 비슷한 방제효과를 보여 미생물 제제로서의 개발 가능성을 보여주었다. In vitro and in vivo activities of a biocontrol agent, Bacillus stearothermophilus strain YC4194 was evaluated for the control of Pythium damping-off of cucumber. B. stearothermophilus YC4194 inhibited germination of cystospores and formation of zoosporangia of Pythium aphanidermatum in vitro. Incorporation of a bentonite and talc based formulation(10<TEX>$^{9}$</TEX> cfu/g) of B. stearothermophilus YC4194 to the nursery soils (10 g/ι soil) resulted In a significant (p=0.01) reduction in the disease severity of cucumber damping-off after inoculation with P. aphanidermatum. The control efficacy of B. stearothermophilus YC4194 formulation was not different from that of the fungicides, dimethomorph, metalaxyl, ethaboxam. When the cucumber plants were transplanted to the soil inoculated with P. aphanidermatum zoospores, the B. stearothermophilus YC4194 maintained the high population density in rhizosphere soil upto 10<TEX>$^{7}$</TEX> cfu/g until 15 days after treatment.

Bacillus stearothermophilus for thermophilic production of l-lactic acid

A process for the continuous production of high purity L-lactic acid in a membrane bioreactor at 65 degrees C has been developed. Two different Bacillus stearothermophilus strains have been tested in batch experiments. Lactic acid yields are between 60 and more than 95% of theoretical yields. The amounts of ethanol, acetate, and formate formed varied between 0 and 0.4, 0 and 0.1, and 0 and 0.5, respectively (mol/mol glucose). All byproducts are valuable and may be separated easily by rectification of the fermentation broth. Complete cell retention enables high volumetric productivity (5 g/Lh), and a minimum of growth supplements. The high temperature of 65 degrees C allows the autoselective fermentation without problems with contamination.

Characterization of a Thermostable Cyclodextrin Glucanotransferase Isolated from Bacillus stearothermophilus ET1

A thermostable cyclodextrin glucanotransferase (CGTase) was isolated from a Bacillus stearothermophilus strain, ET1, which was screened from Korean soil. The corresponding CGTase gene cloned in Escherichia coli shared 84% and 88% identity with CGTase genes from other B. stearothermophilus strains at the nucleotide and amino acid sequence level, respectively. The enzyme was purified to apparent homogeneity by β-cyclodextrin (CD) affinity chromatography and high-performance liquid chromatography. The enzyme had an apparent molecular mass of 66,800 Da and a pI of 5.0. The optimum pH for the enzyme-catalyzed reaction was pH 6.0, and the optimum temperature was observed at 80 °C. Thermostability of the enzyme was enhanced by Ca2+. A 13% (w/v) cornstarch solution was liquefied and converted to CDs solely using this enzyme. The cornstarch conversion rate was 44% and α-, β-, and γ-CDs were produced in the ratio of 4.2:5.9:1. Keywords: Cyclodextrin glucanotransferase; thermostability; cyclodextrin; Bacillus stearothermophilus

Biological indicators for low temperature steam and formaldehyde sterilization: effect of variations in recovery conditions on the response of spores ofBacillus stearothermophilusNCIMB 8224 to low temperature steam and formaldehyde

A.M. WRIGHT, E.V. HOXEY, C.J. SOPER AND D.J.G. DAVIES. 1997. Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml formaldehyde at temperatures between 63° and 83°C. The data indicate that LTSF-exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.

Biological indicators for low temperature steam and formaldehyde sterilization: effect of variations in recovery conditions on the response of spores of Bacillus stearothermophilus NCIMB 8224 to low temperature steam and formaldehyde

Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml-1 formaldehyde at temperatures between 63° and 83°C The data indicate that LTSF-exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.

Nucleotide and amino acid sequences for cytochrome caa3-type oxidase of Bacillus stearothermophilus K1041 and non-Michaelis-type kinetics with cytochrome c

A pseudo-sigmoidal cytochrome c-dependence curve of oxidase activity was observed with cytochrome oxidase from the Bacillus stearothermophilus strain K1041, while the other thermophilic Bacillus PS3 which has been extensively studied possessed normal Michaelis-Menten type kinetics. The genes coding for four subunits of cytochrome caa 3-type oxidase and for heme O synthase were isolated from a genomic DNA library of K1041 by using a PS3 DNA fragment containing the highly-conserved region of the largest subunit as a probe, and sequenced. Most residues in subunits I (COI/ caaB product), III (COIII/ caaC product), and IV (COIV/ caaD product) of K1041 were highly conserved when compared with those of PS3. However, the sequence of K1041 subunit II (COII/ caaA product) was distinctly different from that of the PS3 subunit II. These Bacillus COIIs have an additional sequence for cytochrome c after the Cu A binding protein portion with two transmembrane segments which is homologous to the mitochondrial counterpart, and represents the site of electron ingress. Several charged residues in the vicinity of cytochrome c moiety are replaced by oppositely charged residues. It is likely that these amino acid replacements in subunit II are the cause of the abnormal sigmoidal saturation curve for extrinsic cytochromes c of the K1041 enzyme.

Sequence of the cloned bseCIM gene: M.BseCI reveals high homology to M.BanIII.

The bseCIM gene, encoding M.BseCI methyltransferase (MTase) from a Bacillus stearothermophilus strain, has been previously cloned and expressed in Escherichia coli [Rina and Bouriotis, Gene 133 (1993) 91-94]. The nucleotide (nt) sequence of a 2357-bp BspMII-EcoRI fragment encoding bseCIM has now been determined. The sequence predicts a MTase of 579 amino acids (aa), 66.7 kDa. Comparison of the deduced aa sequence of M.BseCI with sequences of various MTases revealed a significant homology to m6A-MTases, especially to its isoschizomer M.BanIII from Bacillus aneurinolyticus.

Comparative studies of S-layer proteins from Bacillus stearothermophilus strains expressed during growth in continuous culture under oxygen-limited and non-oxygen-limited conditions

The specific properties of S-layer proteins from three different Bacillus stearothermophilus strains revealing oblique, square, or hexagonal lattice symmetry were preserved during growth in continuous culture on complex medium only under oxygen-limited conditions in which glucose was used as the sole carbon source. When oxygen limitation was relieved, amino acids became metabolized, cell density increased, and different S-layer proteins from wild-type strains became rapidly replaced by a new common type of S-layer protein with an apparent subunit molecular weight of 97,000 which assembled into an identical oblique (p2) lattice type. During switching from wild-type strains to variants, patches of the S-layer lattices characteristics for wild-type strains, granular regions, and areas with oblique lattice symmetry could be observed on the surface of individual cells from all organisms. The granular regions apparently consisted of mixtures of the S-layer proteins from the wild-type strains and the newly synthesized p2 S-layer proteins from the variants. S-layer proteins from wild-type strains possessed identical N-terminal regions but led to quite different cleavage products upon peptide mapping, indicating that they are encoded by different genes. Chemical analysis including N-terminal sequencing and peptide mapping showed that the oblique S-layer lattices synthesized under increased oxygen supply were composed of identical protein species.

Cultivation of an L‐lactate dehydrogenase mutant of Bacillus stearothermophilus in continuous culture with cell recycle

Continuous fermentation with cell recycle proved very effective in increasing the ethanol volumetric productivity of the thermophilic facultative anaerobe, Bacillus stearothermophilus strain LLD-15, on sucrose at 70 degrees C. When complete cell recycle was used, cell viability decreased after a few residence times and sucrose consumption was reduced. Operation using a constant bleed rate resulted in greater stability and higher ethanol volumetric productivities. A mathematical model based on maintenance energy requirements provided an adequate description of the system.

Cloning and expression of a pathway for benzene and toluene from Bacillus stearothermophilus.

Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil. The chromosomally-located aromatic pathway from this isolate was cloned into Escherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.

Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production

A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl2.

Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4

A thermophilic Bacillus stearothermophilus strain AP-4 excreting a thermostable alkaline protease, was isolated from a local compost. Maximum activity of protease (250 U/ml) was after 36 h growth in broth at pH 9.0 and at 55°C. The protease was optimally active at pH 9.0 and 55°C and was stable in 5 mM CaCl2. The enzyme was completely inactivated by PMSF, EDTA and β-mercaptoethanol. It is therefore a metal ion-dependent, alkaline, serine protease.

Mutants of Bacillus stearothermophilus defective in the uptake and metabolism of acetate

Summary: Mutants unable to utilize acetate as sole source of carbon and energy were isolated from ethyl-methanesulphonate-mutagenized cultures of a prototrophic Bacillus stearothermophilus strain. Three groups of mutants had identifiable defects. Thirteen of the mutants lacked one or both enzymes of the inducible anaplerotic glyoxylate shunt, and three mutants were auxotrophic for isoleucine and valine when grown on acetate, in a manner analogous to acetohydroxyacid synthase I mutants of enteric bacteria. One mutant was defective specifically in acetate uptake, providing direct evidence for an acetate transport system in B. stearothermophilus. The acetate uptake system was uncoupler-sensitive and had a relatively high K m (in the range 300–350 μm). Escherichia coli was also shown to possess a saturable acetate uptake system, but its K m was much lower (15 μm).

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.

The Metabolism of Aromatic Ring Fission Products by Bacillus stearothermophilus Strain IC3

Bacillus stearothermophilus IC3 degraded the meta cleavage product of catechol, 2-hydroxymuconic semialdehyde, to pyruvate and acetaldehyde via the 4-oxalocrotonate pathway. The pathway was identical to those previously delineated in several mesophilic organisms. However, all the enzymes showed activity at 55 degrees C and other properties (substrate specificities and effects of metal ions) also differed from those displayed by the mesophilic enzymes. All enzymes of this meta cleavage pathway, except the 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase and 4-hydroxy-2-oxovalerate aldolase activities, were induced by growth on phenol.

Production of thermostable, recombinant α-galactosidase suitable for raffinose elimination from sugar beet syrup

Bacillus stearothermophilus strain KVE39, isolated from Icelandic hot springs, was shown to produce two isoenzymes of α-galactosidase and β-galactosidase. The gene for the major α-galactosidase ( agaA) was cloned and expressed in E. coli and the gene product was demonstrated to possess excellent properties for application in D-raffinose removal from low green sugar beet syrup: (a) its activity is not inhibited by D-galactose or sucrose; (b) it is highly stable under process conditions; (c) its pH optimum of activity is close to neutrality. The agaA gene is constitutively expressed in E. coli at a high level: heating of crude extracts from the agaA plasmid-carrying strain and removal of precipitated host proteins by centrifugation results in simple and efficient purification.

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains.

Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique. During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein.