Thermophilic Geobacillus Stearothermophilus Research Articles

Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963.

The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0-0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.

Plant Heat Shock Proteins Are More Effective in Enhancing Recombinant Alcohol Dehydrogenase Activity than Bacterial Ones In Vitro.

Recombinant proteins produced in the cell factories are used in biological research, pharmaceutical production, and biochemical and agricultural applications. Molecular chaperones, such as heat shock proteins (Hsps), are co-expressed with recombinant proteins to enhance their yield, stability, and activity. When Escherichia coli (E. coli) is used as a cell factory, E. coli Hsps are the frequently used co-expression partners. We examined if there are differences in the molecular chaperone activities of plant and bacterial Hsps on recombinant protein activity. We compared the effects of the Hsps from carrot (Daucus carota) and E. coli on enhancing the recombinant alcohol dehydrogenase (ADH) activity and solubility under normal and heat conditions in vitro. His-tagged carrot Hsps (DcHsp17.7 and DcHsp70), E. coli Hsps (IbpA, IbpB, and DnaK), and ADH from a thermophile Geobacillus stearothermophilus were individually cloned in a pET11a or a pET26b vector, introduced into E. coli BL21(DE3), and expressed by isopropyl β-D-1-thiogalactopyranoside treatment (0.5 mM, 16 °C , 20 h). The recombinant proteins were purified using Ni-NTA affinity chromatography and resolved in SDS-PAGE (17%). The recombinant ADH was treated with the individual Hsps or in combination, and the enzyme activity was examined by measuring the NADH product levels at O.D.340. The recombinant ADH was expressed at high levels in E. coli and very thermotolerant when the purified enzyme reacted (up to 70 °C). All five Hsps enhanced the ADH activity under normal and heat conditions in vitro, compared to the control. DcHsp17.7 and DcHsp70 were the most effective for improving the enzyme activity by up to 13.0- and 11.6-fold, respectively, followed by IbpA (8.4-fold), DnaK (6.5-fold), and IbpB (3.4-fold), at 37 °C . Combined incubation of DcHsp17.7-DcHsp70 and DcHsp17.7-DnaK further enhanced the ADH activity by 13.8 and 14.2-fold, respectively. DcHsp70 effectively enhanced ADH's solubility at 37 °C in vitro. Our results suggest that plant Hsps can enhance recombinant protein activity, such as ADH, more effectively than their bacterial counterparts. Identifying effective molecular chaperones in the bacterial and eukaryotic domains will help enhance the production of recombinant proteins inE. coli.

Different Substrate Selectivity and Product Patterns of Immobilized Thermophilic Lipases from Geobacillus stearothermophilus, Anoxybacillus flavithermus, and Thermomyces lanuginosus for Glyceryl Decanoate Synthesis

Lipases can catalyze synthesis reactions in a micro aqueous system, producing useful partial glycerides (mono- and diglycerides), and these compounds are commonly utilized in different products as surfactants. Depending on the microbial sources for lipases, immobilization conditions, and starting substrates for synthesis reaction, the composition and yields of the resulting partial glycerides could be variable. These differences could lead to the final efficacy of partial glycerides as surfactants in targeted products. Therefore, it is necessary to establish a group of immobilized lipases from different microbial sources with information about substrate specificity to produce effective partial glycerides for various product types. Here, lipases from thermophilic Geobacillus stearothermophilus and Anoxybacillus flavithermus were prepared with a simple partial purification method, and after immobilization, these lipases were tested to synthesize partial glycerides using different types of decanoic acids. The distinct product patterns were analyzed using HPLC. Both immobilized lipases showed the highest substrate selectivity to decanoic acids in common, producing mainly glyceryl monodecanoate. However, commercial immobilized lipases from Thermomyces lanuginosus produced the largest glyceryl monodecanoate from methyl decanoate. These results indicate the importance of immobilization conditions like different microbial sources and substrates and the need for their optimal combination.

Production and characterization of rhamnolipid biosurfactant from thermophilic Geobacillus stearothermophilus bacterium isolated from Uhud mountain.

Biosurfactants have been given considerable attention as they are potential candidates for several biotechnological applications. In this study, a promising thermophilic biosurfactant-producing HA-2 was isolated from the volcanic and arid region of Uhud mountain, Madinah, Saudi Arabia. It was identified using 16S rRNA gene sequence analysis. The biosurfactant production ability was screened using different methods such as the drop collapse test, oil spreading test, hemolytic activity test, CTAB test, and emulsification index. The ability of rhamnolipid production by the tested strain was confirmed by the polymerase chain reaction (PCR) of rhlAB. The affinity of thermophilic HA-2 to hydrophobic substrates was also investigated. Optimization of biosurfactant production was conducted. The biological activities of produced surfactant were investigated. The isolated HA-1 was identified as Geobacillus stearothermophilus strain OR911984. It could utilize waste sunflower frying oil (WSFF) oil as a low-cost carbon source. It showed high emulsification activity (52 ± 0.0%) and positive results toward other biosurfactant screening tests. The strain showed high cell adhesion to hexane with 41.2% cell surface hydrophobicity. Fourier-transform infrared (FTIR) spectra indicated the presence of hydrophobic chains that comprise lipids, sugars, and hydrophilic glycolipid components. The optimization results showed the optimal factors included potato peel as a carbon source with 68.8% emulsification activity, yeast extract as a nitrogen source with 60% emulsification activity, a pH of 9 (56.6%), and a temperature of 50° (72%). The kinetics showed that optimum biosurfactant production (572.4 mg/L) was recorded at 5 days of incubation. The produced rhamnolipid biosurfactant showed high antimicrobial activity against some human and plant pathogenic bacterial and fungal isolates and high antioxidant activity (90.4%). In addition, it enhanced wheat (Triticum aestivum) growth, with the greatest enhancement obtained with the 5% concentration. Therefore, thermophilic G. stearothermophilus is a promising rhamnolipid biosurfactant producer that utilizes many organic wastes. The produced biosurfactant could be applied as a promising emulsifier, antimicrobial, antioxidant, and plant growth promoter.

Production and characterization of a highly stable laccase from extreme thermophile Geobacillus stearothermophilus MB600 isolated from hot spring of Gilgit Balitistan (Pakistan)

Extracellular thermophilic multicopper oxidase i.e. laccase was purified, identified and characterized from Geobacillus stearothermophilus MB600. Fast protein liquid chromatography (FPLC) of lacasse showed a prominent peak at 610 nm. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for laccase presented a molecular weight of 59.13 kDa. Analysis via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) confirmed the presence of laccase similar to in Geobacillus thermoleovorans B23 and Geobacillus sp. LEMMY01. Laccase showed Km 0.532 mM for guaiacol and 0.307 mM for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Its optimum activity was witnessed at 90°C, at pH 5.0. Laccase catalytic activity was completely inhibited by sodium dodecyl-sulfate (SDS), sodium azide (NaN3), and ethylenediaminetetraacetic acid (EDTA). Among the metal salts against Cu, Ca, Cd, and Li enhanced while Mg inhibited the enzyme activity. It was stable against Cl, I and solvents (Ethanol > Methanol > Dimethyl sulfoxide > Acetonitrile). Thermophilic laccase effectively degraded 93% of Remazol brilliant blue R, 92% Bisphenol A diglycidyle ether without mediator.

A Method for Rapid Polyethyleneimine-Based Purification of Bacteriophage-Expressed Proteins from Diluted Crude Lysates, Exemplified by Thermostable TP-84 Depolymerase.

Purification of bacteriophage-expressed proteins poses methodological difficulties associated with the need to process entire culture medium volume upon bacteriophage-induced bacterial cell lysis. We have used novel capsule glycosylase-depolymerase (TP84_26 GD) from bacteriophage TP-84, infecting thermophilic Geobacillus stearothermophilus bacteria, as a representative enzyme to develop a method for rapid concentration and purification of the enzyme present in diluted crude host cell lysate. A novel variant of the polyethyleneimine (PEI)-based purification method was devised that offers a fast and effective approach for handling PEI-facilitated purification of bacteriophage-expressed native proteins. Due to the very basic nature of PEI, the method is suitable for proteins interacting with nucleic acids or acidic proteins, where either mixed PEI-DNA or RNA-protein complexes or PEI-acidic protein complexes are reversibly precipitated. (i) The method is of general use, applicable with minor modifications to a variety of bacteriophage cell lysates and proteins. (ii) In the example application, TP84_26 GD was highly purified (over 50%) in a single PEI step; subsequent chromatography yielded a homogeneous enzyme. (iii) The enzyme's properties were examined, revealing the presence of three distinct forms of the TP84_26 GD. These forms included soluble, unbound proteins found in host cell lysate, as well as an integrated form within the TP-84 virion.

Novel extracellular synthesized silver nanoparticles using thermophilic Anoxybacillus flavithermus and Geobacillus stearothermophilus and their evaluation as nanodrugs

In this investigation, two new thermophilic bacteria were isolated. The new isolates were characterized by 16S rRNA, biochemical, morphological, and physiological analyzes and the isolates were identified as Geobacillus stearothermophilus strain Gecek20 and thermophilic Anoxybacillus flavithermus strain Gecek19. Various biological activities of extracellular Ag-NPs synthesized from thermophilic G. stearothermophilus strain Gecek20 and thermophilic A. flavithermus strain Gecek19 were evaluated. The produced NPs were analyzed by SEM, SEM-EDX, and XRD analyses. The antioxidant abilities of new synthesized Ag-NPs from thermophilic G. stearothermophilus strain Gecek20 (T1-Ag-NPs) and new synthesized Ag-NPs from thermophilic A. flavithermus strain Gecek19 (T2-Ag-NPs) were studied by DPPH inhibition and metal chelating ability. The highest DPPH and metal chelating abilities of T1-Ag-NPs and T2-Ag-NPs at 200 mg/L concentration were 93.17 and 90.85%, and 75.80 and 83.64%, respectively. The extracellular green synthesized T1-Ag-NPs and T2-AgN-Ps showed DNA nuclease activity at all tested concentrations. Moreover, both new synthesized Ag-NPs had antimicrobial activity against the strains studied, especially on Gram positive bacteria. T1-Ag-NPs and T2-AgNPs also showed powerful Escherichia coli growth inhibition. The highest biofilm inhibition percentages of T1-Ag-NPs and T2-Ag-NPs against Pseudomonas aeruginosa and Staphylococcus aureus were 100.0%, respectively, at 500 mg/L.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48g/l biomass and 0.32g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73g/l biomass and 0.33g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Integrated lactic acid production from lignocellulosic agricultural wastes under thermal conditions

The production of lactic acid (LA) from agricultural wastes attracts great attention because of the sustainability and abundance of lignocellulosic feedstocks, as well as the increasing demand for biodegradable polylactic acid. In this study, we isolated a thermophilic strain Geobacillus stearothermophilus 2H-3 for use in robust production of L-(+)LA under the optimal conditions of 60 °C, pH 6.5, which were consistent with the whole-cell-based consolidated bio-saccharification (CBS) process. Sugar-rich CBS hydrolysates derived from various agricultural wastes, including corn stover, corncob residue, and wheat straw, were used as the carbon sources for 2H-3 fermentation by directly inoculating 2H-3 cells into the CBS system, without intermediate sterilization, nutrient supplementation, or adjustment of fermentation conditions. Thus, we successfully combined two whole-cell-based steps into a one-pot successive fermentation process to efficiently produce LA with high optical purity (99.5%), titer (51.36 g/L), and yield (0.74 g/gbiomass). This study provides a promising strategy for LA production from lignocellulose through CBS and 2H-3 fermentation integration.

Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent.

The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.

Molecular cloning, characterization, and homology modeling of serine hydroxymethyltransferase from psychrophilic bacterium Psychrobacter sp.

Serine hydroxymethyltransferase (SHMT) plays a significant role in the synthesis of l-serine, purine, and thymidylate, which could be extensively applied in the treatment of cancers and the development of antibiotics. In this study, cloned from Psychrobacter sp. ANT206, a novel cold-adapted SHMT gene (psshmt, 1257 bp) encoding a protein of 418 amino acids was expressed in Escherichia coli. The homology modeling result revealed that PsSHMT owned fewer Proline (Pro) residues and hydrogen bonds compared with its homologs from mesophilic E. coli and thermophilic Geobacillus stearothermophilus. In addition, the molecular weight of the purified recombinant PsSHMT (rPsSHMT) was identified to be 45 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, approximately. The enzymatic characteristics of the cold-adapted rPsSHMT displayed that its optimum temperature and pH were 30°C and 7.5, respectively, and its enzymatic activity could be inhibited by Cu2+ , significantly. rPsSHMT also showed a high kcat value and low ΔG at low temperatures. Furthermore, arginine (Arg) could affect the activity of rPsSHMT and be vital to its active sites. The results of this study reflected that these characteristics of the cold-adapted rPsSHMT made it a remarkable candidate that could be utilized in multiple industrial fields under low temperatures.

Polyacrylamide hydrogel carrier (matrix-type macrogel beads): Improvement in the catalytic behavior, stability, and reusability of industrially valuable xylanase from a thermophile Geobacillus stearothermophilus

• xylanase was immobilized within polyacrylamide matrix-based macrogel beads through entrapment technique. • Optimization of various catalytic parameters of xylanase was executed to attain improved reaction efficiency. • Enhanced long-term stability (reduction of thermal and storage ageing) of immobilized xylanase was achieved. • An excellent reusability of immobilized xylanase proposed its broad utility profile for bioreactors at industrial scale. The solid macrogel beads composed of the polyacrylamide hydrogel matrix are widely opted to use in various bioengineering applications because of chemically inert and electrically neutral behavior. In the current study, biocompatibility and the robust nature of the insoluble polyacrylamide matrix-based macrogel beads were successfully analyzed and proved to be an efficient carrier matrix with 59% immobilization yield of xylanase. Immobilized xylanase indicated remarkable catalytic properties and stability profile with reference to its soluble counterpart. The optimum reaction temperature and pH of immobilized enzyme remained same at 50 °C and 7.0, respectively; however, the catalysis period shifted to 15 min. as related to soluble enzyme (5 min.). The diffusional hindrance of the large molecular mass xylan from the corncob source created a decline in V max of immobilized enzyme from 4772.5 to 451.3 U min −1 ; however, the K m value increased from 0.507 to 0.76 mg min −1 with reference to its soluble counterpart. Immobilized xylanase showed its striking tolerance capability against high temperatures as compared to its soluble part and retained 52% activity at 60 °C after 120 min. About 32% activity of immobilized xylanase attained at 70 °C even after 60 min. Immobilized xylanase within macrogel beads also exhibited good recycling efficiency by retaining 24% activity after 6 consecutive catalytic rotations and revealed its capability to be used as a plausible candidate to proceed various industrial bioprocesses.

Characterization of Lipases from <i>Geobacillus stearothermophilus</i> and <i>Anoxybacillus flavithermus</i>cell Lysates

The aim of this study was to characterize lipases from two thermophilic bacteria, Geobacillus stearothermophilus (GS) and Anoxybacillus flavithermus (AF) in heat treated cell lysates. The pH optimum, pH stability, temperature stability and substrate kinetics and specificity of the lipases were determined. Optimum activity of the lipase from GS (LGS) was observed at pH 7.5, and the optimum activity of the lipase from AF (LAF) was at pH 8.0. LGS was stable up to 70°C after 12 hrs while LAF was stable up to 90°C after 12 hrs. Both enzymes were stable at a pH range of 6 to 8 over 12 h at 4°C. LGS had a highest Vmax value of 22 mM·min-1·mg-1 with p-nitrophenyl acetate while the lowest Km was 0.8 mM with p-nitrophenyl laurate. The highest Vmax of LAF was 2.5 mM·min-1·mg-1 with p-nitrophenyl myristate, and the lowest Km was 0.4 mM with p-nitrophenyl octanoate. LGS preferentially hydrolyzed p-nitrophenyl acetate and p-nitrophenyl octanoate while LAF preferentially hydrolyzed p-nitrophenyl myristate and p-nitrophenyldodecanoate. Lipases from both GS and AF showed characteristics that would be beneficial in food processing.

Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.

Species-specific real-time PCR assay for enumeration of Anoxybacillus flavithermus and Geobacillus stearothermophilus spores in dairy products

The thermophilic spore-forming bacteria, Anoxybacillus flavithermus and Geobacillus stearothermophilus, grow readily, especially on milk-powder processing lines, and are important for processing-plant hygiene management. We developed a real-time PCR assay using SYBR Green I to monitor the spores of these species in dairy products. We designed new primer pairs specific for each species from the nucleotide sequences of the stage 0 sporulation gene A (spo0A). Ethidium monoazide treatment enabled us to quantify only spores. In dairy products with high protein content, treatment with proteinase K allowed precise quantification of spores. This assay was superior for counting spores, having high linearity (r2 = 0.99) and a wide quantification range (101 to 106 spores mL−1). This new method can be applied to quantification of spores in samples of milk, skimmed milk, and cream.

Preconcentrations of Ni(II) and Co(II) by using immobilized thermophilic Geobacillus stearothermophilus SO-20 before ICP-OES determinations

This study deals with the preconcentrations of Ni(II) and Co(II) ions in real samples using the solid phase extraction method (SPE) before their determinations by inductively coupled plasma optical emission spectrometry (ICP-OES). Thermophilic bacterium Geobacillus stearothermophilus SO-20 (Accession number: KJ095002), loaded with Amberlite XAD-4, was utilized as a novel biosorbent. Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscope (SEM) were employed for the investigation of the bacterial surface before and after Ni(II) and Co(II) biosorption. The experimental parameters were examined to find the best conditions. The retained Ni(II) and Co(II) ions on the biosorbent were eluted by using 5.0 ml of 1.0 mol l−1 HCI as the best eluent. The sorption capacities were found to be 16.8 mg g−1 for Ni(II) and 21.6 mg g−1 for Co(II). It was also successfully used for the quantification of Ni(II) and Co(II) in a river water sample, some vegetables and soil.

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

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

Droplet millifluidics for kinetic study of transketolase.

We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions.

Heavy hydrocarbon degradation of crude oil by a novel thermophilic Geobacillus stearothermophilus strain A-2

Thermophilic hydrocarbon-degrading microbial strains are particularly suited to improve the oil recovery process in high temperature reservoirs. In the present study, the effects of the thermophilic hydrocarbon-degrading Geobacillus stearothermophilus strain A-2 on heavy crude oil were investigated in terms of oil emulsification, changes in the oil viscosity and hydrocarbons degradation. Strain A-2 was isolated from the Dagang petroleum reservoir. Strain A-2 has strong surface hydrophobicity, and produces a bioemulsifier. The fermentation broth of strain A-2 caused the oil to disperse, and decreased the oil viscosity from 1540 mPa s to 923 mPa s. Strain A-2 preferentially degrades n-alkanes and aromatic hydrocarbons. In particular strain A-2 prefers the long-chain n-alkanes (>C21), naphthalene and methylated phenanthrene, and the highest degradation rate was 89.83% when cultured with C28 as a sole carbon source. Collectively, Geobacillus stearothermophilus strain A-2 showed its potential application for improving oil exploration in high-temperature oil reservoirs by improving the oil's properties.