Purification and Characterization of Aldehyde Dehydrogenase of Acetobacter aceti

Germination and growth inhibiting activities of surface lipids from 54 Nicotiana species were investigated. Almost half of the extracts were found to have such activities. Among them the surface lipids of N. glutinosa, N. bigelovii, N. sylvestris, N. repanda, N. stocktonii, and N. nesophila were rather strong. Guided by a bioassay using the inhibitory effects on tobacco seed germination and growth, two types of sucrose esters were isolated and identified from the surface lipids of N. glutinosa. The ester positions of each compounds were identified by ¹³C-NMR using deuterium exchange of HO ? OD. The structures were (2,3,4-tri-O-acyl)-α-d-glucopyranosyl)-(3-O-acetyl)-β-d-fructofuranqside (M1) and (2,3,4-tri-O-acyl)-α-d-glucopyranosyl-β-d-fructofuranoside (M2). The main fatty acids of M1 and M2 were acetic (only in M1), propionic, 2-methylbutyric, 4-methylpentanoic, 4-methylhexanoic, 5-methylhexanoic, and octanoic acids. These sucrose esters obtained from N. glutinosa inhibited not only tobacco seed germination and growth but also other plants’ growth.     

Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH₂-terminal β-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively.     

Purification and Properties of Methanol Dehydrogenase and Aldehyde Dehydrogenase from Methylobacillus glycogenes

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (M_r 140,000) was composed of two different subunits (M_r 60,000 and 9,000) forming an α₂β₂ structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (M_r 144,000) was composed of two identical subunits of molecular mass of 72,000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.     

Oxidation of long-chain alkanes by Acetobacter rancens

Summary The degradation of hexadecane and tetradecane by Acetobacter rancens CCM 1774 was investigated. It was found that this strain is able to grow to a limited extent on hexadecane as a carbon source. The occurrence of n-alkanoic acids and alcohols among the reaction products of growing as well as resting cells indicates a monoterminal degradation of long-chain alkanes. Both alkane-grown and glucose-grown resting cells exhibited alkane oxidizing activities which were not influenced by chloramphenicol. This suggested a constitutive nature of the appropriate enzymes.     

Membrane-Associated Quinoprotein Formaldehyde Dehydrogenase from Methylococcus capsulatus Bath

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.     

Nutrition of Acetobacter rancens S3 and F11 Isolated from Tanks for Vinegar Production

The strains S3 and F11 which were isolated respectively from static and submerged tanks for vinegar production were identified as Acetobacter rancens. Neither strain grew in an ammonium defined medium containing ethanol, glucose, glycerol or organic acids as the sole carbon source. When casamino acids were added, they grew luxuriantly with lactate, ethanol or glycerol as the carbon source and less well with acetate or glucose. They grew, forming much acetic acid, in defined ethanol medium when alanine was supplied in place of casamino acids, but strain S3 showed a longer lag time than strain Fl1. This lag time could be shortened by addition of aspartate and glutamate. These amino acids could be replaced by succinate, fumarate, malate, lactate, pyruvate or propionate but not by glucose. Both strains required lactate or pyruvate in defined glucose medium but many other organic acids, which were effective in defined ethanol medium, were ineffective or slightly effective in glucose medium.     

乙醛脱氢酶及其医疗领域研究进展

对乙醛脱氢酶的种类、基本特性、制备、检测及其在医疗领域中应用作了简要的概述,旨在为该酶的进一步研究提供参考。     

Cloning and Nucleotide Sequencing of the Membrane-Bound l-Sorbosone Dehydrogenase Gene of Acetobacter liquefaciens IFO 12258 and Its Expression in Gluconobacter oxydans

Volume 61, no. 2, p. 419, column 1, lines 15-19: this sentence should read as follows. "The alcohol dehydrogenase and glucose dehydrogenase have a common region reported to be related to pyrroloquinoline quinone binding (2, 10), but SNDH does not contain such a region, indicating that SNDH is not a quinoprotein." Page 419, column 2, line 12: "(Table 4)" should read "(Table 3)." [This corrects the article on p. 413 in vol. 61.].     

A Novel Type of D-Mannitol Dehydrogenase from Acetobacter xylinum: Occurrence,Purification, and Basic Properties

We purified a novel type of D-mannitol dehydrogenase, which contains a c-type cytochrome and an unknown chromophore in the soluble fraction of an acetic acid bacterium, Acetobacter xylinum KU-1, to homogeneity. The enzyme showed the maximum activity at pH 5 and 40°C. It was stable up to 60°C at pH 6, and was inhibited by Hg²⁺ and p-quinone (K_i = 0.18 mm). The molecular weight of the enzyme was about 140,000, and those of the subunits were 69,000, 51,000, and 20,000; the enzyme is hetero-trimeric and contained 8 g-atoms of Fe per mole. The α-helix content was estimated to be about 52.9%. The enzyme catalyzed phenazine methosulfate dependent oxidation of d-mannitol with an apparent K_m of 98 μm (for d-mannitol) and V_max of 213 μmol/min/mg. The reduced form of the enzyme showed the absorption maxima at 386, 416, 480, 518, 550, and 586 nm, which are attributable to a c-type cytochrome in the enzyme.     

Characterization of Betaine Aldehyde Dehydrogenase from Cylindrocarpon didymum M-1

To obtain a lipase which effectively hydrolyzes castor oil, bacteria were isolated from 500 soil samples. The best strain was examined; its microbiological characteristics suggested that it belongs to the genus Pseudomonas. A lipase from this strain was purified by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and DEAE-Toyopearl 650 M. The enzyme was purified about 400-fold with a yield of 13%. The purified enzyme was electrophoretically homogeneous and its molecular weight was 30,000. The optimum pH and temperature for the hydrolysis of olive oil emulsion were 7.0 and 60°C. The enzyme was stable up to 35°C at pH 7.0 for 30min and also stable from pH 9.0 to 10.0 at 4°C for 22 hr. The activity was inhibited by Fe³⁺ , Hg²⁺ , pCMB, and anionic surfactants, and enhanced by nonionic surfactants and bile salts. The enzyme efficiently hydrolyzed castor oil.     

Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate, Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the ³⁵S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.     

Subcellular Distribution of Human Brain Aldehyde Dehydrogenase

Abstract: Two human brain surgery biopsies and one autopsy sample were subjected to subcellular fractionation. With either 0.12 or 6 mM-acetaldehyde as substrate, about half of the total aldehyde dehydrogenase activity was found in the mitochondrial (+ synaptosomal) fraction and less activity in the cytosolic, nuclear, and microsomal fractions. High-affinity activity was found only in the mitochondrial fraction. The enzyme in all fractions had a higher affinity for indole-3-acetaldehyde than for acetaldehyde. The kinetic data indicate the presence of several distinct aldehyde dehydrogenase isozymes that have ample capacity to oxidize both aliphatic and aromatic aldehydes in human brain.     

Histochemical Study of Aldehyde Dehydrogenase in the Rat CNS

A quantitative histochemical method was developed to determine aldehyde dehydrogenase (EC 1.2.1.3; ALDH) activity in the CNS. The distribution of ALDH activity in all rat brain and spinal cord regions is described. Among the CNS neuron structures, high enzyme activity was found in receptor and effector neurons, whereas low activity was noted in perikarya of the majority of intermediate neurons, including all aminergic neurons. A positive correlation was demonstrated between the distribution of ALDH activity among rat CNS microregions (our own data) and the density of dopaminergic terminals, dopamine content, and monoamine oxidase activity (literature data) among the same microregions. They may reflect a spatial linkage between ALDH and the predicted sites of natural aldehyde production. Lower enzyme activity was found in phylogenetically younger brain structures. It may explain the differential resistance of CNS structures to ethanol (acetaldehyde). Among the barrier CNS structures, moderate ALDH activity was found in capillaries and surrounding astrocytes and high activity was noted in ependimocytes covering the brain cavities and those of the vascular plexus. This provides realization of the function of ALDH as a brain metabolic barrier for aldehydes.     

梭梭甜菜碱醛脱氢酶基因克隆及序列分析

采用RT-PCR、RACE等方法从超旱生、耐盐植物梭梭(Haloxylon ammodendron)中扩增出BADH基因的cDNA序列(命名为HaBADH),其开放阅读框为1 503 bp,推测的氨基酸序列全长为500个氨基酸残基,并含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).其核苷酸序列与藜科几种盐生植物如盐爪爪(Kalidium foliatum)、中亚滨藜(Atriplex centralasiatica)、三角叶滨藜(Atriplex triangularis)、菠菜(Spinacia oleracea)、山菠菜(Atriplex hortensis)和甜菜(Beta vulgaris)等的相似性均在85%以上,推导编码蛋白的氨基酸序列一致性均在87%以上,表明BADH基因在藜科植物中是一种比较保守的基因.研究结果为进一步从分子水平探明梭梭的抗旱、耐盐机制,挖掘并利用植物抗逆基因奠定基础.     

Archaeal Aldehyde Dehydrogenase ST0064 from Sulfolobus tokodaii,a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase,Is a Succinate Semialdehyde Dehydrogenase

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K _m of 0.0029 mM and k _cat of 30.0 s^?1) with no allosteric regulation. Activity toward GAP was lower (K _m of 4.6 mM and k _cat of 4.77 s^?1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.     

甜菜碱醛脱氢酶在菠菜叶细胞中的定位

将菠菜叶片匀浆后．用差速离心和梯度率心分离叶绿体、过氧物酶体、微粒体等细胞器和１０００００×ｇ上清法部分。用酶活测定法测定各部分甜菜碱醛脱氢酶（ＢＡＤＨ）的活性;用免疫扩散法鉴定各组分的ＢＡＤＨ。除叶绿体外,过氧物酶体、微粒体．以及１０００００×ｇ上清液中也存在ＢＡＤＨ。