Thermoanaerobacter Sp Research Articles

Scalable economic extracellular synthesis of CdS nanostructured particles by a non-pathogenic thermophile

We report microbially facilitated synthesis of cadmium sulfide (CdS) nanostructured particles (NP) using anaerobic, metal-reducing Thermoanaerobacter sp. The extracellular CdS crystallites were <10 nm in size with yields of ~3 g/L of growth medium/month with demonstrated reproducibility and scalability up to 24 L. During synthesis, Thermoanaerobacter cultures reduced thiosulfate and sulfite salts to H₂S, which reacted with Cd²⁺ cations to produce thermodynamically favored NP in a single step at 65 °C with catalytic nucleation on the cell surfaces. Photoluminescence (PL) analysis of dry CdS NP revealed an exciton-dominated PL peak at 440 nm, having a narrow full width at half maximum of 10 nm. A PL spectrum of CdS NP produced by dissimilatory sulfur reducing bacteria was dominated by features associated with radiative exciton relaxation at the surface. High reproducibility of CdS NP PL features important for scale-up conditions was confirmed from test tubes to 24 L batches at a small fraction of the manufacturing cost associated with conventional inorganic NP production processes.

Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9mM hydroquinone, maltodextrin (5%, w/v) in 20mM citrate phosphate buffer, pH 5.5 and 0.025mg/ml toruzyme at 40°C for 24h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8g/100ml which is approximately 1.8 fold higher than that of hydroquinone.

Microevolution from shock to adaptation revealed strategies improving ethanol tolerance and production in Thermoanaerobacter

IntroductionThe molecular links between shock-response and adaptation remain poorly understood, particularly for extremophiles. This has hindered rational engineering of solvent tolerance and correlated traits (e.g., productivity) in extremophiles. To untangle such molecular links, here we established a model that tracked the microevolution from shock to adaptation in thermophilic bacteria.MethodTemporal dynamics of genomes and transcriptomes was tracked for Thermoanaerobacter sp. X514 which under increasing exogenous ethanol evolved from ethanol-sensitive wild-type (Strain X) to tolerance of 2%- (XI) and eventually 6%-ethanol (XII). Based on the reconstructed transcriptional network underlying stress tolerance, genetic engineering was employed to improve ethanol tolerance and production in Thermoanaerobacter.ResultsThe spontaneous genome mutation rate (μg) of Thermoanaerobacter sp. X514, calculated at 0.045, suggested a higher mutation rate in thermophile than previously thought. Transcriptomic comparison revealed that shock-response and adaptation were distinct in nature, whereas the transcriptomes of XII resembled those of the extendedly shocked X. To respond to ethanol shock, X employed fructose-specific phosphotransferase system (PTS), Arginine Deiminase (ADI) pathway, alcohol dehydrogenase (Adh) and a distinct mechanism of V-type ATPase. As an adaptation to exogenous ethanol, XI mobilized resistance-nodulation-cell division (RND) efflux system and Adh, whereas XII, which produced higher ethanol than XI, employed ECF-type ϭ24, an alcohol catabolism operon and phase-specific heat-shock proteins (Hsps), modulated hexose/pentose-transport operon structure and reinforced membrane rigidity. Exploiting these findings, we further showed that ethanol productivity and tolerance can be improved simultaneously by overexpressing adh or ϭ24 in X.ConclusionOur work revealed thermophilic-bacteria specific features of adaptive evolution and demonstrated a rational strategy to engineer co-evolving industrial traits. As improvements of shock-response, stress tolerance and productivity have been crucial aims in industrial applications employing thermophiles, our findings should be valuable not just to the production of ethanol but also to a wide variety of biofuels and biochemicals.

Analyzing Intracellular flux Distributions of Thermoanaerobacter sp.X514 under Various Substrate Conditions by Gibbs Sampling

Thermoanaerobacter sp. X514, capable of fermenting most hexose and pentose to produce ethanol, is of critical importance in industrial bioethanol production. This paper provides a detailed investigation of the intracellular metabolic flux flowing activities of X514 under different conditions, including sole glucose substrate condition, sole xylose substrate condition and mixed glucose and xylose substrates condition by means of Gibbs sampling algorithm and stoichiometric metabolic flux balances. Statistical analysis of the results show that all the flux distributions exhibit Gaussian or truncated Gaussian distributions under the assumption that noise of the system is Gaussian distribution. Pentose phosphate pathway becomes more active when xylose is the sole substrate whereas glycolysis pool is more active under sole glucose substrate condition. Major changes of the fluxes activities occur in the connection flux between glycolysis pool and pentose phosphate metabolite pool, indicating the balances between the need for NADPH, the requirement of ATP as well as the necessity of pyruvate under different substrate conditions. Generation of pyruvate reaches its maximum when sole glucose is fed into the system, indicating the preference of the system on glucose consumption. The whole metabolic flux distributions illustrate vivid information about the central metabolic map of X514.

Regioselective glucosylation of inositols catalyzed by Thermoanaerobacter sp. CGTase

Monoglucosylated products of l-chiro-, d-chiro-, muco-, and allo-inositol were synthesized by regioselective α-d-glucosylation with cyclodextrin glucosyl transferase from Thermoanaerobacter sp. after hydrolysis of by products with Aspergillus niger glucoamylase. While the reactions carried out with d-chiro-, muco-, and allo-inositol resulted in the regioselective formation of monoglucosylated products, two products were obtained in the reaction with l-chiro-inositol. Through the structural characterization of the glucosylated inositols here we demonstrated that the selectivity observed in the glucosylation of several inositols by Thermoanaerobacter sp. CGTase, is analogous to the specificity observed for the glucosylation of β-d-glucopyranose and equivalent glucosides.

English

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from a thermophilic anaerobic bacterium, Thermoanaerobacter sp. P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin epoxy activated-sepharose 6B column chromatography. Enzyme was purified 141 fold and had the specific activity of 143.8 U/mg proteins. Purification yields after ammonium sulfate precipitation and affinity chromatography were 25.8 and 17.8%, respectively. SDS-PAGE analysis showed that enzyme was purified successfully and had a single band. Molecular weight of the enzyme was determined as 68.7 kDa. The enzyme had optimum cyclization activity at 80 to 90°C and hydrolyzing activity at 90°C and maintained 87 and 95% of these activities at 95°C, respectively. Optimal pH was found as 7.0. It retained full activity at 80°C for 4 h. Enzyme was strongly inhibited by HgSO 4 and AgNO 3 . Addition of 1 mM CaCl 2 increased the enzymatic activity up to 7%. This novel enzyme could be a good candidate for industrial applications according to its characteristic found in the current study. Key words: Cyclodextrin glycosyltransferase, cyclodextrin production, Thermoanaerobacter sp. P4, thermophilic, enzyme purification, enzyme characterization.

Purification and characterization of a thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. P4

Purification and characterization of a thermostable cyclodextrin glycosyltransferase from Thermoanaerobacter sp. P4

Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T.thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (>0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.

Immobilisation of CGTase for continuous production of long-carbohydrate-chain alkyl glycosides

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) was covalently immobilised on Eupergit C and used in a packed-bed reactor to investigate the continuous production of long-carbohydrate-chain alkyl glycosides from alpha-cyclodextrin (alpha-CD) and n-dodecyl-(1,4)-beta-maltopyranoside (C(12)G(2)beta). The effects of buffer ion strength and pH, and enzyme loading on the immobilisation yield and the enzyme activity were evaluated. Approximately 98% of the protein and 33% of the total activity were immobilised. At pH 5.15, the enzymatic half-life was 132 min at 60 degrees C and 18 min at 70 degrees C. The immobilised enzyme maintained 60% of its initial activity after 28 days storage at 4 degrees C. The degree of conversion was controlled by simple regulation of the flow rate through the reactor, making it possible to optimise the product distribution. It was possible to achieve a yield of the primary coupling product n-dodecyl-(1,4)-beta-maltooctaoside (C(12)G(8)beta) of about 50%, with a ratio between the primary and the secondary coupling product of about 10. Thermoanaerobacter sp. CGTase (Toruzyme 3.0 L) immobilised on Eupergit C had good operational stability at 60 and 70 degrees C thus showing the advantages of using more thermostable enzymes in biocatalysis. However, this enzyme was unsuitable for the production of C(12)G(8)beta due to extensive disproportionation reactions, giving a broad product range. (C) 2011 Elsevier B.V. All rights reserved. (Less)

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

BackgroundThermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes.Methodology/Principal FindingsHere we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic.Conclusions/SignificanceIn this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically.

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase fromThermoanaerobacter sp.

Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [D-glucopyranosyl-alpha(1,1)alpha-D-glucopyranose] to beta-D-glucose 1-phosphate and D-glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P2(1)2(1)2(1). Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n-octyl-beta-D-glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420 A, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7-8 to 3-4 A resolution. The structure of recombinant TP was determined by molecular replacement to 2.8 A resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase.

Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. Strain 1004-09 isolated from a hot spring in the Baikal rift zone

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.

Microbial formation of lanthanide-substituted magnetites by Thermoanaerobacter sp. TOR-39

The potentially toxic effects of soluble lanthanide (L) ions, although microbially induced mineralization can facilitate the formation of tractable materials, has been one factor preventing the more widespread use of L-ions in biotechnology. Here, we propose a new mixed-L precursor method as compared to the traditional direct addition technique. L (Nd, Gd, Tb, Ho and Er)-substituted magnetites, L( y )Fe(3 - y )O(4) were microbially produced using L-mixed precursors, L( x )Fe(1 - x )OOH, where x = 0.01-0.2. By combining lanthanides into the akaganeite precursor phase, we were able to mitigate some of the toxicity, enabling the microbial formation of L-substituted magnetites using a metal reducing bacterium, Thermoanaerobacter sp. TOR-39. The employment of L-mixed precursors enabled the microbial formation of L-substituted magnetite, nominal composition up to L(0.06)Fe(2.94)O(4), with at least tenfold higher L-concentration than could be obtained when the lanthanides were added as soluble salts. This mixed-precursor method can be used to extend the application of microbially produced L-substituted magnetite, while also mitigating their toxicity.

Kinetic Modeling of Acetophenone Reduction Catalyzed by Alcohol Dehydrogenase from Thermoanaerobacter sp.

NADPH-dependent alcohol dehydrogenase (ADH) from Thermoanaerobacter sp. was kinetically characterized using reduction of acetophenone as a model. To achieve 98% conversion of acetophenone, cofactor regeneration by oxidation of 2-propanol with the same enzyme was used. The enzyme was stable in the batch reactor. It was enantioselective towards (S)-1-phenylethanol (ee>99.5%). Due to its high deactivation in continuously operated stirred tank reactor (kd=0.0141 min-1) there was no way to keep high conversion of acetophenone at 98%. The deactivation occurred in the repetitive batch as well. A mathematical model for the acetophenone reduction with cofactor regeneration describing the behaviour in a batch, repetitive-batch and continuously stirred tank reactor was developed.

Sequence analysis and heterologous expression of the groE genes from Thermoanaerobacter sp. Rt8.G4.

The groE homologous genes of the anaerobic thermophile Thermoanaerobacter sp. Rt8.G4 (TRt) have been isolated, sequenced and analysed. The TRt groES and groEL encode subunits of chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) of 94 and 541 amino acids, respectively, and are arranged in that order forming the open reading frames (ORFs) of a bicistronic operon. A controlling inverted repeat of chaperone expression (CIRCE) element lies between the consensus promoter of the operon and TRt groES. At optimum growth temperature (65 degreesC) the chaperonins of TRt are expressed, but production of Cpn60 increases significantly following temperature increases of 3-10 degreesC. Functionally intact recombinant TRt chaperonins were produced in Escherichia coli. However, owing to codon incompatibility, replacement of consecutive AGA codons in the gene encoding TRt Cpn60 was necessary for optimum expression in this heterologous host.

The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases

The effects of temperature on the kinetic parameters kcat and Km, for three isolates of the highly conserved monomeric enzyme 3-phosphoglycerate kinase (PGK), were investigated in detail using a rapid automated kinetics apparatus. PGK was purified from the thermophilic bacterium Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degrees C), the mesophile Zymomonas mobilis (optimum growth temperature 32 degrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserved nature of the enzyme. The kcat values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhenius equation. Maximum kcat values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the thermophile the temperature of maximum kcat was close to its optimum growth temperature. Km values were in general nearly constant through the lower temperature ranges, but increased substantially as the optimum temperature (highest kcat) was passed. Thermal irreversible denaturation of the PGK proteins was also investigated by measuring loss of activity over time. In a dilute buffer, Arrhenius plots for denaturation were linear, and the calculated apparent energy of activation (Eact) for denaturation for the thermophilic PGK was 600 kJ.mol-1, whereas for the mesophilic enzymes the values were 200-250 kJ.mol-1. In the presence of substrates, a considerable stabilization occurred, and in the case of the Z. mobilis enzyme, the apparent Eact was increased to 480 kJ.mol-1. A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturation rates determined at relevant temperatures, it was clear that kcat values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.

Integration and amplification of a cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 on the Bacillus subtilis chromosome

A plasmid was constructed containing the replication functions of pUC19, and the cgtA gene from Thermoanaerobacter sp. ATCC 53627, flanked by the dal gene from Bacillus subtilis and a sequence downstream from this gene. This was transformed into a Dal- B. subtilis strain, selecting for Dal+ transformants, which contained the cgtA gene in single copy integrated in the B. subtilis chromosome. The gene was subsequently amplified by a method which ensured that there was no functional plasmid replication system on the integrated DNA. The amplified structure was stable in the absence of selection pressure.

Studies of Specific Cyclodextrin Production Starting from Pure Maltooligosaccharides Using Thermoanaerobacter Sp. Cyclodextrin Glycosyltransferase

The behaviour of the highly thermostable Cyclodextrin GlycosylTransferase (CGTase) of Thermoanaerobacter sp., with substrates like pure cyclodextrins and/or short mal-tooligosaccharides was investigated. This enzyme has been found to be able to disproportionate all the saccharides tested (from maltose to maltoheptaose) to produce suitable intermediate substrates for cyclization.The disproportionation of maltose leads to a sequential series of higher degree polymers and cyclodextrins, mainly composed of α-cyclodextrin. However, Thermoanaerobacter sp. CGTase produces mainly β-cyclodextrin starting from the maltooligosaccharides that we tested, while γ-cyclodextrin production can be improved by using longer polymers of glucose (mal-topentaose) and cyclodextrin as substrates. An α/β/γ/7 molar ratio of 4/5/1 was obtained with 10mM of maltopentaose which changed to 9/12/8 with initial addition of 100μM of β-cyclodextrin.

Cloning and nucleotide sequence of a thermostable cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 and its expression in Escherichia coli

A gene, cgtA, encoding an extremely thermostable cyclodextrin glycosyltransferase (CGTase) was cloned from a thermophilic anaerobe, Thermoanaerobacter sp. ATCC 53627, and expressed in Escherichia coli. DNA and protein sequencing revealed that the mature enzyme of 683 amino acid residues (MW 75 kDa) was preceded by a signal peptide of 27 amino acid residues. The sequence of the Thermoanaerobacter CGTase was similar to sequences of Bacillus CGTases, with more than 58% identity, and very similar (89% identity) to a CGTase enzyme from Thermoanaerobacterium thermosulfurogenes.

Thermoanaerobacter sp. CGTase: Its Properties and Application.

A novel CGTase (Cyclomaltodextrin glucanotransferase) has been isolated from a strain of Thermoanaerobacter, a thermophilic anaerobe. The enzyme is extremely heat stable and has a temperature optimum of 90-95°C at pH 6.0. It is active over a broad pH range, and exhibits more than 80% activity from pH 5.0-6.7. In the presence of starch, the enzyme is stable at temperatures in excess of 100°C. In addition to producing cyclodextrins from starch, Thermoanaerobacter sp. CGTase has excellent starch liquefying properties. It is possible to liquefy a 35% DS starch slurry at pH 4. 5, in the absence of calcium, using the "jet cooker" process developed for B. licheniformis α-amylase (105°C for 5 min, 90-95°C for 90 min). Saccharification of the liquefied starch with A. niger glucoamylase can therefore be carried out without further pH adjustment. Some of the properties of Thermoanaerobacter sp. CGTase are described, and examples of the use of the enzyme for cyclodextrin production, intermolecular transglycosylation and starch liquefaction are given. The gene coding for Thermoanaerobacter sp. CGTase has been transfered to a Bacillus host, making large-scale production of the enzyme in commercially acceptable yields possible.