Suitability of thermal plasmas for large-area bacteria inactivation on temperature-sensitive surfaces – first results with Geobacillus stearothermophilus spores

International Nuclear Information System (INIS)

Szulc, M; Schein, S; Schaup, J; Zimmermann, S; Schein, J

2017-01-01

The application of thermal plasma for large-area bacteria inactivation on temperature-sensitive surfaces is not a common one. Nonetheless, there are thermal plasma generators which offer a high sheath homogeneity and have proven to be suitable for treatment of thermally sensitive materials in the past. To investigate the suitability of such plasmas, agar dishes plated with endospores of Geobacillus stearothermophilus have been treated with a long arc plasma generator called LARGE. The achieved results have been compared with a commercially available non-thermal plasma generator. A significant inactivation of the endospores could be observed only after 60 s of treatment with the thermal plasma source. This was not possible with the non-thermal generator. Moreover, no temperature damage or increase of the specimen could be detected. An attempt to determine the main agents responsible for the microbicidal effects have been made – the influence of plasma gas composition, discharge current and treatment time has been investigated. Significant improvements in the disinfection rates after adding small amounts of nitrogen to the plasma gas could be observed. A first discussion regarding the suitability of thermal plasmas for bacteria inactivation has been given. (paper)

Highly Stable l-Lysine 6-Dehydrogenase from the Thermophile Geobacillus stearothermophilus Isolated from a Japanese Hot Spring: Characterization, Gene Cloning and Sequencing, and Expression

Science.gov (United States)

Heydari, Mojgan; Ohshima, Toshihisa; Nunoura-Kominato, Naoki; Sakuraba, Haruhiko

2004-01-01

l-Lysine dehydrogenase, which catalyzes the oxidative deamination of l-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Δ1-piperideine-6-carboxylate, indicating that the enzyme is l-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70°C, respectively. No activity was lost at temperatures up to 65°C in the presence of 5 mM l-lysine. The enzyme was relatively selective for l-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for l-lysine, NAD, and NADP at 50°C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da. PMID:14766574

Sensor kinase KinB and its pathway-associated key factors sense the signal of nutrition starvation in sporulation of Bacillus subtilis.

Science.gov (United States)

Liu, Weipeng; He, Zeying; Gao, Feng; Yan, Jinyuan; Huang, Xiaowei

2018-01-03

Bacillus subtilis responds to environmental stress cues and develops endospores for survival. In the process of endospore formation, sporulation initiation is a vital stage and this stage is governed by autophosphorylation of the sensor histidine kinases. The second major sensor kinase KinB perceives the intracellular changes of GTP and ATP during sporulation. However, determination of the environmental signals as well as its related signaling pathway of KinB requires further elucidation. Our current study found that, contrary to the sporulation failure induced by ΔkinA in the nutrient-rich 2× SG medium, the sensor kinase KinB sensed the environmental cues in the nutrient-poor MM medium. Two other membrane proteins, KapB and KbaA, also responded similarly to the same external signal as KinB. Both KapB and KbaA acted upstream of KinB, but they exerted their regulation upon KinB independently. Furthermore, we demonstrated that both the SH3 domain and the α-helix structure in KapB are required for sensing or transducing the signal of sporulation initiation. Collectively, our work here supplied the direct evidences that KinB and its pathway sense the external signal of nutrient starvation in MM medium, and further analyzes the interrelationship among KinB, KbaA, and KapB. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

Science.gov (United States)

Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Science.gov (United States)

Dror, Adi; Shemesh, Einav; Dayan, Natali

2014-01-01

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

Structure-specificity relationships in Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus T6.

Science.gov (United States)

Lansky, Shifra; Salama, Rachel; Solomon, Hodaya V; Feinberg, Hadar; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

2014-11-01

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer

Biosynthesis of omega-alicyclic fatty acids induced by cyclic precursors and change of membrane fluidity in thermophilic bacteria Geobacillus stearothermophilus and Meiothermus ruber

Czech Academy of Sciences Publication Activity Database

Siřišťová, L.; Luhový, R.; Sigler, Karel; Řezanka, Tomáš

2011-01-01

Roč. 15, č. 3 (2011), 423-429 ISSN 1431-0651 Institutional research plan: CEZ:AV0Z50200510 Keywords : Thermophilic bacteria * Geobacillus * Meiothermus Subject RIV: EE - Microbiology, Virology Impact factor: 2.941, year: 2011

A meta-analysis accounting for sources of variability to estimate heat resistance reference parameters of bacteria using hierarchical Bayesian modeling: Estimation of D at 121.1 °C and pH 7, zT and zpH of Geobacillus stearothermophilus.

Science.gov (United States)

Rigaux, Clémence; Denis, Jean-Baptiste; Albert, Isabelle; Carlin, Frédéric

2013-02-01

Predicting microbial survival requires reference parameters for each micro-organism of concern. When data are abundant and publicly available, a meta-analysis is a useful approach for assessment of these parameters, which can be performed with hierarchical Bayesian modeling. Geobacillus stearothermophilus is a major agent of microbial spoilage of canned foods and is therefore a persistent problem in the food industry. The thermal inactivation parameters of G. stearothermophilus (D(ref), i.e.the decimal reduction time D at the reference temperature 121.1°C and pH 7.0, z(T) and z(pH)) were estimated from a large set of 430 D values mainly collected from scientific literature. Between-study variability hypotheses on the inactivation parameters D(ref), z(T) and z(pH) were explored, using three different hierarchical Bayesian models. Parameter estimations were made using Bayesian inference and the models were compared with a graphical and a Bayesian criterion. Results show the necessity to account for random effects associated with between-study variability. Assuming variability on D(ref), z(T) and z(pH), the resulting distributions for D(ref), z(T) and z(pH) led to a mean of 3.3 min for D(ref) (95% Credible Interval CI=[0.8; 9.6]), to a mean of 9.1°C for z(T) (CI=[5.4; 13.1]) and to a mean of 4.3 pH units for z(pH) (CI=[2.9; 6.3]), in the range pH 3 to pH 7.5. Results are also given separating variability and uncertainty in these distributions, as well as adjusted parametric distributions to facilitate further use of these results in aqueous canned foods such as canned vegetables. Copyright © 2012 Elsevier B.V. All rights reserved.

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

Energy Technology Data Exchange (ETDEWEB)

Jacques, David A.; Streamer, Margaret [School of Molecular and Microbial Biosciences, University of Sydney (Australia); Rowland, Susan L.; King, Glenn F. [Institute of Molecular Biology, University of Queensland (Australia); Guss, J. Mitchell; Trewhella, Jill; Langley, David B., E-mail: d.langley@usyd.edu.au [School of Molecular and Microbial Biosciences, University of Sydney (Australia)

2009-06-01

The crystal structure of Sda, a DNA-replication/damage checkpoint inhibitor of sporulation in B. subtilis, has been solved via the MAD method. The subunit arrangement in the crystal has enabled a reappraisal of previous biophysical data, resulting in a new model for the behaviour of the protein in solution. The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.

Isolation of four hydrocarbon effluent-degrading Bacillaceae species ...

African Journals Online (AJOL)

percentage decreases in total hydrocarbon concentration within 18 days: 98% with Bacillus licheniformis STK08, 87% with Geobacillus stearothermophilus STM04, 80% with Lysinibacillus sphaericus STZ75 and 72% with Bacillus firmus STS84.

Genetic toolbox for controlled expression of functional proteins in Geobacillus spp

DEFF Research Database (Denmark)

Pogrebnyakov, Ivan; Jendresen, Christian Bille; Nielsen, Alex Toftgaard

2017-01-01

Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One...

The Geobacillus pan-genome: implications for the evolution of the genus

Directory of Open Access Journals (Sweden)

Oliver Keoagile Ignatius Bezuidt

2016-05-01

Full Text Available The genus Geobacillus is comprised of a diverse group of spore-forming Gram-positive thermophilic bacterial species and is well known for both its ecological diversity and as a source of novel thermostable enzymes. Although the mechanisms underlying the thermophilicity of the organism and the thermostability of its macromolecules are reasonably well understood, relatively little is known of the evolutionary mechanisms, which underlie the structural and functional properties of members of this genus. In this study, we have compared 29 Geobacillus genomes, with a specific focus on the elements, which comprise the conserved core and flexible genomes. Based on comparisons of conserved core and flexible genomes, we present evidence of habitat delineation with specific Geobacillus genomes linked to specific niches. Interestingly, our analysis has shown that horizontal gene transfer is a major factor deriving the evolution of Geobacillus from Bacillus, with genetic contributions from other phylogenetically distant taxa.

J-GLOBAL MeSH Dictionary: Bacillus stearothermophilus [MeCab user dictionary for science technology term[Archive

Lifescience Database Archive (English)

Full Text Available MeCab user dictionary for science technology term Bacillus stearothermophilus 名詞 一般 * * * * Bacillus stea...rothermophilus ... MeSH D001411 200906079736943583 C LS07 UNKNOWN_2 Bacillus stearothermophilus

Phylogenomic re-assessment of the thermophilic genus Geobacillus.

Science.gov (United States)

Aliyu, Habibu; Lebre, Pedro; Blom, Jochen; Cowan, Don; De Maayer, Pieter

2016-12-01

Geobacillus is a genus of Gram-positive, aerobic, spore-forming obligate thermophiles. The descriptions and subsequent affiliations of the species in the genus have mostly been based on polyphasic taxonomy rules that include traditional sequence-based methods such as DNA-DNA hybridization and comparison of 16S rRNA gene sequences. Currently, there are fifteen validly described species within the genus. The availability of whole genome sequences has provided an opportunity to validate and/or re-assess these conventional estimates of genome relatedness. We have applied whole genome approaches to estimate the phylogenetic relatedness among the sixty-three Geobacillus strains for which genome sequences are currently publicly available, including the type strains of eleven validly described species. The phylogenomic metrics AAI (Average Amino acid Identity), ANI (Average Nucleotide Identity) and dDDH (digital DNA-DNA hybridization) indicated that the current genus Geobacillus is comprised of sixteen distinct genomospecies, including several potentially novel species. Furthermore, a phylogeny constructed on the basis of the core genes identified from the whole genome analyses indicated that the genus clusters into two monophyletic clades that clearly differ in terms of nucleotide base composition. The G+C content ranges for clade I and II were 48.8-53.1% and 42.1-44.4%, respectively. We therefore suggest that the Geobacillus species currently residing within clade II be considered as a new genus. Copyright © 2016 Elsevier GmbH. All rights reserved.

CHARACTERIZATION OF A NEW BACILLUS-STEAROTHERMOPHILUS ISOLATE - A HIGHLY THERMOSTABLE ALPHA-AMYLASE-PRODUCING STRAIN

NARCIS (Netherlands)

WIND, RD; BUITELAAR, RM; EGGINK, G; HUIZING, HJ; DIJKHUIZEN, L

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known alpha-amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable alpha-amylase. The half-time of inactivation of this

Genetic toolbox for controlled expression of functional proteins in Geobacillus spp.

Directory of Open Access Journals (Sweden)

Ivan Pogrebnyakov

Full Text Available Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins.

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia.

Science.gov (United States)

Abd Rahman, Raja Noor Zaliha Raja; Leow, Thean Chor; Salleh, Abu Bakar; Basri, Mahiran

2007-08-10

Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78 degrees C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5-99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification. Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70 degrees C and was also stable up to 60 degrees C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T). Strain T1T was able to secrete extracellular thermostable lipase into culture

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

Directory of Open Access Journals (Sweden)

Salleh Abu

2007-08-01

Full Text Available Abstract Background Thermophilic Bacillus strains of phylogenetic Bacillus rRNA group 5 were described as a new genus Geobacillus. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus Geobacillus have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0 as the major fatty acids. The members of Geobacillus have similarity in their 16S rRNA gene sequences (96.5–99.2%. Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification. Results Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70°C and was also stable up to 60°C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T and Geobacillus kaustophilus (DSM 7263T. Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T. Conclusion Strain T1T was able to secrete extracellular

Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.

Science.gov (United States)

Miñana-Galbis, David; Pinzón, Dora L; Lorén, J Gaspar; Manresa, Angels; Oliart-Ros, Rosa M

2010-07-01

Although Anoxybacillus and Geobacillus, two genera of thermophilic bacteria close to the genus Bacillus, have only been described recently, the number of species in these genera has increased rapidly. Four thermophilic, lipolytic strains (DR01, DR02, DR03 and DR04) isolated from a hot spring in Veracruz (Mexico), which could not be identified phenotypically, were subjected to 16S rRNA gene sequence analysis. Three strains were identified as belonging to the genus Anoxybacillus, but strain DR03 was identified as Geobacillus pallidus. This result led us to perform a phylogenetic analysis of the genera Anoxybacillus and Geobacillus based on 16S rRNA gene sequences from all the type strains of these genera. Phylogenetic trees showed three major clusters, Anoxybacillus-Geobacillus tepidamans, Geobacillus sensu stricto and Geobacillus pallidus, while the 16S rRNA gene sequences of G. pallidus (DR03 and the type strain) showed low similarity to sequences of Anoxybacillus (92.5-95.1 %) and Geobacillus (92.8-94.5 %) species, as well as to Bacillus subtilis (92.2-92.4 %). In addition, G. pallidus could be differentiated from Anoxybacillus and Geobacillus on the basis of DNA G+C content and fatty acid and polar lipid profiles. From these results, it is proposed that Geobacillus pallidus should be classified in a novel genus, for which we propose the name Aeribacillus, as Aeribacillus pallidus gen. nov., comb. nov. The type strain of Aeribacillus pallidus is H12(T) (=ATCC 51176(T) =DSM 3670(T) =LMG 19006(T)).

Complete genome sequence of Geobacillus thermoglucosidasius C56-YS93, a novel biomass degrader isolated from obsidian hot spring in Yellowstone National Park.

Science.gov (United States)

Brumm, Phillip J; Land, Miriam L; Mead, David A

2015-01-01

Geobacillus thermoglucosidasius C56-YS93 was one of several thermophilic organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. Comparison of 16 S rRNA sequences confirmed the classification of the strain as a G. thermoglucosidasius species. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). The genome of G. thermoglucosidasius C56-YS93 consists of one circular chromosome of 3,893,306 bp and two circular plasmids of 80,849 and 19,638 bp and an average G + C content of 43.93 %. G. thermoglucosidasius C56-YS93 possesses a xylan degradation cluster not found in the other G. thermoglucosidasius sequenced strains. This cluster appears to be related to the xylan degradation cluster found in G. stearothermophilus. G. thermoglucosidasius C56-YS93 possesses two plasmids not found in the other two strains. One plasmid contains a novel gene cluster coding for proteins involved in proline degradation and metabolism, the other contains a collection of mostly hypothetical proteins.

JST Thesaurus Headwords and Synonyms: Bacillus stearothermophilus [MeCab user dictionary for science technology term[Archive

Lifescience Database Archive (English)

Full Text Available MeCab user dictionary for science technology term Bacillus stearothermophilus 名詞 一般...ムオーピーエイチアイエルユーエス Thesaurus2015 200906079736943583 C LS07 UNKNOWN_2 Bacillus stearothermophilus

Challenges to validation of a complex nonsterile medical device tray.

Science.gov (United States)

Prince, Daniel; Mastej, Jozef; Hoverman, Isabel; Chatterjee, Raja; Easton, Diana; Behzad, Daniela

2014-01-01

Validation by steam sterilization of reusable medical devices requires careful attention to many parameters that directly influence whether or not complete sterilization occurs. Complex implant/instrument tray systems have a variety of configurations and components. Geobacillus stearothermophilus biological indicators (BIs) are used in overkill cycles to to simulate worst case conditions and are intended to provide substantial sterilization assurance. Survival of G. stearothermophilus spores was linked to steam access and size of load in the chamber. By a small and reproducible margin, it was determined that placement of the trays in a rigid container into minimally loaded chambers were more difficult to completely sterilize than maximally loaded chambers.

STABILIZATION OF BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE BY INTRODUCTION OF PROLINES

NARCIS (Netherlands)

HARDY, F; VRIEND, G; VELTMAN, OR; VANDERVINNE, B; VENEMA, G; EIJSINK, VGH

1993-01-01

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by

Complete genome sequences of Geobacillus sp. Y412MC52, a xylan-degrading strain isolated from obsidian hot spring in Yellowstone National Park.

Science.gov (United States)

Brumm, Phillip; Land, Miriam L; Hauser, Loren J; Jeffries, Cynthia D; Chang, Yun-Juan; Mead, David A

2015-01-01

Geobacillus sp. Y412MC52 was isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute and deposited at the NCBI in December 2011 (CP002835). Based on 16S rRNA genes and average nucleotide identity, Geobacillus sp. Y412MC52 and the related Geobacillus sp. Y412MC61 appear to be members of a new species of Geobacillus. The genome of Geobacillus sp. Y412MC52 consists of one circular chromosome of 3,628,883 bp, an average G + C content of 52 % and one circular plasmid of 45,057 bp and an average G + C content of 45 %. Y412MC52 possesses arabinan, arabinoglucuronoxylan, and aromatic acid degradation clusters for degradation of hemicellulose from biomass. Transport and utilization clusters are also present for other carbohydrates including starch, cellobiose, and α- and β-galactooligosaccharides.

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

Science.gov (United States)

Assareh, Reza; Shahbani Zahiri, Hossein; Akbari Noghabi, Kambiz; Aminzadeh, Saeed; Bakhshi Khaniki, Gholamreza

2012-09-01

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structure based protein engineering of Bacillus stearothermophilus {alpha}-amylase: toward a new substrate specificity

Energy Technology Data Exchange (ETDEWEB)

Rasera, Ana Claudia [Sao Paulo Univ., SP (Brazil). Inst. de Ciencias Biomedicas; Iulek, Jorge [Universidade Estadual de Ponta Grossa, PR (Brazil). Inst. de Quimica; Delboni, Luis Fernando; Barbosa, Valma Martins Barbosa [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica

1997-12-31

Full text. Structural similarity is observed in all members of {alpha}-amylase family but different products are generated during hydrolysis of starch due to different affinities for intermediate dextrins. In order to understand the structural determinants for this property and to introduce different specificity to {alpha}-amylase of Bacillus stearothermophilus we intend to solve the three dimensional structure by X-ray crystallography of the native protein by using synchrotron radiation at Brazilian National Synchrotron Light Laboratory (LNLS). Protein was over expressed in E. coli, purified and crystallization experiments were carried out by using sparse matrix Crystal Screen and Crystal Screen II from Hampton Research (Laguna Hills, CA, USA). Several condition have produced crystals with some defined characteristic: MDP seems to be important to the crystallization: the preferential pH is around 7.5 with organic buffer (HEPES); organic solvent as 2-propanol seems to be also important for the crystallization. On those condition crystals appeared as cluster of needles or small crystals with high number of nucleation. New conditions are being prepared to improve the site and quality of crystals. Data collection of native of Bacillus stearothermophilus {alpha}-amylase will e done at Protein Crystallography Station at LNLS. Crystal structure of mutated {alpha}-amylase bu site direct mutagenesis of residues suggested by the native crystal structure will be obtained. Co-crystallization of Bacillus stearothermophilus {alpha}-amylase and oligosaccharide will be carried out to identify residues involved in the binding site to plan new mutation. In another series of mutation the putative binding site residues, once identified, will be mutated to residues observed in TAKA amylase to confer different specificity to {alpha}-amylase. Based on the available TAKA amylase structure, in the primary sequence homology and in the three dimensional model of Bacillus stearothermophilus

Structure based protein engineering of Bacillus stearothermophilus α-amylase: toward a new substrate specificity

International Nuclear Information System (INIS)

Rasera, Ana Claudia; Iulek, Jorge; Delboni, Luis Fernando; Barbosa, Valma Martins Barbosa

1997-01-01

Full text. Structural similarity is observed in all members of α-amylase family but different products are generated during hydrolysis of starch due to different affinities for intermediate dextrins. In order to understand the structural determinants for this property and to introduce different specificity to α-amylase of Bacillus stearothermophilus we intend to solve the three dimensional structure by X-ray crystallography of the native protein by using synchrotron radiation at Brazilian National Synchrotron Light Laboratory (LNLS). Protein was over expressed in E. coli, purified and crystallization experiments were carried out by using sparse matrix Crystal Screen and Crystal Screen II from Hampton Research (Laguna Hills, CA, USA). Several condition have produced crystals with some defined characteristic: MDP seems to be important to the crystallization: the preferential pH is around 7.5 with organic buffer (HEPES); organic solvent as 2-propanol seems to be also important for the crystallization. On those condition crystals appeared as cluster of needles or small crystals with high number of nucleation. New conditions are being prepared to improve the site and quality of crystals. Data collection of native of Bacillus stearothermophilus α-amylase will e done at Protein Crystallography Station at LNLS. Crystal structure of mutated α-amylase bu site direct mutagenesis of residues suggested by the native crystal structure will be obtained. Co-crystallization of Bacillus stearothermophilus α-amylase and oligosaccharide will be carried out to identify residues involved in the binding site to plan new mutation. In another series of mutation the putative binding site residues, once identified, will be mutated to residues observed in TAKA amylase to confer different specificity to α-amylase. Based on the available TAKA amylase structure, in the primary sequence homology and in the three dimensional model of Bacillus stearothermophilus α-amylase (using Bacillus

21 CFR 184.1012 - α-Amylase enzyme preparation from Bacillus stearothermophilus.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true α-Amylase enzyme preparation from Bacillus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1012 α-Amylase enzyme preparation from Bacillus stearothermophilus. (a) α-Amylase enzyme preparation is obtained from the culture...

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase from Geobacillus kaustophilus

International Nuclear Information System (INIS)

Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Kuroishi, Chizu; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

2007-01-01

DHNA synthetase from G. kaustophilus has been cloned, expressed, purified and crystallized. The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K 2 ) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222 1 , with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 Å. The crystal diffracted to a resolution of 2.2 Å. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

A thermostable Cas9 with increased lifetime in human plasma

OpenAIRE

Harrington, LB; Paez-Espino, D; Staahl, BT; Chen, JS; Ma, E; Kyrpides, NC; Doudna, JA

2017-01-01

© 2017 The Author(s). CRISPR-Cas9 is a powerful technology that has enabled genome editing in a wide range of species. However, the currently developed Cas9 homologs all originate from mesophilic bacteria, making them susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. Here, we show that the Cas9 protein from the thermophilic bacterium Geobacillus stearothermophilus (GeoCas9) catalyzes RNA-guided DNA cleavage at elevated temperatures. GeoCas...

Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus

International Nuclear Information System (INIS)

Suzuki, Kenichiro; Ito, Sohei; Shimizu-Ibuka, Akiko; Sakai, Hiroshi

2005-01-01

This report describes the crystallization and X-ray diffraction data collection of three types (wild-type, W416F/V435W and C9S/C268S) of B. stearothermophilus. Crystals of C9S/C268S belonged to space group P6 2 22 and diffracted to a resolution of 2.4 Å. Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6 2 22, with unit-cell parameters a = b = 145.97, c = 118.03 Å

Sterilization of liquid foods by pulsed electric fields?an innovative ultra-high temperature process

OpenAIRE

Reineke, Kai; Schottroff, Felix; Meneses, Nicolas; Knorr, Dietrich

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm−1), skim milk (0.3% fat; 5.3 mS cm−1) and fresh prepared carrot juice (7.73 mS cm−1). The combination of moderate preheating (70–90°C) ...

Sterilization of liquid foods by pulsed electric fields – an innovative ultra-high temperature process

OpenAIRE

Kai eReineke; Kai eReineke; Felix eSchottroff; Nicolas eMeneses; Nicolas eMeneses; Dietrich eKnorr

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm-1), skim milk (0.3% fat; 5.3 mS cm-1) and fresh prepared carrot juice (7.73 mS cm-1). The combination of moderate preheating (70-90 °C)...

Tailoring nutritional and process variables for hyperproduction of catalase from a novel isolated bacterium Geobacillus sp. BSS-7.

Science.gov (United States)

Kauldhar, Baljinder Singh; Sooch, Balwinder Singh

2016-01-14

Catalase (EC 1.11.1.6) is one of the important industrial enzyme employed in diagnostic and analytical methods in the form of biomarkers and biosensors in addition to their enormous applications in textile, paper, food and pharmaceutical sectors. The present study demonstrates the utility of a newly isolated and adapted strain of genus Geobacillus possessing unique combination of several industrially important extremophilic properties for the hyper production of catalase. The bacterium can grow over a wide range of pH (3-12) and temperature (10-90 °C) with extraordinary capability to produce catalase. A novel extremophilic strain belonging to genus Geobacillus was exploited for the production of catalase by tailoring its nutritional requirements and process variables. One variable at a time traditional approach followed by computational designing was applied to customize the fermentation process. A simple fermentation media containing only three components namely sucrose (0.55 %, w/v), yeast extract (1.0 %, w/v) and BaCl2 (0.08 %, w/v) was designed for the hyperproduction of catalase. A controlled and optimum air supply caused a tremendous increase in the enzyme production on moving the bioprocess from the flask to bioreactor level. The present paper reports high quantum of catalase production (105,000 IU/mg of cells) in a short fermentation time of 12 h. To the best of our knowledge, there is no report in the literature that matches the performance of the developed protocol for the catalase production. This is the first serious study covering intracellular catalase production from thermophilic genus Geobacillus. An increase in intracellular catalase production by 214.72 % was achieved in the optimized medium when transferred from the shake flask to the fermenter level. The extraordinary high production of catalase from Geobacillus sp. BSS-7 makes the isolated strain a prospective candidate for bulk catalase production on an industrial scale.

STABILIZATION OF THE NEUTRAL PROTEASE OF BACILLUS-STEAROTHERMOPHILUS BY REMOVAL OF A BURIED WATER MOLECULE

NARCIS (Netherlands)

VRIEND, G; BERENDSEN, HJC; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G; EIJSINK, VGH

1991-01-01

Using site-directed mutagenesis, Ala166 in the neutral protease of Bacillus stearothermophilus was changed into Ser. Model building and molecular dynamics simulations of the mutant enzyme indicated that the Ser hydroxyl group fits well in a cavity which contains a water molecule in the wild-type

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Science.gov (United States)

Arndt, E; Scholzen, T; Krömer, W; Hatakeyama, T; Kimura, M

1991-06-01

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.

Effect of ionization and nisin on the Bacillus strains and Salmonella Enteritidis inoculated Stearothermophilus

International Nuclear Information System (INIS)

Bouzayen, Sarra

2010-01-01

The antimicrobial effect of nisin (at 1000UI/g), and irradiation (at 1, 3 and 5kGy), against the growth of Salmonella enteritidis (106 ufc/ml) and Bacillus Stearothermophilus (10 6 ufc/ml), inoculated in turkey salami, was studied during storage at 4 degree for 21 days. Treatment of turkey salami with nisin at 1000UI/g did not show any antimicrobial activity against S. Enteritidis with 6.7 pour cent and 0.8 pour cent of reduction after 0 and 21 days of storage respectively, and seems to be insufficient to inhibit B. Stearothermophilus with 23 pour cent and 21 pour cent of reduction after 0 and 21 days of storage respectively. Antimicrobial activities of irradiation were better and proportional to irradiation doses; it shows a reduction of 27 pour cent, 55 pour cent and 67 pour cent by D1, D2 and D3 respectively. The combination of nisin with irradiation at 5kGy showed stronger antimicrobial activities than those obtained by its combination with the first and the second irradiation dose.

Purification and Characterization of a Thermostable Lipase from Geobacillus thermodenitrificans IBRL-nra

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Anuradha Balan

2012-01-01

Full Text Available Thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was purified and characterized. The production of thermostable lipase from Geobacillus thermodenitrificans IBRL-nra was carried out in a shake-flask system at 65°C in cultivation medium containing; glucose 1.0% (w/v; yeast extract 1.25% (w/v; NaCl 0.45% (w/v olive oil 0.1% (v/v with agitation of 200 rpm for 24 hours. The extracted extracellular crude thermostable lipase was purified to homogeneity by using ultrafiltration, Heparin-affinity chromatography, and Sephadex G-100 gel-filtration chromatography by 34 times with a final yield of 9%. The molecular weight of the purified enzyme was estimated to be 30 kDa after SDS-PAGE analysis. The optimal temperature for thermostable lipase was 65°C and it retained its initial activity for 3 hours. Thermostable lipase activity was highest at pH 7.0 and stable for 16 hours at this pH at 65°C. Thermostable lipase showed elevated activity when pretreated with BaCl2, CaCl2, and KCl with 112%, 108%, and 106%, respectively. Lipase hydrolyzed tripalmitin (C16 and olive oil with optimal activity (100% compared to other substrates.

INCREASING THE THERMOSTABILITY OF THE NEUTRAL PROTEINASE OF BACILLUS-STEAROTHERMOPHILUS BY IMPROVEMENT OF INTERNAL HYDROGEN-BONDING

NARCIS (Netherlands)

EIJSINK, VGH; VRIEND, G; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G

1992-01-01

In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594

Directory of Open Access Journals (Sweden)

Raimonda Petkauskaite

Full Text Available Abstract Geobacillus thermodenitrificans DSM 101594 was isolated as a producer of extracellular thermostable pectic polysaccharide degrading enzymes. The completely sequenced genome was 3.6 Mb in length with GC content of 48.86%. A number of genes encoding enzymatic active against the high molecular weight polysaccharides of potential biotechnological importance were identified in the genome.

The caa(3) terminal oxidase of Bacillus stearothermophilus - Transient spectroscopy of electron transfer and ligand binding

NARCIS (Netherlands)

Giuffre, A; DItri, E; Giannini, S; Brunori, M; UbbinkKok, T; Konings, WN; Antonini, G

1996-01-01

The thermophilic bacterium Bacillus stearothermophilus possesses a caa(3)-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. HL, and Konings, W. N. (1989) Ear. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by

Identification, characterization and preliminary X-ray diffraction analysis of the rolling-circle replication initiator protein from plasmid pSTK1

International Nuclear Information System (INIS)

Carr, Stephen B.; Mecia, Lauren B.; Phillips, Simon E. V.; Thomas, Christopher D.

2013-01-01

A proteolytically stable fragment of a plasmid replication initiation protein from the thermophile G. stearothermophilus has been biochemically characterized, crystallized and diffraction data collected to a resolution of 2.5 Å. Antibiotic resistance in bacterial pathogens poses an ever-increasing risk to human health. In antibiotic-resistant strains of Staphylococcus aureus this resistance often resides in extra-chromosomal plasmids, such as those of the pT181 family, which replicate via a rolling-circle mechanism mediated by a plasmid-encoded replication initiation protein. Currently, there is no structural information available for the pT181-family Rep proteins. Here, the crystallization of a catalytically active fragment of a homologous replication initiation protein from the thermophile Geobacillus stearothermophilus responsible for the replication of plasmid pSTK1 is reported. Crystals of the RepSTK1 fragment diffracted to a resolution of 2.5 Å and belonged to space group P2 1 2 1 2 1

Isolation of the phe-operon from G. stearothermophilus comprising the phenol degradative meta-pathway genes and a novel transcriptional regulator

Directory of Open Access Journals (Sweden)

Reiss Monika

2008-11-01

Full Text Available Abstract Background Geobacillus stearothermophilus is able to utilize phenol as a sole carbon source. A DNA fragment encoding a phenol hydroxylase catalyzing the first step in the meta-pathway has been isolated previously. Based on these findings a PCR-based DNA walk was performed initially to isolate a catechol 2,3-dioxygenase for biosensoric applications but was continued to elucidate the organisation of the genes encoding the proteins for the metabolization of phenol. Results A 20.2 kb DNA fragment was isolated as a result of the DNA walk. Fifteen open reading frames residing on a low-copy megaplasmid were identified. Eleven genes are co-transcribed in one polycistronic mRNA as shown by reverse transcription-PCR. Ten genes encode proteins, that are directly linked with the meta-cleavage pathway. The deduced amino acid sequences display similarities to a two-component phenol hydroxylase, a catechol 2,3-dioxygenase, a 4-oxalocrotonate tautomerase, a 2-oxopent-4-dienoate hydratase, a 4-oxalocrotonate decarboxylase, a 4-hydroxy-2-oxovalerate aldolase, an acetaldehyde dehydrogenase, a plant-type ferredoxin involved in the reactivation of extradiol dioxygenases and a novel regulatory protein. The only enzymes missing for the complete mineralization of phenol are a 2-hydroxymuconic acid-6-semialdehyde hydrolase and/or 2-hydroxymuconic acid-6-semialdehyde dehydrogenase. Conclusion Research on the bacterial degradation of aromatic compounds on a sub-cellular level has been more intensively studied in gram-negative organisms than in gram-positive bacteria. Especially regulatory mechanisms in gram-positive (thermophilic prokaryotes remain mostly unknown. We isolated the first complete sequence of an operon from a thermophilic bacterium encoding the meta-pathway genes and analyzed the genetic organization. Moreover, the first transcriptional regulator of the phenol metabolism in gram-positive bacteria was identified. This is a first step to elucidate

Production of alpha-amylase in batch and chemostat culture by bacillus stearothermophilus

Energy Technology Data Exchange (ETDEWEB)

Davis, P E; Cohen, D L; Whitaker, A

1980-01-01

The production of alpha-amylase by a strain of B.stearothermophilus isolated from leaf litter was investigated in a tryptone-maltose medium at 55 degrees in batch and chemostat culture. Amylase production was growth-limited and restricted to the exponential phase in batch culture. The enzyme yield was reduced by 40% when the culture pH was maintained at pH 7.2. Amylase production in chemostat culture was influenced by the growth rate throughout the dilution rate range used.

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

Science.gov (United States)

Huma, Tayyaba; Maryam, Arooma; Rehman, Shahid Ur; Qamar, Muhammad Tahir Ul; Shaheen, Tayyaba; Haque, Asma; Shaheen, Bushra

2014-01-01

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

Effects of gamma- and UV-radiation on DNA synthesis in permeable cells of Bacillus stearothermophilus

International Nuclear Information System (INIS)

Trofimenko, A.F.; Vorob'eva, A.M.; Gaziev, A.I.

1981-01-01

It was shown that the most of the DNA synthesis is repaired in permeable cells of Bacillus stearothermophilus not affected by injurious agents. γ-irradiation stimulates the reparative synthesis and degradation of DNA whereas UV-radiation decreases the activity of these processes. The reason for such an unusual response of thermophiles to irradiation lies perhaps in high temperatures at which the cells exist

Compact solar autoclave based on steam generation using broadband light-harvesting nanoparticles.

Science.gov (United States)

Neumann, Oara; Feronti, Curtis; Neumann, Albert D; Dong, Anjie; Schell, Kevin; Lu, Benjamin; Kim, Eric; Quinn, Mary; Thompson, Shea; Grady, Nathaniel; Nordlander, Peter; Oden, Maria; Halas, Naomi J

2013-07-16

The lack of readily available sterilization processes for medicine and dentistry practices in the developing world is a major risk factor for the propagation of disease. Modern medical facilities in the developed world often use autoclave systems to sterilize medical instruments and equipment and process waste that could contain harmful contagions. Here, we show the use of broadband light-absorbing nanoparticles as solar photothermal heaters, which generate high-temperature steam for a standalone, efficient solar autoclave useful for sanitation of instruments or materials in resource-limited, remote locations. Sterilization was verified using a standard Geobacillus stearothermophilus-based biological indicator.

Compact solar autoclave based on steam generation using broadband light-harvesting nanoparticles

Science.gov (United States)

Neumann, Oara; Feronti, Curtis; Neumann, Albert D.; Dong, Anjie; Schell, Kevin; Lu, Benjamin; Kim, Eric; Quinn, Mary; Thompson, Shea; Grady, Nathaniel; Nordlander, Peter; Oden, Maria; Halas, Naomi J.

2013-01-01

The lack of readily available sterilization processes for medicine and dentistry practices in the developing world is a major risk factor for the propagation of disease. Modern medical facilities in the developed world often use autoclave systems to sterilize medical instruments and equipment and process waste that could contain harmful contagions. Here, we show the use of broadband light-absorbing nanoparticles as solar photothermal heaters, which generate high-temperature steam for a standalone, efficient solar autoclave useful for sanitation of instruments or materials in resource-limited, remote locations. Sterilization was verified using a standard Geobacillus stearothermophilus-based biological indicator. PMID:23836642

Structural studies on a 2,3-diphosphoglycerate independent phosphoglycerate mutase from Bacillus stearothermophilus.

Science.gov (United States)

Chander, M; Setlow, P; Lamani, E; Jedrzejas, M J

1999-06-15

Phosphoglycerate mutase (PGM), an important enzyme in the glycolytic pathway, catalyzes the transfer of a phosphate group between the 2 and the 3 positions of glyceric acid. The gene coding for the 2, 3-diphosphoglycerate independent monomeric PGM from Bacillus stearothermophilus (57 kDa), whose activity is extremely pH sensitive and has an absolute and specific requirement for Mn2+, has been cloned and the enzyme overexpressed and purified to homogeneity. Circular dichroism studies showed at most only small secondary structure changes in the enzyme upon binding to Mn2+ or its 3-phosphoglycerate substrate, but thermal unfolding analyses revealed that Mn2+ but not 3-phosphoglycerate caused a large increase in the enzyme's stability. Diffraction-quality crystals of the enzyme were obtained at neutral pH in the presence of 3-phosphoglyceric acid with ammonium sulfate as the precipitating agent; these crystals diffract X rays to beyond 2.5-A resolution and belong to the orthorhombic space group C2221 with unit cell dimensions, a = 58.42, b = 206.08, c = 124.87 A, and alpha = beta = gamma = 90.0 degrees. The selenomethionyl version of the B. stearothermophilus protein has also been overexpressed, purified, and crystallized. Employing these crystals, the determination of the three-dimensional structure of this PGM by the multiwavelength anomalous dispersion method is in progress. Copyright 1999 Academic Press.

Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry

OpenAIRE

Abol Fotouh, Deyaa M.; Bayoumi, Reda A.; Hassan, Mohamed A.

2016-01-01

Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase pr...

Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy.

Science.gov (United States)

Uzuner, Ugur; Canakci, Sabriye; Bektas, Kadriye Inan; Sapmaz, Merve Tuncel; Belduz, Ali Osman

2017-06-01

Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Expression and characterization of thermostable glycogen branching enzyme from Geobacillus mahadia Geo-05

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Nur Syazwani Mohtar

2016-12-01

Full Text Available The glycogen branching enzyme (EC 2.4.1.18, which catalyses the formation of α-1,6-glycosidic branch points in glycogen structure, is often used to enhance the nutritional value and quality of food and beverages. In order to be applicable in industries, enzymes that are stable and active at high temperature are much desired. Using genome mining, the nucleotide sequence of the branching enzyme gene (glgB was extracted from the Geobacillus mahadia Geo-05 genome sequence provided by the Malaysia Genome Institute. The size of the gene is 2013 bp, and the theoretical molecular weight of the protein is 78.43 kDa. The gene sequence was then used to predict the thermostability, function and the three dimensional structure of the enzyme. The gene was cloned and overexpressed in E. coli to verify the predicted result experimentally. The purified enzyme was used to study the effect of temperature and pH on enzyme activity and stability, and the inhibitory effect by metal ion on enzyme activity. This thermostable glycogen branching enzyme was found to be most active at 55 °C, and the half-life at 60 °C and 70 °C was 24 h and 5 h, respectively. From this research, a thermostable glycogen branching enzyme was successfully isolated from Geobacillus mahadia Geo-05 by genome mining together with molecular biology technique.

Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.

Science.gov (United States)

Amador Espejo, Genaro Gustavo; Hernández-Herrero, M M; Juan, B; Trujillo, A J

2014-12-01

Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk. Copyright © 2014 Elsevier Ltd. All rights reserved.

PRODUCTION AND CHARACTERIZATION OF AN ALKALOTHERMOSTABLE, ORGANIC SOLVENT TOLERANT AND SURFACTANT TOLERANT ESTERASE PRODUCED BY A THERMOPHILIC BACTERIUM GEOBACILLUS SP. AGP-04, ISOLATED FROM BAKRESHWAR HOT SPRING, INDIA

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Amit Ghati

2013-10-01

Full Text Available A thermophilic bacteria, Geobacillus sp. AGP-04, isolated from Surya Kund hot spring, Bakreshwar, West Bengal, India was studied in terms of capability of tributyrin hydrolysis and characterization of its thermostable esterase activity using p-nitrophenyl butyrate (PNPB as substrate. The extracellular crude preparation was characterized in terms of pH and temperature optima and stability, organic solvent tolerance capacity and stability, substrate specificity, surfactant tolerance capacity, kinetic parameters and activation/inhibition behavior towards some metal ions and chemicals. Tributyrin agar assay exhibited that Geobacillus sp. AGP-04 secretes an extracellular esterase. The Vmax and Km values of the esterase were found to be 5099 U/Land 103.5µM, respectively in the presence of PNPB as substrate. The optimum temperature and pH, for Geobacillus sp. AGP-04 esterase was 60oC and 8.0, respectively. Although the enzyme activity was not significantly altered by incubating crude extract solution at 20-70oC for 1 hour, the enzyme activity was fully lost at 90oC for same incubation period. The pH stability profile showed that original crude esterase activity is stable at a broad range (pH 5.0-10.0. Moreover, the enzyme was highly organic solvent and surfactant tolerant. The effect of some chemical on crude esterase activity indicated that Geobacillus sp. AGP-04 produce an esterase which contains a serine residue in active site and for its activity -SH groups are essential. Besides, enzyme production was highly induced if fermentation medium contain polysaccharides and oil as carbon source.

Higher-order structure in the 3'-terminal domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus

DEFF Research Database (Denmark)

Garrett, R A; Christensen, A; Douthwaite, S

1984-01-01

An experimental approach was used to determine, and compare, the higher-order structure within domain VI of the 23 S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus. This domain, which encompasses approximately 300 nucleotides at the 3' end of the RNAs, consists of two large ...

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 beta-xylosidase from Geobacillus thermoleovorans IT-08

NARCIS (Netherlands)

Rohman, Ali; van Oosterwijk, Niels; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

2007-01-01

The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-beta-xylanase and beta-xylosidase. beta-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-beta-xylanase into xylose monomers. The beta-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of

ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

Science.gov (United States)

Yang, Mu; Wang, Ganggang

2016-09-15

The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. Copyright © 2016 Elsevier Inc. All rights reserved.

Highly thermostable xylanase production from a thermophilic Geobacillus sp. strain WSUCF1 utilizing lignocellulosic biomass

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Aditya eBhalla

2015-06-01

Full Text Available AbstractEfficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing hemicellulose component of lignocellulose to xylo-oligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70ºC, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70ºC, respectively. At 70ºC, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, CellicHTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70ºC. High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1.

Science.gov (United States)

Jia, Xianbo; Chen, Jichen; Lin, Chenqiang; Lin, Xinjian

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications.

Cloning, Expression, and Characterization of a Novel Thermophilic Monofunctional Catalase from Geobacillus sp. CHB1

Science.gov (United States)

2016-01-01

Catalases are widely used in many scientific areas. A catalase gene (Kat) from Geobacillus sp. CHB1 encoding a monofunctional catalase was cloned and recombinant expressed in Escherichia coli (E. coli), which was the first time to clone and express this type of catalase of genus Geobacillus strains as far as we know. This Kat gene was 1,467 bp in length and encoded a catalase with 488 amino acid residuals, which is only 81% similar to the previously studied Bacillus sp. catalase in terms of amino acid sequence. Recombinant catalase was highly soluble in E. coli and made up 30% of the total E. coli protein. Fermentation broth of the recombinant E. coli showed a high catalase activity level up to 35,831 U/mL which was only lower than recombinant Bacillus sp. WSHDZ-01 among the reported catalase production strains. The purified recombinant catalase had a specific activity of 40,526 U/mg and K m of 51.1 mM. The optimal reaction temperature of this recombinant enzyme was 60°C to 70°C, and it exhibited high activity over a wide range of reaction temperatures, ranging from 10°C to 90°C. The enzyme retained 94.7% of its residual activity after incubation at 60°C for 1 hour. High yield and excellent thermophilic properties are valuable features for this catalase in industrial applications. PMID:27579320

Thermostable hemicellulases of a bacterium, Geobacillus sp. DC3, isolated from the former Homestake gold mine in Lead, South Dakota.

Science.gov (United States)

Bergdale, Terran E; Hughes, Stephen R; Bang, Sookie S

2014-04-01

A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K m and V max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.

Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

Science.gov (United States)

Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

2010-06-01

D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

Science.gov (United States)

Oh, Hyo-Jung; Kim, Hye-Jung; Oh, Deok-Kun

2006-02-01

Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

Alkane inducible proteins in Geobacillus thermoleovorans B23

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Kato Tomohisa

2009-03-01

Full Text Available Abstract Background Initial step of β-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution. Results An extremely thermophilic and alkane degrading Geobacillus thermoleovorans B23 was previously isolated from a deep subsurface oil reservoir in Japan. In the present study, we identified novel membrane proteins (P16, P21 and superoxide dismutase (P24 whose production levels were significantly increased upon alkane degradation. Unlike other bacteria acyl-CoA oxidase and catalase activities were also increased in strain B23 by addition of alkane. Conclusion We first suggested that peroxisomal β-oxidation system exists in bacteria. This eukaryotic-type alkane degradation pathway in thermophilic bacterial cells might be a vestige of primitive living cell systems that had evolved into eukaryotes.

Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass.

Science.gov (United States)

Potprommanee, Laddawan; Wang, Xiao-Qin; Han, Ye-Ju; Nyobe, Didonc; Peng, Yen-Ping; Huang, Qing; Liu, Jing-Yong; Liao, Yu-Ling; Chang, Ken-Lin

2017-01-01

A themophilic cellulase-producing bacterium was isolated from a hot spring district and identified as Geobacillus sp. HTA426. The cellulase enzyme produced by the Geobacillus sp. HTA426 was purified through ammonium sulfate precipitation and ion exchange chromatography, with the recovery yield and fold purification of 10.14% and 5.12, respectively. The purified cellulase has a molecular weight of 40 kDa. The optimum temperature and pH for carboxymethyl cellulase (CMCase) activity of the purified cellulase were 60°C and pH 7.0, respectively. The enzyme was also stable over a wide temperature range of 50°C to 70°C after 5 h of incubation. Moreover, the strain HTA426 was able to grow and produce cellulase on alkali-treated sugarcane bagasse, rice straw and water hyacinth as carbon sources. Enzymatic hydrolysis of sugarcane bagasse, which was regarded as the most effective carbon source for cellulase production (CMCase activity = 103.67 U/mL), followed by rice straw (74.70 U/mL) and water hyacinth (51.10 U/mL). This strain producing an efficient thermostable cellulose is a potential candidate for developing a more efficient and cost-effective process for converting lignocellulosic biomass into biofuel and other industrial process.

Sterilization of single-use helical stone baskets: an experimental study

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Cely Barreto da Silva

2011-03-01

Full Text Available Objectives: To experimentally evaluate the efficacy of a standard sterilization protocol employed during reuse of disposable helical stone baskets. Methods: Study performed on 20 helical stone baskets: 10 were used in the initial validation process, contaminated with Escherichia coli ATCC 25922 and imprinted on Müeller-Hinton media; 10 catheters were contaminated with Geobacillus stearothermophilus ATCC 7953, processed, inoculated in TSB and incubated in a water bath at a temperature of 55ºC. Bacterial growth was evaluated after 1, 3, 5 and 7 days. After sterilization, stone baskets were also opened and closed 40 times to check for functional problems. All plastic and basket parts were carefully checked for damages. Results: After the 72-hour incubation period, there was growth of E. coli ATCC 25922 in 100% of imprints. After the sterilization process and up to 7 days incubation period on a blood agar plate, there was no growth of G. stearothermophilus ATCC 7953 or any other bacteria. There were no functional problems or damage to baskets after the sterilization process. Conclusion: The ethylene oxide system is efficacious and safe for sterilization of disposable helical stone baskets. However, further clinical studies are required and should provide more safety information.

Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders.

Science.gov (United States)

Sadiq, Faizan A; Flint, Steve; Yuan, Lei; Li, Yun; Liu, TongJie; He, GuoQing

2017-12-04

Biofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3logCFU/cm 2 , respectively. Number of cells recovered from biofilms on 1cm 2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk. Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript

Parameters for Novel Production of Fruity Floral Fragrance Ester (Geranyl Butyrate) by Locally Isolated Lipase Geobacillus thermodenitrificans nr68 (LGT)

Science.gov (United States)

Nik Raikhan, N. H.

2018-05-01

Geranyl butyrate has been synthesized successfully using our locally isolated lipase Geobacillus thermodenitrificans nr68 (LGT) as the fragrance ester with aim to be used in a nanotechnology fragrance application. We have used and modified few parameters from the previous research and then, continued with optimization of the synthesis by looking into degree of esterification and water content in the system. Butyric acid (C4), stearic acid (C18: 0), caprylic acid (C8), linolenic acid (C18: 3), myristic acid (C14), linoleic acid (C18: 2) and oleic acid (C18: 1) were used in the substrate selection. The yield of geranyl butyrate before the optimization was 31.68±0.01%. The optimum parameters for the synthesis of geranyl butyrate were recorded as temperature of 65°C, shaking rate at 200 rpm, 5.0 ml of geraniol and 0.40 ml of butyric acid and 4.0 ml of n-butanol and 0.40 ml of oleic acid. After the optimization, geranyl butyrate synthesis was increased by 297% as to compare with the value before the parameters were optimized. We also have significantly reduced water content as a byproduct of the esterification and managed to run the system a success. The ability thermotolerant lipase from Geobacillus thermodenitrificans (LGT) in this synthesis is novel to Malaysian fragrance industry.

Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain

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Xiao Zijun

2012-12-01

Full Text Available Abstract Background Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination. Results This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography–mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. α-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C. Conclusions Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its

Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77

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Wajdi Thebti

2016-01-01

Full Text Available A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

OpenAIRE

Sakoda, H; Imanaka, T

1992-01-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those cata...

Modelling of the acid base properties of two thermophilic bacteria at different growth times

Science.gov (United States)

Heinrich, Hannah T. M.; Bremer, Phil J.; McQuillan, A. James; Daughney, Christopher J.

2008-09-01

Acid-base titrations and electrophoretic mobility measurements were conducted on the thermophilic bacteria Anoxybacillus flavithermus and Geobacillus stearothermophilus at two different growth times corresponding to exponential and stationary/death phase. The data showed significant differences between the two investigated growth times for both bacterial species. In stationary/death phase samples, cells were disrupted and their buffering capacity was lower than that of exponential phase cells. For G. stearothermophilus the electrophoretic mobility profiles changed dramatically. Chemical equilibrium models were developed to simultaneously describe the data from the titrations and the electrophoretic mobility measurements. A simple approach was developed to determine confidence intervals for the overall variance between the model and the experimental data, in order to identify statistically significant changes in model fit and thereby select the simplest model that was able to adequately describe each data set. Exponential phase cells of the investigated thermophiles had a higher total site concentration than the average found for mesophilic bacteria (based on a previously published generalised model for the acid-base behaviour of mesophiles), whereas the opposite was true for cells in stationary/death phase. The results of this study indicate that growth phase is an important parameter that can affect ion binding by bacteria, that growth phase should be considered when developing or employing chemical models for bacteria-bearing systems.

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus

International Nuclear Information System (INIS)

McAdam, M.E.; Lavelle, F.; Fox, R.A.; Fielden, E.M.

1977-01-01

The mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus has been shown to involve a 'fast cycle' and a 'slow cycle' (McAdam, M.E., Fox, R.A., Lavelle, F., and Fielden, E.M., Biochem. J.; 165:71 (1977)). Further properties of the enzyme are now considered. Pulse-radiolysis studies, under conditions of low substrate concentration to enzyme concentration (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5 to 10.2). Activity was unaffected by the addition of H 2 O 2 or NaN 3 but slightly decreased by KCN. Both H 2 O 2 and the reducing radical anion CO 2 sup(-.) caused a decrease in A 480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5 to 55 0 C), and as temperature increased the slow catalytic cycle became relatively more important. Arrhenius parameters of the rate constants were estimated. The possible identity of the various forms of the enzyme is considered. (author)

Sterilization of liquid foods by pulsed electric fields – an innovative ultra-high temperature process

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Kai eReineke

2015-05-01

Full Text Available The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm-1, skim milk (0.3% fat; 5.3 mS cm-1 and fresh prepared carrot juice (7.73 mS cm-1. The combination of moderate preheating (70-90 °C and an insulated PEF-chamber, combined with a holding tube (65 cm and a heat exchanger for cooling, enabled a rapid heat up to 105-140 °C (measured above the PEF chamber within 92.2-368.9 µs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using Comsol Multiphysics combined with a Matlab routine.A preheating of saline water to 70 °C with a flow rate of 5 l h-1, a frequency of 150 Hz and an energy input of 226.5 kJ kg-1, resulted in a measured outlet temperature of 117 °C and a 4.67 log10 inactivation of Bacillus subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for Geobacillus stearothermophilus spores in saline water. A preheating to 95 °C and an energy input of 144 kJ kg-1 resulted in an outlet temperature of 126 °C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10 was achieved during the thermal treatment.Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring

Geobacillus thermoglucosidasius Endospores Function as Nuclei for the Formation of Single Calcite Crystals

Science.gov (United States)

Murai, Rie

2013-01-01

Geobacillus thermoglucosidasius colonies were placed on an agar hydrogel containing acetate, calcium ions, and magnesium ions, resulting in the formation of single calcite crystals (calcites) within and peripheral to the plating area or parent colony. Microscopic observation of purified calcites placed on the surface of soybean casein digest (SCD) nutrient medium revealed interior crevices from which bacterial colonies originated. Calcites formed on the gel contained [1-13C]- and [2-13C]acetate, demonstrating that G. thermoglucosidasius utilizes carbon derived from acetate for calcite formation. During calcite formation, vegetative cells swam away from the parent colony in the hydrogel. Hard-agar hydrogel inhibited the formation of calcites peripheral to the parent colony. The calcite dissolved completely in 1 M HCl, with production of bubbles, and the remaining endospore-like particles were easily stained with Brilliant green dye. The presence of DNA and protein in calcites was demonstrated by electrophoresis. We propose that endospores initiate the nucleation of calcites. Endospores of G. thermoglucosidasius remain alive and encapsulated in calcites. PMID:23455343

Survival and Adaptation of the Thermophilic Species Geobacillus thermantarcticus in Simulated Spatial Conditions

Science.gov (United States)

Di Donato, Paola; Romano, Ida; Mastascusa, Vincenza; Poli, Annarita; Orlando, Pierangelo; Pugliese, Mariagabriella; Nicolaus, Barbara

2018-03-01

Astrobiology studies the origin and evolution of life on Earth and in the universe. According to the panspermia theory, life on Earth could have emerged from bacterial species transported by meteorites, that were able to adapt and proliferate on our planet. Therefore, the study of extremophiles, i.e. bacterial species able to live in extreme terrestrial environments, can be relevant to Astrobiology studies. In this work we described the ability of the thermophilic species Geobacillus thermantarcticus to survive after exposition to simulated spatial conditions including temperature's variation, desiccation, X-rays and UVC irradiation. The response to the exposition to the space conditions was assessed at a molecular level by studying the changes in the morphology, the lipid and protein patterns, the nucleic acids. G. thermantarcticus survived to the exposition to all the stressing conditions examined, since it was able to restart cellular growth in comparable levels to control experiments carried out in the optimal growth conditions. Survival was elicited by changing proteins and lipids distribution, and by protecting the DNA's integrity.

Rational design of Bacillus stearothermophilus US100 L-arabinose isomerase: potential applications for D-tagatose production.

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Rhimi, Moez; Aghajari, Nushin; Juy, Michel; Chouayekh, Hichem; Maguin, Emmanuelle; Haser, Richard; Bejar, Samir

2009-05-01

L-arabinose isomerases catalyze the bioconversion of D-galactose into D-tagatose. With the aim of producing an enzyme optimized for D-tagatose production, three Bacillus stearothermophilus US100 L-arabinose isomerase mutants were constructed, purified and characterized. Our results indicate that mutant Q268K was significantly more acidotolerant and more stable at acidic pH than the wild-type enzyme. The N175H mutant has a broad optimal temperature range from 50 to 65 degrees C. With the aim of constructing an acidotolerant mutant working at relatively low temperatures we generated the Q268K/N175H construct. This double mutant displays an optimal pH in the range 6.0-7.0 and an optimal activity around 50-65 degrees C, temperatures at which the enzyme was stable without addition of metal ions.

Pressure-assisted thermal sterilization of soup

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Shibeshi, Kidane; Farid, Mohammed M.

2010-12-01

The overall efficiency of an existing scale-up pressure-assisted thermal sterilization (PATS) unit was investigated with regards to inactivation of Geobacillus stearothermophilus spores suspended in pumpkin soup. The PATS unit is a double pipe heat exchanger in which the soup is pumped into its inner high pressure tube and constrained by two high pressure valves, while steam is continuously passed through the annular region to heat the content. The technology is based on pressure generation by thermal expansion of the liquid in an enclosure. In this work, the addition of an air line to push the treated liquid food out of the existing PATS unit has improved the overall quality of the treated samples, as evidenced by achieving higher log reduction of the spores. Compared with thermal processing, the application of PATS shows the potential for lowering the thermal treatment temperature, offering improved food quality.

Tracking spore-forming bacteria in food: from natural biodiversity to selection by processes.

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Postollec, Florence; Mathot, Anne-Gabrielle; Bernard, Muriel; Divanac'h, Marie-Laure; Pavan, Sonia; Sohier, Danièle

2012-08-01

Sporeforming bacteria are ubiquitous in the environment and exhibit a wide range of diversity leading to their natural prevalence in foodstuff. The state of the art of sporeformer prevalence in ingredients and food was investigated using a multiparametric PCR-based tool that enables simultaneous detection and identification of various genera and species mostly encountered in food, i.e., Alicyclobacillus, Anoxybacillus flavithermus, Bacillus, B. cereus group, B. licheniformis, B. pumilus, B. sporothermodurans, B. subtilis, Brevibacillus laterosporus, Clostridium, Geobacillus stearothermophilus, Moorella and Paenibacillus species. In addition, 16S rDNA sequencing was used to extend identification to other possibly present contaminants. A total of 90 food products, with or without visible trace of spoilage were analysed, i.e., 30 egg-based products, 30 milk and dairy products and 30 canned food and ingredients. Results indicated that most samples contained one or several of the targeted genera and species. For all three tested food categories, 30 to 40% of products were contaminated with both Bacillus and Clostridium. The percentage of contaminations associated with Clostridium or Bacillus represented 100% in raw materials, 72% in dehydrated ingredients and 80% in processed foods. In the last two product types, additional thermophilic contaminants were identified (A. flavithermus, Geobacillus spp., Thermoanaerobacterium spp. and Moorella spp.). These results suggest that selection, and therefore the observed (re)-emergence of unexpected sporeforming contaminants in food might be favoured by the use of given food ingredients and food processing technologies. Copyright © 2012 Elsevier B.V. All rights reserved.

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus

International Nuclear Information System (INIS)

McAdam, M.E.; Fox, R.A.; Lavelle, F.; Fielden, E.M.

1977-01-01

The enzymic reaction mechanism of a manganese-containing superoxide dismutase from Bacillus stearothermophilus was studied by using pulse radiolysis. During catalysis (pH 8.9; 25 0 C), changes occurring in the kinetics of substrate disappearance and in the visible absorption of the enzyme at 480 nm established that the simple two-step mechanism found for copper- and iron-containing superoxide dismutases was not involved. At a low ratio ( 2 sup(-.) was close to exponential, whereas at much higher ratios (> 100) the observed decay was predominantly zero-order. The simplest interpretation of the results invokes a rapid one-electron oxidation-reduction cycle ('the fast cycle') and, concurrently, a slower reaction giving a form of the enzyme that is essentially unreactive towards O 2 sup(-.) but which undergoes a first-order decay to yield fully active native enzyme ('the slow cycle'). The fast cycle involved the native enzyme Esub(A) and a form of the enzyme Esub(B) which could be obtained also be treating the form Esub(A) with H 2 O 2 . Computer calculations made with such a simple model predicted behaviour in excellent agreement with the observed results. (author)

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

International Nuclear Information System (INIS)

Faraci, W.S.; Walsh, C.T.

1988-01-01

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L → D and D→ L directions for all three enzymes to assess the degree to which abstraction of the α-proton or protonation of substrate PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of α- 3 H from substrate to product and solvent exchange/substrate conversion experiments in 3 H 2 O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

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Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Amylase Zymography.

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Andrades, Adarelys; Contreras, Lellys M

2017-01-01

Amylase zymography was carried out for the detection of amylases produced by a Geobacillus stearothermophilus strain isolated from the Thermal Center "Las Trincheras" in Venezuela. Zymography is an electrophoretic technique used to study hydrolases by means of thin gels containing copolymerized-specific substrates, under nonreducing conditions. In this study, 0.1% starch was incorporated into the gel as substrate. The formation of clear zones against a dark background in the gel stained with iodine indicated the presence of amylolytic activity. The thermophilic bacteria released several extracellular amylases to a selective growth medium supplemented with 1% soluble starch at 55 °C after 40 h incubation. The amylolytic enzymes showed an optimum temperature of 60 °C and an optimum pH at 6.0. The amylases were partially purified by cold acetone precipitation followed by two chromatographic techniques. These purified amylases showed different molecular masses which were determined by sodium dodecyl sulfate gel electrophoresis and confirmed by zymography.

Tryptophan Oxidative Metabolism Catalyzed by : A Thermophile Isolated from Kuwait Soil Contaminated with Petroleum Hydrocarbons

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Jassim M. Al-Hassan

2011-01-01

Full Text Available Tryptophan metabolism has been extensively studied in humans as well as in soil. Its metabolism takes place mainly through kynurenine pathway yielding hydroxylated, deaminated and many other products of physiological significance. However, tryptophan metabolism has not been studied in an isolated thermophilic bacterium. Geobacillus stearothermophilus is a local thermophile isolated from Kuwait desert soil contaminated with petroleum hydrocarbons. The bacterium grows well at 65 °C in 0.05 M phosphate buffer (pH 7, when supplied with organic compounds as a carbon source and has a good potential for transformation of steroids and related molecules. In the present study, we used tryptophan ethyl ester as a carbon source for the bacterium to study the catabolism of the amino acid at pH 5 and pH 7. In this endeavor, we have resolved twenty one transformation products of tryptophan by GC/LC and have identified them through their mass spectral fragmentation.

Immobilization of Lipase from Geobacillus sp. and Its Application in Synthesis of Methyl Salicylate.

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Bhardwaj, Kamal Kumar; Saun, Nitin Kumar; Gupta, Reena

2017-04-03

The present study showed unique properties of an alkaline, thermophilic lipase of Geobacillus sp. which was isolated from soil of hot spring. The study was aimed to investigate the optimum immobilization conditions of lipase onto silica gel matrix (100-200 mesh) by surface adsorption method and its application in the synthesis of methyl salicylate. Lipase immobilized by surface adsorption onto silica pretreated with 4% glutaraldehyde showed 74.67% binding of protein and the optimum binding time for glutaraldehyde was found to be 2 h. The enzyme showed maximum activity at temperature 55°C, incubation time of 10 min at pH 9.5 of Tris buffer (0.1 M). Free as well as immobilized lipase was more specific to p-NPP (20 mM). All the metal ions and detergents used had inhibitory effect on free as well as immobilized enzyme. The silica immobilized enzyme was reused for hydrolysis and it retained almost 40.78% of its original activity up to 4 th cycle. On optimizing different parameters such as molar ratio, incubation time, temperature, amount of enzyme, amount of molecular sieve, the % yield of methyl salicylate was found to be 82.94.

High Quality Draft Genomes of the Type Strains Geobacillus thermocatenulatus DSM 730T, G. uzenensis DSM 23175T And Parageobacillus galactosidasius DSM 18751T.

Science.gov (United States)

Ramaloko, Winnie Thabisa; Koen, Nadine; Polliack, Shamara; Aliyu, Habibu; Lebre, Pedro Humberto; Mohr, Teresa; Oswald, Florian; Zwick, Michaela; Zeigler, Daniel Ray; Neumann, Anke; Syldatk, Christoph; Cowan, Don Arthur; De Maayer, Pieter

2018-01-01

The thermophilic 'Geobacilli' are important sources of thermostable enzymes and other biotechnologically relevant macromolecules. The present work reports the high quality draft genome sequences of previously unsequenced type strains of Geobacillus uzenensis (DSM 23175 T ), G. thermocatenulatus (DSM 730 T ) and Parageobacillus galactosidasius (DSM 18751 T ). Phylogenomic analyses revealed that DSM 18751 T and DSM 23175 T represent later heterotypic synonyms of P. toebii and G. subterraneus , respectively, while DSM 730 T represents the type strain for the species G. thermocatenulatus . These genome sequences will contribute towards a deeper understanding of the ecological and biological diversity and the biotechnological exploitation of the 'geobacilli'.

CLONING, PURIFICATION AND CHARACTERIZATION OF HALOTOLERANT XYLANASE FROM Geobacillus Thermodenitrificans C5

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Muhammad Irfan

2016-06-01

Full Text Available High levels of extracellular xylanase activity (994.50 IU/ml produced by Geobacillus thermodenitrificans C5 originated gene was detected when it was expressed in E. coli BL21 host. Thermostable xylanase (GthC5Xyl was purified to homogeneity and showed a molecular mass of approximately 44 kDa according to SDS-PAGE. The specific activity of the purified GthC5Xyl was up to 1243.125IU/mg with a 9.89-fold purification. The activity of GthC5Xyl was stimulated by CoCl2, MnSO4, CuSO4, MnCl2 but was inhibited by FeSO4, Hg, CaSO4. GthC5Xyl showed resistant to SDS, Tween 20, Triton X-100, β- Mercaptoethanol, PMSF, DTT, NEM and DEPC, SDS, and EDTA. A greater affinity for oat spelt xylan was exhibited by GthC5Xyl with maximum enzymatic activity at 60°C and 6.0 pH. The activity portrayed by GthC5Xyl was found to be acellulytic with stability at high temperature (70°C-80°C and low pH (4.0 to 8.0. Xylobiose and xylopentose were the end products of proficient oat spelt xylanase hydrolysis by GthC5Xyl. High SDS resistance and broader stability of GthC5Xyl proves it to be worthy of impending application in numerous industrial processes especially textile, detergents and animal feed industry.

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator.

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Dlugokenski, Regina E F; Sella, Sandra R B R; Guizelini, Belquis P; Vandenberghe, Luciana P S; Woiciechowski, Adenise L; Soccol, Carlos R; Minozzo, João C

2011-04-01

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D(121 °C) value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D(121 °C)=2.9±0.5 min and Usk=12.7±2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D(121 °C) value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product. © Springer-Verlag 2011

Prevalence of Clostridium botulinum and thermophilic heat-resistant spores in raw carrots and green beans used in French canning industry.

Science.gov (United States)

Sevenier, V; Delannoy, S; André, S; Fach, P; Remize, F

2012-04-16

Two categories of vegetables (carrots and green beans) that are widely used in the manufacture of canned food were surveyed for their spore contamination. Samples were recovered from 10 manufactures spread over all producing areas in France. Two samples over 316 raw vegetables collected were found positive for botulinum neurotoxin producing Clostridia spores as tested by PCR-based GeneDisc assay. Both positive samplestested positive for the type B neurotoxin gene (bont/B). In parallel, heat-resistant spores of thermophilic bacteria that are likely to be associated with canned food spoilage after prolonged incubation at 55 °C were surveyed after specific enrichment. Prevalence varied between 1.6% for Moorella thermoacetica/thermoautotrophica in green bean samples and 8.6% for either Geobacillus stearothermophilus or Thermoanaerobacterium spp. in carrot samples. Vegetable preparation, e.g. washing and edge cutting, considerably reduced spore contamination levels. These data constitute the first wide examination of vegetables specifically cultivated for industrialpurposes for their contamination by spores of thermophilic bacterial species. Copyright © 2012 Elsevier B.V. All rights reserved.

PCR detection of thermophilic spore-forming bacteria involved in canned food spoilage.

Science.gov (United States)

Prevost, S; Andre, S; Remize, F

2010-12-01

Thermophilic bacteria that form highly heat-resistant spores constitute an important group of spoilage bacteria of low-acid canned food. A PCR assay was developed in order to rapidly trace these bacteria. Three PCR primer pairs were designed from rRNA gene sequences. These primers were evaluated for the specificity and the sensitivity of detection. Two primer pairs allowed detection at the species level of Geobacillus stearothermophilus and Moorella thermoacetica/thermoautrophica. The other pair allowed group-specific detection of anaerobic thermophilic bacteria of the genera Thermoanaerobacterium, Thermoanaerobacter, Caldanerobium and Caldanaerobacter. After a single enrichment step, these PCR assays allowed the detection of 28 thermophiles from 34 cans of spoiled low-acid food. In addition, 13 ingredients were screened for the presence of these bacteria. This PCR assay serves as a detection method for strains able to spoil low-acid canned food treated at 55°C. It will lead to better reactivity in the canning industry. Raw materials and ingredients might be qualified not only for quantitative spore contamination, but also for qualitative contamination by highly heat-resistant spores.

Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae

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Eivind B. Drejer

2018-05-01

Full Text Available Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B. methanolicus, B. coagulans, B. smithii, B. licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.

Phenolic acids and flavonoids of peanut by-products: Antioxidant capacity and antimicrobial effects.

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de Camargo, Adriano Costa; Regitano-d'Arce, Marisa Aparecida Bismara; Rasera, Gabriela Boscariol; Canniatti-Brazaca, Solange Guidolin; do Prado-Silva, Leonardo; Alvarenga, Verônica Ortiz; Sant'Ana, Anderson S; Shahidi, Fereidoon

2017-12-15

Peanut skin (PS) and meal from dry-blanched peanuts (MDBP) were evaluated as sources of phenolic compounds. PS rendered the highest total phenolic content, antioxidant capacity towards ABTS radical cation, DPPH and hydroxyl radicals as well as reducing power. Phenolic acids were present in PS and MDBP whereas proanthocyanidins and monomeric flavonoids were found only in PS as identified by HPLC-DAD-ESI-MS n . Procyanidin-rich extracts prevented oxidation in non-irradiated and gamma-irradiated fish model system. Both extracts inhibited the growth of gram-positive (Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Geobacillus stearothermophilus) and gram-negative bacteria (Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli). Regardless of the strain, phenolic acid-rich extracts showed the lowest minimum inhibitory capacity (MIC); therefore presenting higher antibacterial effect. The MIC of phenolic acid-rich extracts (24-49μgphenolics/mL) was higher but comparable to Ampicillin (10μg/mL). Thus, phenolics in PS and MDBP may serve as antioxidants and antimicrobial compounds. Copyright © 2017. Published by Elsevier Ltd.

Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

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Yu Zhang

Full Text Available Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL from Geobacillus kaustophilus HTA426 (GkaP exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry.

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Abol Fotouh, Deyaa M; Bayoumi, Reda A; Hassan, Mohamed A

2016-01-01

Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v), respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v), 4% (v/v), and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5%) or the sole crude enzyme (8.9%). It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards.

Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry

Directory of Open Access Journals (Sweden)

Deyaa M. Abol Fotouh

2016-01-01

Full Text Available Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v, respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v, 4% (v/v, and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5% or the sole crude enzyme (8.9%. It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards.

Added value of experts' knowledge to improve a quantitative microbial exposure assessment model--Application to aseptic-UHT food products.

Science.gov (United States)

Pujol, Laure; Johnson, Nicholas Brian; Magras, Catherine; Albert, Isabelle; Membré, Jeanne-Marie

2015-10-15

In a previous study, a quantitative microbial exposure assessment (QMEA) model applied to an aseptic-UHT food process was developed [Pujol, L., Albert, I., Magras, C., Johnson, N. B., Membré, J. M. Probabilistic exposure assessment model to estimate aseptic UHT product failure rate. 2015 International Journal of Food Microbiology. 192, 124-141]. It quantified Sterility Failure Rate (SFR) associated with Bacillus cereus and Geobacillus stearothermophilus per process module (nine modules in total from raw material reception to end-product storage). Previously, the probabilistic model inputs were set by experts (using knowledge and in-house data). However, only the variability dimension was taken into account. The model was then improved using expert elicitation knowledge in two ways. First, the model was refined by adding the uncertainty dimension to the probabilistic inputs, enabling to set a second order Monte Carlo analysis. The eight following inputs, and their impact on SFR, are presented in detail in this present study: D-value for each bacteria of interest (B. cereus and G. stearothermophilus) associated with the inactivation model for the UHT treatment step, i.e., two inputs; log reduction (decimal reduction) number associated with the inactivation model for the packaging sterilization step for each bacterium and each part of the packaging (product container and sealing component), i.e., four inputs; and bacterial spore air load of the aseptic tank and the filler cabinet rooms, i.e., two inputs. Second, the model was improved by leveraging expert knowledge to develop further the existing model. The proportion of bacteria in the product which settled on surface of pipes (between the UHT treatment and the aseptic tank on one hand, and between the aseptic tank and the filler cabinet on the other hand) leading to a possible biofilm formation for each bacterium, was better characterized. It was modeled as a function of the hygienic design level of the aseptic

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

International Nuclear Information System (INIS)

Hwa Park, K.; Jeong Kim, M.; Seob Lee, H.; Kim, D.; Soo Han, N.; Robyt, J.F.

1998-01-01

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an α-(1-6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached α-(1-6) to d-glucose, d-mannose, d-galactose, and methyl α-d-glucopyranoside. With d-fructopyranose and d-xylopyranose, PTS was linked α-(1-5) and α-(1-4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of α-(1-3) and/or α-(1-4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked α-(1-4) to the glucose residue. α,α-Trehalose gave two major products with PTS linked α-(1-6) and α-(1-4). Maltitol gave two major products with PTS linked α-(1-6) and α-(1-4) to the glucopyranose residue. Raffinose gave two major products with PTS linked α-(1-6) and α-(1-4) to the d-galactopyranose residue. Maltotriose gave two major products with PTS linked α-(1-6) and α-(1-4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked α-(1-5) as the major product and d-glucitol gave PTS linked α-(1-6) as the only product. The structures of the transfer products were determined using thin layer-chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13 C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was d-glucitol. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Transglycosylation reactions of Bacillus stearothermophilus maltogenic amylase with acarbose and various acceptors

Energy Technology Data Exchange (ETDEWEB)

Hwa Park, K; Jeong Kim, M; Seob Lee, H; Kim, D [Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon (Korea, Republic of); Soo Han, N; Robyt, J F [Laboratory for Carbohydrate Chemistry and Enzymology, Department of Biochemistry and Biophysics, Iowa State University, Ames, IA (United States)

1998-12-15

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an {alpha}-(1-6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached {alpha}-(1-6) to d-glucose, d-mannose, d-galactose, and methyl {alpha}-d-glucopyranoside. With d-fructopyranose and d-xylopyranose, PTS was linked {alpha}-(1-5) and {alpha}-(1-4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of {alpha}-(1-3) and/or {alpha}-(1-4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked {alpha}-(1-4) to the glucose residue. {alpha},{alpha}-Trehalose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4). Maltitol gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the glucopyranose residue. Raffinose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the d-galactopyranose residue. Maltotriose gave two major products with PTS linked {alpha}-(1-6) and {alpha}-(1-4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked {alpha}-(1-5) as the major product and d-glucitol gave PTS linked {alpha}-(1-6) as the only product. The structures of the transfer products were determined using thin layer-chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and {sup 13}C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was d-glucitol. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

Science.gov (United States)

Kimura, M; Kimura, J; Hatakeyama, T

1988-11-21

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

Thermostable, alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus.

Science.gov (United States)

Ben Bacha, Abir; Moubayed, Nadine M S; Abid, Islam

2015-04-01

Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

Crystallization, characterization and preliminary X-ray crystallographic analysis of GK2848, a putative carbonic anhydrase of Geobacillus kaustophilus

International Nuclear Information System (INIS)

Ragunathan, Preethi; Raghunath, Gokul; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Kumarevel, Thirumananseri; Ponnuraj, Karthe

2013-01-01

The expression, purification, characterization and crystallization of GK2848, a carbonic anhydrase from G. kaustophilus, are described. The crystals diffracted to a resolution of 2.70 Å. GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni–NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2 1 2 1 2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur–Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized

Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivo and in vitro

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Korkmaz, Nuriye; Ostermann, Kai; Rödel, Gerhard

2011-03-01

Surface layer proteins have the appealing property to self-assemble in nanosized arrays in solution and on solid substrates. In this work, we characterize the formation of assembly structures of the recombinant surface layer protein SbsC of Geobacillus stearothermophilus ATTC 12980, which was tagged with enhanced green fluorescent protein and expressed in the yeast Saccharomyces cerevisiae. The tubular structures formed by the protein in vivo are retained upon bursting the cells by osmotic shock; however, their average length is decreased. During dialysis, monomers obtained by treatment with chaotropic chemicals recrystallize again to form tube-like structures. This process is strictly dependent on calcium (Ca2 + ) ions, with an optimal concentration of 10 mM. Further increase of the Ca2 + concentration results in multiple non-productive nucleation points. We further show that the lengths of the S-layer assemblies increase with time and can be controlled by pH. After 48 h, the average length at pH 9.0 is 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications.

Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

Science.gov (United States)

Sakudo, Akikazu; Toyokawa, Yoichi; Nakamura, Tetsuji; Yagyu, Yoshihito; Imanishi, Yuichiro

2017-01-01

Gas plasma, produced by a short high‑voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme‑linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2‑4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75‑142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO‑), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Energy Technology Data Exchange (ETDEWEB)

Kolodkin-Gal, I; Elsholz, AKW; Muth, C; Girguis, PR; Kolter, R; Losick, R

2013-04-29

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa(3) and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD(+))/NADH ratio via binding of NAD(+) to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Science.gov (United States)

Kolodkin-Gal, Ilana; Elsholz, Alexander K.W.; Muth, Christine; Girguis, Peter R.; Kolter, Roberto; Losick, Richard

2013-01-01

Bacillus subtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration. PMID:23599347

Synthesis of cinnamyl alcohol from cinnamaldehyde with Bacillus stearothermophilus alcohol dehydrogenase as the isolated enzyme and in recombinant E. coli cells.

Science.gov (United States)

Pennacchio, Angela; Rossi, Mosè; Raia, Carlo A

2013-07-01

The synthesis of the aroma chemical cinnamyl alcohol (CMO) by means of enzymatic reduction of cinnamaldehyde (CMA) was investigated using NADH-dependent alcohol dehydrogenase from Bacillus stearothermophilus both as an isolated enzyme, and in recombinant Escherichia coli whole cells. The influence of parameters such as reaction time and cofactor, substrate, co-substrate 2-propanol and biocatalyst concentrations on the bioreduction reaction was investigated and an efficient and sustainable one-phase system developed. The reduction of CMA (0.5 g/L, 3.8 mmol/L) by the isolated enzyme occurred in 3 h at 50 °C with 97% conversion, and yielded high purity CMO (≥98%) with a yield of 88% and a productivity of 50 g/genzyme. The reduction of 12.5 g/L (94 mmol/L) CMA by whole cells in 6 h, at 37 °C and no requirement of external cofactor occurred with 97% conversion, 82% yield of 98% pure alcohol and a productivity of 34 mg/gwet cell weight. The results demonstrate the microbial system as a practical and efficient method for larger-scale synthesis of CMO.

Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

International Nuclear Information System (INIS)

Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

2007-01-01

The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4 3 2 1 2, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2 1 , with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis.

Science.gov (United States)

Grau, Roberto R; de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Donato, Verónica; Hölscher, Theresa; Boland, Wilhelm; Kuipers, Oscar P; Kovács, Ákos T

2015-07-07

Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. Alternation between motile and sessile behaviors is central to bacterial adaptation, survival, and colonization. However, how is the collective

Gold nanoparticles synthesized by Geobacillus sp. strain ID17 a thermophilic bacterium isolated from Deception Island, Antarctica

Science.gov (United States)

2013-01-01

Background The use of microorganisms in the synthesis of nanoparticles emerges as an eco-friendly and exciting approach, for production of nanoparticles due to its low energy requirement, environmental compatibility, reduced costs of manufacture, scalability, and nanoparticle stabilization compared with the chemical synthesis. Results The production of gold nanoparticles by the thermophilic bacterium Geobacillus sp. strain ID17 is reported in this study. Cells exposed to Au3+ turned from colourless into an intense purple colour. This change of colour indicates the accumulation of intracellular gold nanoparticles. Elemental analysis of particles composition was verified using TEM and EDX analysis. The intracellular localization and particles size were verified by TEM showing two different types of particles of predominant quasi-hexagonal shape with size ranging from 5–50 nm. The mayority of them were between 10‒20 nm in size. FT-IR was utilized to characterize the chemical surface of gold nanoparticles. This assay supports the idea of a protein type of compound on the surface of biosynthesized gold nanoparticles. Reductase activity involved in the synthesis of gold nanoparticles has been previously reported to be present in others microorganisms. This reduction using NADH as substrate was tested in ID17. Crude extracts of the microorganism could catalyze the NADH-dependent Au3+ reduction. Conclusions Our results strongly suggest that the biosynthesis of gold nanoparticles by ID17 is mediated by enzymes and NADH as a cofactor for this biological transformation. PMID:23919572

Molecular Dynamic Simulation of Space and Earth-Grown Crystal Structures of Thermostable T1 Lipase Geobacillus zalihae Revealed a Better Structure.

Science.gov (United States)

Ishak, Siti Nor Hasmah; Aris, Sayangku Nor Ariati Mohamad; Halim, Khairul Bariyyah Abd; Ali, Mohd Shukuri Mohamad; Leow, Thean Chor; Kamarudin, Nor Hafizah Ahmad; Masomian, Malihe; Rahman, Raja Noor Zaliha Raja Abd

2017-09-25

Less sedimentation and convection in a microgravity environment has become a well-suited condition for growing high quality protein crystals. Thermostable T1 lipase derived from bacterium Geobacillus zalihae has been crystallized using the counter diffusion method under space and earth conditions. Preliminary study using YASARA molecular modeling structure program for both structures showed differences in number of hydrogen bond, ionic interaction, and conformation. The space-grown crystal structure contains more hydrogen bonds as compared with the earth-grown crystal structure. A molecular dynamics simulation study was used to provide insight on the fluctuations and conformational changes of both T1 lipase structures. The analysis of root mean square deviation (RMSD), radius of gyration, and root mean square fluctuation (RMSF) showed that space-grown structure is more stable than the earth-grown structure. Space-structure also showed more hydrogen bonds and ion interactions compared to the earth-grown structure. Further analysis also revealed that the space-grown structure has long-lived interactions, hence it is considered as the more stable structure. This study provides the conformational dynamics of T1 lipase crystal structure grown in space and earth condition.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

Science.gov (United States)

Sumida, Yosuke; Iwai, Sachio; Nishiya, Yoshiaki; Kumagai, Shinya; Yamada, Toshihide; Azuma, Masayuki

2018-03-01

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of pH on Thermoanaerobacterium thermosaccharolyticum DSM 571 growth, spore heat resistance and recovery.

Science.gov (United States)

Mtimet, Narjes; Guégan, Stéphanie; Durand, Lucile; Mathot, Anne-Gabrielle; Venaille, Laurent; Leguérinel, Ivan; Coroller, Louis; Couvert, Olivier

2016-05-01

Thermophilic spore-forming bacteria are potential contaminants in several industrial sectors involving high temperatures (40-65 °C) in the manufacturing process. Among those thermophilic spore-forming bacteria, Thermoanaerobacterium thermosaccharolyticum, called "the swelling canned food spoiler", has generated interest over the last decade in the food sector. The aim of this study was to investigate and to model pH effect on growth, heat resistance and recovery abilities after a heat-treatment of T. thermosaccharolyticum DSM 571. Growth and sporulation were conducted on reinforced clostridium media and liver broth respectively. The highest spore heat resistances and the greatest recovery ability after a heat-treatment were obtained at pH condition allowing maximal growth rate. Growth and sporulation boundaries were estimated, then models using growth limits as main parameters were extended to describe and quantify the effect of pH on recovery of injured spores after a heat-treatment. So, cardinal values were used as a single set of parameters to describe growth, sporulation and recovery abilities. Besides, this work suggests that T. thermosaccharolyticum preserve its ability for germination and outgrowth after a heat-treatment at a low pH where other high resistant spore-forming bacteria like Geobacillus stearothermophilus are unable to grow. Copyright © 2015 Elsevier Ltd. All rights reserved.

Computational design of a Diels-Alderase from a thermophilic esterase: the importance of dynamics

Science.gov (United States)

Linder, Mats; Johansson, Adam Johannes; Olsson, Tjelvar S. G.; Liebeschuetz, John; Brinck, Tore

2012-09-01

A novel computational Diels-Alderase design, based on a relatively rare form of carboxylesterase from Geobacillus stearothermophilus, is presented and theoretically evaluated. The structure was found by mining the PDB for a suitable oxyanion hole-containing structure, followed by a combinatorial approach to find suitable substrates and rational mutations. Four lead designs were selected and thoroughly modeled to obtain realistic estimates of substrate binding and prearrangement. Molecular dynamics simulations and DFT calculations were used to optimize and estimate binding affinity and activation energies. A large quantum chemical model was used to capture the salient interactions in the crucial transition state (TS). Our quantitative estimation of kinetic parameters was validated against four experimentally characterized Diels-Alderases with good results. The final designs in this work are predicted to have rate enhancements of ≈103-106 and high predicted proficiencies. This work emphasizes the importance of considering protein dynamics in the design approach, and provides a quantitative estimate of the how the TS stabilization observed in most de novo and redesigned enzymes is decreased compared to a minimal, `ideal' model. The presented design is highly interesting for further optimization and applications since it is based on a thermophilic enzyme ( T opt = 70 °C).

Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest.

Science.gov (United States)

Klämpfl, Tobias G; Isbary, Georg; Shimizu, Tetsuji; Li, Yang-Fang; Zimmermann, Julia L; Stolz, Wilhelm; Schlegel, Jürgen; Morfill, Gregor E; Schmidt, Hans-Ulrich

2012-08-01

Physical cold atmospheric surface microdischarge (SMD) plasma operating in ambient air has promising properties for the sterilization of sensitive medical devices where conventional methods are not applicable. Furthermore, SMD plasma could revolutionize the field of disinfection at health care facilities. The antimicrobial effects on Gram-negative and Gram-positive bacteria of clinical relevance, as well as the fungus Candida albicans, were tested. Thirty seconds of plasma treatment led to a 4 to 6 log(10) CFU reduction on agar plates. C. albicans was the hardest to inactivate. The sterilizing effect on standard bioindicators (bacterial endospores) was evaluated on dry test specimens that were wrapped in Tyvek coupons. The experimental D(23)(°)(C) values for Bacillus subtilis, Bacillus pumilus, Bacillus atrophaeus, and Geobacillus stearothermophilus were determined as 0.3 min, 0.5 min, 0.6 min, and 0.9 min, respectively. These decimal reduction times (D values) are distinctly lower than D values obtained with other reference methods. Importantly, the high inactivation rate was independent of the material of the test specimen. Possible inactivation mechanisms for relevant microorganisms are briefly discussed, emphasizing the important role of neutral reactive plasma species and pointing to recent diagnostic methods that will contribute to a better understanding of the strong biocidal effect of SMD air plasma.

Utilization of α-amylase enzyme from Bacillus stearothermophilus RSAII1B for maltodextrin production from sago starch

Science.gov (United States)

Arfah, R. A.; Ahmad, A.; Dali, S.; Djide, M. N.; Mahdalia; Arif, A. R.

2018-03-01

The dried sago flour derived from Palopo contains 28.80% amylose and 91.23% total carbohydrate. Based on the data, sago starch has the potential to become an alternative raw material for themaltodextrin production. Maltodextrin is one of the starch derivative products produced by hydrolysis process using the α-amylase enzyme with amaximum DE (dextrose equivalent) value of 20. The use of maltodextrin for food and pharmaceutical industries is increasing because of maltodextrin is widely used as thickener filler, surfactant and sugar substitute in milk powder. The aims of this study are to optimize the addition of enzyme concentration and hydrolysis time of α -amylase enzyme to obtain high quality ofmaltodextrin This study also aimed to characterization the obtained maltodextrin. The first step was isolation and purification α-amylase from the isolate of Bacillus stearothermophilus RSAII1B, followed by determination of the α-amylase concentration (0.05%, 0.07% and 0.09%) in 2.0% starch substrate, and the hydrolysis time ofα-amilase (60, 90, 120, 240 minutes). Maltodextrin characters observed were dextrose equivalent (DE), reducing sugar, moisture content, pH changes, color, solubility, viscosity, and total plate count (TPC). The results showed that the value of DE was 12.31, reducing sugar was 11.4%; water content was 10.92%; pH was 4.85; The color of maltodextrin powder was white bone color; solubility was 153.2 g/L; Viscositywas 210-240 cps, TPCwas 380 cfu/g. Maltodextrins produced from sago starch using the α-amylase enzyme from B.stearothermophillus RSAIIm met the quality requirements of SNI 7599: 2010.

Purification and characterization of an L-arabinose isomerase from an isolated strain of Geobacillus thermodenitrificans producing D-tagatose.

Science.gov (United States)

Kim, Hye-Jung; Oh, Deok-Kun

2005-11-04

The araA gene, encoding l-arabinose isomerase (AI), from the thermophilic bacterium Geobacillus thermodenitrificans was cloned and expressed in Escherichia coli. Recombinant AI was isolated with a final purity of about 97% and a final specific activity of 2.10 U/mg. The molecular mass of the purified AI was estimated to be about 230 kDa to be a tetramer composed of identical subunits. The AI exhibited maximum activity at 70 degrees C and pH 8.5 in the presence of Mn2+. The enzyme was stable at temperatures below 60 degrees C and within the pH range 7.5-8.0. d-Galactose and l-arabinose as substrate were isomerized with high activities. Ribitol was the strongest competitive inhibitor of AI with a Ki of 5.5mM. The apparent Km and Vmax for L-arabinose were 142 mM and 86 U/mg, respectively, whereas those for d-galactose were 408 mM and 6.9 U/mg, respectively. The catalytic efficiency (kcat/Km) was 48 mM(-1)min(-1) for L-arabinose and 0.5mM(-1)min(-1) for D-galactose. Mn2+ was a competitive activator and increased the thermal stability of the AI. The D-tagatose yield produced by AI from d-galactose was 46% without the addition of Mn2+ and 48% with Mn2+ after 300 min at 65 degrees C.

Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain

Energy Technology Data Exchange (ETDEWEB)

Tang, Yinjie J.; Sapra, Rajat; Joyner, Dominique; Hazen, Terry C.; Myers, Samuel; Reichmuth, David; Blanch, Harvey; Keasling, Jay D.

2009-01-20

A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and istolerant to high ethanol concentrations (10percent, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and 13C-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications. Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner?Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions (including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not be accuratelydetermined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on a normalized glucose uptake rate of 100 units (g DCW)-1 h-1) through the TCA cycle and oxidative pentose phosphate pathway were reduced from 64+-3 to 25+-2 and from 30+-2 to 19+-2, respectively. The carbon flux under micro-aerobic growth was directed formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38+-0.07 mol mol-1 glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG reduces the maximum ethanol yieldby approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.

The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

International Nuclear Information System (INIS)

Agarkar, Vinod B.; Kimani, Serah W.; Cowan, Donald A.; Sayed, Muhammed F.-R.; Sewell, B. Trevor

2006-01-01

The amidase from G. pallidus RAPc8, a moderate thermophile, converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned, expressed and purified, and then crystallized using the hanging-drop vapour-diffusion method. The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2 M sodium citrate, 400 mM NaCl, 100 mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96 Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4 2 32, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved

Enantioselectivity and Thermostability of a Novel Hyperhermotolerant Lipase from Geobacillus Thermodenitrificans nr68 (Lip.nr-68) on Secondary Racemic Alcohols Acetylation

Science.gov (United States)

Nik Him, N. R.; Ibrahim, D.

2018-05-01

In our previous work, a new lipase enzyme has been purified from a species identified as a Gram negative Geobacillus thermodenitrificans nr68, isolated from a hot spring in Malaysia with growth temperature of 48°C. This new lipase, called Lip.nr-68 has been characterized as a hyperthermotolerant protein with high stability at 65°C and has been showing excellent characteristics that are very much comparable yet better than some of those of well-known industrially-used lipases. It shows high activity against long-chain triglycerides with molecular weight of the purified enzyme estimated to be 33.5 kDa using SDS-PAGE analysis. This paper is focusing on hyperthermotolerant Lip.nr-68 performance in promoting for enantioselectivity activities towards three secondary racemic alcohols namely 1-phenylethanol, 1-cyclohexilethanol and 1-(naft-2-il) ethanol by acetylation with vinyl acetate. Lip.nr-68 has been confirmed to show high and usual enantioselectivitiy according to the Kazlauskas Rule towards all secondary racemic alcohols and has significantly approved as an enantiomer selective biocatalyst towards 1-phenylethanol and 1-cyclohexylethanol at 65°C. Lip.nr-68 has showed a reduction of (R) and (S) enantiomers as well as the production of 68-98% ee and almost 94% yield of 3-4 mg/ml for 1-cyclohexilethanol.

Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria.

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Shcherbakov, D; Dontsova, M; Tribus, M; Garber, M; Piendl, W

2006-01-01

The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called 'L12 stalk' on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the 'L12 stalk'. The 'L12 stalk' plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.

The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications

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María José Grande Burgos

2014-12-01

Full Text Available Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria. The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.

The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications.

Science.gov (United States)

Grande Burgos, María José; Pulido, Rubén Pérez; Del Carmen López Aguayo, María; Gálvez, Antonio; Lucas, Rosario

2014-12-08

Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica) and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria). The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.

Contamination pathways of spore-forming bacteria in a vegetable cannery.

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Durand, Loïc; Planchon, Stella; Guinebretiere, Marie-Hélène; André, Stéphane; Carlin, Frédéric; Remize, Fabienne

2015-06-02

Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement of Vivarium Biodecontamination through Data-acquisition Systems and Automation.

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Devan, Shakthi Rk; Vasu, Suresh; Mallikarjuna, Yogesha; Ponraj, Ramkumar; Kamath, Gireesh; Poosala, Suresh

2018-03-01

Biodecontamination is important for eliminating pathogens at research animal facilities, thereby preventing contamination within barrier systems. We enhanced our facility's standard biodecontamination method to replace the traditional foggers, and the new system was used effectively after creating bypass ducts in HVAC units so that individual rooms could be isolated. The entire system was controlled by inhouse-developed supervisory control and data-acquisition software that supported multiple cycles of decontamination by equipment, which had different decontamination capacities, operated in parallel, and used different agents, including H2O2 vapor and ClO2 gas. The process was validated according to facility mapping, and effectiveness was assessed by using biologic (Geobacillus stearothermophilus) and chemical indicator strips, which were positioned before decontamination, and by sampling contact plates after the completion of each cycle. The results of biologic indicators showed 6-log reduction in microbial counts after successful decontamination cycles for both agents and found to be compatible with clean-room panels including commonly used materials in vivarium such as racks, cages, trolleys, cage changing stations, biosafety cabinets, refrigerators and other equipment in both procedure and animal rooms. In conclusion, the automated process enabled users to perform effective decontamination through multiple cycles with realtime documentation and provided additional capability to deal with potential outbreaks. Enabling software integration of automation improved quality-control systems in our vivarium.

Sterilization Effect of Wet Oxygen Plasma in the Bubbling Method.

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Tamazawa, Kaoru; Shintani, Hideharu; Tamazawa, Yoshinori; Shimauchi, Hidetoshi

2015-01-01

A new low-temperature sterilization method to replace the ethylene oxide gas sterilization is needed. Strong bactericidal effects of OH and O2H radicals are well known. The purpose of this study was to evaluate the sterilization effect of wet oxygen ("O2+H2O") plasma in the bubbling method, confirming the effect of humidity. Sterility assurance was confirmed by using a biological indicator (Geobacillus stearothermophilus ATCC7953, Namsa, USA). One hundred and eight samples (10(5) spores/carrier) were divided into three groups of 36 in each for treatment with a different type of gas (O2, O2+H2O, Air+H2O). Plasma processing was conducted using a plasma ashing apparatus (13.56 MHz, PACK-3(®), Y. A. C., Japan) under various gas pressures (13, 25, 50 Pa) and gas flows (50, 100, 200 sccm). Fixed plasma treatment parameters were power at 150 W, temperature of 60 ℃, treatment time of 10 min. The samples after treatment were incubated in trypticase soy broth at 58 ℃ for 72 h. The negative culture rate in the "O2+H2O" group was significantly (Mantel-Haenszel procedure, pbubbling method which is the method of introducing vapor into the chamber. The bubbling method seems able to generate OH and O2H radicals in a stable way.

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

International Nuclear Information System (INIS)

Lin, Yen-Chen; Naveen, Vankadari; Hsiao, Chwan-Deng

2016-01-01

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

Energy Technology Data Exchange (ETDEWEB)

Lin, Yen-Chen [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Naveen, Vankadari [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Hsiao, Chwan-Deng, E-mail: hsiao@gate.sinica.edu.tw [Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan (China); Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China)

2016-04-22

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, the path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.

A Duo of Potassium-Responsive Histidine Kinases Govern the Multicellular Destiny of Bacillus subtilis

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de Oña, Paula; Kunert, Maritta; Leñini, Cecilia; Gallegos-Monterrosa, Ramses; Mhatre, Eisha; Vileta, Darío; Hölscher, Theresa; Kuipers, Oscar P.

2015-01-01

ABSTRACT Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host. PMID:26152584

Increased Production of Food-Grade d-Tagatose from d-Galactose by Permeabilized and Immobilized Cells of Corynebacterium glutamicum, a GRAS Host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans.

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Shin, Kyung-Chul; Sim, Dong-Hyun; Seo, Min-Ju; Oh, Deok-Kun

2016-11-02

The generally recognized as safe microorganism Corynebacterium glutamicum expressing Geobacillus thermodenitrificans d-galactose isomerase (d-GaI) was an efficient host for the production of d-tagatose, a functional sweetener. The d-tagatose production at 500 g/L d-galactose by the host was 1.4-fold higher than that by Escherichia coli expressing d-GaI. The d-tagatose-producing activity of permeabilized C. glutamicum (PCG) cells treated with 1% (w/v) Triton X-100 was 2.1-fold higher than that of untreated cells. Permeabilized and immobilized C. glutamicum (PICG) cells in 3% (w/v) alginate showed a 3.1-fold longer half-life at 50 °C and 3.1-fold higher total d-tagatose concentration in repeated batch reactions than PCG cells. PICG cells, which produced 165 g/L d-tagatose after 3 h, with a conversion of 55% (w/w) and a productivity of 55 g/L/h, showed significantly higher d-tagatose productivity than that reported for other cells. Thus, d-tagatose production by PICG cells may be an economical process to produce food-grade d-tagatose.

Sterilization of liquid foods by pulsed electric fields–an innovative ultra-high temperature process

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Reineke, Kai; Schottroff, Felix; Meneses, Nicolas; Knorr, Dietrich

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm−1), skim milk (0.3% fat; 5.3 mS cm−1) and fresh prepared carrot juice (7.73 mS cm−1). The combination of moderate preheating (70–90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105–140°C (measured above the PEF chamber) within 92.2–368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h−1, a frequency of 150 Hz and an energy input of 226.5 kJ kg−1, resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg−1 resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature

Sterilization of liquid foods by pulsed electric fields-an innovative ultra-high temperature process.

Science.gov (United States)

Reineke, Kai; Schottroff, Felix; Meneses, Nicolas; Knorr, Dietrich

2015-01-01

The intention of this study was to investigate the inactivation of endospores by a combined thermal and pulsed electric field (PEF) treatment. Therefore, self-cultivated spores of Bacillus subtilis and commercial Geobacillus stearothermophilus spores with certified heat resistance were utilized. Spores of both strains were suspended in saline water (5.3 mS cm(-1)), skim milk (0.3% fat; 5.3 mS cm(-1)) and fresh prepared carrot juice (7.73 mS cm(-1)). The combination of moderate preheating (70-90°C) and an insulated PEF-chamber, combined with a holding tube (65 cm) and a heat exchanger for cooling, enabled a rapid heat up to 105-140°C (measured above the PEF chamber) within 92.2-368.9 μs. To compare the PEF process with a pure thermal inactivation, each spore suspension was heat treated in thin glass capillaries and D-values from 90 to 130°C and its corresponding z-values were calculated. For a comparison of the inactivation data, F-values for the temperature fields of both processes were calculated by using computational fluid dynamics (CFD). A preheating of saline water to 70°C with a flow rate of 5 l h(-1), a frequency of 150 Hz and an energy input of 226.5 kJ kg(-1), resulted in a measured outlet temperature of 117°C and a 4.67 log10 inactivation of B. subtilis. The thermal process with identical F-value caused only a 3.71 log10 inactivation. This synergism of moderate preheating and PEF was even more pronounced for G. stearothermophilus spores in saline water. A preheating to 95°C and an energy input of 144 kJ kg(-1) resulted in an outlet temperature of 126°C and a 3.28 log10 inactivation, whereas nearly no inactivation (0.2 log10) was achieved during the thermal treatment. Hence, the PEF technology was evaluated as an alternative ultra-high temperature process. However, for an industrial scale application of this process for sterilization, optimization of the treatment chamber design is needed to reduce the occurring inhomogeneous temperature fields.

Cloning and sequencing of the gene coding for alcohol dehydrogenase of Bacillus stearothermophilus and rational shift of the optimum pH.

Science.gov (United States)

Sakoda, H; Imanaka, T

1992-02-01

Using Bacillus subtilis as a host and pTB524 as a vector plasmid, we cloned the thermostable alcohol dehydrogenase (ADH-T) gene (adhT) from Bacillus stearothermophilus NCA1503 and determined its nucleotide sequence. The deduced amino acid sequence (337 amino acids) was compared with the sequences of ADHs from four different origins. The amino acid residues responsible for the catalytic activity of horse liver ADH had been clarified on the basis of three-dimensional structure. Since those catalytic amino acid residues were fairly conserved in ADH-T and other ADHs, ADH-T was inferred to have basically the same proton release system as horse liver ADH. The putative proton release system of ADH-T was elucidated by introducing point mutations at the catalytic amino acid residues, Cys-38 (cysteine at position 38), Thr-40, and His-43, with site-directed mutagenesis. The mutant enzyme Thr-40-Ser (Thr-40 was replaced by serine) showed a little lower level of activity than wild-type ADH-T did. The result indicates that the OH group of serine instead of threonine can also be used for the catalytic activity. To change the pKa value of the putative system, His-43 was replaced by the more basic amino acid arginine. As a result, the optimum pH of the mutant enzyme His-43-Arg was shifted from 7.8 (wild-type enzyme) to 9.0. His-43-Arg exhibited a higher level of activity than wild-type enzyme at the optimum pH.

Improvement of Thermal Stability via Outer-Loop Ion Pair Interaction of Mutated T1 Lipase from Geobacillus zalihae Strain T1

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Mahiran Basri

2012-01-01

Full Text Available Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The Tm for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher Tm as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

NCBI nr-aa BLAST: CBRC-OLAT-20-0007 [SEVENS

Lifescience Database Archive (English)

Full Text Available ry transduction system [Geobacillus thermodenitrificans NG80-2] gb|ABO65713.1| Transcriptional regulatory co...mponent of sensory transduction system [Geobacillus thermodenitrificans NG80-2] YP_001124458.1 5.1 27% ...

Development of eco-friendly process for the production of bioethanol from banana peel using inhouse developed cocktail of thermo-alkali-stable depolymerizing enzymes.

Science.gov (United States)

Prakash, Heena; Chauhan, Prakram Singh; General, Thiyam; Sharma, A K

2018-03-29

Conversion of agro-industrial wastes to energy is an innovative approach for waste valorization and management which also mitigates environmental pollution. In this view, present study investigated the feasibility of producing bioethanol from banana peels using cocktail of depolymerizing enzyme/s. We isolated Geobacillus stearothermophilus HPA19 from natural resource which produces cocktail of thermo-alkali-stable xylano-pectino-cellulolytic enzyme/s using wheat bran within 24 h. The optimal temperature and pH for xylanase, filter paper cellulase and pectinase were 80, 70 and 80 °C, and 9.0, 8.0 and 9.0, respectively. Cocktail enzymes showed stability at high temperature (80 °C) and pH (10.0). Ni 2+ and Zn 2+ promoted the relative activity of xylanase and FPase, whereas Na + , Ca 2+ and K + promoted pectinase activity. Cocktail was assessed in saccharification of banana peel. Reducing sugar obtained (37.06 mg ml -1 ) after one variable at a time (OVAT) method is greatly influenced by enzyme dose. Further, response surface methodology was used to optimize saccharification leading to twofold increase in reducing sugar. Maximum ethanol production (21.1 gl -1 ) was achieved through fermentation giving the efficiency of 76.5% within 30 h. Hence utilization of waste biomass for production of value-added products through biotechnological intervention not only helps to combat environmental pollution but also contributes significantly to the economy.

Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

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Wim Reybroeck

Full Text Available Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Structure of the large terminase from a hyperthermophilic virus reveals a unique mechanism for oligomerization and ATP hydrolysis.

Science.gov (United States)

Xu, Rui-Gang; Jenkins, Huw T; Antson, Alfred A; Greive, Sandra J

2017-12-15

The crystal structure of the large terminase from the Geobacillus stearothermophilus bacteriophage D6E shows a unique relative orientation of the N-terminal adenosine triphosphatase (ATPase) and C-terminal nuclease domains. This monomeric 'initiation' state with the two domains 'locked' together is stabilized via a conserved C-terminal arm, which may interact with the portal protein during motor assembly, as predicted for several bacteriophages. Further work supports the formation of an active oligomeric state: (i) AUC data demonstrate the presence of oligomers; (ii) mutational analysis reveals a trans-arginine finger, R158, indispensable for ATP hydrolysis; (iii) the location of this arginine is conserved with the HerA/FtsK ATPase superfamily; (iv) a molecular docking model of the pentamer is compatible with the location of the identified arginine finger. However, this pentameric model is structurally incompatible with the monomeric 'initiation' state and is supported by the observed increase in kcat of ATP hydrolysis, from 7.8 ± 0.1 min-1 to 457.7 ± 9.2 min-1 upon removal of the C-terminal nuclease domain. Taken together, these structural, biophysical and biochemical data suggest a model where transition from the 'initiation' state into a catalytically competent pentameric state, is accompanied by substantial domain rearrangements, triggered by the removal of the C-terminal arm from the ATPase active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Creation of metal-independent hyperthermophilic L-arabinose isomerase by homologous recombination.

Science.gov (United States)

Hong, Young-Ho; Lee, Dong-Woo; Pyun, Yu-Ryang; Lee, Sung Haeng

2011-12-28

Hyperthermophilic L-arabinose isomerases (AIs) are useful in the commercial production of D-tagatose as a low-calorie bulk sweetener. Their catalysis and thermostability are highly dependent on metals, which is a major drawback in food applications. To study the role of metal ions in the thermostability and catalysis of hyperthermophilic AI, four enzyme chimeras were generated by PCR-based hybridization to replace the variable N- and C-terminal regions of hyperthermophilic Thermotoga maritima AI (TMAI) and thermophilic Geobacillus stearothermophilus AI (GSAI) with those of the homologous mesophilic Bacillus halodurans AI (BHAI). Unlike Mn(2+)-dependent TMAI, the GSAI- and TMAI-based hybrids with the 72 C-terminal residues of BHAI were not metal-dependent for catalytic activity. By contrast, the catalytic activities of the TMAI- and GSAI-based hybrids containing the N-terminus (residues 1-89) of BHAI were significantly enhanced by metals, but their thermostabilities were poor even in the presence of Mn(2+), indicating that the effects of metals on catalysis and thermostability involve different structural regions. Moreover, in contrast to the C-terminal truncate (Δ20 residues) of GSAI, the N-terminal truncate (Δ7 residues) exhibited no activity due to loss of its native structure. The data thus strongly suggest that the metal dependence of the catalysis and thermostability of hyperthermophilic AIs evolved separately to optimize their activity and thermostability at elevated temperatures. This may provide effective target regions for engineering, thereby meeting industrial demands for the production of d-tagatose.

NCBI nr-aa BLAST: CBRC-SARA-01-1309 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-SARA-01-1309 ref|YP_001125845.1| hypothetical protein GTNG_1736 [Geobacillus thermoden...itrificans NG80-2] gb|ABO67100.1| Conserved hypothetical protein [Geobacillus thermodenitrificans NG80-2] YP_001125845.1 0.47 23% ...

NCBI nr-aa BLAST: CBRC-DDIS-05-0159 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-DDIS-05-0159 ref|YP_001124941.1| Amino acid permease [Geobacillus thermodenitr...ificans NG80-2] gb|ABO66196.1| Amino acid permease [Geobacillus thermodenitrificans NG80-2] YP_001124941.1 4e-49 39% ...

NCBI nr-aa BLAST: CBRC-AGAM-07-0029 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-AGAM-07-0029 ref|YP_001126159.1| Lantibiotic mersacidin modifying enzyme [Geobacillus thermoden...itrificans NG80-2] gb|ABO67414.1| Lantibiotic mersacidin modifying enzyme [Geobacillus thermodenitrificans NG80-2] YP_001126159.1 1e-17 24% ...

NCBI nr-aa BLAST: CBRC-ACAR-01-0779 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-ACAR-01-0779 ref|YP_001127455.1| Putative sigma-B regulator [Geobacillus thermoden...itrificans NG80-2] gb|ABO68710.1| Putative sigma-B regulator [Geobacillus thermodenitrificans NG80-2] YP_001127455.1 5.6 32% ...

NCBI nr-aa BLAST: CBRC-LAFR-01-0478 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-LAFR-01-0478 ref|YP_001124451.1| Carbon starvation-induced protein [Geobacillus thermoden...itrificans NG80-2] gb|ABO65706.1| Carbon starvation-induced protein [Geobacillus thermodenitrificans NG80-2] YP_001124451.1 0.40 30% ...

NCBI nr-aa BLAST: CBRC-XTRO-01-0514 [SEVENS

Lifescience Database Archive (English)

Full Text Available modenitrificans NG80-2] gb|ABO66879.1| Branched-chain amino acid ABC transporter (permease) [Geobacillus thermodenitrificans NG80-2] YP_001125624.1 2e-97 62% ... ...CBRC-XTRO-01-0514 ref|YP_001125624.1| Branched-chain amino acid ABC transporter (permease) [Geobacillus ther

NCBI nr-aa BLAST: CBRC-MDOM-01-0519 [SEVENS

Lifescience Database Archive (English)

Full Text Available CBRC-MDOM-01-0519 ref|YP_001126904.1| prophage LambdaBa01, membrane protein, putative [Geobacillus thermoden...itrificans NG80-2] gb|ABO68159.1| Prophage LambdaBa01, membrane protein, putative [Geobacillus thermodenitrificans NG80-2] YP_001126904.1 8e-12 24% ...

New pressure and temperature effects on bacterial spores

Science.gov (United States)

Mathys, A.; Heinz, V.; Knorr, D.

2008-07-01

The mechanism of inactivation of bacterial spores by heat and pressure is still a matter of discussion. Obviously, the change of the dissociation equilibrium under pressure and temperature plays a dominant role in inactivation of microorganisms. Heat and pressure inactivation of Geobacillus. stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed this view. Thermal inactivation in ACES buffer at 122°C resulted in higher logarithmic reductions. Contrary, after pressure treatment at 900 MPa with 80°C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure. Due to preparation, storage and handling of highly concentrated spore suspensions the clumping and the formation of aggregates can hardly be avoided. Consequently, the impact of the agglomeration size distribution on the quantitative assessment of G. stearothermophilus spore inactivation was determined by using a three-fold dynamic optical backreflexion measurement. Two limiting cases have been discriminated in mathematical modelling: three dimensional, spherical packing for maximum spore count and two dimensional, circular packing for minimum spore count of a particular agglomerate. Thermal inactivation studies have been carried out in thin glass capillaries, where by using numerical simulations the non isothermal conditions were modelled and taken into account. It is shown that the shoulder formation often found in thermal spore inactivation can sufficiently be described by first-order inactivation kinetics when the agglomeration size is considered. In case of high pressure inactivation agglomerations could be strongly changed by high forces at compression and especially decompression phase. The physiological response of Bacillus licheniformis spores to high pressure was investigated using multiparameter flow cytometry. Spores were treated by high pressure at 150 MPa with 37�

New pressure and temperature effects on bacterial spores

Energy Technology Data Exchange (ETDEWEB)

Mathys, A; Knorr, D [Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Koenigin-Luise-Str. 22, D-14195 Berlin (Germany); Heinz, V [German Institute of Food Technology, p. o. box 1165, D-49601, Quackenbrueck (Germany)], E-mail: alexander.mathys@tu-berlin.de

2008-07-15

The mechanism of inactivation of bacterial spores by heat and pressure is still a matter of discussion. Obviously, the change of the dissociation equilibrium under pressure and temperature plays a dominant role in inactivation of microorganisms. Heat and pressure inactivation of Geobacillus. stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed this view. Thermal inactivation in ACES buffer at 122 deg. C resulted in higher logarithmic reductions. Contrary, after pressure treatment at 900 MPa with 80 deg. C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure. Due to preparation, storage and handling of highly concentrated spore suspensions the clumping and the formation of aggregates can hardly be avoided. Consequently, the impact of the agglomeration size distribution on the quantitative assessment of G. stearothermophilus spore inactivation was determined by using a three-fold dynamic optical backreflexion measurement. Two limiting cases have been discriminated in mathematical modelling: three dimensional, spherical packing for maximum spore count and two dimensional, circular packing for minimum spore count of a particular agglomerate. Thermal inactivation studies have been carried out in thin glass capillaries, where by using numerical simulations the non isothermal conditions were modelled and taken into account. It is shown that the shoulder formation often found in thermal spore inactivation can sufficiently be described by first-order inactivation kinetics when the agglomeration size is considered. In case of high pressure inactivation agglomerations could be strongly changed by high forces at compression and especially decompression phase. The physiological response of Bacillus licheniformis spores to high pressure was investigated using multiparameter flow cytometry. Spores were treated by high pressure at 150 MPa

New pressure and temperature effects on bacterial spores

International Nuclear Information System (INIS)

Mathys, A; Knorr, D; Heinz, V

2008-01-01

The mechanism of inactivation of bacterial spores by heat and pressure is still a matter of discussion. Obviously, the change of the dissociation equilibrium under pressure and temperature plays a dominant role in inactivation of microorganisms. Heat and pressure inactivation of Geobacillus. stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed this view. Thermal inactivation in ACES buffer at 122 deg. C resulted in higher logarithmic reductions. Contrary, after pressure treatment at 900 MPa with 80 deg. C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure. Due to preparation, storage and handling of highly concentrated spore suspensions the clumping and the formation of aggregates can hardly be avoided. Consequently, the impact of the agglomeration size distribution on the quantitative assessment of G. stearothermophilus spore inactivation was determined by using a three-fold dynamic optical backreflexion measurement. Two limiting cases have been discriminated in mathematical modelling: three dimensional, spherical packing for maximum spore count and two dimensional, circular packing for minimum spore count of a particular agglomerate. Thermal inactivation studies have been carried out in thin glass capillaries, where by using numerical simulations the non isothermal conditions were modelled and taken into account. It is shown that the shoulder formation often found in thermal spore inactivation can sufficiently be described by first-order inactivation kinetics when the agglomeration size is considered. In case of high pressure inactivation agglomerations could be strongly changed by high forces at compression and especially decompression phase. The physiological response of Bacillus licheniformis spores to high pressure was investigated using multiparameter flow cytometry. Spores were treated by high pressure at 150 MPa

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone.

Science.gov (United States)

Uma Maheswar Rao, J L; Satyanarayana, T

2004-01-01

Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.

Comparison of different decontaminant delivery methods for sterilizing unoccupied commercial airliner cabins

Energy Technology Data Exchange (ETDEWEB)

Chen, Xi; Chen, Qingyan [National Air Transport Center of Excellence for Research in the Intermodal Transport Environment (RITE), School of Mechanical Engineering, Purdue University, 585 Purdue Mall, West Lafayette, IN 47905 (United States)

2010-09-15

Effective decontamination is crucial if an airliner cabin is contaminated by biological contaminants, such as infectious disease viruses or intentionally released biological agents. This study used computational fluid dynamics (CFD) method as a tool and vaporized hydrogen peroxide (VHP) as an exemplary decontaminant and Geobacillus stearothermophilus spores as a simulant contaminant to investigate three VHP delivery methods for sterilizing two different airliner cabins. The CFD first determined the airflow and the transient distributions of the contaminant and decontaminant in cabins. Auxiliary equations were implemented into the CFD model for evaluating efficacy of the sterilization process. The improved CFD model was validated by the measured airflow and simulated contaminant distributions obtained from a cabin mockup and the measured efficacy data from the literature. The three decontaminant delivery methods were (1) to supply the mixed VHP and air through the environmental control system of a cabin, (2) to send mixed VHP and air through a front door and to extract them from a back door of a cabin, and (3) to send directly VHP to a cabin and enhance the mixing with air in the cabin by fans. The two air cabins studied were a single-aisle and a twin-aisle airliner one. The results show that the second decontaminant delivery method (displacement method) was the best because the VHP distributions in the cabins were most uniform, the sterilization time was moderate, and the corrosion risk was low. The method displaced the existing air by the air/disinfectant solution, rather than dispersive mixing as the other two methods. (author)

Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

Science.gov (United States)

Zhou, Li; Deng, Can; Cui, Wen-Jing; Liu, Zhong-Mei; Zhou, Zhe-Min

2016-01-01

L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33 °C with a 1-h thermo-induction at 42 °C followed by an oxygen-limited phase at 42 °C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3 g biomass/100 g glucose during the aerobic growth phase and 96 g alanine/100 g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8 g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g/l h, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

A selection that reports on protein-protein interactions within a thermophilic bacterium.

Science.gov (United States)

Nguyen, Peter Q; Silberg, Jonathan J

2010-07-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions.

Isolation of bacteria from mechanic workshops' soil environment ...

African Journals Online (AJOL)

isolation of Bacillus Stearothermophilus (8.3%) and Cyanobacteria (1.7%) from the sites sampled. The number of viable bacterial growth of B. Stearothermophilus and Cyanobacteria were enumerated and expressed in colony forming units. Agbani had bacteria densities of 5 x 104, 1.25 x 104 and 6.25 x 105 from the three ...

Binding site of ribosomal proteins on prokaryotic 5S ribonucleic acids: a study with ribonucleases

DEFF Research Database (Denmark)

Douthwaite, S; Christensen, A; Garrett, R A

1982-01-01

The binding sites of ribosomal proteins L18 and L25 on 5S RNA from Escherichia coli were probed with ribonucleases A, T1, and T2 and a double helix specific cobra venom endonuclease. The results for the protein-RNA complexes, which were compared with those for the free RNA [Douthwaite, S...... stearothermophilus 5S RNA. Several protein-induced changes in the RNA structures were identified; some are possibly allosteric in nature. The two prokaryotic 5S RNAs were also incubated with total 50S subunit proteins from E. coli and B. stearothermophilus ribosomes. Homologous and heterologous reconstitution....... stearothermophilus 5S RNA, which may have been due to a third ribosomal protein L5....

Determination of decimal reduction time (D value) of chemical agents used in hospitals for disinfection purposes

Science.gov (United States)

Mazzola, Priscila Gava; Penna, Thereza Christina Vessoni; da S Martins, Alzira M

2003-01-01

Background Prior to the selection of disinfectants for low, intermediate and high (sterilizing) levels, the decimal reduction time, D-value, for the most common and persistent bacteria identified at a health care facility should be determined. Methods The D-value was determined by inoculating 100 mL of disinfecting solution with 1 mL of a bacterial suspension (104 – 105 CFU/mL for vegetative and spore forms). At regular intervals, 1 mL aliquots of this mixture were transferred to 8 mL of growth media containing a neutralizing agent, and incubated at optimal conditions for the microorganism. Results The highest D-values for various bacteria were determined for the following solutions: (i) 0.1% sodium dichloroisocyanurate (pH 7.0) – E. coli and A. calcoaceticus (D = 5.9 min); (ii) sodium hypochlorite (pH 7.0) at 0.025% for B. stearothermophilus (D = 24 min), E. coli and E. cloacae (D = 7.5 min); at 0.05% for B. stearothermophilus (D = 9.4 min) and E. coli (D = 6.1 min) and 0.1% for B. stearothermophilus (D = 3.5 min) and B. subtilis (D = 3.2 min); (iii) 2.0% glutaraldehyde (pH 7.4) – B. stearothermophilus, B. subtilis (D = 25 min) and E. coli (D = 7.1 min); (iv) 0.5% formaldehyde (pH 6.5) – B. subtilis (D = 11.8 min), B. stearothermophilus (D = 10.9 min) and A. calcoaceticus (D = 5.2 min); (v) 2.0% chlorhexidine (pH 6.2) – B. stearothermophilus (D = 9.1 min), and at 0.4% for E. cloacae (D = 8.3 min); (vi) 1.0% Minncare® (peracetic acid and hydrogen peroxide, pH 2.3) – B. stearothermophilus (D = 9.1 min) and E. coli (D = 6.7 min). Conclusions The suspension studies were an indication of the disinfectant efficacy on a surface. The data in this study reflect the formulations used and may vary from product to product. The expected effectiveness from the studied formulations showed that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasizes the importance and need to develop routine and novel programs to

Determination of decimal reduction time (D value of chemical agents used in hospitals for disinfection purposes

Directory of Open Access Journals (Sweden)

da S Martins Alzira M

2003-10-01

Full Text Available Abstract Background Prior to the selection of disinfectants for low, intermediate and high (sterilizing levels, the decimal reduction time, D-value, for the most common and persistent bacteria identified at a health care facility should be determined. Methods The D-value was determined by inoculating 100 mL of disinfecting solution with 1 mL of a bacterial suspension (104 – 105 CFU/mL for vegetative and spore forms. At regular intervals, 1 mL aliquots of this mixture were transferred to 8 mL of growth media containing a neutralizing agent, and incubated at optimal conditions for the microorganism. Results The highest D-values for various bacteria were determined for the following solutions: (i 0.1% sodium dichloroisocyanurate (pH 7.0 – E. coli and A. calcoaceticus (D = 5.9 min; (ii sodium hypochlorite (pH 7.0 at 0.025% for B. stearothermophilus (D = 24 min, E. coli and E. cloacae (D = 7.5 min; at 0.05% for B. stearothermophilus (D = 9.4 min and E. coli (D = 6.1 min and 0.1% for B. stearothermophilus (D = 3.5 min and B. subtilis (D = 3.2 min; (iii 2.0% glutaraldehyde (pH 7.4 – B. stearothermophilus, B. subtilis (D = 25 min and E. coli (D = 7.1 min; (iv 0.5% formaldehyde (pH 6.5 – B. subtilis (D = 11.8 min, B. stearothermophilus (D = 10.9 min and A. calcoaceticus (D = 5.2 min; (v 2.0% chlorhexidine (pH 6.2 – B. stearothermophilus (D = 9.1 min, and at 0.4% for E. cloacae (D = 8.3 min; (vi 1.0% Minncare® (peracetic acid and hydrogen peroxide, pH 2.3 – B. stearothermophilus (D = 9.1 min and E. coli (D = 6.7 min. Conclusions The suspension studies were an indication of the disinfectant efficacy on a surface. The data in this study reflect the formulations used and may vary from product to product. The expected effectiveness from the studied formulations showed that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasizes the importance and need to develop routine and novel programs to

A comparative study of pectinolytic enzyme production by Bacillus ...

African Journals Online (AJOL)

The effect of temperature on the pectinase of the three isolates showed that B. stearothermophilus had optimum temperature at 60°C while B. cereus and B. subtilis both showed optimum activity at 50°C. On the effect of pH, B. stearothermophilus, B. cereus and B. subtilis showed optimum pectinase activities at pH 7.5, 8.0 ...

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

Science.gov (United States)

Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

2007-07-01

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Analytical strategy for the detection of antibiotic residues in sheep and goat’s milk

International Nuclear Information System (INIS)

Beltrán, M.C.; Althaus, R.L.; Molina, A.; Berruga, M.I.; Molina, M.P.

2015-01-01

The use of antibiotics to treat mastitis and other infectious diseases in dairy sheep and goats is a widespread practice nowadays that can, when not properly applied, result in the contamination of the milk supply. Spanish legislation establishes the control of the presence of antibiotic residues in sheep and goat’s milk using screening methods that detect, at least, beta-lactam drugs. Microbial inhibitor tests using Geobacillus stearothermophilus var. calidolactis and specific receptor-binding assays are most widely employed for this purpose. The detection rates of screening tests routinely used in Spain have been calculated considering the frequency of use of veterinary drugs commonly applied in ovine and caprine livestock to treat and prevent mastitis as well as the test sensitivity toward these substances at safety levels. In general, the use of a single test allows detecting 62.8-82.4% of the antibiotics employed. For sheep milk, the total detection range achieved with microbial tests was significantly higher than that reached with rapid receptor tests. However, no significant differences between the two types of methods were found when goat's milk was analysed. In both types of milk, the simultaneous use of two screening tests with a different analytical basis increases the total detection range significantly, reaching values ≥ 90% in some cases (81.5-90.1% for sheep and 84.7-92.6% for goats). However, the periodical use of screening tests able to detect quinolones, macrolides or aminoglycosides would be recommended to carry out more efficient screening and ensure the safety of milk and dairy products from sheep and goats. (Author)

Analytical strategy for the detection of antibiotic residues in sheep and goat’s milk

Energy Technology Data Exchange (ETDEWEB)

Beltrán, M.C.; Althaus, R.L.; Molina, A.; Berruga, M.I.; Molina, M.P.

2015-07-01

The use of antibiotics to treat mastitis and other infectious diseases in dairy sheep and goats is a widespread practice nowadays that can, when not properly applied, result in the contamination of the milk supply. Spanish legislation establishes the control of the presence of antibiotic residues in sheep and goat’s milk using screening methods that detect, at least, beta-lactam drugs. Microbial inhibitor tests using Geobacillus stearothermophilus var. calidolactis and specific receptor-binding assays are most widely employed for this purpose. The detection rates of screening tests routinely used in Spain have been calculated considering the frequency of use of veterinary drugs commonly applied in ovine and caprine livestock to treat and prevent mastitis as well as the test sensitivity toward these substances at safety levels. In general, the use of a single test allows detecting 62.8-82.4% of the antibiotics employed. For sheep milk, the total detection range achieved with microbial tests was significantly higher than that reached with rapid receptor tests. However, no significant differences between the two types of methods were found when goat's milk was analysed. In both types of milk, the simultaneous use of two screening tests with a different analytical basis increases the total detection range significantly, reaching values ≥ 90% in some cases (81.5-90.1% for sheep and 84.7-92.6% for goats). However, the periodical use of screening tests able to detect quinolones, macrolides or aminoglycosides would be recommended to carry out more efficient screening and ensure the safety of milk and dairy products from sheep and goats. (Author)

Analytical strategy for the detection of antibiotic residues in sheep and goat’s milk

Directory of Open Access Journals (Sweden)

M. Carmen Beltrán

2015-03-01

Full Text Available The use of antibiotics to treat mastitis and other infectious diseases in dairy sheep and goats is a widespread practice nowadays that can, when not properly applied, result in the contamination of the milk supply. Spanish legislation establishes the control of the presence of antibiotic residues in sheep and goat’s milk using screening methods that detect, at least, beta-lactam drugs. Microbial inhibitor tests using Geobacillus stearothermophilus var. calidolactis and specific receptor-binding assays are most widely employed for this purpose. The detection rates of screening tests routinely used in Spain have been calculated considering the frequency of use of veterinary drugs commonly applied in ovine and caprine livestock to treat and prevent mastitis as well as the test sensitivity toward these substances at safety levels. In general, the use of a single test allows detecting 62.8-82.4% of the antibiotics employed. For sheep milk, the total detection range achieved with microbial tests was significantly higher than that reached with rapid receptor tests. However, no significant differences between the two types of methods were found when goat’s milk was analysed. In both types of milk, the simultaneous use of two screening tests with a different analytical basis increases the total detection range significantly, reaching values ≥ 90% in some cases (81.5-90.1% for sheep and 84.7-92.6% for goats. However, the periodical use of screening tests able to detect quinolones, macrolides or aminoglycosides would be recommended to carry out more efficient screening and ensure the safety of milk and dairy products from sheep and goats.

Microbial ecology of two hot springs of Sikkim: Predominate population and geochemistry.

Science.gov (United States)

Najar, Ishfaq Nabi; Sherpa, Mingma Thundu; Das, Sayak; Das, Saurav; Thakur, Nagendra

2018-10-01

Northeastern regions of India are known for their floral and faunal biodiversity. Especially the state of Sikkim lies in the eastern Himalayan ecological hotspot region. The state harbors many sulfur rich hot springs which have therapeutic and spiritual values. However, these hot springs are yet to be explored for their microbial ecology. The development of neo generation techniques such as metagenomics has provided an opportunity for inclusive study of microbial community of different environment. The present study describes the microbial diversity in two hot springs of Sikkim that is Polok and Borong with the assist of culture dependent and culture independent approaches. The culture independent techniques used in this study were next generation sequencing (NGS) and Phospholipid Fatty Acid Analysis (PLFA). Having relatively distinct geochemistry both the hot springs are thermophilic environments with the temperature range of 50-77 °C and pH range of 5-8. Metagenomic data revealed the dominance of bacteria over archaea. The most abundant phyla were Proteobacteria and Bacteroidetes although other phyla were also present such as Acidobacteria, Nitrospirae, Firmicutes, Proteobacteria, Parcubacteria and Spirochaetes. The PLFA studies have shown the abundance of Gram Positive bacteria followed by Gram negative bacteria. The culture dependent technique was correlative with PLFA studies. Most abundant bacteria as isolated and identified were Gram-positive genus Geobacillus and Anoxybacillus. The genus Geobacillus has been reported for the first time in North-Eastern states of India. The Geobacillus species obtained from the concerned hot springs were Geobacillus toebii, Geobacillus lituanicus, Geobacillus Kaustophillus and the Anoxybacillus species includes Anoxybacillus gonensis and Anoxybacillus Caldiproteolyticus. The distribution of major genera and their statistical correlation analyses with the geochemistry of the springs predicted that the temperature, p

Comparative Amino Acids Studies on Phac Synthases and Proteases as Well as Establishing a New Trend in Experimental Design

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Amro Abd al fattah Amara

2012-04-01

Full Text Available ABSTRACT: A question addressed in this study is: why similar enzymes are classified into different subclasses? As an example, PhaC synthases are classified according to four different classes (I, II, III and IV. To answer this question we proposed that besides the catalytic residues, the overall amino acids (AAs present are responsible for the differences observed. The AAs’ composition affects the structure/function/substrate specificity (SFS of these enzymes. The differences between the classes in various PhaC synthases and proteases were analysed to support our argument. Homology and phylogenic tree of some selected PhaC synthases of different strains (representing the four classes were demonstrated. The properties of a specific class of enzyme could not be changed into those of another by changing the catalytic residues. Moreover, these differences could not be detected from the proteins’ 3D structures, despite clear differences at the AAs level. Another question was also addressed: could we benefit from the various existing protein databases in the field of biotechnology? To answer this, we introduced a model for an Experimental Design based on the information in the protein database (for strains available in our lab regarding their ability to degrade castor oil. Two enzymes in the phenol degradation pathway, phenol 2-monooxygenase and catechol 1,2-dioxygenase, and a lipase enzyme were analysed. These enzymes were screened and analysed according to the BLAST-protein database and BRENDA. The comprehensive enzyme information system compared six strains against each other, including: Pseudomonas aeruginosa, Bacillus subtilis, Bacillus pumilus, Bacillus thuringiensis, Bacillus licheniformis, and Geobacillus stearothermophilus. Only P. aeruginosa proved to have the three required enzymes and was suitable for the production of lipases from castor oil (crude castor oil is usually contaminated with phenol as indicated by the databases. In

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions.

Science.gov (United States)

Ploss, Tina N; Reilman, Ewoud; Monteferrante, Carmine G; Denham, Emma L; Piersma, Sjouke; Lingner, Anja; Vehmaanperä, Jari; Lorenz, Patrick; van Dijl, Jan Maarten

2016-03-29

Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M. Here we show that activity of the htrB promoter as induced by overproduction of AmyM was "noisy", which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions. Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

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Shelly J Krebs

Full Text Available Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab responses toward conserved regions of the viral Envelope (Env. However, the generation of neutralizing Abs (NAbs targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

Evaluation of modified stainless steel surfaces targeted to reduce biofilm formation by common milk sporeformers.

Science.gov (United States)

Jindal, Shivali; Anand, Sanjeev; Huang, Kang; Goddard, Julie; Metzger, Lloyd; Amamcharla, Jayendra

2016-12-01

The development of bacterial biofilms on stainless steel (SS) surfaces poses a great threat to the quality of milk and other dairy products as the biofilm-embedded bacteria can survive thermal processing. Established biofilms offer cleaning challenges because they are resistant to most of the regular cleaning protocols. Sporeforming thermoduric organisms entrapped within biofilm matrix can also form heat-resistant spores, and may result in a long-term persistent contamination. The main objective of this study was to evaluate the efficacy of different nonfouling coatings [AMC 18 (Advanced Materials Components Express, Lemont, PA), Dursan (SilcoTek Corporation, Bellefonte, PA), Ni-P-polytetrafluoroethylene (PTFE, Avtec Finishing Systems, New Hope, MN), and Lectrofluor 641 (General Magnaplate Corporation, Linden, NJ)] on SS plate heat exchanger surfaces, to resist the formation of bacterial biofilms. It was hypothesized that modified SS surfaces would promote a lesser amount of deposit buildup and bacterial adhesion as compared with the native SS surface. Vegetative cells of aerobic sporeformers, Geobacillus stearothermophilus (ATCC 15952), Bacillus licheniformis (ATCC 6634), and Bacillus sporothermodurans (DSM 10599), were used to study biofilm development on the modified and native SS surfaces. The adherence of these organisms, though influenced by surface energy and hydrophobicity, exhibited no apparent relation with surface roughness. The Ni-P-PTFE coating exhibited the least bacterial attachment and milk solid deposition, and hence, was the most resistant to biofilm formation. Scanning electron microscopy, which was used to visualize the extent of biofilm formation on modified and native SS surfaces, also revealed lower bacterial attachment on the Ni-P-PTFE as compared with the native SS surface. This study thus provides evidence of reduced biofilm formation on the modified SS surfaces. Copyright © 2016 American Dairy Science Association. Published by Elsevier

Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

Science.gov (United States)

Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

2016-01-01

Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

Destruction of Spores on Building Decontamination Residue in a Commercial Autoclave▿

Science.gov (United States)

Lemieux, P.; Sieber, R.; Osborne, A.; Woodard, A.

2006-01-01

The U.S. Environmental Protection Agency conducted an experiment to evaluate the effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR). The BDR was intended to simulate porous materials removed from a building deliberately contaminated with biological agents such as Bacillus anthracis (anthrax) in a terrorist attack. The purpose of the tests was to assess whether the standard operating procedure for a commercial autoclave provided sufficiently robust conditions to adequately destroy bacterial spores bound to the BDR. In this study we investigated the effects of several variables related to autoclaving BDR, including time, temperature, pressure, item type, moisture content, packing density, packing orientation, autoclave bag integrity, and autoclave process sequence. The test team created simulated BDR from wallboard, ceiling tiles, carpet, and upholstered furniture, and embedded in the BDR were Geobacillus stearothermophilus biological indicator (BI) strips containing 106 spores and thermocouples to obtain time and temperature profile data associated with each BI strip. The results indicated that a single standard autoclave cycle did not effectively decontaminate the BDR. Autoclave cycles consisting of 120 min at 31.5 lb/in2 and 275°F and 75 min at 45 lb/in2 and 292°F effectively decontaminated the BDR material. Two sequential standard autoclave cycles consisting of 40 min at 31.5 lb/in2 and 275°F proved to be particularly effective, probably because the second cycle's evacuation step pulled the condensed water out of the pores of the materials, allowing better steam penetration. The results also indicated that the packing density and material type of the BDR in the autoclave could have a significant impact on the effectiveness of the decontamination process. PMID:17012597

Destruction of spores on building decontamination residue in a commercial autoclave.

Science.gov (United States)

Lemieux, P; Sieber, R; Osborne, A; Woodard, A

2006-12-01

The U.S. Environmental Protection Agency conducted an experiment to evaluate the effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR). The BDR was intended to simulate porous materials removed from a building deliberately contaminated with biological agents such as Bacillus anthracis (anthrax) in a terrorist attack. The purpose of the tests was to assess whether the standard operating procedure for a commercial autoclave provided sufficiently robust conditions to adequately destroy bacterial spores bound to the BDR. In this study we investigated the effects of several variables related to autoclaving BDR, including time, temperature, pressure, item type, moisture content, packing density, packing orientation, autoclave bag integrity, and autoclave process sequence. The test team created simulated BDR from wallboard, ceiling tiles, carpet, and upholstered furniture, and embedded in the BDR were Geobacillus stearothermophilus biological indicator (BI) strips containing 10(6) spores and thermocouples to obtain time and temperature profile data associated with each BI strip. The results indicated that a single standard autoclave cycle did not effectively decontaminate the BDR. Autoclave cycles consisting of 120 min at 31.5 lb/in2 and 275 degrees F and 75 min at 45 lb/in2 and 292 degrees F effectively decontaminated the BDR material. Two sequential standard autoclave cycles consisting of 40 min at 31.5 lb/in2 and 275 degrees F proved to be particularly effective, probably because the second cycle's evacuation step pulled the condensed water out of the pores of the materials, allowing better steam penetration. The results also indicated that the packing density and material type of the BDR in the autoclave could have a significant impact on the effectiveness of the decontamination process.

ORF Alignment: NC_006510 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available tor ... [Geobacillus kaustophilus HTA426] ... Length = 108 ... Query: 293 IVRRARQYIQKHFSQSDLTLESV...AQFLNVSPVYLSRIIKQELGISFIHLLAKTRMEKAAE 352 ... IVRRARQYIQKHFSQSDLTLESVAQFLN...VSPVYLSRIIKQELGISFIHLLAKTRMEKAAE Sbjct: 1 ... IVRRARQYIQKHFSQSDLTLESVAQFLNVSPVYLSRIIKQELGISFIHLLAKTRMEKAAE 60 ...

Screening and characterization of thermo-active enzymes of biotechnological interest produced by thermophilic Bacillus isolated from hot springs in Tunisia.

Science.gov (United States)

Thebti, Wajdi; Riahi, Yosra; Gharsalli, Rawand; Belhadj, Omrane

2016-01-01

As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest, we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 - proteases, 43 - cellulases, 31 - xylanases and 37 - mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geobacillus toebii.

ORF Alignment: NC_006510 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available ... [Geobacillus kaustophilus HTA426] ... Length = 152 ... Query: 1 ... MRAVIQRAKEAKVTVGGEVVGAIDAGFVVLLGITHEDTEDDA...AYLAEKIAHLRVFEDEDG 60 ... MRAVIQRAKEAKVTVGGEVVGAIDAGFVVLLGITHEDTEDDA...AYLAEKIAHLRVFEDEDG Sbjct: 1 ... MRAVIQRAKEAKVTVGGEVVGAIDAGFVVLLGITHEDTEDDAAYLAEKIAHLRVFEDEDG 60 ... Query: 1

Global transport of thermophilic bacteria in atmospheric dust.

Science.gov (United States)

Perfumo, Amedea; Marchant, Roger

2010-04-01

Aerosols from dust storms generated in the Sahara-Sahel desert area of Africa are transported north over Europe and periodically result in dry dust precipitation in the Mediterranean region. Samples of dust collected in Turkey and Greece following two distinct desert storm events contained viable thermophilic organisms of the genus Geobacillus, namely G. thermoglucosidasius and G. thermodenitrificans, and the recently reclassified Aeribacillus pallidus (formerly Geobacillus pallidus). We present here evidence that African dust storms create an atmospheric bridge between distant geographical regions and that they are also probably the source of thermophilic geobacilli later deposited over northern Europe by rainfall or dust plumes themselves. The same organisms (99% similarity in the 16S rDNA sequence) were found in dust collected in the Mediterranean region and inhabiting cool soils in Northern Ireland. This study also contributes new insights to the taxonomic identification of Geobacillus sp. Attempts to identify these organisms using 16S rRNA gene sequences have revealed that they contain multiple and diverse copies of the ribosomal RNA operon (up to 10 copies with nine different sequences), which dictates care in interpreting data about the systematics of this genus. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

Esterilização em Ortodontia: eficácia do esterilizador com esferas de vidro Sterilization in Orthodontics: effectiveness of glass bead sterilizers

Directory of Open Access Journals (Sweden)

Sônia Rodrigues Dutra

2008-08-01

Full Text Available OBJETIVO: avaliar a efetividade do esterilizador com esferas de vidro Steri® 350, quanto ao controle de infecção das partes ativas dos alicates ortodônticos. METODOLOGIA: foram utilizados nove alicates ortodônticos, previamente esterilizados em autoclave à temperatura de 121ºC, durante 20 minutos. Posteriormente, as partes ativas dos alicates foram contaminadas com Bacillus stearothermophilus. Logo após, as pontas dos alicates foram colocadas no Steri® 350, durante os períodos de 3, 5, 10, 15, 20, 30 e 40 segundos, em temperatura de 255ºC, para avaliar a eficácia da esterilização. RESULTADOS E CONCLUSÕES: o esterilizador com esferas de vidro mostrou-se eficaz no controle do crescimento de Bacillus stearothermophilus nas partes ativas dos alicates ortodônticos, a partir de 10 segundos de exposição à temperatura de 255ºC.AIM: The aim of this research was to evaluate the efficiency of Steri® 350glass bead sterilizer, on the infection control of the active part of the orthodontic pliers. METHODS: Nine orthodontic pliers were sterilized in the steam autoclave at 121ºC in 20 minutes and subsequently the active part of each plier was dipped in a culture for Bacillus stearothermophilus. Later, the active part of each plier was putted into the Steri® 350 in the following periods: 3, 5, 10, 15, 20, 30, and 40 seconds at 255ºC to evaluate the efficiency of this method in the infection control. RESULTS AND CONCLUSIONS: This sterilization method has been effective to control the presence of Bacillus stearothermophilus on the active part of orthodontic pliers at 255ºC in 10 seconds.

Fluorescent S-layer fusion proteins

International Nuclear Information System (INIS)

Kainz, B.

2010-01-01

This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 ±0.5 nm, b=14 ±0.4 nm, g=80 ±1 o ). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 - pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 ±0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology. (author) [de

Heat Melt Compaction as an Effective Treatment for Eliminating Microorganisms from Solid Waste

Science.gov (United States)

Hummerick, Mary P.; Strayer, Richard F.; McCoy, Lashelle E.; Richards, Jeffrey T.; Ruby, Anna Maria; Wheeler, Ray; Fisher, John

2013-01-01

One of the technologies being tested at Ames Research Center as part of the logistics and repurposing project is heat melt compaction (HMC) of solid waste to reduce volume, remove water and render a biologically stable and safe product. Studies at Kennedy Space Center have focused on the efficacy of the heat melt compaction process for killing microorganisms in waste and specific compacter operation protocols, i.e., time and temperature required to achieve a sterile, stable product. The work. reported here includes a controlled study to examine the survival and potential re-growth of specific microorganisms over a 6-month period of storage after heating and compaction. Before heating and compaction, ersatz solid wastes were inoculated with Bacillus amyloliquefaciens and Rhodotorula mucilaginosa, previously isolated from recovered space shuttle mission food and packaging waste. Compacted HMC tiles were sampled for microbiological analysis at time points between 0 and 180 days of storage in a controlled environment chamber. In addition, biological indicator strips containing spores of Bacillus atrophaeus and Geobacillus stearothermophilus were imbedded in trash to assess the efficacy of the HMC process to achieve sterilization. Analysis of several tiles compacted at 180deg C for times of 40 minutes to over 2 hours detected organisms in all tile samples with the exception of one exposed to 180deg C for approximately 2 hours. Neither of the inoculated organisms was recovered, and the biological indicator strips were negative for growth in all tiles indicating at least local sterilization of tile areas. The findings suggest that minimum time/temperature combination is required for complete sterilization. Microbial analysis of tiles processed at lower temperatures from 130deg C-150deg C at varying times will be discussed, as well as analysis of the bacteria and fungi present on the compactor hardware as a result of exposure to the waste and the surrounding environment

Isolation and Phylogenetic Analysis of Thermophile Community Within Tanjung Sakti Hot Spring, South Sumatera, Indonesia

Directory of Open Access Journals (Sweden)

Heni Yohandini

2015-07-01

Full Text Available A community of thermophiles within Tanjung Sakti Hot Spring (South Sumatera have been cultivated and identified based on 16S ribosomal RNA gene sequence. The hot spring has temperature 80 °C–91 °C and pH 7–8. We used a simple method for culturing the microbes, by enriching the spring water with nutrient broth media. Phylogenetic analysis showed that the method could recover microbes, which clustered within four distinct taxonomic groups: Anoxybacillus, Geobacillus, Brevibacillus, and Bacillus. These microbes closely related to Anoxybacillus rupiensis, Anoxybacillus flavithermus, Geobacillus pallidus, Brevibacillus thermoruber, Bacillus licheniformis, and Bacillus thermoamylovorans. The 16S ribosomal RNA gene sequence of one isolate only had 96% similarity with Brevibacillus sequence in GenBank.

A Novel Spectroscopic Methodology for the Investigation of Individual Bacillus Spores

National Research Council Canada - National Science Library

Alexander, Troy A; Pellegrino, Paul; Gillespie, James B

2005-01-01

A methodology has been developed for the investigation of bacterial spores. Specifically, this method has been used to probe the spore coat composition of two different Bacillus stearothermophilus variants...

ORF Alignment: NC_006509 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_006509 gi|56410456 >1n62C 1 286 1 287 4e-70 ... ref|YP_145830.1| nicotine dehydrog...enase chain A [Geobacillus kaustophilus HTA426] ... dbj|BAD74262.1| nicotine dehydrogenase chain A ...

Cloning and characterization of a carboxylesterase from Bacillus coagulans 81-11

CSIR Research Space (South Africa)

Mnisi, SM

2005-04-01

Full Text Available significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-XG included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p...

Production and characterization of lipase from Bacillus ...

African Journals Online (AJOL)

Jane

2011-10-10

Oct 10, 2011 ... properties of fats at high temperature and increased ..... Effect of growth medium pH on lipase activity, protein concentration and B. stearothermophilus growth. .... inactivation after 30 min of incubation in 10 mM Cu+2 ions.

Aerobic Denitrification as an Innovative Method for In-Situ Biological Remediation of Contaminated Subsurface Sites

Science.gov (United States)

1989-01-01

roseus Bacillus stearothermophilus Micrococcus varians Paracoccus denitrificans Bacillus coagulans Paracoccus halodenitrificans Bacillus flrmus Bacillus ...Geodermatophilus Plesiomonas Arachnia Haemophilus Propionibacterium Arthrobacter Halobacteriua Proteus Bacillus Ifalocuccus Pseudoraonas Bacteroides...Mycobacterium peregrinum Nocardia opaca Chromobacterium violaceum Bacillus subtllis Nocardia atlantica Bacillus licheniformis Flavobacterium

Comprehensive microbial analysis of combined mesophilic anaerobic-thermophilic aerobic process treating high-strength food wastewater

DEFF Research Database (Denmark)

Jang, Hyun Min; Ha, Jeong Hyub; Park, Jong Moon

2015-01-01

of phylum Actinobacteria in both R1 and R2, and a predominance of phyla Synergistetes and Firmicutes in R3 during Run II. Furthermore, R1 and R2 shared genera (Prevotella, Aminobacterium, Geobacillus and Unclassified Actinobacteria), which suggests synergy between mesophilic anaerobic digestion...

77 FR 48165 - Cooperative Research and Development Agreement (CRADA) Opportunity With the Department of...

Science.gov (United States)

2012-08-13

... perform in-place decontamination of heating, ventilation, and cooling (HVAC) HEPA filtration systems. The... target agents (FMDV and ASFV) and test microorganisms (Bacillus subtilis, Vaccinia Virus, Geobacillius stearothermophilus, and potentially other commercially available spore strips) will be used to test the efficacy of...

The Efficacy of Ultraviolet Radiation for Sterilizing Tools Used for Surgically Implanting Transmitters into Fish

Energy Technology Data Exchange (ETDEWEB)

Walker, Ricardo W.; Markillie, Lye Meng; Colotelo, Alison HA; Gay, Marybeth E.; Woodley, Christa M.; Brown, Richard S.

2013-02-28

Telemetry is frequently used to examine the behavior of fish, and the transmitters used are normally surgically implanted into the coelom of fish. Implantation requires the use of surgical tools such as scalpels, forceps, needle holders, and sutures. When several fish are implanted consecutively for large telemetry studies, it is common for surgical tools to be sterilized or, at minimum, disinfected between each use so that pathogens that may be present are not spread among fish. However, autoclaving tools can take a long period of time, and chemical sterilants or disinfectants can be harmful to both humans and fish and have varied effectiveness. Ultraviolet (UV) radiation is commonly used to disinfect water in aquaculture facilities. However, this technology has not been widely used to sterilize tools for surgical implantation of transmitters in fish. To determine its efficacy for this application, Pacific Northwest National Laboratory researchers used UV radiation to disinfect surgical tools (i.e., forceps, needle holder, stab scalpel, and suture) that were exposed to one of four aquatic organisms that typically lead to negative health issues for salmonids. These organisms included Aeromonas salmonicida, Flavobacterium psychrophilum, Renibacterium salmoninarum, and Saprolegnia parasitica. Surgical tools were exposed to the bacteria by dipping them into a confluent suspension of three varying concentrations (i.e., low, medium, high). After exposure to the bacterial culture, tools were placed into a mobile Millipore UV sterilization apparatus. The tools were then exposed for three different time periods—2, 5, or 15 min. S. parasitica, a water mold, was tested using an agar plate method and forceps-pinch method. UV light exposures of 5 and 15 min were effective at killing all four organisms. UV light was also effective at killing Geobacillus stearothermophilus, the organism used as a biological indicator to verify effectiveness of steam sterilizers. These

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry

DEFF Research Database (Denmark)

Kirpekar, F; Douthwaite, S; Roepstorff, P

2000-01-01

RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus...... that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated......, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here....

EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

NARCIS (Netherlands)

EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

1992-01-01

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

Microwave oven irradiation as a method for bacterial decontamination in a clinical microbiology laboratory

International Nuclear Information System (INIS)

Latimer, J.M.; Matsen, J.M.

1977-01-01

Exposure of 10 frequently isolated clinical pathogens to microwave irradiation resulted in total sterilization with 60 s. Time exposure experiments done with commercially prepared test strips containing Bacillus stearothermophilus spores indicated that 5-min exposure was adequate to ensure sterility of small, contaminated loads

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir

OpenAIRE

Sevasti Filippidou; Marion Jaussi; Thomas Junier; Tina Wunderlin; Nicole Jeanneret; Simona Regenspurg; Po-E Li; Chien-Chi Lo; Shannon Johnson; Kim McMurry; Cheryl D. Gleasner; Momchilo Vuyisich; Patrick S. Chain; Pilar Junier

2015-01-01

The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera.

Nutritional optimization for anaerobic growth of Bacillus steaothermophilus LLD-16

Directory of Open Access Journals (Sweden)

Muhammad Javed

2016-04-01

Full Text Available In this study, a range of nutritional supplements including twenty amino acids, major vitamins and four nucleic acid bases were exploited as added-value supplements for the growth of a lactate-minus (ldh mutant Bacillus stearothermophilus LLD-16 under anaerobic environment. The chemostat studies revealed that five amino acids that includes aspartate, glutamate, isoleucine, methionine, and serine were essential for persuaded growth of B. stearothermophilus LLD-16. The anaerobic batch studies showed that a number of nutritional supplements, such as, p-aminobenzoic acid (PABA, folic acid, pantothenic acid, adenine, glycine, leucine, tryptophan, proline, alanine and α-ketoglutarate, when added individually, improved the biomass levels. In contrast, the higher concentrations of cyanocobalamine or biotin, guanine, uracil and isoleucine were found inhibitory. Furthermore, the study explains why the highest biomass formation cannot necessarily be achieved on the richest mixture of amino acids, and the inadequacy of the biosynthetic machinery is very much dependent on the growth conditions of the microorganism.

Détection biologique des résidus d'antibiotiques dans le lait et ...

African Journals Online (AJOL)

SARAH

Published online at www.m.elewa.org on 31st March 2015 ... supermarkets then analyzed by the method using Bacillus subtilis ATCC 6633 and Geobacillus ..... children with and without diarrhea in Burkina ... from “zoom-koom” beverage and ice in ... residues in market milk. ... environment: Food safety and public health.

Smallpox and pan-Orthodox Virus Detection by Real-Time 3’-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms

Science.gov (United States)

2003-11-08

Bacillus anthracis BA0068 Ames Sterne SPS 97.13.213 Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus macerans Bacillus ...megaterium Bacillus polymyxa Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis subsp. niger Bacillus thuringiensis Bacillus popilliae...varicella- zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving:

Determination of kinetic parameters of α-amylase producing ...

African Journals Online (AJOL)

GREGO

2007-03-19

Mar 19, 2007 ... Bernfeld P (1955). Amylases alpha and beta. Methods in enzymology. Academic Press, New York, pp. 140-146. Brigidi P, González-Vera A, Rossi M (1997). Study of stability of recombinant plasmids during the continuous culture of Bacillus stearothermophilus NUB3621 in nonselective medium. Biotechnol.

Genome Sequence of Aeribacillus pallidus Strain GS3372, an Endospore-Forming Bacterium Isolated in a Deep Geothermal Reservoir.

Science.gov (United States)

Filippidou, Sevasti; Jaussi, Marion; Junier, Thomas; Wunderlin, Tina; Jeanneret, Nicole; Regenspurg, Simona; Li, Po-E; Lo, Chien-Chi; Johnson, Shannon; McMurry, Kim; Gleasner, Cheryl D; Vuyisich, Momchilo; Chain, Patrick S; Junier, Pilar

2015-08-27

The genome of strain GS3372 is the first publicly available strain of Aeribacillus pallidus. This endospore-forming thermophilic strain was isolated from a deep geothermal reservoir. The availability of this genome can contribute to the clarification of the taxonomy of the closely related Anoxybacillus, Geobacillus, and Aeribacillus genera. Copyright © 2015 Filippidou et al.

The role of heat resistance in thermorestoration of hydrated bacterial spores

International Nuclear Information System (INIS)

Friedman, Y.S.; Grecz, N.

1973-01-01

This study for the first time presents evidence of the distinct role played in thermorestoration by cellular determinants such as the resistance to heat and radiation, and the ionic state of spores. In the past only radiochemical determinants associated with radical annealment have been studied in hydrated systems. The basic heat resistance of spores plays a significant role in the precipitous drop in spore survival due to 0.45 Mrad radiation plus heat above 65-75 0 C for B.cereus and 75-95 0 C for B.stearothermophilus. The effect of the spores radiation resistance was not distinct except in the frozen state and at the saturation plateau of thermorestoration where the radiation resistant B.cereus showed ca. 1 log cycle higher survival than the radiation sensitive B.stearothermophilus. When spores are chemically converted into their H + and Ca ++ ionic forms, the H + spores are distinctly more responsive than Ca ++ spores to processes of radical annealment responsible for thermorestoration in hydrated spore systems. At temperatures of extensive thermorestoration of water radicals, H + spores showed higher survival than Ca ++ spores. (F.J.)

Microbiological evaluation of the steam sterilization of assembled laparoscopic instruments.

Science.gov (United States)

Camargo, Tamara Carolina de; Graziano, Kazuko Uchikawa; Almeida, Alda Graciele Claudio Dos Santos; Suzuki, Karina; Silva, Cely Barreto da; Pinto, Flávia Morais Gomes

2016-11-21

de caseína soja, incubado a 56oC por 21 dias. Não havendo crescimento, foram submetidos a um choque térmico de 80oC, por 20 minutos e reincubados por 72 horas. Tamanho da amostra, 185 pinças e 185 trocartes, com poder de 95%. Os experimentos foram acompanhados dos grupos controle negativo comparativo (5 pinças e 5 trocartes com contaminação desafio, esterilizados desmontados) e positivo (30 suportes de papel filtro, não esterilizados), submetidos aos mesmos procedimentos de incubação. não houve nenhum crescimento microbiano nos grupos experimental e controle negativo. Os resultados do controle positivo foram satisfatórios. este estudo forneceu fortes evidências científicas para sustentar a segurança da prática de esterilização a vapor do instrumental laparoscópico montado. evaluar la seguridad de la esterilización a través de vapor, de instrumental laparoscópico previamente montado con desafío de contaminación. estudio experimental en laboratorio, cuyo cuerpo de prueba fueron trócarte y pinza laparoscópica. Se utilizó esporas Geobacillus stearothermophilus ATCC-7953, con población microbiana de 106UFC/soporte de papel filtro, removidos del indicador biológico. Tres de ellos fueron introducidos en el interior de cada instrumento, en el momento del montaje, los que fueron esterilizados a vapor saturado bajo presión, 134oC por 5 minutos. Después de la esterilización, el instrumental fue desmontado y cada soporte de papel filtro fue inoculado en medio de una cultura de caseína y soya, incubado a 56oC por 21 días. No habiendo crecimiento, fueron sometidos a un choque térmico de 80oC, por 20 minutos y nuevamente incubados por 72 horas. La muestra estuvo constituida por 185 pinzas y 185 trócartes, con poder de 95%. Los experimentos fueron acompañados en los grupos: control negativo comparativo (5 pinzas y 5 trócartes con contaminación desafío, esterilizados desmontados) y positivo (30 soportes de papel filtro, no esterilizados

The evaluation of consolidated bioprocessing as a strategy for production of fuels and chemicals from lignocellulose

OpenAIRE

Hussein, Ali

2015-01-01

Cellulosic biomass is one of the most abundant industrial waste products and an appealing substrate for biorefining strategies to produce biofuels by fermentation. The metabolic engineering of fermentative bacteria, such as the thermophile Geobacillus thermoglucosidasius, for high bioethanol yield is well characterised. This has been traditionally facilitated by an economically inefficient multistep process referred to as separate hydrolysis and fermentation (SHF), in which the enzymatic hydr...

Lignocellulolytic capacities of Geobacillus thermodenitrificans: towards consolidated bioprocessing

NARCIS (Netherlands)

Daas, Martinus J.A.

2017-01-01

The growing demand for consumables and energy, combined with increasing consciousness over environmental issues like global warming, faces us with the challenge to find alternatives for fossil resources. Alternative production methods for energy, like windmills, solar panels and hydroelectricity

Improving the Efficiency of New Automatic Dishwashing Detergent Formulation by Addition of Thermostable Lipase, Protease and Amylase

Directory of Open Access Journals (Sweden)

Ashwini Naganthran

2017-09-01

Full Text Available The use of T1 lipase in automatic dishwashing detergent (ADD is well established, but efficiency in hard water is very low. A new enzymatic environmentally-friendly dishwashing was formulated to be efficient in both soft and hard water. Thermostable enzymes such as T1 lipase from Geobacillus strain T1, Rand protease from Bacillus subtilis strain Rand, and Maltogenic amylase from Geobacillus sp. SK70 were produced and evaluated for an automatic dishwashing detergent formulation. The components of the new ADD were optimized for compatibility with these three enzymes. In compatibility tests of the enzymes with different components, several criteria were considered. The enzymes were mostly stable in non-ionic surfactants, especially polyhydric alcohols, Glucopon UP 600, and in a mixture of sodium carbonate and glycine (30:70 buffer at a pH of 9.25. Sodium polyacrylate and sodium citrate were used in the ADD formulation as a dispersing agent and a builder, respectively. Dishwashing performance of the formulated ADDs was evaluated in terms of percent of soil removed using the Leenert‘s Improved Detergency Tester. The results showed that the combination of different hydrolysis enzymes could improve the washing efficiency of formulated ADD compared to the commercial ADD “Finish” at 40 and 50 C.

Symmetric Allosteric Mechanism of Hexameric Escherichia coli Arginine Repressor Exploits Competition between L-Arginine Ligands and Resident Arginine Residues

Czech Academy of Sciences Publication Activity Database

Strawn, R.; Melicherčík, Milan; Green, M.; Stockner, T.; Carey, J.; Ettrich, Rüdiger

2010-01-01

Roč. 6, č. 6 (2010), s. 1-12 ISSN 1553-734X R&D Projects: GA MŠk(CZ) LC06010; GA ČR GAP207/10/1934 Institutional research plan: CEZ:AV0Z60870520 Keywords : molecular-dynamics simulations * free-energy calculations * structural basis * DNA-binding domain * bacillus-stearothermophilus * T4 lysozyme * proteins * hemoglobin * model * affinity Subject RIV: EH - Ecology, Behaviour Impact factor: 5.515, year: 2010

Microbial System for Identification of Antibiotic Residues in Milk

OpenAIRE

Nagel, Orlando Guillermo; Molina Pons, Mª Pilar; Althaus, Rafael Lisandro

2011-01-01

[EN] The aim of this study was to evaluate the ResScreen (R) microbiological system for the identification of antibiotic residues in milk. This microbiological system consists of two methods, the BT (betalactams and tetracyclines) and BS (betalactams and sulfamides) bioassays, containing spores of G. stearothermophilus subsp. calidolactis, culture media and indicators (acid-base and redox). The detection limits of 29 antimicrobial agents were calculated using a logistic regression model. ...

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming β-cyano-L-alanine

International Nuclear Information System (INIS)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru; Kobayashi, Michihiko; Shimizu, Sakayu

2003-01-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable β-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of β-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various β-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the β-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the β-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed β-cyano-L-alanine synthase. Heat stable β-cyano-L-alanine synthase can be applied to the synthesis of [4- 11 C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

Purification, characterization and gene cloning of thermostable O-acetyl-L-serine sulfhydrylase forming {beta}-cyano-L-alanine

Energy Technology Data Exchange (ETDEWEB)

Omura, Hironori; Yoshida, Toyokazu; Nagasawa, Toru [Gifu Univ. (Japan). Dept. of Biomolecular Science; Kuroda, Masako [Ikeda Food Research Co., Ltd., Fukuyama, Hiroshima (Japan); Kobayashi, Michihiko; Shimizu, Sakayu [Kyoto Univ. (Japan). Agricultural Sciences

2003-10-01

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable {beta}-cyano-L-alanine synthase. The enzyme catalyzes the synthesis of {beta}-cyano-L-alanine from O-acetyl-L-serine and cyanide ions. The purified enzyme has a molecular mass of approximately 70 kDa and consists of two identical sub-units. It was stable in the pH range of 6.0 to 10.0 and up to 70degC. The enzyme also catalyzes the synthesis of various {beta}-substituted-L-alanine derivatives from O-acetyl-L-serine and nucleophilic reagents. The gene encoding the {beta}-cyano-L-alanine synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the {beta}-cyano-L-alanine synthase of the bacterium is O-acetyl-L-serine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli JM109. The transformed E. coli cells overexpressed {beta}-cyano-L-alanine synthase. Heat stable {beta}-cyano-L-alanine synthase can be applied to the synthesis of [4-{sup 11}C]L-2,4-diaminobutyric acid as a tracer for positron emission tomography. (author)

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

OpenAIRE

Hansen, M A; Kirpekar, F; Ritterbusch, W; Vester, B

2002-01-01

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-...

食品細菌に対する香辛料エキスの抗菌性

OpenAIRE

Liu, Zhi-He; Nakano, Hiroyuki

1996-01-01

The present study was designed to identify spices and herbs that possess antibacterial activity and to apply for control of microorganisms in food. Alcoholic extracts of seventeen spices and five herbs were prepared and examined for growth inhibition of several kinds of food-related bacteria in culture media. Bacillus stearothermophilus, which produces heat-resistant spores, was highly sensitive to most of the spices tested. Both germination of spores and outgrowth of vegetative cells of this...

The crystal structure of a multifunctional protein: Phosphoglucose isomerase/autocrine motility factor/neuroleukin

OpenAIRE

Sun, Yuh-Ju; Chou, Chia-Cheng; Chen, Wei-Shone; Wu, Rong-Tsun; Meng, Menghsiao; Hsiao, Chwan-Deng

1999-01-01

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm tha...

Enrichment dynamics of Listeria monocytogenes and the associated microbiome from naturally contaminated ice cream linked to a listeriosis outbreak.

Science.gov (United States)

Ottesen, Andrea; Ramachandran, Padmini; Reed, Elizabeth; White, James R; Hasan, Nur; Subramanian, Poorani; Ryan, Gina; Jarvis, Karen; Grim, Christopher; Daquiqan, Ninalynn; Hanes, Darcy; Allard, Marc; Colwell, Rita; Brown, Eric; Chen, Yi

2016-11-16

Microbiota that co-enrich during efforts to recover pathogens from foodborne outbreaks interfere with efficient detection and recovery. Here, dynamics of co-enriching microbiota during recovery of Listeria monocytogenes from naturally contaminated ice cream samples linked to an outbreak are described for three different initial enrichment formulations used by the Food and Drug Administration (FDA), the International Organization of Standardization (ISO), and the United States Department of Agriculture (USDA). Enrichment cultures were analyzed using DNA extraction and sequencing from samples taken every 4 h throughout 48 h of enrichment. Resphera Insight and CosmosID analysis tools were employed for high-resolution profiling of 16S rRNA amplicons and whole genome shotgun data, respectively. During enrichment, other bacterial taxa were identified, including Anoxybacillus, Geobacillus, Serratia, Pseudomonas, Erwinia, and Streptococcus spp. Surprisingly, incidence of L. monocytogenes was proportionally greater at hour 0 than when tested 4, 8, and 12 h later with all three enrichment schemes. The corresponding increase in Anoxybacillus and Geobacillus spp.indicated these taxa co-enriched in competition with L. monocytogenes during early enrichment hours. L. monocytogenes became dominant after 24 h in all three enrichments. DNA sequences obtained from shotgun metagenomic data of Listeria monocytogenes at 48 h were assembled to produce a consensus draft genome which appeared to have a similar tracking utility to pure culture isolates of L. monocytogenes. All three methods performed equally well for enrichment of Listeria monocytogenes. The observation of potential competitive exclusion of L. mono by Anoxybacillus and Geobacillus in early enrichment hours provided novel information that may be used to further optimize enrichment formulations. Application of Resphera Insight for high-resolution analysis of 16S amplicon sequences accurately identified L. monocytogenes

Complete Genome Sequence of Paenibacillus strain Y4.12MC10, a Novel Paenibacillus lautus strain Isolated from Obsidian Hot Spring in Yellowstone National Park.

Science.gov (United States)

Mead, David A; Lucas, Susan; Copeland, Alex; Lapidus, Alla; Cheng, Jan-Feng; Bruce, David C; Goodwin, Lynne A; Pitluck, Sam; Chertkov, Olga; Zhang, Xiaojing; Detter, John C; Han, Cliff S; Tapia, Roxanne; Land, Miriam; Hauser, Loren J; Chang, Yun-Juan; Kyrpides, Nikos C; Ivanova, Natalia N; Ovchinnikova, Galina; Woyke, Tanja; Brumm, Catherine; Hochstein, Rebecca; Schoenfeld, Thomas; Brumm, Phillip

2012-07-30

Paenibacillus sp.Y412MC10 was one of a number of organisms isolated from Obsidian Hot Spring, Yellowstone National Park, Montana, USA under permit from the National Park Service. The isolate was initially classified as a Geobacillus sp. Y412MC10 based on its isolation conditions and similarity to other organisms isolated from hot springs at Yellowstone National Park. Comparison of 16 S rRNA sequences within the Bacillales indicated that Geobacillus sp.Y412MC10 clustered with Paenibacillus species, and the organism was most closely related to Paenibacillus lautus. Lucigen Corp. prepared genomic DNA and the genome was sequenced, assembled, and annotated by the DOE Joint Genome Institute. The genome sequence was deposited at the NCBI in October 2009 (NC_013406). The genome of Paenibacillus sp. Y412MC10 consists of one circular chromosome of 7,121,665 bp with an average G+C content of 51.2%. Comparison to other Paenibacillus species shows the organism lacks nitrogen fixation, antibiotic production and social interaction genes reported in other paenibacilli. The Y412MC10 genome shows a high level of synteny and homology to the draft sequence of Paenibacillus sp. HGF5, an organism from the Human Microbiome Project (HMP) Reference Genomes. This, combined with genomic CAZyme analysis, suggests an intestinal, rather than environmental origin for Y412MC10.

Choice of sterilizing/disinfecting agent: determination of the Decimal ReductionTime (D-Value

Directory of Open Access Journals (Sweden)

Priscila Gava Mazzola

2009-12-01

Full Text Available Efforts to diminish the transmission of infections include programs in which disinfectants play a crucial role. Hospital surfaces and medical devices are potential sources of cross contamination, and each instrument, surface or area in a health care unit can be responsible for spread of infection. The decimal reduction time was used to study and compare the behavior of selected strains of microorganisms. The highest D-values for various bacteria were obtained for the following solutions: (i 0.1% sodium dichloroisocyanurate (pH 7.0 - E. coli and A. calcoaceticus (D = 5.9 min; (ii sodium hypochlorite (pH 7.0 at 0.025% for B. stearothermophilus (D = 24 min, E. coli and E. cloacae (D = 7.5 min; at 0.05% for B. stearothermophilus (D = 9.4 min and E. coli (D = 6.1 min. The suspension studies were an indication of the disinfectant efficacy on a surface. The data in this study reflect the formulations used and may vary from product to product. The expected effectiveness from the studied formulations shows that the tested agents can be recommended for surface disinfection as stated in present guidelines and emphasize the importance and need to develop routine and novel programs to evaluate product utility.Esforços para diminuir o risco de transmissões de infecções incluem programas nos quais os desinfetantes desempenham papel crucial. As superfícies de materiais médico-hospitalares, se não estiverem diretamente ligados à transmissão de doenças, podem contribuir, potencialmente, para uma contaminação cruzada secundária. Cada instrumento ou superfície do estabelecimento do ambiente de saúde que entra em contato com um paciente é um disseminador potencial de infecção. Para estudar e comparar o comportamento dos microrganismos selecionados foram realizados ensaios de determinação do tempo de redução decimal. Os maiores valores D determinados, foram: (i 0,1% dicloroisocianurato de sódio (NaDCC (pH 7.0 - E. coli e A. calcoaceticus (D = 5

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

OpenAIRE

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-01-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic ...

Characterization of Thermostable Cellulases Produced by Bacillus and Geobacillus Strains

Science.gov (United States)

Bacterial community composition of thermophilic (60 deg C) mixed cellulose-enrichment cultures was examined by constructing a 16S rDNA clone library which demonstrated major lineages affiliated to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria. A tot...

Amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui.

Science.gov (United States)

Hatakeyama, T; Hatakeyama, T

1990-07-06

The complete amino acid sequences of the ribosomal proteins HL30 and HmaL5 from the archaebacterium Halobacterium marismortui were determined. Protein HL30 was found to be acetylated at its N-terminal amino acid and shows homology to the eukaryotic ribosomal proteins YL34 from yeast and RL31 from rat. Protein HmaL5 was homologous to the protein L5 from Escherichia coli and Bacillus stearothermophilus as well as to YL16 from yeast. HmaL5 shows more similarities to its eukaryotic counterpart than to eubacterial ones.

Environmental microbiology as related to planetary quarantine. [synergetic effect of heat and radiation

Science.gov (United States)

Pflug, I. J.

1973-01-01

The mechanistic basis of the synergetic effect of combined heat and radiation on microbial destruction was analyzed and results show that radiation intensity, temperature, and relative humidity are the determining factors. Dry heat resistance evaluation for selected bacterial spore crops indicates that different strains of Bacillus stearothermophilus demonstrate marked differences in resistance. Preliminary work to determine the effects of storage time, suspending medium, storage temperature and spore crop cleaning procedures on dry heat survival characteristics of Bacillus subtilis var. Niger, and dry heat resistance of natural microflora in soil particles is also reported.

Pre-treatment Of Thermoduric Spores In Co2 Modified Atmosphere And Their Survivability During Food Extrusion [pré-tratamento De Esporos Termodúricos Em Atmosfera Modificada De Co2 E Sua Sobrevivência Ao Processo De Extrusão

OpenAIRE

Fraiha M.; Ferraz A.C.O.; Biagi J.D.

2011-01-01

The aim of this experiment was to evaluate how susceptible spores become to mechanical damage during food extrusion after being submitted to CO2. B. stearothermophilus spores sowed to corn and soy mix were submitted to 99% CO2 for 10 days and extruded in a single-screw extruder. The treatments were: T1 - spore-containing samples, extruded at screw rotational speed of 65 rpm and barrel wall temperature of 80 °C; T2 - as T1, except for screw rotational speed of 150 rpm; and T3 - as T2, except t...

Dynamic sterilization of titanium implants with ultraviolet light

International Nuclear Information System (INIS)

Singh, S.; Schaaf, N.G.

1989-01-01

All implantable devices must be sterile. However, autoclaves produce poor surface properties that jeopardize the integration process. The application of a modified ultraviolet light source has proven to enhance bioreactivity by controlling surface properties, but it lacks validation of its sterilization capabilities. Forty-eight titanium implants were contaminated with spores of the biological indicator Bacillus stearothermophilus and subjected to dynamic sterilization by ultraviolet light. Forty-seven of the implants were successfully sterilized, as indicated by not producing turbidity in a suitable growth medium. This sterilization technique only requires a 20-second exposure to achieve sterility

Dicty_cDB: Contig-U16358-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 01389_2365( CP001389 |pid:none) Rhizobium sp. NGR234, complete ... 90 1e-33 AM494972_195( AM494972 |pid:none) Leishmania braziliensi...81( CT005272 |pid:none) Leishmania major strain Friedlin... 94 2e-33 AP009152_1947( AP009152 |pid:none) Kocu...1053_2521( CP001053 |pid:none) Burkholderia phytofirmans PsJN ... 245 e-100 BA000011_1082( BA000011 |pid:none) Thermoplasma volcani...baalenii PYR-1... 228 6e-94 CP000325_2196( CP000325 |pid:none) Mycobacterium ulcerans... ... 140 2e-49 CP000557_1254( CP000557 |pid:none) Geobacillus thermodenitrificans

Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

Science.gov (United States)

Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

2014-07-01

The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui.

Science.gov (United States)

Bergmann, U; Wittmann-Liebold, B

1993-03-23

50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A). The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli [Walleczek, J., Martin, T., Redl, B., Stöffler-Meilicke, M., & Stöffler, G. (1989) Biochemistry 28, 4099-4105] and from Bacillus stearothermophilus [Brockmöller, J., & Kamp, R. M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935]. To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography. After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides. The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent. This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A. Comparison of our cross-linking results with those obtained with B. stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.

Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry.

Science.gov (United States)

Kirpekar, F; Douthwaite, S; Roepstorff, P

2000-02-01

We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS). After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry. A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides. Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications. A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD). This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui. One S. acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32. The modified C is located in a region that is clearly conserved with respect to both sequence and position in B. stearothermophilus and H. halobium and to some degree also in H. marismortui. However, no analogous modification was identified in the latter three organisms. We further find that the 5' end of H. halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated. The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses. Discrepancies with two of the published 5S rRNA sequences were identified and are reported here.

Secondary structure of prokaryotic 5S ribosomal ribonucleic acids: a study with ribonucleases

DEFF Research Database (Denmark)

Douthwaite, S; Garrett, R A

1981-01-01

The structures of 5S ribosomal RNAs from Escherichia coli and Bacillus stearothermophilus were examined by using ribonucleases A, T1, and T2 and a double helix specific cobra venom ribonuclease. By using both 5' and 3'-32P-end labeling methods and selecting for digested but intact 5S RNA molecules...... evidence for three of the helical regions of the Fox and Woese model of 5S RNA [Fox, G. E., & Woese, C. (1975) Nature (London) 256, 505] and support other important structural features which include a nucleotide looped out from a helical region which has been proposed as a recognition site for protein L18....

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

Science.gov (United States)

Glotz, C; Müssig, J; Gewitz, H S; Makowski, I; Arad, T; Yonath, A; Wittmann, H G

1987-11-01

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Desenvolvimento de processo termico otimizado para mosto de caldo de cana na fermentação alcoolica

OpenAIRE

Jonas Nolasco Junior

2005-01-01

Resumo: Nesta pesquisa é proposto um processo de tratamento térmico do mosto, com máxima preservação do conteúdo em açúcares fermentescíveis (sacarose, glicose e frutose), a fim de promover a inativação térmica de seus contaminantes bacterianos e por extensão os da fermentação alcoólica. Com esse objetivo foram examinadas as cinéticas de degradação térmica da sacarose, glicose, frutose e açúcares redutores totais (ART) (110 ? 140ºC) e também dos esporos de B. stearothermophilus (98 ? 130ºC), ...

Identification of thermophilic bacterial strains producing thermotolerant hydrolytic enzymes from manure compost.

Science.gov (United States)

Charbonneau, David M; Meddeb-Mouelhi, Fatma; Boissinot, Maurice; Sirois, Marc; Beauregard, Marc

2012-03-01

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60-65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.

Extremophiles survival to simulated space conditions: an astrobiology model study.

Science.gov (United States)

Mastascusa, V; Romano, I; Di Donato, P; Poli, A; Della Corte, V; Rotundi, A; Bussoletti, E; Quarto, M; Pugliese, M; Nicolaus, B

2014-09-01

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

Disinfection/sterilization of extracted teeth for dental student use.

Science.gov (United States)

Dominici, J T; Eleazer, P D; Clark, S J; Staat, R H; Scheetz, J P

2001-11-01

Extracted human teeth are used in many preclinical courses. While there has been no report of disease transmission with extracted teeth, sterilization of teeth used in the teaching laboratory should be a concern. The purpose of this study was to determine the effectiveness of different sterilization/disinfection methods of extracted human teeth using Bacillus stearothermophilus, a bacteria resistant to heat and frequently used to test sterilizers. In this study, 110 extracted molars with no carious lesions were collected and stored in buffered saline. An endodontic occlusal access preparation was cut into the pulp chamber of each tooth. Pulp tissue in the chamber was removed with a broach. Approximately 1 x 10(5) B. stearothermophilus endospores in culture medium were injected into the pulp chamber, sealed with Cavit G, and then placed in sterile saline for twelve hours. Ten teeth were placed into each of eleven groups. Seven groups were immersed for one week in one of the following solutions: a) sterile saline (control group), b) 5.25% NaOCl, c) 2.6% NaOCl, d) 1% NaOCl, e) 10% buffered formalin, f) 2% gluteraldehyde, g) 0.28% quaternary ammonium. Four additional groups were treated by h) 10% formalin for two days, i) 10% formalin for four days, j) autoclaving at 240 degrees F and 20 psi for twenty minutes, and k) autoclaving at 240 degrees F and twenty psi for forty minutes. Each tooth was then aseptically split and placed in an individual test tube with growth medium. Samples were examined for evidence of growth (turbidity) at forty-eight hours. Only autoclaving for forty minutes at 240 degrees F and 20 psi or soaking in 10 percent formalin for one week were 100 percent effective in preventing growth. A chi-square analysis of the data indicates these two methods were significantly better than all other methods (p<0.001).

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

Science.gov (United States)

Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

2010-04-01

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Evaluation of cellulose-binding domain fused to a lipase for the lipase immobilization.

Science.gov (United States)

Hwang, Sangpill; Ahn, Jungoh; Lee, Sumin; Lee, Tai Gyu; Haam, Seungjoo; Lee, Kangtaek; Ahn, Ik-Sung; Jung, Joon-Ki

2004-04-01

A cellulose-binding domain (CBD) fragment of a cellulase gene of Trichoderma hazianum was fused to a lipase gene of Bacillus stearothermophilus L1 to make a gene cluster for CBD-BSL lipase. The specific activity of CBD-BSL lipase for oil hydrolysis increased by 33% after being immobilized on Avicel (microcrystalline cellulose), whereas those of CBD-BSL lipase and BSL lipase decreased by 16% and 54%, respectively, after being immobilized on silica gel. Although the loss of activity of an enzyme immobilized by adsorption has been reported previously, the loss of activity of the CBD-BSL lipase immobilized on Avicel was less than 3% after 12 h due to the irreversible binding of CBD to Avicel.

Effect of Hyperbaric Carbon Dioxide on Spores and Vegetative Cells of Bacillus stearothermophilus

Science.gov (United States)

1994-05-01

thyme, parsley, mint, and spoiled apple juice were killed by a 30-minute exposure to 800 psi C02 at elevated temperature (45°C). In the present...under both aerobic and anaerobic conditions, and ferments sugars (2). Spores of BS may be present in "commercially sterile" foods and may be...spores at 55°C is less than 5.4. Cross et al (5) reported that germination of four strains of Bacillus in a yeast -dextrose broth medium was inhibited

A multi-enzymatic cascade reaction for the stereoselective production of γ-oxyfunctionalyzed amino acids

Directory of Open Access Journals (Sweden)

Junichi eEnoki

2016-04-01

Full Text Available A stereoselective three-enzyme cascade for synthesis of diasteromerically pure γ-oxyfunctionalized α-amino acids was developed. By coupling a dynamic kinetic resolution using an N-acylamino acid racemase and an L-selective aminoacylase from Geobacillus thermoglucosidasius with a stereoselective isoleucine dioxygenase from Bacillus thuringiensis, diastereomerically pure oxidized amino acids were produced from racemic N-acetylamino acids. The three enzymes differ in their optimal temperature and pH-spectra. Their different metal cofactor dependencies lead to inhibitory effects. Under optimized conditions, racemic N-acetylmethionine was quantitatively converted into L-methionine-(S-sulfoxide with 97% conversion and 95% de. The combination of these three different biocatalysts allows the direct synthesis of diastereopure oxyfunctionalized amino acids from inexpensive racemic starting material.

Construction and evaluation of an exopolysaccharide-producing engineered bacterial strain by protoplast fusion for microbial enhanced oil recovery.

Science.gov (United States)

Sun, Shanshan; Luo, Yijing; Cao, Siyuan; Li, Wenhong; Zhang, Zhongzhi; Jiang, Lingxi; Dong, Hanping; Yu, Li; Wu, Wei-Min

2013-09-01

Enterobacter cloacae strain JD, which produces water-insoluble biopolymers at optimal temperature of 30°C, and a thermophilic Geobacillus strain were used to construct an engineered strain for exopolysaccharide production at high temperatures by protoplast fusion. The obtained fusant strain ZR3 produced exopolysaccharides at up to 45°C with optimal growth temperature at 35°C. The fusant produced exopolysaccharides of approximately 7.5 g/L or more at pH between 7.0 and 9.0. The feasibility of the enhancement of crude oil recovery with the fusant was tested in a sand-packed column at 40°C. The results demonstrated that bioaugmentation of the fusant was promising approach for MEOR. Mass growth of the fusant was confirmed in fermentor tests. Copyright © 2013 Elsevier Ltd. All rights reserved.

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui.

Science.gov (United States)

Hatakeyama, T; Hatakeyama, T; Kimura, M

1988-11-21

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13,538, 16,812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.

Validation of the sterilization process for radiopharmaceuticals and materials with humid heat

International Nuclear Information System (INIS)

Robles, Anita; Moore, Mariel; Morote, Mario; Guevara, Buenaventura; Castro, Delcy; Paragulla, Wilson; Martinez, Ramos; Ocana, Elias; Novoa, Carlos

2014-01-01

A validation protocol has been designed and applied for the sterilization process of radiopharmaceuticals and materials, with humid heat for sodium pertechnetate Tc-99m injection solution (placebo) and materials, in compliance with good manufacturing practices for pharmaceutical products. The sterilization cycle set for each load is developed, according to the following parameters: 121 o C ± 1 o C (temperature), 15 ± 0.5 psi (pressure) and an exposure time of 20 and 15 minutes, respectively. The results in the penetration test with load, F0 values were higher than 20 minutes at 121 o C and for the biological challenge by biological indicators (Bacillus stearothermophilus) was negative in colder spots, in three consecutive runs. The sterilization process for each load and equipment has been validated to meet the established acceptance criteria. (authors).

Use of extremophilic bacteria for second generation bioethanol production

DEFF Research Database (Denmark)

Tomás, Ana Faria; Karakashev, Dimitar Borisov; Angelidaki, Irini

production from food crops, such as corn (starch) or sugar cane (sucrose) is already an established process, with the USA and Brazil supplying 86% of the market. The major challenge remains in the use of different waste sources – agricultural, forestry, animal and household waste - as a feedstock....... The recalcitrance of these materials and their diverse sugar composition make the industrial yeast strains currently used unsuitable for a second generation bioethanol production process. One of the alternative strategies is the use of extreme thermophilic microorganisms. Currently, selected members from the genera...... Clostridium, Thermoanaerobacter, Geobacillus and Thermoanaerobacterium are among the best candidates. A new strain of Thermoanaerobacter, closely related to T. italicus and T. mathranii, has achieved 0.43 gethanol/gxylose, which is 83% of the theoretical yield of ethanol based on xylose and the highest value...

Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm, Eudrilus eugeniae Kinb.

Science.gov (United States)

Vasantha-Srinivasan, Prabhakaran; Senthil-Nathan, Sengottayan; Thanigaivel, Annamalai; Edwin, Edward-Sam; Ponsankar, Athirstam; Selin-Rani, Selvaraj; Pradeepa, Venkatraman; Sakthi-Bhagavathy, Muthiah; Kalaivani, Kandaswamy; Hunter, Wayne B; Duraipandiyan, Veeramuthu; Al-Dhabi, Naif Abdullah

2016-07-01

Evaluations of biological effects of (Pb-CVO) the crude volatile oil of Piper betle leaves on the tobacco cutworm Spodoptera litura were conducted. Pb-CVO was subjected to GC-MS analysis and twenty vital compounds were isolated from the betel leaf oil. Pb-CVO was tested at four different concentrations (0.25, 0.5, 1.0 and 1.5%) against S. litura. The treated insects exhibited dose depended mortality. The mortality rate was significantly higher at the 1.0 and 1.5% Pb-CVO. The LC50 (Lethal concentration) were observed at 0.48% Pb-CVO. Larval and pupal durations increased in all treatment concentrations (0.25, 0.3, 0.4 and 0.5%) whereas, pupal weight decreased compared to control. Adult longevity of S. litura was reduced in all treatments but predominantly in the 0.4 and 0.5% Pb-CVO. Correspondingly, mean fecundity rate was reduced at all concentrations compared to control. Histological studies of larvae mid-gut profiles of S. litura were severely damaged in 1.0 and 1.5% and showed abnormalities in mid-gut cells with 0.25 and 0.5% Pb-CVO treatments. Earthworm toxicity illustrated that 0.1% of chemical insecticides (monocrotophos and cypermethrin) varied widely in their contact toxicities compared to 0.5 and 1.0% Pb-CVO and control in both contact filter paper and artificial soil test. These findings suggest that twenty essential compounds of betel leaf oil were significant inhibitors of the development and caused behavioral changes of S. litura. Treatment with betel leaf oil at these concentrations had no adverse effect on earthworm populations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of a new device for sterilizing dental high-speed handpieces

DEFF Research Database (Denmark)

Larsen, T; Andersen, H K; Fiehn, N E

1997-01-01

Dental high-speed turbines and handpieces can take up and expel microorganisms during operation and thus need regular sterilization. This study established a method for validating devices used to sterilize high-speed turbines and handpieces. The air and water channels and turbine chambers were...... contaminated with suspensions of Streptococcus salivarius or endospores of Bacillus stearothermophilus. The effect of flushing and/or autoclaving performed by a new device combining both procedures was evaluated by counting the number of viable bacteria recovered from these devices. Further, the effect...... on clinically used handpieces was evaluated. In an initial experiment, the device partially reduced S. salivarius, and the endospores survived. In a second experiment, a 5 to 6 log reduction of S. salivarius in air and water channels was obtained. No growth was observed in clinically used high-speed handpieces...

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

Science.gov (United States)

Nicolas, Pierre; Repoila, Francis; Bardowski, Jacek; Aymerich, Stéphane

2017-01-01

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks. PMID:28723971

Development of a high temperature microbial fermentation process for butanol

Energy Technology Data Exchange (ETDEWEB)

Jeor, Jeffery D. St. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Reed, David W. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Daubaras, Dayna L. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Thompson, Vicki S. [Idaho National Lab. (INL), Idaho Falls, ID (United States)

2015-08-01

Transforming renewable biomass into cost-competitive high-performance biofuels and bioproducts is key to the U.S. future energy and chemical needs. Butanol production by microbial fermentation for chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process could decrease energy costs, capital cost, give higher butanol production, and allow for continuous fermentation. In this paper, we describe our approach to genetically transform Geobacillus caldoxylosiliticus, using a pUCG18 plasmid, for potential insertion of a butanol production pathway. Transformation methods tested were electroporation of electrocompetent cells, ternary conjugation with E. coli donor and helper strains, and protoplast fusion. These methods have not been successful using the current plasmid. Growth controls show cells survive the various methods tested, suggesting the possibility of transformation inhibition from a DNA restriction modification system in G. caldoxylosiliticus, as reported in the literature.

Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

CSIR Research Space (South Africa)

Williamson, DS

2010-09-01

Full Text Available . In common with the plant nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. Electron microscopy and image classification showed complexes having crescentlike, “c-shaped”, circular and “figure...

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins.

Science.gov (United States)

Dijk, J; van den Broek, R; Nasiulas, G; Beck, A; Reinhardt, R; Wittmann-Liebold, B

1987-08-01

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.

In vitro production of thymine dimer by ultroviolet irradiation of DNA from mesophilic and thermophilic bacteria

International Nuclear Information System (INIS)

Yein, F.S.; Stenesh, J.

1989-01-01

Thymine dimer was produced in vitro by ultraviolet irradiation of DNA, isolated from the mesophile Bacillus licheniformis and the thermophile B. stearothermophilus. Irradiation was performed at three different temperaturs (35, 45 and 55 C) and the thymine dimer was isolated and determined. An HPLC procedure was developed that permitted temperature was greater for the thermophile than for the mesophile. Formation of thymine dimer increased with temperature for both organisms but more so for the thermophile; over the temperature range of 35-55 C, the average increase in thymine dimer production for the themrophile was about 4-times that for the mesophile. The melting out temperature, as a function of increasing irradiation temperature, was essentially unchanged for the mesophilic DNA, but decreased progressively for the thermophilic DNA. These results are discussed in terms of the macromolecular theory of to the macromolecular theory of the thermophily. (author). 31 refs.; 4 figs.; 3 tabs

Development of a High Temperature Microbial Fermentation Processfor Butanol Production

Energy Technology Data Exchange (ETDEWEB)

Jeor, Jeffery D. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Reed, David W. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Daubaras, Dayna L. [Idaho National Lab. (INL), Idaho Falls, ID (United States); Thompson, Vicki S. [Idaho National Lab. (INL), Idaho Falls, ID (United States)

2016-06-01

Transforming renewable biomass into cost competitive high-performance biofuels and bioproducts is key to US energy security. Butanol production by microbial fermentation and chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process can facilitate butanol recovery up to 40%, by using gas stripping. Other benefits of fermentation at high temperatures are optimal hydrolysis rates in the saccharification of biomass which leads to maximized butanol production, decrease in energy costs associated with reactor cooling and capital cost associated with reactor design, and a decrease in contamination and cost for maintaining a sterile environment. Butanol stripping at elevated temperatures gives higher butanol production through constant removal and continuous fermentation. We describe methods used in an attempt to genetically prepare Geobacillus caldoxylosiliticus for insertion of a butanol pathway. Methods used were electroporation of electrocompetent cells, ternary conjugation with E. coli, and protoplast fusion.

Development of a High Temperature Microbial Fermentation Processfor Butanol Production

International Nuclear Information System (INIS)

Jeor, Jeffery D.; Reed, David W.; Daubaras, Dayna L.; Thompson, Vicki S.

2016-01-01

Transforming renewable biomass into cost competitive high-performance biofuels and bioproducts is key to US energy security. Butanol production by microbial fermentation and chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process can facilitate butanol recovery up to 40%, by using gas stripping. Other benefits of fermentation at high temperatures are optimal hydrolysis rates in the saccharification of biomass which leads to maximized butanol production, decrease in energy costs associated with reactor cooling and capital cost associated with reactor design, and a decrease in contamination and cost for maintaining a sterile environment. Butanol stripping at elevated temperatures gives higher butanol production through constant removal and continuous fermentation. We describe methods used in an attempt to genetically prepare Geobacillus caldoxylosiliticus for insertion of a butanol pathway. Methods used were electroporation of electrocompetent cells, ternary conjugation with E. coli, and protoplast fusion.

Development of a biotechnological process for the production of high quality linen fibers.

Science.gov (United States)

Valladares Juárez, Ana Gabriela; Rost, Gernot; Heitmann, Uwe; Heger, Egon; Müller, Rudolf

2011-10-01

A novel biotechnological process for the production of high-quality flax fibers was developed. In this process, decorticated fibers from green flax were washed with 0.5% soda solution and treated with the pectinolytic strain Geobacillus thermoglucosidasius PB94A. Before drying the fibers, they were treated with the textile softener Adulcinol BUN. If the fibers contained contaminant shives, a bleaching step with hydrogen peroxide was performed before the softener treatment. In experiments where fibers were treated by the new process, and in which the bacterial solutions were reused seven times, the fiber quality was similar in all batches. The resolution of the treated fibers was 2.7 ± 0.4 and the fineness was 11.1 ± 1.1 dtex, while the starting material had a resolution of 7.3 and a fineness of 37 dtex. The new biotechnological treatment eliminates the weather-associated risks of the traditional fiber retting completely and produces consistently high-quality fibers that can be used to produce fine linen yarns.

High production of D-tagatose by the addition of boric acid.

Science.gov (United States)

Lim, Byung-Chul; Kim, Hye-Jung; Oh, Deok-Kun

2007-01-01

An L-arabinose isomerase mutant enzyme from Geobacillus thermodenitrificans was used to catalyze the isomerization of D-galactose to D-tagatose with boric acid. Maximum production of D-tagatose occurred at pH 8.5-9.0, 60 degrees C, and 0.4 molar ratio of boric acid to D-galactose, and the production increased with increasing enzyme concentration. Under the optimum conditions, the enzyme (10.8 units/mL) converted 300 g/L D-galactose to 230 g/L D-tagatose for 20 h with a yield of 77% (w/w); the production and conversion yield with boric acid were 1.5-fold and 24% higher than without boric acid, respectively. In 24 h, the enzyme produced 370 g/L D-tagatose from 500 g/L D-galactose with boric acid, corresponding to a conversion yield of 74% (w/w) and a production rate of 15.4 g/L.h. The production and yield of D-tagatose obtained in this study are unprecedented.

Bio-affinity mediated immobilization of lipase onto magnetic cellulose nanospheres for high yield biodiesel in one time addition of methanol.

Science.gov (United States)

Bandikari, Ramesh; Qian, Jiaxin; Baskaran, Ram; Liu, Ziduo; Wu, Gaobing

2018-02-01

To synthesis biodiesel from palm oil in one-time addition of methanol and solvent-free medium using CBD fused with C-terminal of lipase from G. stearothermophilus (GSlip-CBD) was immobilized onto magnetic cellulose nanosphere (MCNS). The immobilized matrix traits were preconceived by FT-IR, TEM and XRD. Perceptible biodiesel yield 98 and 73% was synthesized by GSlip-CBD-MCNS in 4 h and GSlip-MCNS in 6 h under the optimized conditions of oil:methanol ratio (1:3.5), temperature (55 and 50 °C) and enzyme loading (15 U). Intriguingly, the operational stability of GSlip-CBD-MCNS was an easily attainable owing to the magnetic properties and could be reused up to 8th and19th cycles with 94 and 45% of biodiesel yield respectively, compared to GSlip-MCNS. Thus GSlip-CBD-MCNS could be a potential biocatalyst for higher yield of biodiesel and reusability in one step addition of methanol. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative economic assessment of ethanol production under mesophilic and thermophilic conditions

International Nuclear Information System (INIS)

Mistry, P.B.

1991-01-01

Key technical factors affecting the economics of bioethanol production are critically analyzed with special reference to the relative merits of thermophilic and mesophilic fermentation. A number of novel process schemes to take advantage of thermophilic operation are discussed. Analysis of the capital and operating costs for a range of flowsheets then provides a basis for critical study. Estimates for thermophilic production are compared with those for a sugar cane based mesophilic process (using S. cerevisiae). For the thermophilic fermentation, the basic kinetic and yield constants are based on projected values for a strain of B. stearothermophilus. Compared to mesophilic operation, thermophilic operation results in reduced capital, operating and feed costs. The feed cost still accounts for a large proportion (75%) of the total production cost. However, on a feed-cost-free basis, a reduction in production cost of up to 32% could be realized by changing to thermophilic operation from existing yeast-based processes, after minor process modifications. 20 refs., 10 figs., 8 tabs

The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin.

Science.gov (United States)

Sun, Y J; Chou, C C; Chen, W S; Wu, R T; Meng, M; Hsiao, C D

1999-05-11

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-A resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted alpha/beta structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

Biodegradation of phenanthrene in bioaugmented microcosm by consortium ASP developed from coastal sediment of Alang-Sosiya ship breaking yard.

Science.gov (United States)

Patel, Vilas; Patel, Janki; Madamwar, Datta

2013-09-15

A phenanthrene-degrading bacterial consortium (ASP) was developed using sediment from the Alang-Sosiya shipbreaking yard at Gujarat, India. 16S rRNA gene-based molecular analyses revealed that the bacterial consortium consisted of six bacterial strains: Bacillus sp. ASP1, Pseudomonas sp. ASP2, Stenotrophomonas maltophilia strain ASP3, Staphylococcus sp. ASP4, Geobacillus sp. ASP5 and Alcaligenes sp. ASP6. The consortium was able to degrade 300 ppm of phenanthrene and 1000 ppm of naphthalene within 120 h and 48 h, respectively. Tween 80 showed a positive effect on phenanthrene degradation. The consortium was able to consume maximum phenanthrene at the rate of 46 mg/h/l and degrade phenanthrene in the presence of other petroleum hydrocarbons. A microcosm study was conducted to test the consortium's bioremediation potential. Phenanthrene degradation increased from 61% to 94% in sediment bioaugmented with the consortium. Simultaneously, bacterial counts and dehydrogenase activities also increased in the bioaugmented sediment. These results suggest that microbial consortium bioaugmentation may be a promising technology for bioremediation. Copyright © 2013 Elsevier Ltd. All rights reserved.

X-ray Crystallographic Study of the Drug Target Phosphoglycerate Mutase from Leishmania mexicana, A Cobalt-dependent Enzyme

International Nuclear Information System (INIS)

Poolpem, Buabal; Fortergill-gillmor, Linda; Michel, Poul; Wallkinchow, Malcolm

2005-10-01

Crystal structures of Leishmania mexicana iPGAM show a mixture of substrate (3PGA) and product (2PGA) in the active sites which occupy essentially the same position. Lm iPGAM requires Co 2 + ions as cofactors, but not Mn 2 + or Zn 2 +. Comparison of Lm iPGAM and the well-defined structure of Bacillus stearothermophilus iPGAM that requires Mn 2 + shows that the metal requirement of iPGAMs can be discriminated by the existence of an extra residue at Tyr210 (Lm) that causes His360 to adopt a position where it can form a H-bond with the phospho group of the substrate/product. These changes in active site structure cause differences in the active site preferences of each of the iPGAMs from both organisms for particular metals. Metal reactivation experiments show that manganese inhibits Lm iPGAM, whereas the zinc inhibitory effect is unclear. Manganese or zinc substitutions in both metal sites cause changes in metal geometry leading to loss of enzyme activity

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

Science.gov (United States)

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase.

Science.gov (United States)

Rhimi, Moez; Chouayekh, Hichem; Gouillouard, Isabelle; Maguin, Emmanuelle; Bejar, Samir

2011-02-01

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process. Copyright © 2010 Elsevier Ltd. All rights reserved.

Spectroscopic studies and antibacterial activities of some new 16-membered octaazamacrocyclic complexes derived from thiocarbohydrazide and pentane-2,4-dione

Science.gov (United States)

Singh, D. P.; Kumar, Krishan; Chopra, Rimpi Mehani ne'e.

2011-02-01

A series of macrocyclic complexes of the type [M(C 12H 20N 8S 2)X 2]; where M = Co(II), Ni(II), Cu(II), Zn(II); X = Cl -, NO 3-, CH 3COO - has been synthesized by template condensation of thiocarbohydrazide and pentane-2,4-dione in the presence of divalent metal salts in methanolic medium. The complexes have been characterized with the help of elemental analyses, conductance measurements, magnetic measurements, electronic, NMR, IR, EPR and MS spectral studies. The low value of molar conductance indicates them to be non-electrolytes. On the basis of various studies a distorted octahedral geometry may be proposed for all of these complexes. These metal complexes were also tested for their in vitro antibacterial activities against some Gram-positive bacterial strains, i.e., Bacillus subtilis, Bacillus stearothermophilus and two Gram-negative bacterial strains, i.e., Escherichia coli, Pseudomonas putida. The results obtained were compared with standard antibiotics, Chloramphenicol and Streptomycin and found that some of the synthesized complexes show good antibacterial activities as compared to the standard antibiotics.

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov

Science.gov (United States)

Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.; Deinhard, G.; Poralla, K.

1992-01-01

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria Bac. stearothermophilus exposed to. gamma. -, UV-radiation or methylnitrosourea

Energy Technology Data Exchange (ETDEWEB)

Fomenko, L A; Kuznetsovea, E A; Gaziev, A I

1984-07-01

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to ..gamma..-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S/sub 1/-nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria.

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria Bac. stearothermophilus exposed to γ-, UV-radiation or methylnitrosourea

International Nuclear Information System (INIS)

Fomenko, L.A.; Kuznetsovea, E.A.; Gaziev, A.I.

1984-01-01

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to γ-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S 1 -nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria. (orig.)

Thermostability of Multidomain Proteins: Elongation Factors EF-Tu from Escherichia coli and Bacillus stearothermophilus and Their Chimeric Forms

Czech Academy of Sciences Publication Activity Database

Šanderová, Hana; Hůlková, Marta; Maloň, Petr; Kepková, M.; Jonák, Jiří

2004-01-01

Roč. 13, č. 1 (2004), s. 89-99 ISSN 0961-8368 R&D Projects: GA AV ČR IPP1050128; GA ČR GA204/98/0863; GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : elongation factor EF-Tu, thermostability, chimeric protein Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.116, year: 2004

Characterization Of A Novel Hydrolytic Enzyme Producing Thermophilic Bacterium Isolated From The Hot Spring Of Azad Kashmir-Pakistan

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Sana Zahoor

Full Text Available ABSTRACT A thermophilic bacterium (TP-2 was isolated from the Tatta Pani hot spring in Azad Kashmir and was characterized using phenotypic and genotypic characters. The strain developed cream colored, round, smooth, flat and slimy colonies while the cells were Gram positive rods that ranged in size from about 2.1-3.6 μm to 0.2-0.3 μm in width. Sequence analysis of its 16S rRNA gene showed that isolate TP-2 had 89% homology with Geobacillus debilis. It grew within pH range of 5.5 to 8.5 with optimum growth at pH 7.0. The isolate showed optimum growth at 65ºC and gave positive results for gelatin hydrolysis (GEL, ortho nitrophenyl-β-D-galactopyranosidase (ONPG, and nitrate production and produced acid from sucrose, glucose and maltose. It utilized glucose, fructose, maltose, lactose, sucrose, xylan, starch, filter paper and carboxymethylcellulose as sole carbon source. Isolate TP-2 produced significant amount of industrially important enzymes i.e. extracellular α-amylase, CMCase, FPase, Xylanase, Protease and Lipase and intracellular CMCase and FPase.

Properties of a novel thermostable glucose isomerase mined from Thermus oshimai and its application to preparation of high fructose corn syrup.

Science.gov (United States)

Jia, Dong-Xu; Zhou, Lin; Zheng, Yu-Guo

2017-04-01

Glucose isomerase (GI) is used in vitro to convert d-glucose to d-fructose, which is capable of commercial producing high fructose corn syrup (HFCS). To manufacture HFCS at elevated temperature and reduce the cost of enriching syrups, novel refractory GIs from Thermoanaerobacterium xylanolyticum (TxGI), Thermus oshimai (ToGI), Geobacillus thermocatenulatus (GtGI) and Thermoanaerobacter siderophilus (TsGI) were screened via genome mining approach. The enzymatic characteristics research showed that ToGI had higher catalytic efficiency and superior thermostability toward d-glucose among the screened GIs. Its optimum temperature reached 95°C and could retain more than 80% of initial activity in the presence of 20mM Mn 2+ at 85°C for 48h. The K m and k cat /K m values for ToGI were 81.46mM and 21.77min -1 mM -1 , respectively. Furthermore, the maximum conversion yield of 400g/L d-glucose to d-fructose at 85°C was 52.16%. Considering its excellent high thermostability and ameliorable application performance, ToGI might be promising for realization of future industrial production of HFCS at elevated temperature. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of modification with 1,4-α-glucan branching enzyme on the rheological properties of cassava starch.

Science.gov (United States)

Li, Yadi; Li, Caiming; Gu, Zhengbiao; Hong, Yan; Cheng, Li; Li, Zhaofeng

2017-10-01

Steady and dynamic shear measurements were used to investigate the rheological properties of cassava starches modified using the 1,4-α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02. GBE treatment lowered the hysteresis loop areas, the activation energy (E a ) values and the parameters in rheological models of cassava starch pastes. Moreover, GBE treatment increased its storage (G') and loss (G″) moduli, and decreased their tan δ (ratio of G″/G') values and frequency-dependencies. Scanning electron microscopic studies showed the selective and particular attack of GBE on starch granules, and X-ray diffraction analyses showed that GBE treatment produces significant structural changes in amylose and amylopectin. These changes demonstrate that GBE modification produces cassava starch with a more structured network and improved stability towards mechanical processing. Differential scanning calorimetric analysis and temperature sweeps indicated greater resistance to granule rupture, higher gel rigidity, and a large decrease in the rate of initial conformational ordering with increasing GBE treatment time. Pronounced changes in rheological parameters revealed that GBE modification enhances the stability of cassava starch and its applicability in the food processing industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Gene adaptation to extreme environments

International Nuclear Information System (INIS)

Marlaire, P.; Rodriguez, V.; Kerner, N.

2005-01-01

Full text: This work is oriented to the study of gene adaptation to extreme conditions, such as the hydrothermal system located in Copahue, Neuquen, Argentina. The organisms living there develop under two pressure selection conditions: the high temperature of thermal water and the strong impact of ultraviolet (UV) radiation. Several microorganisms found in this region were isolated and different colonies resistant to UV radiation were selected, a Geobacillus thermoleovorans strain identified through 16S RNA sequence, being the most remarkable. A gene library was prepared out of this strain with UV sensitive bacteria BH200 (uvrA::Tn10). A number of clones were isolated by means of UV selection, the most outstanding being a gene carrier able to codify for the guanosine monophosphate synthetase enzyme (GMPs). The suitability of said enzyme was proved by means of additional assays performed on ght 1 bacteria (guaA26::Tn 10) which lacked the enzyme. A transcript of 1100 pb was detected through Northern Blot. The result was consistent with that obtained for the mapping of the starting transcription site. The cloned GMPs produces an increase in growth speed and a greater biomass in BH200 bacteria. (author)

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost.

Science.gov (United States)

Fong, Jiunn C N; Svenson, Charles J; Nakasugi, Kenlee; Leong, Caine T C; Bowman, John P; Chen, Betty; Glenn, Dianne R; Neilan, Brett A; Rogers, Peter L

2006-10-01

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50-80 degrees C and pH 6.0-8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA-DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542(T)). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.

Cloning and characterization of the .I.str ./I.operon and elongation factor Tu expression in .I.Bacillus stearothermophilus./I..

Czech Academy of Sciences Publication Activity Database

Krásný, Libor; Vacík, Tomáš; Fučík, Vladimír; Jonák, Jiří

2000-01-01

Roč. 182, č. 21 (2000), s. 6114-6122 ISSN 0021-9193 R&D Projects: GA ČR GA204/98/0863 Grant - others:HHMI(US) 75195-540305 Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.506, year: 2000

Monitoring, Verification, and Treatment of Infectious Wastes and Their Optimal Management in the Hospitals of Qom City, Iran

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Mohammad Fahiminia

2016-08-01

Full Text Available Background and Objectives: Given that no comprehensive studies have yet been conducted on treatment of infectious wastes in hospitals of Qom City, this research was performed with the purpose of investigating the treatment methods used in these hospitals and monitoring the performance of waste elimination devices. Methods: Required information was obtained through in-person visit and observing the current situation, and the variables affecting waste treatment were extracted based on the type of treatment systems, and were collected, and accordingly, biological monitoring tests were designed for the studied hospitals. The data were analyzed using Fisher's exact test. Results: In this study, from 9 active hospitals in Qom Province, only 3 hospitals were equipped with waste treatment system. In hospital A, growth of Bacillus stearothermophilus spore were observed in 6.25% of the samples, while no microbial growth was recorded in hospital B. The initial investment to buy the machine in hospital A was about four times than that of hospital B. Conclusion: The findings of this study showed that treatment device of hospital B is more appropriate compared to the devices of hospital A due to complete destruction of spores, lower cost (for purchase, and maintenance. .

Isolation and Characterization of an α-Glucosidase Inhibitor from Musa spp. (Baxijiao Flowers

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Zhanwu Sheng

2014-07-01

Full Text Available The use of α-glucosidase inhibitors is considered to be an effective strategy in the treatment of diabetes. Using a bioassay-guided fractionation technique, five Bacillus stearothermophilus α-glucosidase inhibitors were isolated from the flowers of Musa spp. (Baxijiao. Using NMR spectroscopy analysis they were identified as vanillic acid (1, ferulic acid (2, β-sitosterol (3, daucosterol (4 and 9-(4′-hydroxyphenyl-2-methoxyphenalen-1-one (5. The half maximal inhibitory concentration (IC50 values of compounds 1–5 were 2004.58, 1258.35, 283.67, 247.35 and 3.86 mg/L, respectively. Compared to a known α-glucosidase inhibitor (acarbose, IC50 = 999.31 mg/L, compounds 3, 4 and 5 showed a strong α-glucosidase inhibitory effect. A Lineweaver-Burk plot indicated that compound 5 is a mixed-competitive inhibitor, while compounds 3 and 4 are competitive inhibitors. The inhibition constants (Ki of compounds 3, 4 and 5 were 20.09, 2.34 and 4.40 mg/L, respectively. Taken together, these data show that the compounds 3, 4 and 5 are potent α-glucosidase inhibitors.

Kinetic analysis of photochemical sterilization of thermoduric bacterial spores in slurry of semiconductor catalyst particles with aeration. Handotai shokubai ryushi no tsuki kendakukei ni okeru tainetsusei saikin hoshi no hikari sakkin to sono sokudoronteki kaiseki

Energy Technology Data Exchange (ETDEWEB)

Tone, S; Taya, M; Kato, S; Horie, Y; Ashikaga, Y [Osaka Univ., Osaka (Japan). Faculty of Engineering and Science; Joo, Hyunkyu [Konkuk Univ., Seoul (Korea, Republic of)

1993-11-10

To study the utilization of the photochemical sterilization of thermoduric bacterial spores B. stearothermophilus with photocatalysis of TiO2 particles, light irradiation experiments are conducted under various operational conditions. TiO2 particles and dissolved oxygen resulting from aeration must coexist to accelerate the rate of spore sterilization under light irradiation of a high pressure mercury lamp. The sterilization rate increases with larger average light intensity in the reactor, and it is estimated that the dissolved oxygen contributes to the formation of oxidative radicals which become the attacking species. The maximal rate of spore sterilization is obtained under the condition of TiO2 concentration of 5[times]10[sup -2]kg[center dot]m[sup -3]. By combining a second-order rate equation, wherein the spores' death caused by the oxidative radicals formed by TiO2 photocatalysis is taken into consideration, and single-hit multitarget model, wherein the target damaged by the oxidizer in the spores is assumed, the sterilization velocity can be described well, and the effect of light intensity and the obtained rate parameters are correlated well. 13 refs., 7 figs., 3 tabs.

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

Science.gov (United States)

Hatakeyama, T; Kimura, M

1988-03-15

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

Science.gov (United States)

Hansen, M A; Kirpekar, F; Ritterbusch, W; Vester, B

2002-02-01

Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-directed RNase H digestion. A novel method of mapping modifications by analysis of short, nucleotide-specific, RNase digestion fragments with Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was utilized. The MALDI-MS data were complemented by two primer extension techniques using reverse transcriptase. One technique utilizes decreasing concentrations of deoxynucleotide triphosphates to map 2'-O-ribose methylations. In the other, the rRNA is chemically modified, followed by mild alkaline hydrolysis to map pseudouridines (psis). A total of 10 posttranscriptionally methylated nucleotides and 6 psis were detected in the five organisms. Eight of the methylated nucleotides and one psi have not been reported previously. The distribution of modified nucleotides and their locations on the surface of the ribosomal peptidyl transferase cleft suggests functional importance.

DETECTION OF LASALOCID RESIDUES IN THE TISSUES OF BROILER CHICKENS BY A NEW SCREENING TEST TOTAL ANTIBIOTICS

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Martin Levkut, ml.

2011-04-01

Full Text Available The aim of the present study was to evaluate the microbial growth inhibition test Total antibiotics for the screening of lasalocid residues in the tissues of broiler chickens after its oral administration in medicated feed. The residues were investigated throughout the 5-day withdrawal period /WP/ and also on day 6 representing the first day following the WP. All broiler chicken tissues were positive for lasalocid. The breast muscle was positive (the presence of residues at/above the detection limit /LOD/ of method up to day 1 of the WP, the thigh muscle, gizzard, heart, skin and fat up to day 3 of the WP and the liver and kidneys up to day 4 of the WP. When evaluating the dubious results (the presence of residues just below the LOD of method, the breast muscle was suspect positive up to day 3 of the WP and the gizzard, skin and fat up to day 4 of the WP. No positive or dubious results were detected on day 5 of the WP. The LOD of Bacillus stearothermophilus var. calidolactis for maduramycin was 500 µg.l-1.doi:10.5219/140

Purification, crystallization and preliminary X-ray crystallographic analysis of the archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3

Energy Technology Data Exchange (ETDEWEB)

Lokanath, Neratur K.; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

2006-08-01

The archaeal phosphoglycerate mutase PH0037 from P. horikoshii OT3 has been crystallized in space group R32, with unit-cell parameters a = 155.62, c = 230.35 Å. A 2.2 Å resolution data was collected at SPring-8 beamline BL26B1. Phosphoglycerate mutases catalyze the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in glycolysis and gluconeogenesis pathways. The archaeal phosphoglycerate mutase PH0037 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to a resolution of 2.2 Å has been collected and processed in space group R32. Assuming the presence of a dimer in the asymmetric unit, the V{sub M} value is calculated to be 3.0 Å{sup 3} Da{sup −1}, consistent with the dynamic light-scattering experiment result, which shows a dimeric state of the protein in solution. Molecular-replacement trials using the crystal structure of Bacilllus stearothermophilus phosphoglycerate mutase as a search model did not provide a satisfactory solution, indicating substantially different structures of these two phophoglycerate mutases.

Comparison of four microbiological inhibition tests for the screening of antimicrobial residues in the tissues of food-producing animals

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Zuzana Gondová

2014-10-01

Full Text Available The study compares two existing microbiological inhibition tests, Screening Test for Antibiotic Residues (STAR and Premi®Test with two recently introduced tests, Nouws Antibiotic Test (NAT and Total Antibiotics for the screening of antimicrobial residues in the tissues of food-producing animals. In the negative or positive sample classification based on inhibition of the growth of test strain sensitive to many antibiotics and sulphonamides, out of 142 samples obtained from slaughterhouses and retail operations, 39 samples yielded a positive result in one or more tests: 4 samples in four tests, 14 samples in three tests, 13 samples in two tests, and 8 samples in one test. As for the numbers of observed positive samples, the descending sequence of tests was: STAR, Total Antibiotics, Premi®Test, NAT. The growth inhibition was observed in three out of seven test strains, namely Bacillus cereus ATCC 11778, Kocuria rhizophila ATCC 9341, and Bacillus stearothermophilus var. calidolactis. Considering the test strains sensitivity and no inhibition on the Bacillus pumilus NCIMB 10822 NAT test plates, our preliminary conclusion is that the animal samples are suspected for the presence of tetracycline, macrolide, and b-lactam antibiotics.

Sterilization by oxygen plasma

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Moreira, Adir Jose; Mansano, Ronaldo Domingues; Andreoli Pinto, Terezinha de Jesus; Ruas, Ronaldo; Silva Zambon, Luis da; Silva, Monica Valero da; Verdonck, Patrick Bernard

2004-07-31

The use of polymeric medical devices has stimulated the development of new sterilization methods. The traditional techniques rely on ethylene oxide, but there are many questions concerning the carcinogenic properties of the ethylene oxide residues adsorbed on the materials after processing. Another common technique is the gamma irradiation process, but it is costly, its safe operation requires an isolated site and it also affects the bulk properties of the polymers. The use of a gas plasma is an elegant alternative sterilization technique. The plasma promotes an efficient inactivation of the micro-organisms, minimises the damage to the materials and presents very little danger for personnel and the environment. Pure oxygen reactive ion etching type of plasmas were applied to inactivate a biologic indicator, the Bacillus stearothermophilus, to confirm the efficiency of this process. The sterilization processes took a short time, in a few minutes the mortality was complete. In situ analysis of the micro-organisms' inactivating time was possible using emission spectrophotometry. The increase in the intensity of the 777.5 nm oxygen line shows the end of the oxidation of the biologic materials. The results were also observed and corroborated by scanning electron microscopy.

Degradation of diclorinated dibenzo-P-dioxin by a cell free extract from Geobacillus midosuji SH2B-J2

International Nuclear Information System (INIS)

Nakamura, M.; Otsuka, Y.; Katayama, Y.; Takahashi, A.

2009-01-01

Polychlorinated dibenzo-p-dioxins (PCDDs) have been widespread environmental contaminants formed unintentionally as a by-products during the bleaching of pulp and paper, the manufacture of pesticides, and the incineration of halogen containing chemicals. PCDDs and PCDFs contamination has led to serious social problem because of their toxicity mutagenic and carcinogenic properties. (Author)

Evaluation of sensitivity of modified star protocol microbiological method for beta-lactame antibiotics detection in raw cow milk

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Borović Branka

2013-01-01

previously inoculated G.stearothermophilus ATCC 10149 strain. Aliquots of 30 _l of working solution at 0.5, 1 and 1.5 MRL concentration level, for each antibiotic, were inflicted on two paper disks placed on inoculated Kundrat agar surface. Petri plates with Kundrat agar previously inoculated with G.stearothermophilus , on which the samples were deposited, were incubated for 12-15h at 55oC. The obtained width of microorganisms growth inhibition zone, that is supposed to be at least 2.0 mm, measured from the disc edge, demonstrated the capability to detect all the tested 7 antibiotics from the beta lactam group at a level below the MRLs. Consequently, this proves that use of this method it is possible to meet the demands of Regulative Commission EC No. 37/2010. [Projekat Ministarstva nauke Republike Srbije, br. III-46009

Viabilidade celular de Saccharomyces cerevisiae cultivada em associação com bactérias contaminantes da fermentação alcoólica Cellular viability of Saccharomyces cerevisiae cultivated in association with contaminant bacteria of alcoholic fermentation

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Thais de Paula Nobre

2007-03-01

Full Text Available O objetivo deste trabalho foi estudar a influência de bactérias dos gêneros Bacillus e Lactobacillus, bem como de seus produtos metabólicos, na redução da viabilidade celular de leveduras Saccharomyces cerevisiae. As bactérias Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum e Lactobacillus plantarum foram cultivadas em associação com a levedura S. cerevisiae (cepa Y-904 por 72 horas a 32 °C, sob agitação. A viabilidade celular, a taxa de brotamento e a população de células de S. cerevisiae e a acidez total, a acidez volátil e o pH dos meios de cultivos foram determinados às 0, 24, 48 e 72 horas do cultivo misto. As culturas de bactérias foram tratadas através do calor, de agente antimicrobiano e de irradiação. Os resultados mostraram que apenas os meios de cultivo mais acidificados, contaminados com as bactérias ativas L. fermentum e B. subtilis, provocaram redução na viabilidade celular de S. cerevisiae. Excetuando a bactéria B. subtilis tratada com radiação gama, as demais bactérias tratadas pelos diferentes processos (calor, irradiação e antimicrobiano não causaram diminuição da viabilidade celular e da população de S. cerevisiae, indicando que a presença isolada dos metabólitos celulares dessas bactérias não foi suficiente para reduzir a porcentagem de células vivas de S. cerevisiae.The aim of this project was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products to decrease the cellular viability of the yeast Saccharomyces cerevisiae. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast S. cerevisiae (strain Y-904 for 72 hours at 32 ºC under agitation. The cellular viability, budding rate and population of S. Cerevisiae and the total acidity, volatile acidity and pH of culture medium were

The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach.

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Jose Sergio Hleap

Full Text Available The Glycoside Hydrolase Family 13 (GH13 is both evolutionarily diverse and relevant to many industrial applications. Its members hydrolyze starch into smaller carbohydrates and members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We find that the TIM-barrel (a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone, common to all amylases is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated α-amylase protein from Geobacillus thermoleovorans (PDB code: 4E2O; α-1,4-glucan-4-glucanohydrolase; EC 3.2.1.1. Overall, this paper demonstrates the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

Aplikasi Micro Morr E-3360 sebagai Bahan Bioremedian Tumpahan Minyak di Laut

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Endar Marraskuranto

2012-12-01

Full Text Available Penelitian ini bertujuan untuk menguji kelayakan Micro Morr E-3360 sebagai bahan bioremedian tumpahan minyak di laut pada skala laboratorium. Kelayakan didasari pada efektivitas pengurangan kadar Total Petroleum Hydrocarbon (TPH, toksisitas air laut hasil bioremediasi dan residu media imobilisasi dari produk. Analisis kadar TPH dilakukan dengan menggunakan spektrofotometer UV-Vis dan tingkat toksisitas diukur menggunakan metode Brine Shrimp Lethality Test (BSLT. Identifikasi konsorsium bakteri dilakukan pada media imobilisasi dengan analisis sekuens gen 16S rDNA. Penelitian dilakukan dengan lima uji coba dalam beaker, yaitu air laut dan minyak 2% w/w dari air laut (M; air laut, minyak 2% w/w dari air laut, bioremedian 10% w/w dari minyak, dan nutrien (P; kontrol air laut (AL; kontrol air laut dan bioremedian 10% w/w dari minyak (B; serta air laut dan nutrien (K. Hasil analisis menunjukkan bahwa konsorsium bakteri terdiri dari 3 spesies bakteri, yaitu Bacillus licheniformis, B. thermoamylovorans, dan Geobacillus pallidus. Bioremedian ini mampu menurunkan kadar TPH dalam air sebesar 77% dan tingkat toksisitas sebesar 88% selama 20 hari. Kadar residu minyak dalam media imobilisasi juga rendah, yaitu di bawah 2 ppm. Namun, penurunan kadar oksigen dan peningkatan turbiditas yang signifikan (P<0,05 dapat menjadi faktor utama kematian 50% populasi A. salina.

Unraveling the lipolytic activity of thermophilic bacteria isolated from a volcanic environment.

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Stathopoulou, Panagiota M; Savvides, Alexander L; Karagouni, Amalia D; Hatzinikolaou, Dimitris G

2013-01-01

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70-80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.

Microbial diversity in an Armenian geothermal spring assessed by molecular and culture-based methods.

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Panosyan, Hovik; Birkeland, Nils-Kåre

2014-11-01

The phylogenetic diversity of the prokaryotic community thriving in the Arzakan hot spring in Armenia was studied using molecular and culture-based methods. A sequence analysis of 16S rRNA gene clone libraries demonstrated the presence of a diversity of microorganisms belonging to the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, Firmicutes, Bacteroidetes phyla, and Cyanobacteria. Proteobacteria was the dominant group, representing 52% of the bacterial clones. Denaturing gradient gel electrophoresis profiles of the bacterial 16S rRNA gene fragments also indicated the abundance of Proteobacteria, Bacteroidetes, and Cyanobacteria populations. Most of the sequences were most closely related to uncultivated microorganisms and shared less than 96% similarity with their closest matches in GenBank, indicating that this spring harbors a unique community of novel microbial species or genera. The majority of the sequences of an archaeal 16S rRNA gene library, generated from a methanogenic enrichment, were close relatives of members of the genus Methanoculleus. Aerobic endospore-forming bacteria mainly belonging to Bacillus and Geobacillus were detected only by culture-dependent methods. Three isolates were successfully obtained having 99, 96, and 96% 16S rRNA gene sequence similarities to Arcobacter sp., Methylocaldum sp., and Methanoculleus sp., respectively. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Degradation of lignocelluloses in rice straw by BMC-9, a composite microbial system.

Science.gov (United States)

Zhao, Hongyan; Yu, Hairu; Yuan, Xufeng; Piao, Renzhe; Li, Hulin; Wang, Xiaofen; Cui, Zongjun

2014-05-01

To evaluate the potential utility of pretreatment of raw biomass with a complex microbial system, we investigated the degradation of rice straw by BMC-9, a lignocellulose decomposition strain obtained from a biogas slurry compost environment. The degradation characteristics and corresponding changes in the bacterial community were assessed. The results showed that rapid degradation occurred from day 0 to day 9, with a peak total biomass bacterium concentration of 3.3 × 10(8) copies/ml on day 1. The pH of the fermentation broth declined initially and then increased, and the mass of rice straw decreased steadily. The highest concentrations of volatile fatty acid contents (0.291 mg/l lactic acid, 0.31 mg/l formic acid, 1.93 mg/l acetic acid, and 0.73 mg/l propionic acid) as well as the highest xylanse activity (1.79 U/ml) and carboxymethyl cellulase activity (0.37 U/ml) occurred on day 9. The greatest diversity among the microbial community also occurred on day 9, with the presence of bacteria belonging to Clostridium sp., Bacillus sp., and Geobacillus sp. Together, our results indicate that BMC-9 has a strong ability to rapidly degrade the lignocelluloses of rice straw under relatively inexpensive conditions, and the optimum fermentation time is 9 days.

Unraveling the Lipolytic Activity of Thermophilic Bacteria Isolated from a Volcanic Environment

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Panagiota M. Stathopoulou

2013-01-01

Full Text Available In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70–80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.

Lysinibacillus fusiformis M5 Induces Increased Complexity in Bacillus subtilis 168 Colony Biofilms via Hypoxanthine.

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Gallegos-Monterrosa, Ramses; Kankel, Stefanie; Götze, Sebastian; Barnett, Robert; Stallforth, Pierre; Kovács, Ákos T

2017-11-15

In recent years, biofilms have become a central subject of research in the fields of microbiology, medicine, agriculture, and systems biology, among others. The sociomicrobiology of multispecies biofilms, however, is still poorly understood. Here, we report a screening system that allowed us to identify soil bacteria which induce architectural changes in biofilm colonies when cocultured with Bacillus subtilis We identified the soil bacterium Lysinibacillus fusiformis M5 as an inducer of wrinkle formation in B. subtilis colonies mediated by a diffusible signaling molecule. This compound was isolated by bioassay-guided chromatographic fractionation. The elicitor was identified to be the purine hypoxanthine using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. We show that the induction of wrinkle formation by hypoxanthine is not dependent on signal recognition by the histidine kinases KinA, KinB, KinC, and KinD, which are generally involved in phosphorylation of the master regulator Spo0A. Likewise, we show that hypoxanthine signaling does not induce the expression of biofilm matrix-related operons epsABCDEFGHIJKLMNO and tasA-sipW-tapA Finally, we demonstrate that the purine permease PbuO, but not PbuG, is necessary for hypoxanthine to induce an increase in wrinkle formation of B. subtilis biofilm colonies. Our results suggest that hypoxanthine-stimulated wrinkle development is not due to a direct induction of biofilm-related gene expression but rather is caused by the excess of hypoxanthine within B. subtilis cells, which may lead to cell stress and death. IMPORTANCE Biofilms are a bacterial lifestyle with high relevance regarding diverse human activities. Biofilms can be beneficial, for instance, in crop protection. In nature, biofilms are commonly found as multispecies communities displaying complex social behaviors and characteristics. The study of interspecies interactions will thus lead to a better understanding and use of biofilms as they

Comparative study on disinfection potency of spore forming bacteria by electron-beam irradiation and gamma-ray irradiation

International Nuclear Information System (INIS)

Takizawa, Hironobu; Suzuki, Satoru; Suzuki, Tetsuya; Takama, Kozo; Hayashi, Toru; Yasumoto, Kyoden.

1990-01-01

Along with gamma-ray irradiation, electron-beam irradiation (EB) is a method to disinfect microorganisms which cause food decomposition and food-poisoning. The present study was undertaken to compare sterilization efficacy of EB and gamma-ray irradiation on bacterial spores and vegetative cells under various conditions. Spores of Bacillus pumilus, a marker strain for irradiation study, and Bacillus stearothermophilus known as a thermophilic bacteria were irradiated by electron-beam and gamma-ray separately at irradiation dose of 0 to 10 kGy on combination of wet/dry and aerobic/anaerobic conditions. Sterilization effect of irradiation on spores was evaluated by colony counting on agar plates. Results showed that both EB and gamma-ray irradiation gave sufficient sterilization effect on spores, and the sterilization effect increased exponentially with irradiation dose. The sterilization effect of gamma-ray irradiation was higher than that of EB in all cases. Higher disinfection effect was observed under aerobic condition. The present study suggests that oxygen supply in EB is more important than gamma-ray irradiation. No results suggesting that chlorine ion at 0.1 ppm (as available chlorine concentration) enhanced the sterilization efficacy of either EB or gamma-ray irradiation was obtained under any conditions examined. (author)

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

Science.gov (United States)

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-12-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

The primary structures of ribosomal proteins S14 and S16 from the archaebacterium Halobacterium marismortui. Comparison with eubacterial and eukaryotic ribosomal proteins.

Science.gov (United States)

Kimura, J; Kimura, M

1987-09-05

The amino acid sequences of two ribosomal proteins, S14 and S16, from the archaebacterium Halobacterium marismortui have been determined. Sequence data were obtained by the manual and solid-phase sequencing of peptides derived from enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease as well as by chemical cleavage with cyanogen bromide. Proteins S14 and S16 contain 109 and 126 amino acid residues and have Mr values of 11,964 and 13,515, respectively. Comparison of the sequences with those of ribosomal proteins from other organisms demonstrates that S14 has a significant homology with the rat liver ribosomal protein S11 (36% identity) as well as with the Escherichia coli ribosomal protein S17 (37%), and that S16 is related to the yeast ribosomal protein YS22 (40%) and proteins S8 from E. coli (28%) and Bacillus stearothermophilus (30%). A comparison of the amino acid residues in the homologous regions of halophilic and nonhalophilic ribosomal proteins reveals that halophilic proteins have more glutamic acids, asparatic acids, prolines, and alanines, and less lysines, arginines, and isoleucines than their nonhalophilic counterparts. These amino acid substitutions probably contribute to the structural stability of halophilic ribosomal proteins.

Thermal sterilization of heat-sensitive products using high-temperature short-time sterilization.

Science.gov (United States)

Mann, A; Kiefer, M; Leuenberger, H

2001-03-01

High-temperature short-time (HTST) sterilization with a continuous-flow sterilizer, developed for this study, was evaluated. The evaluation was performed with respect to (a) the chemical degradation of two heat-sensitive drugs in HTST range (140-160 degrees C) and (b) the microbiological effect of HTST sterilization. Degradation kinetics of two heat-sensitive drugs showed that a high peak temperature sterilization process resulted in less chemical degradation for the same microbiological effect than a low peak temperature process. Both drugs investigated could be sterilized with acceptable degradation at HTST conditions. For the evaluation of the microbiological effect, Bacillus stearothermophilus ATCC 7953 spores were used as indicator bacteria. Indicator spore kinetics (D(T), z value, k, and E(a)), were determined in the HTST range. A comparison between the Bigelow model (z value concept) and the Arrhenius model, used to describe the temperature coefficient of the microbial inactivation, demonstrated that the Bigelow model is more accurate in prediction of D(T) values in the HTST range. The temperature coefficient decreased with increasing temperature. The influence of Ca(2+) ions and pH value on the heat resistance of the indicator spores, which is known under typical sterilization conditions, did not change under HTST conditions.

Comparative study on disinfection potency of spore forming bacteria by electron-beam irradiation and gamma-ray irradiation

Energy Technology Data Exchange (ETDEWEB)

Takizawa, Hironobu; Suzuki, Satoru; Suzuki, Tetsuya; Takama, Kozo [Hokkaido Univ., Hakodate (Japan). Faculty of Fisheries; Hayashi, Toru; Yasumoto, Kyoden

1990-10-01

Along with gamma-ray irradiation, electron-beam irradiation (EB) is a method to disinfect microorganisms which cause food decomposition and food-poisoning. The present study was undertaken to compare sterilization efficacy of EB and gamma-ray irradiation on bacterial spores and vegetative cells under various conditions. Spores of Bacillus pumilus, a marker strain for irradiation study, and Bacillus stearothermophilus known as a thermophilic bacteria were irradiated by electron-beam and gamma-ray separately at irradiation dose of 0 to 10 kGy on combination of wet/dry and aerobic/anaerobic conditions. Sterilization effect of irradiation on spores was evaluated by colony counting on agar plates. Results showed that both EB and gamma-ray irradiation gave sufficient sterilization effect on spores, and the sterilization effect increased exponentially with irradiation dose. The sterilization effect of gamma-ray irradiation was higher than that of EB in all cases. Higher disinfection effect was observed under aerobic condition. The present study suggests that oxygen supply in EB is more important than gamma-ray irradiation. No results suggesting that chlorine ion at 0.1 ppm (as available chlorine concentration) enhanced the sterilization efficacy of either EB or gamma-ray irradiation was obtained under any conditions examined. (author).

The prevalence and control of Bacillus and related spore-forming bacteria in the dairy industry

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Nidhi eGopal

2015-12-01

Full Text Available Milk produced in udder cells is sterile but due to its high nutrient content, it can be a good growth substrate for contaminating bacteria. The quality of milk is monitored via somatic cell counts and total bacterial counts, with prescribed regulatory limits to ensure quality and safety. Bacterial contaminants can cause disease, or spoilage of milk and its secondary products. Aerobic spore-forming bacteria, such as those from the genera Sporosarcina, Paenisporosarcina, Brevibacillus, Paenibacillus, Geobacillus and Bacillus, are a particular concern in this regard as they are able to survive industrial pasteurisation and form biofilms within pipes and stainless steel equipment. These single or multiple-species biofilms become a reservoir of spoilage microorganisms and a cycle of contamination can be initiated. Indeed, previous studies have highlighted that these microorganisms are highly prevalent in dead ends, corners, cracks, crevices, gaskets, valves and the joints of stainless steel equipment used in the dairy manufacturing plants. Hence, adequate monitoring and control measures are essential to prevent spoilage and ensure consumer safety. Common controlling approaches include specific cleaning-in-place processes, chemical and biological biocides and other novel methods. In this review, we highlight the problems caused by these microorganisms, and discuss issues relating to their prevalence, monitoring thereof and control with respect to the dairy industry.

Fatty acids and survival of bacteria in Hammam Pharaon springs, Egypt

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Yehia A. Osman

2018-06-01

Full Text Available A great lack of knowledge of Hammam Pharaon's microbial community; the most famous hot spring in Sinai, Egypt, derived this work. Three different hyperthermophilic bacterial were isolated from vents in the area, where the temperature was above 80 °C. Response Surface Methodology algorithm such as Central Composite Design determined the optimum cultivation conditions for these isolates. Accordingly, the best growth conditions were at 70 °C and at neutral to slightly acidic pH values. The constructed phylogenetic tree built using the 16S rRNA gene sequences has shown that the isolated strains (HM101, HM102 and HM103 belong to Geobacillus, Rhodothermus and Thermus bacteria, respectively. The fatty acid profiles, an indicative of thermotolerance, dominated by the short chain Dodecanoic acid (Lauric acid; (12:0, which represented about 40% of the total fatty acid contents for each of the three isolates. The enzymatic capabilities of the three strains were determined and α-amylase was found to be the most prominent one. Our own data had led us to conclude that the length of the fatty acid chain and the degree of saturation could be species specific. Moreover, the biotechnological potentials of these local isolates could contribute to fighting viral diseases and/or improve their amylolytic activities for sugar industry; where thermotolerance is really an important factor.

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

Science.gov (United States)

Adachi, Daisuke; Koh, FookHee; Hama, Shinji; Ogino, Chiaki; Kondo, Akihiko

2013-05-10

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production. Copyright © 2013 Elsevier Inc. All rights reserved.

Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

Science.gov (United States)

Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

2012-03-13

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

Science.gov (United States)

Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

2012-01-01

We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

Production of the blood pressure lowing peptides from brown alga ( Undaria pinnatifida)

Science.gov (United States)

Minoru, Sato; Takashi, Oba; Takao, Hosokawa; Toshiyasu, Yamaguchi; Toshiki, Nakano; Tadao, Saito; Koji, Muramoto; Takashi, Kahara; Katsura, Funayama; Akio, Kobayashi; Takahisa, Nakano

2005-07-01

Brown alga ( Undaria pinnatifida) was treated with alginate lyase and hydrolyzed using 17 kinds of proteases and the inhibitory activity of the hydrolysates for the angiotensin-I-converting enzyme (ACE) was measured. Four hydrolysates with potent ACE-inhibitory activity were administered singly and orally to spontaneously hypertensive rats (SHRs). The systolic blood pressure of SHRs decreases significantly after single oral administration of the brown alga hydrolysates by protease S ‘Amano’ (from Bacillus stearothermophilus) at the concentration of 10 (mg protein) (kg body weight)-1. In the 17 weeks of feeding experiment, 7-week-old SHRs were fed standard diet supplemented with the brown alga hydrolysates for 10 weeks. In SHRs fed 1.0 and 0.1% brown alga hydrolysates, elevating of systolic bloodpressure was significantly suppressed for 7 weeks. To elucidate the active components, the brown alga hydrolysates were fractionated by 1-butanol extraction and HPLC on a reverse-phase column. Seven kinds of ACE-inhibitory peptides were isolated and identified by amino acid composition analysis, sequence analysis, and LC-MS with the results Val-Tyr, Ile-Tyr, Ala-Trp, Phe-Tyr, Val-Trp, Ile-Trp, and Leu-Trp. Each peptide was determined to have an antihypertensive effect after a single oral administration in SHRs. The brown alga hydrolysates were also confirmed to decrease the blood pressure in humans.

Pre-treatment of thermoduric spores in CO2 modified atmosphere and their survivability during food extrusion

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Marcos Fraiha

2011-03-01

Full Text Available The aim of this experiment was to evaluate how susceptible spores become to mechanical damage during food extrusion after being submitted to CO2. B. stearothermophilus spores sowed to corn and soy mix were submitted to 99% CO2 for 10 days and extruded in a single-screw extruder. The treatments were: T1 - spore-containing samples, extruded at screw rotational speed of 65 rpm and barrel wall temperature of 80 °C; T2 - as T1, except for screw rotational speed of 150 rpm; and T3 - as T2, except that samples were submitted to the modified atmosphere. The results for cell viability, minimum and maximum residence times, and static pressure were T1 - 19.90 ± 3.24%, 123.3 ± 14.50 seconds; 203.3 ± 14.05 seconds; 2.217 ± 62 kPa; T2 - 21.42 ± 8.24%, 70.00 ± 5.77 seconds; 170.00 ± 4.67 seconds; 2.310 ± 107 kPa; and T3 - 11.06 ± 2.46%, 86.00 ± 7.23 seconds; 186.00 ± 7.50 seconds; 2.403 ± 93 kPa, respectively. It was concluded that the extrusion process did reduce the cell count. However, screw rotational speed variation or CO2 pre-treatment did not affect cell viability.

Validação do processamento térmico de um produto protéico vegetal enlatado Thermal processing validation of a canned vegetal protein product

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Gladistone Carvalho Santos Filho

2003-12-01

Full Text Available A validação de processos nas indústrias alimentícia e farmacêutica é uma das principais ferramentas da garantia de qualidade, tornando os produtos seguros, eficazes e confiáveis.Realizou-se a validação física e biológica da esterilização terminal de um produto protéico constituído de proteína texturizada de soja, proteína de trigo (glúten, gordura vegetal hidrogenada e condimentos, envasado em latas metálicas de 850 gramas.A validação física do autoclave foi executada em três ciclos na câmara com 12 sensores de temperatura. Confirmou-se para os pontos geometricamente distribuídos no interior do autoclave que a diferença máxima de temperatura entre um ponto e outro foi de 1,0 ºC em relação à temperatura média da câmara (121 ºC. Nos ciclos realizados com carga máxima, o menor valor F121ºC10ºC (64,86 minutos garantiu S.A.L.(Sterility Assurance Level ou nível de garantia de esterilidade de pelo menos 10-12 em relação ao indicador biológico de Bacillus stearothermophilus ATCC 7953 (valor D121ºC de 3,68 minutos e população média de 1,00 x 10(6 esporos por fita.Para todas as operações de esterilização a presença de esporos sobreviventes no indicador biológico não foi detectada.Apesar de não existir margem de segurança definida para esterilização de alimentos enlatados, a validação periódica de um autoclave proporcionou maior confiabilidade na avaliação da esterilidade, que o simples teste de incubação por amostragem de produtos acabados, recomendado pelo Ministério da Saúde do Brasil.Process validation in the Food and Pharmaceutics Industries is one of the most important tools meant to guarantee quality, making safe and trustworthy products. Physical and biological validations were performed in the terminal sterilization of a canned vegetal protein product. Physical validation of the autoclave has been proceeded in three empty chamber cycles and 12 temperature sensors. It has been

Pesquisa de resíduos de antibióticos em leite pasteurizado tipo C, comercializado na cidade de Salvador.

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G. M. S. Barros

2005-03-01

Full Text Available RESUMO: O leite é um alimento de alto valor nutritivo, amplamente consumido pelo homem, sendo considerado como um dos principais alimentos da dieta das crianças. Por ser considerado um alimento completo em nutrientes facilmente assimiláveis, torna-se um excelente meio de cultura para a maioria dos microrganismos encontrados na natureza. No leite nunca se deve tolerar inibidores ou substâncias para aumentar sua capacidade de conservação. A incorporação de antibióticos no leite pode ocorrer acidentalmente, como é o caso dos medicamentos ou colocados como adulterantes. Os efeitos que estas drogas causam no homem, podem levar de um simples processo alérgico até um choque anafilático, além das grandes perdas de partidas de leite em indústrias de laticínios, trazendo prejuízos econômicos. Durante o período de outubro de 1998 a março de 1999 foram analisadas 26 amostras de leite pasteurizado “tipo C” de apenas uma marca, utilizando o método ADM (Antimicrobial Diffusion Method. As amostras foram coletadas em vários estabelecimentos comerciais da cidade de Salvador-Ba. Os resultados evidenciaram que 38,5% das amostras apresentaram-se em desacordo com as normas estabelecidas pelo RIISPOA (Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal no Art. 514, parágrafo único, que proíbe o uso de substâncias químicas na conservação do leite. PALAVRAS CHAVE: leite pasteurizado, resíduos antibióticos, Bacillus stearothermophilus, var. calidolactis. SUMMARY: Milk is a food of high nutritional value. It is widely consumed by human beings and it is considered as one of the main aliments of children diet. It is regarded as a complete nurishment in easily assimilable nutrients, thus it becomes an excelent culture medium for most organisms in nature. There must not be any tolerance for inibitors or substances to increase its conservation

Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases.

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Malihe Masomian

Full Text Available Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

Production and properties of two novel exopolysaccharides synthesized by a thermophilic bacterium Aeribacillus pallidus 418.

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Radchenkova, Nadja; Vassilev, Spasen; Panchev, Ivan; Anzelmo, Gianluca; Tomova, Iva; Nicolaus, Barbara; Kuncheva, Margarita; Petrov, Kaloyan; Kambourova, Margarita

2013-09-01

Synthesis of innovative exocellular polysaccharides (EPSs) was reported for few thermophilic microorganisms as one of the mechanisms for surviving at high temperature. Thermophilic aerobic spore-forming bacteria able to produce exopolysaccharides were isolated from hydrothermal springs in Bulgaria. They were referred to four species, such as Aeribacillus pallidus, Geobacillus toebii, Brevibacillus thermoruber, and Anoxybacillus kestanbolensis. The highest production was established for the strain 418, whose phylogenetic and phenotypic properties referred it to the species A. pallidus. Maltose and NH4Cl were observed to be correspondingly the best carbon and nitrogen sources and production yield was increased more than twofold in the process of culture condition optimization. After purification of the polymer fraction, a presence of two different EPSs, electroneutral EPS 1 and negatively charged EPS 2, in a relative weight ratio 3:2.2 was established. They were heteropolysaccharides consisting of unusual high variety of sugars (six for EPS 1 and seven for EPS 2). Six of the sugars were common for both EPSs. The main sugar in EPS 1 was mannose (69.3 %); smaller quantities of glucose (11.2 %), galactosamine (6.3 %), glucosamine (5.4 %), galactose (4.7 %), and ribose (2.9 %) were also identified. The main sugar in EPS 2 was also mannose (33.9 %), followed by galactose (17.9 %), glucose (15.5 %), galactosamine (11.7 %), glucosamine (8.1 %), ribose (5.3 %), and arabinose (4.9 %). Both polymers showed high molecular weight and high thermostability.

Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus

Energy Technology Data Exchange (ETDEWEB)

Saw, Jimmy H [Los Alamos National Laboratory; Mountain, Bruce W [NEW ZEALAND; Feng, Lu [NANKAI UNIV; Omelchenko, Marina V [NCBI/NLM/NIH; Hou, Shaobin [UNIV OF HAWAII; Saito, Jennifer A [UNIV OF HAWAII; Stott, Matthew B [NEW ZEALAND; Li, Dan [NANKAI UNIV; Zhao, Guang [NANKAI UNIV; Wu, Junli [NANKAI UNIV; Galperin, Michael Y [NCBI/NLM/NIH; Koonin, Eugene V [NCBI/NLM/NIH; Makarova, Kira S [NCBI/NLM/NIH; Wolf, Yuri I [NCBI/NLM/NIH; Rigden, Daniel J [UNIV OF LIVERPOOL; Dunfield, Peter F [UNIV OF CALGARY; Wang, Lei [NANKAI UNIV; Alam, Maqsudul [UNIV OF HAWAII

2008-01-01

Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats, such as geothermal hot springs and manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life. We report here the complete genome sequence of A. flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres. Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

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Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

2018-07-02

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

AFNOR validation of Premi Test, a microbiological-based screening tube-test for the detection of antimicrobial residues in animal muscle tissue.

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Gaudin, Valerie; Juhel-Gaugain, Murielle; Morétain, Jean-Pierre; Sanders, Pascal

2008-12-01

Premi Test contains viable spores of a strain of Bacillus stearothermophilus which is sensitive to antimicrobial residues, such as beta-lactams, tetracyclines, macrolides and sulphonamides. The growth of the strain is inhibited by the presence of antimicrobial residues in muscle tissue samples. Premi Test was validated according to AFNOR rules (French Association for Normalisation). The AFNOR validation was based on the comparison of reference methods (French Official method, i.e. four plate test (FPT) and the STAR protocol (five plate test)) with the alternative method (Premi Test). A preliminary study was conducted in an expert laboratory (Community Reference Laboratory, CRL) on both spiked and incurred samples (field samples). Several method performance criteria (sensitivity, specificity, relative accuracy) were estimated and are discussed, in addition to detection capabilities. Adequate agreement was found between the alternative method and the reference methods. However, Premi Test was more sensitive to beta-lactams and sulphonamides than the FPT. Subsequently, a collaborative study with 11 laboratories was organised by the CRL. Blank and spiked meat juice samples were sent to participants. The expert laboratory (CRL) statistically analysed the results. It was concluded that Premi Test could be used for the routine determination of antimicrobial residues in muscle of different animal origin with acceptable analytical performance. The detection capabilities of Premi Test for beta-lactams (amoxicillin, ceftiofur), one macrolide (tylosin) and tetracycline were at the level of the respective maximum residue limits (MRL) in muscle samples or even lower.

Sequence analysis of the aminoacylase-1 family. A new proposed signature for metalloexopeptidases.

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Biagini, A; Puigserver, A

2001-03-01

The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

Differential scanning calorimetry of bacteria.

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Miles, C A; Mackey, B M; Parsons, S E

1986-04-01

Thermograms obtained by differential scanning calorimetry of a range of bacteria of different heat resistances were compared. Equations were derived to calculate the rate at which the numbers of viable organisms in a calorimeter decline as the temperature is raised at a constant rate. Vegetative bacteria scanned at 10 degrees C min-1 showed multi-peaked thermograms with four major peaks (denoted m, n, p and q) occurring in the regions 68-73, 77-84, 89-99 and 105-110 degrees C respectively. Exceptions were that peak m (the largest peak) occurred at 79-82 degrees C in Bacillus stearothermophilus and an additional peak, r, was detected in Escherichia coli at 119 degrees C. At temperatures below the main peak m there were major differences in thermograms between species. There was a direct relationship between the onset of thermal denaturation and the thermoresistance of different organisms. Heat-sensitive organisms displayed thermogram features which were absent in the more heat-resistant types. When samples were cooled to 5 degrees C and re-heated, a small endothermic peak, pr, was observed at the same temperature as p. Peaks p and pr were identified as the melting endotherms of DNA. In all vegetative organisms examined, maximum death rates, computed from published D and z values, occurred at temperatures above the onset of thermal denaturation, i.e. cell death and irreversible denaturation of cell components occurred within the same temperature range.

Making soy sauce from defatted soybean meal without the mejus process by submerged cultivation using thermophilic bacteria.

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Hur, Jeong Min; Park, Doo Hyun

2015-08-01

The diversity of thermophilic bacteria was not significantly altered while growing in a defatted soybean meal (DFSM) slurry at 60 °C for 10, 20, and 30 days. Five species of thermophilic bacteria, which belong to the genera Aeribacillus (temperature gradient gel electrophoresis [TGGE] band no. 1), Saccharococcus (TGGE band no. 2), Geobacillus (TGGE band no. 3), Bacillus (TGGE band no. 4), and Anoxybacillus (TGGE band no. 5), were detected in the fermenting DFSM slurry. The cell-free culture fluid obtained from the fermenting DFSM slurry on day 14 hydrolyzed starch and soy protein at 60 °C but not at 30 °C. Soy sauce (test soy sauce) was prepared from the fermented DFSM slurry after a 30 day cultivation at 60 °C and a 60 day ripening at 45 °C. Free amino acid (AA) and organic acid contents in the soy sauce increased in proportion to the fermentation period, whereas ammonium decreased proportionally. Mg and Ca contained in the soy sauce decreased proportionally during fermentation and were lower than those in the non-fermented DFSM extract (control). Spectral absorbance of soy sauce prepared from the fermented DFSM slurry was maximal at 430 nm and increased slightly in proportion to the fermentation period. The aroma and flavor of the test soy sauce were significantly different from those of traditional Korean soy sauce. Conclusively, soy sauce may be prepared directly from the fermented DFSM slurry without meju-preparing process and fermentation period may be a factor for control of soy sauce quality.

Large-Scale Purification, Characterization, and Spore Outgrowth Inhibitory Effect of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361.

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Wang, Gaoyan; Manns, David C; Guron, Giselle K; Churey, John J; Worobo, Randy W

2014-06-01

Large-scale purification of the highly hydrophobic bacteriocin thurincin H was accomplished via a novel and simple two-step method: ammonia sulfate precipitation and C18 solid-phase extraction. The inhibition spectrum and stability of thurincin H as well as its antagonistic activity against Bacillus cereus F4552 spores were further characterized. In the purification method, secreted proteins contained in the supernatant of a 40 h incubated culture of B. thuringiensis SF361 were precipitated by 68 % ammonia sulfate and purified by reverse-phase chromatography, with a yield of 18.53 mg/l of pure thurincin H. Silver-stained SDS-PAGE, high-performance liquid chromatography, and liquid chromatography-mass spectrometry confirmed the high purity of the prepared sample. Thurincin H exhibited a broad antimicrobial activity against 22 tested bacterial strains among six different genera including Bacillus, Carnobacterium, Geobacillus, Enterococcus, Listeria, and Staphylococcus. There was no detectable activity against any of the selected yeast or fungi. The bacteriocin activity was stable for 30 min at 50 °C and decreased to undetectable levels within 10 min at temperatures above 80 °C. Thurincin H is also stable from pH 2-7 for at least 24 h at room temperature. Thurincin H is germicidal against B. cereus spores in brain heart infusion broth, but not in Tris-NaCl buffer. The efficient purification method enables the large-scale production of pure thurincin H. The broad inhibitory spectrum of this bacteriocin may be of interest as a potential natural biopreservative in the food industry, particularly in post-processed and ready-to-eat food.

Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

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You, Di; Yao, Li-Li; Huang, Dan; Escalante-Semerena, Jorge C; Ye, Bang-Ce

2014-09-01

Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The efficacy of chemical agents in cleaning and disinfection programs

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Silva Martins Alzira

2001-09-01

Full Text Available Abstract Background Due to the growing number of outbreaks of infection in hospital nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Method For the purpose of evaluating the efficacy of a chemical agent, the minimum inhibitory concentration (MIC was reached by the classic method of successive broth dilutions. The reference bacteria utilized were Bacillus subtilis var. globigii ATCC 9372, Bacillus stearothermophilus ATCC 7953, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923. The strains of Enterobacter cloacae IAL 1976 (Adolfo Lutz Institute, Serratia marcescens IAL 1478 and Acinetobactev calcoaceticus IAL 124 (ATCC 19606, were isolated from material collected from babies involved in outbreaks of infection in hospital nurseries. Results The MIC intervals, which reduced bacteria populations over 08 log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs; 63 to 10000 mg/L of chlorhexidine digluconate; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of isopropanol or ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs; 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Conclusions Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus, was observed to show resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens.

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past.

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Judit Ribera

Full Text Available A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past.

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Ribera, Judit; Estupiñán, Mónica; Fuentes, Alba; Fillat, Amanda; Martínez, Josefina; Diaz, Pilar

2017-01-01

A search for extremophile enzymes from ancient volcanic soils in El Hierro Island (Canary Islands, Spain) allowed isolation of a microbial sporulated strain collection from which several enzymatic activities were tested. Isolates were obtained after sample cultivation under several conditions of nutrient contents and temperature. Among the bacterial isolates, supernatants from the strain designated JR3 displayed high esterase activity at temperatures ranging from 30 to 100°C, suggesting the presence of at least a hyper-thermophilic extracellular lipase. Sequence alignment of known thermophilic lipases allowed design of degenerated consensus primers for amplification and cloning of the corresponding lipase, named LipJ. However, the cloned enzyme displayed maximum activity at 30°C and pH 7, showing a different profile from that observed in supernatants of the parental strain. Sequence analysis of the cloned protein showed a pentapeptide motif -GHSMG- distinct from that of thermophilic lipases, and much closer to that of esterases. Nevertheless, the 3D structural model of LipJ displayed the same folding as that of thermophilic lipases, suggesting a common evolutionary origin. A phylogenetic study confirmed this possibility, positioning LipJ as a new member of the thermophilic family of bacterial lipases I.5. However, LipJ clusters in a clade close but separated from that of Geobacillus sp. thermophilic lipases. Comprehensive analysis of the cloned enzyme suggests a common origin of LipJ and other bacterial thermophilic lipases, and highlights the most probable divergent evolutionary pathway followed by LipJ, which during the harsh past times would have probably been a thermophilic enzyme, having lost these properties when the environment changed to more benign conditions.

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

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Melih Koc

2015-12-01

Full Text Available Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg. The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

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Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

A large collapsed-state RNA can exhibit simple exponential single-molecule dynamics.

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Smith, Glenna J; Lee, Kang Taek; Qu, Xiaohui; Xie, Zheng; Pesic, Jelena; Sosnick, Tobin R; Pan, Tao; Scherer, Norbert F

2008-05-09

The process of large RNA folding is believed to proceed from many collapsed structures to a unique functional structure requiring precise organization of nucleotides. The diversity of possible structures and stabilities of large RNAs could result in non-exponential folding kinetics (e.g. stretched exponential) under conditions where the molecules have not achieved their native state. We describe a single-molecule fluorescence resonance energy transfer (FRET) study of the collapsed-state region of the free energy landscape of the catalytic domain of RNase P RNA from Bacillus stearothermophilus (C(thermo)). Ensemble measurements have shown that this 260 residue RNA folds cooperatively to its native state at >or=1 mM Mg(2+), but little is known about the conformational dynamics at lower ionic strength. Our measurements of equilibrium conformational fluctuations reveal simple exponential kinetics that reflect a small number of discrete states instead of the expected inhomogeneous dynamics. The distribution of discrete dwell times, collected from an "ensemble" of 300 single molecules at each of a series of Mg(2+) concentrations, fit well to a double exponential, which indicates that the RNA conformational changes can be described as a four-state system. This finding is somewhat unexpected under [Mg(2+)] conditions in which this RNA does not achieve its native state. Observation of discrete well-defined conformations in this large RNA that are stable on the seconds timescale at low [Mg(2+)] (<0.1 mM) suggests that even at low ionic strength, with a tremendous number of possible (weak) interactions, a few critical interactions may produce deep energy wells that allow for rapid averaging of motions within each well, and yield kinetics that are relatively simple.

Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel

International Nuclear Information System (INIS)

Ishii, Yoshitaka; Markus, Michelle A.; Tycko, Robert

2001-01-01

Water-soluble biological macromolecules can be weakly aligned by dissolution in a strained, hydrated gel such as cross-linked polyacrylamide, an effect termed 'strain-induced alignment in a gel' (SAG). SAG induces nonzero nuclear magnetic dipole-dipole couplings that can be measured in high-resolution NMR spectra and used as structural constraints. The dependence of experimental 15 N- 1 H dipolar couplings extracted from two-dimensional heteronuclear single quantum coherence (HSQC) spectra on several properties of compressed polyacrylamide, including the extent of compression, the polyacrylamide concentration, and the cross-link density, is reported for the B1 immunoglobulin binding domain of streptococcal protein G (protein G/B1, 57 residues). It is shown that the magnitude of macromolecular alignment can be widely varied by adjusting these properties, although the orientation and asymmetry of the alignment tensor are not affected significantly. The dependence of the 15 N relaxation times T 1 and T 2 of protein G/B1 on polyacrylamide concentration are also reported. In addition, the results of 15 N relaxation and HSQC experiments on the RNA binding domain of prokaryotic protein S4 from Bacillus stearothermophilus (S4 Δ41, residues 43-200) in a compressed polyacrylamide gel are presented. These results demonstrate the applicability of SAG to proteins of higher molecular weight and greater complexity. A modified in-phase/anti-phase (IPAP) HSQC technique is described that suppresses natural-abundance 15 N background signals from amide groups in polyacrylamide, resulting in cleaner HSQC spectra in SAG experiments. The mechanism of protein alignment in strained polyacrylamide gels is contrasted with that in liquid crystalline media

Metodología para la validación del llenado aséptico en un proceso de liofilización Methodology for the validation of the aseptic filling in a lyophilization process

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Cindy Marlene Fernández López

2007-04-01

Full Text Available Se presentó la metodología propuesta para la validación de un llenado aséptico en un proceso de liofilización. Para esto se realizaron 3 pruebas en las cuales se envasaron 3 lotes de 15 000 viales cada uno, con caldo casoy que se incubaron a 22,5 ± 2,5 ºC y 32,5 ± 2,5 ºC por 14 días respectivamente, con el fin de detectar la posibilidad de contaminación microbiana en la realización del proceso. Durante las 3 pruebas se realizó el monitoreo microbiológico de ambientes, superficies y operarios; en el muestreo se utilizó Petrifilm para detección de mesófilos aerobios y levaduras, procedimiento ejecutado en condiciones de reposo y operación. Así mismo, se efectuó el conteo de partículas en el área y pruebas de esterilidad para los materiales de envase (tapones y viales; finalmente, se diseñó la metodología de validación para la esterilización del liofilizador y se empleó como microorganismo indicador a Bacillus stearothermophilus. El diseño de la prueba se realizó teniendo en cuenta las recomendaciones de la Food Drug Administration (FDA y la Organización Mundial de la Salud (OMS para productos de administración parenteral. Estas son las organizaciones de referencia para la producción y control de calidad que utiliza la empresa de productos veterinarios en las que se hizo la prueba. Los viales envasados se leyeron por turbidez (presencia de contaminación y los criterios de aceptación se establecieron por las 2 entidades nombradas anteriormente. Finalmente, se indicó un plan de monitoreo microbiológico para el área evaluada en el cual se incluyeron puntos de muestreo estratégicos; así como la realización de procedimientos operativos estándar específicos para esta área.The methodology proposed for the validation of an aseptic filling in a lyophilization process was presented. To this end, 3 tests were made in which 3 batches of 15 000 vials each one were filled with casoy broth. They were incubated at 22.5 �

Comprehensive microbial analysis of combined mesophilic anaerobic-thermophilic aerobic process treating high-strength food wastewater.

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Jang, Hyun Min; Ha, Jeong Hyub; Park, Jong Moon; Kim, Mi-Sun; Sommer, Sven G

2015-04-15

A combined mesophilic anaerobic-thermophilic aerobic process was used to treat high-strength food wastewater in this study. During the experimental period, most of solid residue from the mesophilic anaerobic reactor (R1) was separated by centrifugation and introduced into the thermophilic aerobic reactor (R2) for further digestion. Then, thermophilic aerobically-digested sludge was reintroduced into R1 to enhance reactor performance. The combined process was operated with two different Runs: Run I with hydraulic retention time (HRT) = 40 d (corresponding OLR = 3.5 kg COD/m(3) d) and Run II with HRT = 20 d (corresponding OLR = 7 kg COD/m(3)). For a comparison, a single-stage mesophilic anaerobic reactor (R3) was operated concurrently with same OLRs and HRTs as the combined process. During the overall digestion, all reactors showed high stability without pH control. The combined process demonstrated significantly higher organic matter removal efficiencies (over 90%) of TS, VS and COD and methane production than did R3. Quantitative real-time PCR (qPCR) results indicated that higher populations of both bacteria and archaea were maintained in R1 than in R3. Pyrosequencing analysis revealed relatively high abundance of phylum Actinobacteria in both R1 and R2, and a predominance of phyla Synergistetes and Firmicutes in R3 during Run II. Furthermore, R1 and R2 shared genera (Prevotella, Aminobacterium, Geobacillus and Unclassified Actinobacteria), which suggests synergy between mesophilic anaerobic digestion and thermophilic aerobic digestion. For archaea, in R1 methanogenic archaea shifted from genus Methanosaeta to Methanosarcina, whereas genera Methanosaeta, Methanobacterium and Methanoculleus were predominant in R3. The results demonstrated dynamics of key microbial populations that were highly consistent with an enhanced reactor performance of the combined process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Diversity of Metabolically Active Bacteria in Water-Flooded High-Temperature Heavy Oil Reservoir

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Tamara N. Nazina

2017-04-01

Full Text Available The goal of this work was to study the overall genomic diversity of microorganisms of the Dagang high-temperature oilfield (PRC and to characterize the metabolically active fraction of these populations. At this water-flooded oilfield, the microbial community of formation water from the near-bottom zone of an injection well where the most active microbial processes of oil degradation occur was investigated using molecular, cultural, radiotracer, and physicochemical techniques. The samples of microbial DNA and RNA from back-flushed water were used to obtain the clone libraries for the 16S rRNA gene and cDNA of 16S rRNA, respectively. The DNA-derived clone libraries were found to contain bacterial and archaeal 16S rRNA genes and the alkB genes encoding alkane monooxygenases similar to those encoded by alkB-geo1 and alkB-geo6 of geobacilli. The 16S rRNA genes of methanogens (Methanomethylovorans, Methanoculleus, Methanolinea, Methanothrix, and Methanocalculus were predominant in the DNA-derived library of Archaea cloned sequences; among the bacterial sequences, the 16S rRNA genes of members of the genus Geobacillus were the most numerous. The RNA-derived library contained only bacterial cDNA of the 16S rRNA sequences belonging to metabolically active aerobic organotrophic bacteria (Tepidimonas, Pseudomonas, Acinetobacter, as well as of denitrifying (Azoarcus, Tepidiphilus, Calditerrivibrio, fermenting (Bellilinea, iron-reducing (Geobacter, and sulfate- and sulfur-reducing bacteria (Desulfomicrobium, Desulfuromonas. The presence of the microorganisms of the main functional groups revealed by molecular techniques was confirmed by the results of cultural, radioisotope, and geochemical research. Functioning of the mesophilic and thermophilic branches was shown for the microbial food chain of the near-bottom zone of the injection well, which included the microorganisms of the carbon, sulfur, iron, and nitrogen cycles.

A review of the microbiology of the Rehai geothermal field in Tengchong, Yunnan Province, China

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Brian P. Hedlund

2012-05-01

Full Text Available The Rehai Geothermal Field, located in Tengchong County, in central-western Yunnan Province, is the largest and most intensively studied geothermal field in China. A wide physicochemical diversity of springs (ambient to ∼97 °C; pH from ≤1.8 to ≥9.3 provides a multitude of niches for extremophilic microorganisms. A variety of studies have focused on the cultivation, identification, basic physiology, taxonomy, and biotechnological potential of thermophilic microorganisms from Rehai. Thermophilic bacteria isolated from Rehai belong to the phyla Firmicutes and Deinococcus-Thermus. Firmicutes include neutrophilic or alkaliphilic Anoxybacillus, Bacillus, Caldalkalibacillus, Caldanaerobacter, Laceyella, and Geobacillus, as well as thermoacidophilic Alicyclobacillus and Sulfobacillus. Isolates from the Deinococcus-Thermus phylum include several Meiothermus and Thermus species. Many of these bacteria synthesize thermostable polymer-degrading enzymes that may be useful for biotechnology. The thermoacidophilic archaea Acidianus, Metallosphaera, and Sulfolobus have also been isolated and studied. A few studies have reported the isolation of thermophilic viruses belonging to Siphoviridae (TTSP4 and TTSP10 and Fuselloviridae (STSV1 infecting Thermus spp. and Sulfolobus spp., respectively. More recently, cultivation-independent studies using 16S rRNA gene sequences, shotgun metagenomics, or “functional gene” sequences have revealed a much broader diversity of microorganisms than represented in culture. Studies of the gene and mRNA encoding the large subunit of the ammonia monooxygenase (amoA of ammonia-oxidizing Archaea (AOA and the tetraether lipid crenarchaeol, a potential biomarker for AOA, suggest a wide diversity, but possibly low abundance, of thermophilic AOA in Rehai. Finally, we introduce the Tengchong Partnerships in International Research and Education (PIRE project, an international collaboration between Chinese and U.S. scientists with

Thermophilic spore-forming bacteria isolated from spoiled canned food and their heat resistance. Results of a French ten-year survey.

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André, S; Zuber, F; Remize, F

2013-07-15

Thermal processing of Low Acid Canned Foods (LACF), which are safe and shelf-stable at ambient temperature for several years, results in heat inactivation of all vegetative microorganisms and the partial or total inactivation of spores. Good Manufacturing Hygienic Practices include stability tests for managing the pathogen risk related to surviving mesophilic bacterial spores. LACF are also often submitted to additional incubation conditions, typically 55 °C for 7 days, to monitor spoilage by thermophiles. In this study we identified the bacterial species responsible for non-stability after prolonged at 55 °C of incubation of LACF from 455 samples collected from 122 French canneries over 10 years. Bacteria were identified by microsequencing or a recent developed tool for group-specific PCR detection (SporeTraQ™). A single species was identified for 93% of examined samples. Three genera were responsible for more than 80% of all non-stability cases: mostly Moorella (36%) and Geobacillus (35%), and less frequently Thermoanaerobacterium (10%). The other most frequent bacterial genera identified were Bacillus, Thermoanaerobacter, Caldanaerobius, Anoxybacillus, Paenibacillus and Clostridium. Species frequency was dependent on food category, i.e. vegetables, ready-made meals containing meat, seafood or other recipes, products containing fatty duck, and related to the intensity of the thermal treatment applied in these food categories. The spore heat resistance parameters (D or δ and z values) from 36 strains isolated in this study were determined. Taken together, our results single out the species most suitable for use as indicators for thermal process settings. This extensively-documented survey of the species that cause non-stability at 55 °C in LACF will help canneries to improve the management of microbial contamination. Copyright © 2013 Elsevier B.V. All rights reserved.

The bacteriological composition of biomass recovered by flushing an operational drinking water distribution system.

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Douterelo, I; Husband, S; Boxall, J B

2014-05-01

This study investigates the influence of pipe characteristics on the bacteriological composition of material mobilised from a drinking water distribution system (DWDS) and the impact of biofilm removal on water quality. Hydrants in a single UK Distribution Management Area (DMA) with both polyethylene and cast iron pipe sections were subjected to incremental increases in flow to mobilise material from the pipe walls. Turbidity was monitored during these operations and water samples were collected for physico-chemical and bacteriological analysis. DNA was extracted from the material mobilised into the bulk water before and during flushing. Bacterial tag-encoded 454 pyrosequencing was then used to characterize the bacterial communities present in this material. Turbidity values were high in the samples from cast iron pipes. Iron, aluminium, manganese and phosphate concentrations were found to correlate to observed turbidity. The bacterial community composition of the material mobilised from the pipes was significantly different between plastic and cast iron pipe sections (p < 0.5). High relative abundances of Alphaproteobacteria (23.3%), Clostridia (10.3%) and Actinobacteria (10.3%) were detected in the material removed from plastic pipes. Sequences related to Alphaproteobacteria (22.8%), Bacilli (16.6%), and Gammaproteobacteria (1.4%) were predominant in the samples obtained from cast iron pipes. The highest species richness and diversity were found in the samples from material mobilised from plastic pipes. Spirochaeta spp., Methylobacterium spp. Clostridium spp. and Desulfobacterium spp., were the most represented genera in the material obtained prior to and during the flushing of the plastic pipes. In cast iron pipes a high relative abundance of bacteria able to utilise different iron and manganese compounds were found such as Lysinibacillus spp., Geobacillus spp. and Magnetobacterium spp. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phosphinothricin Acetyltransferases Identified Using In Vivo, In Vitro, and Bioinformatic Analyses

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VanDrisse, Chelsey M.; Hentchel, Kristy L.

2016-01-01

ABSTRACT Acetylation of small molecules is widespread in nature, and in some cases, cells use this process to detoxify harmful chemicals. Streptomyces species utilize a Gcn5 N-acetyltransferase (GNAT), known as Bar, to acetylate and detoxify a self-produced toxin, phosphinothricin (PPT), a glutamate analogue. Bar homologues, such as MddA from Salmonella enterica, acetylate methionine analogues such as methionine sulfoximine (MSX) and methionine sulfone (MSO), but not PPT, even though Bar homologues are annotated as PPT acetyltransferases. S. enterica was used as a heterologous host to determine whether or not putative PPT acetyltransferases from various sources could acetylate PPT, MSX, and MSO. In vitro and in vivo analyses identified substrates acetylated by putative PPT acetyltransferases from Deinococcus radiodurans (DR_1057 and DR_1182) and Geobacillus kaustophilus (GK0593 and GK2920). In vivo, synthesis of DR_1182, GK0593, and GK2920 blocked the inhibitory effects of PPT, MSX, and MSO. In contrast, DR_1057 did not detoxify any of the above substrates. Results of in vitro studies were consistent with the in vivo results. In addition, phylogenetic analyses were used to predict the functionality of annotated PPT acetyltransferases in Burkholderia xenovorans, Bacillus subtilis, Staphylococcus aureus, Acinetobacter baylyi, and Escherichia coli. IMPORTANCE The work reported here provides an example of the use of a heterologous system for the identification of enzyme function. Many members of this superfamily of proteins do not have a known function, or it has been annotated solely on the basis of sequence homology to previously characterized enzymes. The critical role of Gcn5 N-acetyltransferases (GNATs) in the modulation of central metabolic processes, and in controlling metabolic stress, necessitates approaches that can reveal their physiological role. The combination of in vivo, in vitro, and bioinformatics approaches reported here identified GNATs that can

The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

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Arzu Coleri Cihan

2012-03-01

Full Text Available Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%, the facultative thermophiles (14% and the mesophiles (12%. These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16, Brevibacillus (13, Paenibacillus (1 and Thermoactinomycetes (2 were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6, B. lichenformis (3, B. subtilis (3, B. agri (3, B. smithii (2, T. vulgaris (2 and finally P. barengoltzii (1. In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.

Structural and functional implications in the eubacterial ribosome as revealed by protein-rRNA and antibiotic contact sites.

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Wittmann-Liebold, B; Uhlein, M; Urlaub, H; Müller, E C; Otto, A; Bischof, O

1995-01-01

Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2-iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, and L36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at the peptide level in several proteins that were found to constitute the antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, AND L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA-peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 with a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.

Characterization and modelling of VanT: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174.

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Arias, C A; Martín-Martinez, M; Blundell, T L; Arthur, M; Courvalin, P; Reynolds, P E

1999-03-01

Sequence determination of a region downstream from the vanXYc gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota.

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Pence, Matthew G; Choi, Jeong-Yun; Egli, Martin; Guengerich, F Peter

2010-12-24

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

Simultaneous quantification of ten constituents of Xanthoceras sorbifolia Bunge using UHPLC-MS methods and evaluation of their radical scavenging, DNA scission protective, and α-glucosidase inhibitory activities.

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Zhang, Yu; Ma, Jian-Nan; Ma, Chun-Li; Qi, Zhi; Ma, Chao-Mei

2015-11-01

The present study was designed to investigate the bioactive constituents of Xanthoceras sorbifolia in terms of amounts and their antioxidant, DNA scission protection, and α-glucosidase inhibitory activities. Simultaneous quantification of 10 X. sorbifolia constituents was carried out by a newly established ultra-high performance liquid chromatography-quadrupole mass spectrometry method (UHPLC-MS). The antioxidant activities were evaluated by measuring DPPH radical scavenging and DNA scission protective activities. The α-glucosidase inhibitory activities were investigated by using an assay with α-glucosidase from Bacillus Stearothermophilus and disaccharidases from mouse intestine. We found that the wood of X. sorbifolia was rich in phenolic compounds with the contents of catechin, epicatechin, myricetin, and dihydromyricetin being 0.12-0.19, 1.94-2.16, 0.77-0.91, and 6.76-7.89 mg·g(-1), respectively. The four constituents strongly scavenged DPPH radicals (with EC50 being 4.2, 3.8 and 5.7 μg·mL(-1), respectively) and remarkably protected peroxyl radical-induced DNA strand scission (92.10%, 94.66%, 75.44% and 89.95% of protection, respectively, at a concentration of 10 μmol·L(-1)). A dimeric flavan 3-ol, epigallocatechin-(4β→8, 2β→O-7)-epicatechin potently inhibited α-glucosidase with an IC50 value being as low as 1.2 μg·mL(-1). The established UHPLC-MS method could serve as a quality control tool for X. sorbifolia. In conclusion, the high contents of antioxidant and α-glucosidase inhibitory constituents in X. sorbifolia support its use as complementation of other therapeutic agents for metabolic disorders, such as diabetes and hypertension. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

The role of water radicals in thermorestoration of bacterial spores

International Nuclear Information System (INIS)

Friedman, Y.S.; Grecz, N.

1974-01-01

Fully hydrated bacterial spores exposed to 0.45 Mrad showed a characteristic pattern of survival associated with thermorestoration. When temperature during radiation was controlled at -15 0 to +120 0 C, the lowest viable cell counts were at 0 0 C. Above 0 0 C radiosurvival gradually increased by 2 to 3 log cycles reaching peak at 75 0 C (Bacillus cereus T heat sensitive spores) and at 95 0 C (B.stearothermophilus, heat resistant spores). Simultaneously high survival was observed in the solidly frozen state at -15 0 C to -5 0 C since harmful radicals produced by radiation were trapped in ice. Radiation modifying effects, i.e., protection by 2M ethanol (a scavenger of OH radicals) and sensitization by 1M sodium nitrate (a scavenger of H radicals and hydrated electrons), were studied. The results with ethanol and nitrate confirm the idea that in aqueous sytems below 50 0 C the lethal action is due to oxidizing OH radicals known to attack cell DNA. However, the reversal of scavenger actions above 50 0 C indicates that at those high temperatures lethal effects may also involve the reducing H and esub(aq), which at lower temperatures appear not to affect spore survival though they are known to attack proteins. In this case, it is proposed that radiation inactivation of spores at temperatures below 50 0 C is due to DNA damage inflicted by OH radicals whereas spore death above 50 0 C seems to involve protein /enzyme/ inactivation due to a combined action of heat plus reducing (H, esub(aq)) as well as oxidizing (OH) radical species. From the practical point of view it is important that normally radioprotective effects of such substances as ethanol or ground beef are progressively lost when radiation is carried out at temperatures above 50 0 C. (F.J.)

Microbiological study of a crushed 'core heart' packaged under sterile atmosphere and isolation of different bacterial strains from the Callovo-Oxfordian argillaceous rock (Bure, France)

International Nuclear Information System (INIS)

Mayeux, B.; Daumas, S.; Vinsot, A.; Labat, M.

2010-01-01

packed into aluminium bags, which were also flushed and sealed. The samples were shipped to the IRD Laboratory at ambient temperature. Core opening and sub-sampling was conducted within 36 hours after the core was retrieved from the borehole. All tools were sterilized either by autoclaving or flaming with ethanol. These precautions allow sampling the 'core heart' free of any presence of foreign microorganisms in the sample. The core heart was crushed aseptically using a sterilized mortar and pestle in anaerobic conditions. The powder was hydrated with sterile minimal mineral medium (MM) with a low salt concentration. The clay suspension obtained was enriched in different conditions to study the present microflora: - Aerobic: MM with yeast extract or glucose. - Fermentative: MM with yeast extract or glucose but without any presence of oxygen. - Sulfate-reducing: MM with Sodium-sulfate and lactate or acetate (as carbon source). - Thio-sulfato-reducing: MM with Sodium-thiosulfate and lactate or acetate. - Auto-trophic: MM with very low concentration of acetate (to enhance the start of the growth) and with an H 2 /CO 2 (80/20%) atmosphere (CO 2 as source of carbon and H 2 as source of energy). These cultures were performed at 30 deg. C for the mesophilic and 60 deg. C to study the thermophilic microorganisms. As some of the enhancements had been positive, an isolation phase of these various bacteria was performed in Petri dishes for aerobes and Roll-tube for anaerobes (the Roll-Tubes were made from agar poured along the glass-wall and were sterilely and anaerobically closed). Different aerobic strains were selected in which the 16S rRNA gene (ubiquitous gene of the bacteria) was sequenced. The results showed the presence of several strains phylogenetically close to aerobic spore-forming genera (Bacillus, Brevibacillus, Geobacillus) and a non spore-forming strain phylogenetically close to the genus Pseudomonas. One of these isolated strains is a thermo

The role of water radicals in thermorestoration of bacterial spores

Energy Technology Data Exchange (ETDEWEB)

Friedman, Y S; Grecz, N [Illinois Inst. of Tech., Chicago (USA). Dept. of Biology

1974-01-01

Fully hydrated bacterial spores exposed to 0.45 Mrad showed a characteristic pattern of survival associated with thermorestoration. When temperature during radiation was controlled at -15/sup 0/ to +120/sup 0/C, the lowest viable cell counts were at 0/sup 0/C. Above 0/sup 0/C radiosurvival gradually increased by 2 to 3 log cycles reaching peak at 75/sup 0/C (Bacillus cereus T heat sensitive spores) and at 95/sup 0/C (B.stearothermophilus, heat resistant spores). Simultaneously high survival was observed in the solidly frozen state at -15/sup 0/C to -5/sup 0/C since harmful radicals produced by radiation were trapped in ice. Radiation modifying effects, i.e., protection by 2M ethanol (a scavenger of OH radicals) and sensitization by 1M sodium nitrate (a scavenger of H radicals and hydrated electrons), were studied. The results with ethanol and nitrate confirm the idea that in aqueous sytems below 50/sup 0/C the lethal action is due to oxidizing OH radicals known to attack cell DNA. However, the reversal of scavenger actions above 50/sup 0/C indicates that at those high temperatures lethal effects may also involve the reducing H and esub(aq), which at lower temperatures appear not to affect spore survival though they are known to attack proteins. In this case, it is proposed that radiation inactivation of spores at temperatures below 50/sup 0/C is due to DNA damage inflicted by OH radicals whereas spore death above 50/sup 0/C seems to involve protein /enzyme/ inactivation due to a combined action of heat plus reducing (H, esub(aq)) as well as oxidizing (OH) radical species. From the practical point of view it is important that normally radioprotective effects of such substances as ethanol or ground beef are progressively lost when radiation is carried out at temperatures above 50/sup 0/C.

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

Science.gov (United States)

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double

Selection of rhizosphere local microbial as bioactive inoculant based on irradiated compost

International Nuclear Information System (INIS)

Dadang Sudrajat; Nana Mulyana; Arief Adhari

2014-01-01

One of the main components of carrier based on irradiation compost for bio organic fertilizer is a potential microbial isolates role in nutrient supply and growth hormone. This research was conducted to obtain microbial isolates from plant root zone (rhizosphere), further isolation and selection in order to obtain potential isolates capable of nitrogen fixation (N 2 ), resulting in growth hormone (Indole Acetic Acid), and phosphate solubilizing. Selected potential isolates used as bioactive microbial inoculants formulation in irradiation compost based. Forty eight (48) rhizosphere samples were collected from different areas of West and Central Java. One hundred sixteen (116) isolates have been characterized for their morphological, cultural, staining and biochemical characteristics. Isolates have been selected for further screening of PGPR traits. Parameters assessed were Indole Acetic Acid (IAA) content analysis with colorimetric methods, dinitrogen fixation using gas chromatography, phosphate solubility test qualitatively (in the media pikovskaya) and quantitative assay of dissolved phosphate (spectrophotometry). Evaluation of the ability of selected isolates on the growth of corn plants were done in pots. The isolates will be used as inoculant consortium base on compost irradiation. The selection obtained eight (8) bacterial isolates identified as Bacillus circulans (3 isolates), Bacillus stearothermophilus (1 isolate), Azotobacter sp (3 isolates), Pseudomonas diminuta (1 isolate). The highest phosphate released (91,21 mg/l) was by BD2 isolate (Bacillus circulan) with a holo zone size (1.32 cm) on Pikovskaya agar medium. Isolate of Pseudomonas diminuta (KACI) was capable to produce the highest IAA hormone (74.34 μg/ml). The highest nitrogen (N 2 ) fixation activity was shown by Azotobacter sp isolates (KDB2) at a rate of 235.05 nmol/hour. The viability test showed that all selected isolates in compost irradiation carrier slightly decreased after 3 months of

Feasibility of a combined carrier test for disinfectants: studies with a mixture of five types of microorganisms.

Science.gov (United States)

Best, M; Springthorpe, V S; Sattar, S A

1994-06-01

There is mounting concern regarding the efficacy of many germicides on the market because officially recognized germicidal tests for various classes of microorganisms vary widely and often lack reproducibility and proper quantitation. We report here a carrier method for simultaneously and quantitatively assessing the efficacy of liquid chemical germicides against a mixture of microorganisms of varying degrees of resistance. In the test, each small glass cup (10 mm wide x 14 mm long) was contaminated with 10 microliters of a standardized mixture of Staphylococcus aureus, Mycobacterium bovis bacille Calmette-Guérin, Trichophyton mentagrophytes spores, Sabin poliovirus type 1, and Bacillus stearothermophilus spores in 5% fetal bovine serum. The inoculum was dried for 60 minutes under ambient conditions and covered with 60 microliters of the disinfectant under test or a balanced salt solution control for the desired contact time. The carrier was then placed in 2940 microliters of an eluent and the eluates assayed separately for the five microorganisms. Tap water was used to dilute the test product as needed. Of the 11 products tested, 2% alkaline glutaraldehyde, 0.6% sodium hypochlorite (about 5000 ppm free chlorine), and a 0.4% quarternary ammonium compound containing 23% hydrochloric acid were effective against all five challenge organisms. A hard-surface spray containing 0.1% o-phenylphenol with 79% ethanol was effective against all but bacterial spores; 70% (volume/volume) ethanol alone and povidone-iodine (1% available iodine) were effective against S. aureus, the mycobacterium, and the fungus; a 3% solution of peroxygen compounds was effective only against S. aureus and the poliovirus; 1.5% chlorhexidine gluconate, 0.06% quaternary ammonia compound, and 0.03% o-phenylphenol + 0.03% p-tertiary amylphenol could inactivate nothing but S. aureus; and 3% hydrogen peroxide was ineffective in all tests. This method shows promise for use with various classes of

InXy and SeXy, compact heterologous reporter proteins for mammalian cells.

Science.gov (United States)

Fluri, David A; Kelm, Jens M; Lesage, Guillaume; Baba, Marie Daoud-El; Fussenegger, Martin

2007-10-15

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream

ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9.

Science.gov (United States)

Zhou, Xuan; Zhang, Nan; Xia, Liming; Li, Qing; Shao, Jiahui; Shen, Qirong; Zhang, Ruifu

2018-04-15

Efficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficial Bacillus strains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD + /NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of the qoxABCD and ctaCDEF operons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governing Bacillus biofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and other Bacillus rhizobacteria used in agriculture. IMPORTANCE Bacillus spp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand, Bacillus is the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555-565, 2015, https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and

Crystal structures of MW1337R and lin2004: Representatives of a novel protein family that adopt a four-helical bundle fold

Energy Technology Data Exchange (ETDEWEB)

Kozbial, Piotr; Xu, Qingping; Chiu, Hsiu-Ju; McMullan, Daniel; Krishna, S. Sri; Miller, Mitchell D.; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Axelrod, Herbert L.; Carlton, Dennis; Clayton, Thomas; Deller, Marc; Duan, Lian; Elias, Ylva; Elsliger, Marc-André; Feuerhelm, Julie; Grzechnik, Slawomir K.; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Koesema, Eric; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Murphy, Kevin D.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Reyes, Ron; Rife, Christopher L.; Spraggon, Glen; Trout, Christina V.; ban den Bedem, Henry; Weekes, Dana; White, Aprilfawn; Wolf, Guenter; Zubieta, Chloe; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A. (Scripps); (SSRL); (JCSG); (UCSD); (Burnham)

2009-08-28

To extend the structural coverage of proteins with unknown functions, we targeted a novel protein family (Pfam accession number PF08807, DUF1798) for which we proposed and determined the structures of two representative members. The MW1337R gene of Staphylococcus aureus subsp. aureus Rosenbach (Wood 46) encodes a protein with a molecular weight of 13.8 kDa (residues 1-116) and a calculated isoelectric point of 5.15. The lin2004 gene of the nonspore-forming bacterium Listeria innocua Clip11262 encodes a protein with a molecular weight of 14.6 kDa (residues 1-121) and a calculated isoelectric point of 5.45. MW1337R and lin2004, as well as their homologs, which, so far, have been found only in Bacillus, Staphylococcus, Listeria, and related genera (Geobacillus, Exiguobacterium, and Oceanobacillus), have unknown functions and are annotated as hypothetical proteins. The genomic contexts of MW1337R and lin2004 are similar and conserved in related species. In prokaryotic genomes, most often, functionally interacting proteins are coded by genes, which are colocated in conserved operons. Proteins from the same operon as MW1337R and lin2004 either have unknown functions (i.e., belong to DUF1273, Pfam accession number PF06908) or are similar to ypsB from Bacillus subtilis. The function of ypsB is unclear, although it has a strong similarity to the N-terminal region of DivIVA, which was characterized as a bifunctional protein with distinct roles during vegetative growth and sporulation. In addition, members of the DUF1273 family display distant sequence similarity with the DprA/Smf protein, which acts downstream of the DNA uptake machinery, possibly in conjunction with RecA. The RecA activities in Bacillus subtilis are modulated by RecU Holliday-junction resolvase. In all analyzed cases, the gene coding for RecU is in the vicinity of MW1337R, lin2004, or their orthologs, but on a different operon located in the complementary DNA strand. Here, we report the crystal structures

Genome-Centric Analysis of a Thermophilic and Cellulolytic Bacterial Consortium Derived from Composting

Science.gov (United States)

Lemos, Leandro N.; Pereira, Roberta V.; Quaggio, Ronaldo B.; Martins, Layla F.; Moura, Livia M. S.; da Silva, Amanda R.; Antunes, Luciana P.; da Silva, Aline M.; Setubal, João C.

2017-01-01

Microbial consortia selected from complex lignocellulolytic microbial communities are promising alternatives to deconstruct plant waste, since synergistic action of different enzymes is required for full degradation of plant biomass in biorefining applications. Culture enrichment also facilitates the study of interactions among consortium members, and can be a good source of novel microbial species. Here, we used a sample from a plant waste composting operation in the São Paulo Zoo (Brazil) as inoculum to obtain a thermophilic aerobic consortium enriched through multiple passages at 60°C in carboxymethylcellulose as sole carbon source. The microbial community composition of this consortium was investigated by shotgun metagenomics and genome-centric analysis. Six near-complete (over 90%) genomes were reconstructed. Similarity and phylogenetic analyses show that four of these six genomes are novel, with the following hypothesized identifications: a new Thermobacillus species; the first Bacillus thermozeamaize genome (for which currently only 16S sequences are available) or else the first representative of a new family in the Bacillales order; the first representative of a new genus in the Paenibacillaceae family; and the first representative of a new deep-branching family in the Clostridia class. The reconstructed genomes from known species were identified as Geobacillus thermoglucosidasius and Caldibacillus debilis. The metabolic potential of these recovered genomes based on COG and CAZy analyses show that these genomes encode several glycoside hydrolases (GHs) as well as other genes related to lignocellulose breakdown. The new Thermobacillus species stands out for being the richest in diversity and abundance of GHs, possessing the greatest potential for biomass degradation among the six recovered genomes. We also investigated the presence and activity of the organisms corresponding to these genomes in the composting operation from which the consortium was built

Reclassification of Bacillus beijingensis Qiu et al. 2009 and Bacillus ginsengi Qiu et al. 2009 as Bhargavaea beijingensis comb. nov. and Bhargavaea ginsengi comb. nov. and emended description of the genus Bhargavaea.

Science.gov (United States)

Verma, Pankaj; Pandey, Prashant Kumar; Gupta, Arvind Kumar; Seong, Chi Nam; Park, Seong Chan; Choe, Han Na; Baik, Keun Sik; Patole, Milind Shivaji; Shouche, Yogesh Shreepad

2012-10-01

We have carried out a polyphasic taxonomic characterization of Bacillus beijingensis DSM 19037(T) and Bacillus ginsengi DSM 19038(T), which are closely related phylogenetically to Bhargavaea cecembensis LMG 24411(T). All three strains are Gram-stain-positive, non-motile, moderately halotolerant and non-spore-forming. 16S rRNA gene sequence analyses showed that the strains constituted a coherent cluster, with sequence similarities between 99.7 and 98.7 %. The percentage similarity on the basis of amino acid sequences deduced from partial gyrB gene nucleotide sequences of these three type strains was 96.1-92.7 %. Phylogenetic trees based on the 16S rRNA gene and GyrB amino acid sequences, obtained by using three different algorithms, were consistent and showed that these three species constituted a deeply rooted cluster separated from the clades represented by the genera Bacillus, Planococcus, Planomicrobium, Sporosarcina, Lysinibacillus, Viridibacillus, Kurthia and Geobacillus, supporting their placement in the genus Bhargavaea. All three type strains have menaquinone MK-8 as the major respiratory quinone and showed similar fatty acid profiles. The main polar lipids present in the three type strains were diphosphatidylglycerol and phosphatidylglycerol, and the three strains showed peptidoglycan type A4α with L-lysine as the diagnostic diamino acid. The DNA G+C contents of Bacillus beijingensis DSM 19037(T), Bacillus ginsengi DSM 19038(T) and Bhargavaea cecembensis LMG 24411(T) were 53.1, 50.2 and 53.7 mol%, respectively. The level of DNA-DNA hybridization among the three strains was 57-39 %, indicating that they are members of different species of the genus Bhargavaea. The phenotypic data are consistent with the placement of these three species in a single genus and support their differentiation at the species level. On the basis of these data, we have emended the description of the genus Bhargavaea and propose the reclassification of Bacillus beijingensis

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

Science.gov (United States)

Arias, C A; Weisner, J; Blackburn, J M; Reynolds, P E

2000-07-01

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Bacterial Populations Associated with Smokeless Tobacco Products

Science.gov (United States)

Han, Jing; Sanad, Yasser M.; Deck, Joanna; Sutherland, John B.; Li, Zhong; Walters, Matthew J.; Duran, Norma; Holman, Matthew R.

2016-01-01

ABSTRACT There are an estimated 8 million users of smokeless tobacco products (STPs) in the United States, and yet limited data on microbial populations within these products exist. To better understand the potential microbiological risks associated with STP use, a study was conducted to provide a baseline microbiological profile of STPs. A total of 90 samples, representing 15 common STPs, were purchased in metropolitan areas in Little Rock, AR, and Washington, DC, in November 2012, March 2013, and July 2013. Bacterial populations were evaluated using culture, pyrosequencing, and denaturing gradient gel electrophoresis (DGGE). Moist-snuff products exhibited higher levels of bacteria (average of 1.05 × 106 CFU/g STP) and diversity of bacterial populations than snus (average of 8.33 × 101 CFU/g STP) and some chewing tobacco products (average of 2.54 × 105 CFU/g STP). The most common species identified by culturing were Bacillus pumilus, B. licheniformis, B. safensis, and B. subtilis, followed by members of the genera Oceanobacillus, Staphylococcus, and Tetragenococcus. Pyrosequencing analyses of the 16S rRNA genes identified the genera Tetragenococcus, Carnobacterium, Lactobacillus, Geobacillus, Bacillus, and Staphylococcus as the predominant taxa. Several species identified are of possible concern due to their potential to cause opportunistic infections and reported abilities to reduce nitrates to nitrites, which may be an important step in the formation of carcinogenic tobacco-specific N′-nitrosamines. This report provides a microbiological baseline to help fill knowledge gaps associated with microbiological risks of STPs and to inform potential regulations regarding manufacture and testing of STPs. IMPORTANCE It is estimated that there 8 million users of smokeless tobacco products (STPs) in the United States; however, there are limited data on microbial populations that exist within these products. The current study was undertaken to better understand the

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases

Science.gov (United States)

Vriend, Gert; Eijsink, Vincent

1993-08-01

of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Cellular viability of Saccharomyces cerevisiae cultivated in association with contaminates bacteria of alcoholic fermentation

International Nuclear Information System (INIS)

Nobre, Thais de Paula

2005-01-01

The aim of this work was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products, in reduction of cellular viability of Saccharomyces cerevisiae, when in mixed culture of yeast and active and treated bacteria. Also was to evaluated an alternative medium (MCC) for the cultivation of bacteria and yeast, constituted of sugarcane juice diluted to 5 deg Brix and supplemented with yeast extract and peptone. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast Saccharomyces cerevisiae (strain Y-904) for 72 h on 32 deg C, under agitation. The cellular viability, budding rate and population of S. cerevisiae, the total acidity, volatile acidity and pH of culture were determined from 0, 24, 48 e 72 h of mixed culture. Also were determined the initial and final of microorganism population across the pour plate method, in traditional culture medium (PCA for Bacillus, MRS-agar for Lactobacillus and YEPD-agar for yeast S. cerevisiae) and in medium constituted of sugarcane juice. The bacteria cultures were treated by heat sterilization (120 deg C for 20 minutes), antibacterial agent (Kamoran HJ in concentration 3,0 ppm) or irradiation (radiation gamma, with doses of 5,0 kGy for Lactobacillus and 15,0 kGy for Bacillus). The results of the present research showed that just the culture mediums more acids (with higher concentrations of total and volatile acidity, and smaller values of pH), contaminated with active bacteria L. fermentum and B. subtilis, caused reduction on yeast cellular viability. Except the bacteria B. subtilis treated with radiation, the others bacteria treated by different procedures (heat, radiation e antibacterial) did not cause reduction on yeast cellular viability and population, indicating that the isolated presence of the cellular metabolic of theses bacteria was not enough to reduce the

Cellular viability of Saccharomyces cerevisiae cultivated in association with contaminates bacteria of alcoholic fermentation;Viabilidade celular de Saccharomyces cerevisiae cultivada em associacao com bacterias contaminantes da fermentacao alcoolica

Energy Technology Data Exchange (ETDEWEB)

Nobre, Thais de Paula

2005-07-01

The aim of this work was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products, in reduction of cellular viability of Saccharomyces cerevisiae, when in mixed culture of yeast and active and treated bacteria. Also was to evaluated an alternative medium (MCC) for the cultivation of bacteria and yeast, constituted of sugarcane juice diluted to 5 deg Brix and supplemented with yeast extract and peptone. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast Saccharomyces cerevisiae (strain Y-904) for 72 h on 32 deg C, under agitation. The cellular viability, budding rate and population of S. cerevisiae, the total acidity, volatile acidity and pH of culture were determined from 0, 24, 48 e 72 h of mixed culture. Also were determined the initial and final of microorganism population across the pour plate method, in traditional culture medium (PCA for Bacillus, MRS-agar for Lactobacillus and YEPD-agar for yeast S. cerevisiae) and in medium constituted of sugarcane juice. The bacteria cultures were treated by heat sterilization (120 deg C for 20 minutes), antibacterial agent (Kamoran HJ in concentration 3,0 ppm) or irradiation (radiation gamma, with doses of 5,0 kGy for Lactobacillus and 15,0 kGy for Bacillus). The results of the present research showed that just the culture mediums more acids (with higher concentrations of total and volatile acidity, and smaller values of pH), contaminated with active bacteria L. fermentum and B. subtilis, caused reduction on yeast cellular viability. Except the bacteria B. subtilis treated with radiation, the others bacteria treated by different procedures (heat, radiation e antibacterial) did not cause reduction on yeast cellular viability and population, indicating that the isolated presence of the cellular metabolic of theses bacteria was not enough to reduce the

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Energy Technology Data Exchange (ETDEWEB)

Cook, William J; Senkovich, Olga; Chattopadhyay, Debasish; (UAB)

2009-06-08

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2{angstrom} resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Directory of Open Access Journals (Sweden)

Chattopadhyay Debasish

2009-02-01

Full Text Available Abstract Background The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. Results We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Å resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the non-covalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in

Lid opening and conformational stability of T1 Lipase is mediated by increasing chain length polar solvents

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Jonathan Maiangwa

2017-05-01

Full Text Available The dynamics and conformational landscape of proteins in organic solvents are events of potential interest in nonaqueous process catalysis. Conformational changes, folding transitions, and stability often correspond to structural rearrangements that alter contacts between solvent molecules and amino acid residues. However, in nonaqueous enzymology, organic solvents limit stability and further application of proteins. In the present study, molecular dynamics (MD of a thermostable Geobacillus zalihae T1 lipase was performed in different chain length polar organic solvents (methanol, ethanol, propanol, butanol, and pentanol and water mixture systems to a concentration of 50%. On the basis of the MD results, the structural deviations of the backbone atoms elucidated the dynamic effects of water/organic solvent mixtures on the equilibrium state of the protein simulations in decreasing solvent polarity. The results show that the solvent mixture gives rise to deviations in enzyme structure from the native one simulated in water. The drop in the flexibility in H2O, MtOH, EtOH and PrOH simulation mixtures shows that greater motions of residues were influenced in BtOH and PtOH simulation mixtures. Comparing the root mean square fluctuations value with the accessible solvent area (SASA for every residue showed an almost correspondingly high SASA value of residues to high flexibility and low SASA value to low flexibility. The study further revealed that the organic solvents influenced the formation of more hydrogen bonds in MtOH, EtOH and PrOH and thus, it is assumed that increased intraprotein hydrogen bonding is ultimately correlated to the stability of the protein. However, the solvent accessibility analysis showed that in all solvent systems, hydrophobic residues were exposed and polar residues tended to be buried away from the solvent. Distance variation of the tetrahedral intermediate packing of the active pocket was not conserved in organic solvent

[Microbial resistance to formaldehyde. I. Comparative quantitative studies in some selected species of vegetative bacteria, bacterial spores, fungi, bacteriophages and viruses].

Science.gov (United States)

Spicher, G; Peters, J

1976-12-01

The resistence of different microorganisms to formaldehyde was determined. As test objects served gram-negative and gram-positive vegetative germs (Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella paratyphi-B, Staphylococcus aureus, Streptococcus faecalis), bacterial spores (Bacillus cereus, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis), fungi (Aspergillus niger, Candida albicans), bacteriophages (Escherichia coli phages, T1, T2, T3), and viruses (adenovirus, poliomyelitis virus, vaccinia virus). For the studies, suspensions of germs were exposed at identical temperature (20 degrees C) and pH (7.0). The microbicidal effect of formaldehyde was measured by the decrease of the proportion of germs capable of multiplication in the suspension (lg (N/N0); where: N0 equals initial number of germs capable of multiplication; N equals number of germs capable of multiplication after exposure to formaldehyde). For all germs the dependence of the microbicidal effect on the concentration of formaldehyde was determined. In all experiments, the duration of exposure was two hours. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella paratyphi-B were found to be more susceptible than Staphylococcus aureus (vf. Fig. 1 A). The strains of Pseudomonas aeruginosa used were widely varying as to their susceptibility. To obtain equal microbicidal effects, concentrations of formaldehyde almost three times as high had to be used for the most resistant strain than were necessary for the most susceptible strain of Pseudomonas aeruginosa. All strains of Klebsiella pneumoniae examined were found to have an identical resistence to formaldehyde. Streptococcus faecalis was even more resistant to formaldehyde than Staphylococcus aureus. In the case of Streptococcus faecalis, a concentration of formaldehyde about three times as high had to be used to obtain microbicidal effects of identical magnitude. For the killing of Candida albicans cells concentrations of

Improvement of the energy balances, material balances and emission balances in the production of bioethanol from renewable raw materials; Verbesserung der Energie-, Stoff- und Emissionsbilanzen bei der Bioethanolproduktion aus nachwachsenden Rohstoffen

Energy Technology Data Exchange (ETDEWEB)

Fleischer, Sven

2010-07-01

process that uses stillage as well as recoverable straw and corn silage to produce biogas and is additionally supplied with heat and electricity from fossil sources, can reduce the CO{sub 2}-emissions to about 71%. If this number is expressed as CO{sub 2}-equipollents ca. 8.8t CO{sub 2}/(ha . a) can be avoided. This can be realised because 50.55GJ/(ha . a) ethanol and non-purified biogas [145.92GJ/(ha . a)] can be obtained. If such a process is run self-sustaining using part of the biogas to supply the distillery process with heat and electricity, a considerable higher CO{sub 2}-reduction of about 81% can be obtained. This excellent result corresponds to an avoidance of 13.2t CO{sub 2}/(ha . a). Also 50.55GJ/(ha . a) ethanol and non-purified 116.29GJ/(ha . a) biogas can be obtained as energy products. The data presented are based on laboratory tests and measurements in a pilot plant distillery without any energy recovery. If such processes are implemented in industrial production plants that can profitably realise energy recovery, further improvements of CO{sub 2}-reduction combined with a lower demand for utilities (steam, electricity, compressed air, etc.) should be possible. In addition, if other fermentation organisms like xylose and arabinose fermenting yeasts or the new geobacillus bacteria TM 242 (TMO renewables) are adopted to a stable fermentation process, the ethanol production could be improved even further. Apart from the process using starchy and lignocellulosic raw material for ethanol production, further improvements of the so-called 1st generation process were explored in laboratory tests. The impact of enhanced yeast nitrogen supply as well as increased fermentation temperature for higher biochemical reaction rates was assessed. But in contrast to results from other studies, these measures did not result in positive effects on fermentation speed. The only measure that could improve the 1st generation ethanol process by saving energy during the mash