Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity. (1/32)

Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.  (+info)

Oligomeric integrity--the structural key to thermal stability in bacterial alcohol dehydrogenases. (2/32)

Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures. The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 A, respectively. Comparison of these two very similar structures (RMS difference in Calpha = 0.8 A) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.  (+info)

Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium. (3/32)

A thermophilic, anaerobic, spore-forming, dissimilatory Fe(III)-reducing bacterium, designated strain SR4T, was isolated from sediment of newly formed hydrothermal vents in the area of the eruption of Karymsky volcano on the Kamchatka peninsula. Cells of strain SR4T were straight-to-curved, peritrichous rods, 0.4-0.6 micron in diameter and 3.5-9.0 microns in length, and exhibited a slight tumbling motility. Strain SR4T formed round, refractile, heat-resistant endospores in terminally swollen sporangia. The temperature range for growth was 39-78 degrees C, with an optimum at 69-71 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.3-6.5. Strain SR4T grew anaerobically with peptone as carbon source. Amorphous iron(III) oxide present in the medium stimulated the growth of strain SR4T; cell numbers increased with the concomitant accumulation of Fe(II). In the presence of Fe(III), strain SR4T grew on H2/CO2 and utilized molecular hydrogen. Strain SR4T reduced 9,10-anthraquinone-2,6-disulfonic acid, sulfite, thiosulfate, elemental sulfur and MnO2. Strain SR4T did not reduce nitrate or sulfate and was not capable of growth with O2. The fermentation products from glucose were ethanol, lactate, H2 and CO2. The G + C content of DNA was 32 mol%. 16S rDNA sequence analysis placed the organism in the genus Thermoanaerobacter. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain SR4T (= DSM 12299T) should be assigned to a new species, Thermoanaerobacter siderophilus sp. nov.  (+info)

High-affinity maltose binding and transport by the thermophilic anaerobe Thermoanaerobacter ethanolicus 39E. (4/32)

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [(14)C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and alpha-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and alpha-trehalose.  (+info)

High resistance to oxygen radicals and heat is caused by a galactoglycerolipid in Microbacterium sp. M874. (5/32)

Microbacterium sp. M874 produced a glyceroglycolipid, di-O-12-methyl-tetradecanoyl-3-O-beta-D-galactopyranosyl-sn-glycerol, at about the 50 microM level. Though the strain was highly resistant to tertiary-butyl hydroperoxide (tBHP) in a glycolipid-productive medium, the resistance was reduced in a nonproductive medium. Exogenous addition of the glycolipid to the nonproductive culture restored the resistance. This addition also increased the resistance to heat, ethanol, and 4-chloro-1-naphthol, in which oxygen radicals might participate. The parallel relationship found in strain M874 mutants between glycolipid productivity and resistance to tBHP or heat suggested that the resistance was mainly caused by the glycolipid. On addition of the glycolipid to a glycolipid-nonproductive culture, it was immediately incorporated into the cells and functioned as an anti-oxygen radical reagent. Thereafter, its intracellular level remained largely unchanged for at least 5 h, even in the presence of tBHP, and its activity was maintained. The glycolipid at 142 microM was sufficient to prevent the cytotoxicity induced by 88.9 mM tBHP. The glycolipid production was not induced by pretreatment with a low level of tBHP or a sublethal heat shock. In brief, the glycolipid might play an essential role in the prevention of damage by oxygen radicals in the glycolipid-producing bacterium.  (+info)

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus. (6/32)

A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.  (+info)

Strain selection in carbon-limited chemostats affects reproducibility of Thermoanaerobacter ethanolicus fermentations. (7/32)

We found that the reproducibility of chemostat trials can be improved by using chemostat-adapted strains. Our experimental findings are consistent with adaptation that involves an improvement in culture fitness and an alteration of the fermentation genotype.  (+info)

Tepidimicrobium ferriphilum gen. nov., sp. nov., a novel moderately thermophilic, Fe(III)-reducing bacterium of the order Clostridiales. (8/32)

A moderately thermophilic, anaerobic bacterium (strain SB91T) was isolated from a freshwater hot spring at Barguzin Valley, Buryatiya, Russia. Cells of strain SB91T were straight to slightly curved rods, 0.5-0.6 microm in diameter and 3.0-7.0 mum in length. Formation of endospores was not observed. The temperature range for growth was 26-62 degrees C, with an optimum at 50 degrees C. The pH range for growth was 5.5-9.5, with an optimum at pH 7.5-8.0. The substrates utilized by strain SB91T in the presence of 9,10-anthraquinone 2,6-disulfonate included peptone, tryptone, Casamino acids, yeast extract, beef extract, casein hydrolysate, alanine plus glycine, alanine plus proline, L-valine and n-propanol. Carbohydrates were not utilized. Strain SB91T reduced amorphous Fe(III) oxide, Fe(III) citrate, Fe(III) EDTA or Fe(III) nitrilotriacetate with peptone, L-valine or n-propanol as an electron donor. Strain SB91T reduced 9,10-anthraquinone 2,6-disulfonate, thiosulfate, elemental sulfur, fumarate and selenite. Strain SB91T survived after exposure to gamma-radiation at a dose of 5.4 kGy. The G+C content of the DNA of strain SB91T was 33 mol%. Analysis of the 16S rRNA gene sequence revealed that the isolated organism belonged to cluster XII of the clostridia. On the basis of its physiological properties and the results of phylogenetic analyses, it is proposed that strain SB91T represents the sole species of a novel genus, Tepidimicrobium; the name Tepidimicrobium ferriphilum gen. nov., sp. nov. is proposed, with strain SB91T (=DSM 16624T=VKM B-2348T) as the type strain.  (+info)