Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase.

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a beta-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of alpha-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.     

Three different coupled enzymatic systems for in situ regeneration of NADPH

Three different coupled enzymatic systems used in the reduction of sulcatone by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), were kinetically compared. The first one involved the use of TBADH for both the principal and recycling reactions and 2-propanol 20%, v/v as the recycling substrate. The other two were based on the use of a different enzyme, glucose- or glucose-6-phosphate dehydrogenases, for in situ regeneration of NADPH. The coupled-substrate approach achieved 100% of conversion against 84% of the other two systems.     

Soybean alcohol dehydrogenase

Alcohol dehydrogenase was prepared from germinating soybean seeds. Specific activity was increased from 511 to 31316 units. The coenzyme is NAD with a K_m of 10^?4M. Allyl alcohol is oxidized faster than ethanol; with the latter substrate, the K_m is 1.3 × 10^?2M, and the pH optimum 8.7. The enzyme catalyses acetaldehyde reduction, with a K_m of 10^?2M and a pH opt of 7.1. The MW is 53(±5) × 10^?3.     

Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (< 5%). Site-directed mutagenesis to replace any one of the three putative zinc ligands of TBADH, Cys 37, His 59, or Asp 150, with the non-chelating residue, alanine, abolished not only the metal-binding capacity of the enzyme but also its catalytic activity, without affecting the overall secondary structure of the enzyme. Replacing the three putative catalytic zinc ligands of TBADH with the respective chelating residues serine, glutamine, or cysteine damaged the zinc-binding capacity of the mutated enzyme and resulted in a loss of catalytic activity that was partially restored by adding excess zinc to the reaction. The results imply that the zinc atom in TBADH is catalytic rather than structural and verify the involvement of Cys 37, His 59, and Asp 150 of TBADH in zinc coordination.     

Aromatic alcohol dehydrogenase from potato tubers

The purification of NADP specific aromatic alcohol dehydrogenase is reported. The properties of the enzyme suggest that it should be classified as E.C. 1.1.1.2. Kinetic constants for a number of substrates are reported. The relative rates of reaction of a variety of substituted benzaldehydes have been found to correlate with Hammett's sigma values yielding a biphasic relationship. The physiological significance of the enzyme is briefly discussed.     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

The presence of an NADP-dependent alcohol dehydrogenase activity in Acinetobacter calcoaceticus

Abstract A soluble NADP-dependent alcohol dehydrogenase activity (EC 1.1.1.2) was found in all five strains of Acinetobacter calcoaceticus tested. In A. calcoaceticus NCIB8250, this dehydrogenase was not induced by growth on ethanol, but was present at approximately the same specific activity when this strain was grown on a variety of carbon sources. The specific activity of the NADP-dependent alcohol dehydrogenase is about 10% of the activity of the NAD-dependent alcohol dehydrogenase found in bacteria grown on ethanol. The distinct biochemical properties of the NADP-dependent dehydrogenase showed that this activity was not due to lack of nucleotide specificity of the NAD-dependent dehydrogenase.     

Aliphatic alcohol dehydrogenase from potato tubers

Potato tubers are shown to contain at least 3 alcohol dehydrogenases, one active with NAD and aliphatic alcohols, one active with NADP and terpene alcohols and one active with NADP and aromatic alcohols. The purification of the aliphatic alcohol dehydrogenase is described and its activity with a wide range of substrates is reported. On the basis of substrate specificity, the enzyme is shown to resemble yeast alcohol dehydrogenase rather than liver alcohol dehydrogenase. The enzyme shows high activity with and high affinity for ethanol, activity and affinity decline as the chain length is increased from ethanol to butanol, but a further increase in chain length leads to increased affinity for the alcohol. The physiological significance of the results is briefly discussed.     

An intergenic alcohol dehydrogenase isozyme in sunflowers

The isozymes of alcohol dehydrogenase (ADH; E.C. 1.1.1.1) in wild and cultivated sunflower (Helianthus annuus) seeds can be resolved electrophoretically into 12 bands. The slowest- and probably the fastest-migrating sets of three are allozymic products of two genes, Adh ₁ and Adh ₂ , each having two alleles, F (for fast) and S (for slow). Evidence from dissociation-recombination experiments utilizing bands excised from starch gels indicates that an intermediately-migrating isozyme is a dimeric intergenic product consisting of ADH-1F and ADH-2S subunits. The hybrid isozyme was unstable in vitro in that its monomers spontaneously dissociated and recombined to produce ADH-1FF and ADH-2SS isozymes. The molecular weights of the hybrid as well as the parental isozymes were estimated at approximately 98,000.Supported by a Graduate School Research grant of the University of Kansas and by NSF grant GB-35853.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Expression of alcohol dehydrogenase in rice embryos under anoxia

Summary Alcohol dehydrogenase (ADH) activity was present in roots and shoots of 48-h rice embryos and rose in response to anoxia. The increase was accompanied by changes in the ADH isozyme pattern. Translatable levels of mRNA for two ADH peptides increases as early as 1 h after the beginning of anoxic treatment. Adh mRNA was detected in aerobically grown rice embryos by hybridization to maize Adh1 cDNA: its level increased significantly after 3 h of anoxia.     

聚乙烯醇脱氢酶的提取及检测

为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶（PVADH）的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂炕基苯磺酸钠（LAS）和溴化十六烷基吡啶（CPB）,酶活力比LAS和CPB提取后所得酶活力分别提高246．5％和831．3％。而非离子型表面活性剂中,Triton X-100与Tween80相比,所得最高酶活提高了101．4％。Triton X—100浓度和提取时间对测定有明显影响,以1％Triton X-100提取18h为宜,最高比酶活达14．9U／g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60．6％和126．5％;酶液与吡咯喹啉醌（PQQ）预先保温对检测该酶活性是十分重要的,可使酶活性提高59．1％.在检测系统中加入的KCN、CaCl2和PQQ的适宜浓度分别为1．ommol／L、0．5mmol／L和2μmol／L,可使测定酶活分别提高37．1％、38．7％和214．0％．     

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S-carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.     

Induction of alcohol dehydrogenase by plant hormones in alfalfa seedlings

Six-day-old alfalfa (Medicago sativa L.) seedlings were treated with auxin, abscisic acid (ABA), cytokinin and gibberellin to determine the effect of these plant hormones on induction of alcohol dehydrogenase (ADH). The ADH activity was increased at concentrations greater than 1 M for auxin and ABA and 3 M for cytokinin, respectively, and all increases were found within 6 h after treatments. However, ADH activity remained almost unchanged in the seedlings treated with gibberellin. At 100 M doses, the activities in the seedlings were 4.0-, 3.6- and 2.1-fold greater than that of non-treated seedlings for auxin, ABA and cytokinin, respectively.     

Co-factor specificity of plant alcohol dehydrogenase

Extracts of seventeen plant tissues show alcohol dehydrogenase activity in the presence of both NADH and NADPH. Using extracts of melon fruits, attempts have been made to separate these two activities by applying a range of chromatographic and electrophoretic techniques but these proved unsuccessful. Evidence from kinetic measurements involving assays of equimolar concentrations of the two co-factors suggests that in the enzyme from the melon there is but a single catalytic site which will accept either co-factor.     

Immobilization of yeast alcohol dehydrogenase on magnetic nanoparticles for improving its stability

Yeast alcohol dehydrogenase (YADH) was immobilized covalently on Fe₃O₄ magnetic nanoparticles (10.6 nm) via carbodiimide activation. The immobilization process did not affect the size and structure of magnetic nanoparticles. The YADH-immobilized magnetic nanoparticles were superparamagnetic with a saturation magnetization of 61 emu g^–1, only slightly lower than that of the naked ones (63 emu g^–1). Compared to the free enzyme, the immobilized YADH retained 62% activity and showed a 10-fold increased stability and a 2.7-fold increased activity at pH 5. For the reduction of 2-butanone by immobilized YADH, the activation energies within 25–45 °C, the maximum specific activity, and the Michaelis constants for NADH and 2-butanone were 27 J mol^–1, 0.23 mol min^–1 mg^–1, 0.62 mM, and 0.43 M, respectively. These results indicated a structural change of YADH with a decrease in affinity for NADH and 2-butanone after immobilization compared to the free enzyme.     

Polymorphism of horse liver alcohol dehydrogenase

The properties of the most cathodal component of horse liver alcohol dehydrogenase (isozyme SS) have been found to vary. The variability is dependent on the livers from which the enzyme is isolated rather than on the purification procedure. Two distinct preparations, differing in catalytic properties, have been obtained and named S-type and A-type preparations. The preparations can be distinguished from each other by the ratio of activity with acetaldehyde to activity with the steroidal ketone 5_β-dihydrotestosterone. This ratio is about one for the S-type and twenty for the A-type preparations.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.