Physiological Function of Alcohol Dehydrogenases and Long-Chain (C30) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C₃₀ fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).     

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus.

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.     

Electrotransformation of Thermoanaerobacter ethanolicus JW200

Electrotransformation of Thermoanaerobacter ethanolicus JW200 was achieved using the plasmid, pTE16, and a pUC-based suicide vector, pTEA2. The construct pTE16 is based on the Escherichia coli-Clostridium perfringens shuttle vector pJIR715 and contains a thermostable chloramphenicol (Cm) resistance cassette. Evidence supporting transformation was provided by extracting plasmid pTE16 from presumptive transformants of T. ethanolicus and by PCR specific to the chloramphenicol acetyltransferase (cat) gene on the vector pTEA2. Transformation frequencies of plasmid pTE16 and pTEA2 were 50 ± 7.4 and 30 ± 4.2 transformants per μg plasmid DNA. The results provide the first unequivocal gene transfer method functional in T. ethanolicus.     

Ethanol fermentation characteristics of Thermoanaerobacter ethanolicus

Thermoanaerobacter ethanolicus is an extreme thermophilic non-spore forming ethanol-producing anaerobic bacterium. Minimum nutrient requirements and optimum growth conditions have been established. An optimum yeast extract-glucose ratio for ethanol yield has also been determined. Initial medium pH, optimally 7.5–8.0, significantly affected the amount of ethanol formed. Maximum specific growth rate was found to be 0.22 h^?1at pH 7.5 and 69°C. Ethanol concentration up to 11 g l^?1at pH 7.5 and 69°C was used to characterize ethanol inhibition. The growth kinetics of T. ethanolicus were characterized in terms of environmental parameters. Substrate utilization, ethanol formation and inhibition by both sugar and ethanol were also quantified.     

嗜热厌氧乙醇菌JW200转化条件的研究

摘要 嗜热厌氧乙醇菌遗传转化系统的缺少,制约了对该菌理论基础和应用领域的进一步研究。利用聚乙二醇（PEG6000）转化和电转化技术国际首次实现了嗜热厌氧乙醇菌JW200外源基因的导入。PEG转化效率很低,因此选择对电转化条件进行优化,转化效率从4±3.2个转化子/μg质粒DNA提高到50±7.4个转化子/μg质粒DNA。实验表明获得较高的转化效率的必要条件是在细胞密度为OD660 0.2时添加甘氨酸与蔗糖后继续培养2h以及细胞在电击前的收集与洗涤保持低温。本研究为利用基因工程手段改造嗜热厌氧乙醇菌和从分子水平研究胞内乙醇代谢途径奠定了基础。     

Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Alcohol Dehydrogenases from Strawberry Seeds

Two alcohol dehydrogenases (alcohol: NAD oxidoreductase, EC 1.1.1.1 and alcohol: NADP oxidoreductase, EC 1.1.1.2) were partially purified from extracts of strawberry seeds by conventional methods. Some of physical, chemical and kinetic properties of the enzymes are described. On the basis of gel filtration, the molecular weights were estimated to be approximately 78,000 for NAD-dependent enzyme and 82,000 for NADP-dependent enzyme. Thiol-reacting compounds inhibited both enzymes. NAD-dependent alcohol dehydrogenase reacted only with aliphatic alcohols and aldehydes, while aromatic and terpene alcohols and aldehydes were the better substrates for NADP-dependent alcohol dehydrogenase than aliphatic alcohols and aldehydes.     

Improved purification and biochemical characterization of extracellular amylopullulanase from Thermoanaerobacter ethanolicus 39E

A maltose-limited chemostat culture was used to investigate the expression and excretion of amylopullulanase by Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). In maltose-limited continuous culture, amylopullulanase was produced and secreted at tenfold higher levels than in batch culture. The extracellular amylopullulanase was purified to homonogeneity by using an inhibitor-linked affinity column matrix. The purified amylopullulanase had a specific activity of 480 units (U)/mg protein for pullulanase and 175 U/mg protein for -amylase. -Cyclodextrin inhibited both -amylase and pullulanase activities, with a substrate inhibition constant (K _i) of 0.065 mg/ml.Amylopullulanase had a relative molecular mass (M_r) of 140 000 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and an M_r of 133 000 using gel-filtration chromatography. The N-terminal sequence of the enzyme was Glu-Thr-Asp-Thr-Ala-Pro-Ala. The purified enzyme displayed Michaelis constant (K _m) values of 0.35 mg/ml for pullulan and 1.00 mg/ml for amylose. The enzyme had an isoelectric point (pI) of 4.0, and displayed an optimum pH for stability and activity of 6.2 and 5.5, respectively. The enzyme was stable up to 85° C in the presence of Ca²⁺, and had a half-life of 40 min at 90° C (pH 6.2). Ca²⁺ was required for thermal stability, but not for activity. Amylose, glycogen, and amylopectin were degrade to maltose, maltotriose, and maltotetraose, whereas only maltotriose was formed from pullulan. Correspondence to: J. G. Zeikus     

Cloning and analysis of the xylAB operon and characterization of xylose isomerase from Thermoanaerobacter ethanolicus

Three genes, xylA-like, xylA and xylB, were cloned and sequenced from the chromosome of Thermoanaerobacter ethanolicus JW200. xylA and xylB share an operon and encode xylose isomerase and xylulokinase, respectively. The xylA-like gene locates upstream of xylAB operon and encodes a hypothetical protein that lacks xylose isomerase activity. The xylose isomerase was expressed in Escherichia coli and purified by heat treatment and an ion-exchange chromatography. The enzyme had highest activity at 85°C and pH 7.0, and a half-life for 1 h at 85°C. The K (m) and V (max) values for xylose were 11 mM and 25 U/mg, respectively. The high level of expression, easy purification, and thermostability of the XylA from T. ethanolicus JW200 suggests industrial usefulness.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophile Clostridium beijerinckii (CBADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26 °C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed in Escherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii

Summary Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophileClostridium beijerinckii (CBADH) and the other from the extreme thermophileThermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26°C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed inEscherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Induction and Regeneration of Autoplasts from Clostridium thermohydrosulfuricum JW102 and Thermoanaerobacter ethanolicus JW200

Autolysis was induced to form stable, cell wall-free cells of Clostridium thermohydrosulfuricum JW102 and Thermoanaerobacter ethanolicus JW200, using a complex medium containing glycine (0.4% wt/vol) and/or sucrose or glycerol (10% wt/vol) at an optimum temperature of 64°C. Autoplasts of both bacteria were grown as L-phase colonies on solid medium; more than 50% of these colonies regenerated to the walled form during prolonged incubation. The removal of the cell wall was confirmed by electron microscopy.     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

Identification,molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus

Biotechnology Letters - We identified a new glucoamylase (TeGA) from Thermoanaerobacter ethanolicus, a thermophilic anaerobic bacterium. Structural studies suggest that TeGA belongs to the family...     

Purification and Properties of Alcohol Dehydrogenase from Tea Seeds

The process of antigen presentation is not well understood. We screened for drugs that distinguish presentation of allogeneic class 2 antigens and exogenous antigens. When spleen cells were used as antigen presenting cells (APC), leupeptin and antipain preferentially inhibited allogeneic class 2 presentation, while they did not affect presentation of exogenous antigen and T cell growth. In contrast, they inhibited both presentations when spleen adherent cells (SAC) were used as APC. Our results suggest that SAC (mainly macrophages) and splenic B cells use different pathways to present exogenous antigens and that pathways to present allogeneic class 2 molecules are similar.     

Thermoanaerobacter ethanolicus Growth and Product Yield from Elevated Levels of Xylose or Glucose in Continuous Cultures

The performance of Thermoanaerobacter ethanolicus was evaluated in continuous culture with media containing concentrations of xylose (8 to 20 g/liter) greater than those previously reported. The ethanol yield declined from to 0.42 to 0.29 g of ethanol per g of xylose consumed when input xylose was increased from 4 to 20 g/liter. Yields of both total C₂ and C₃ products from consumed xylose and of cell biomass from ATP produced declined as the input xylose concentration was increased, which was not the case when glucose was the substrate. This suggested that yeast extract functioned as a significant energy and carbon source for cells in fermentations of xylose but not of glucose. The feasibility of this interpretation was confirmed by (i) the calculation of the products theoretically obtainable from yeast extract and (ii) the observation of significant quantities of fermentation products in inoculated sugar-free media. Markedly different patterns of metabolism for the two sugar substrates were also evidenced by the cell yield for glucose being twice that of xylose at elevated sugar concentrations. It was noted that caution must be exerted when results obtained at low xylose concentrations are extrapolated to predict those which can be obtained at higher concentrations.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.