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1.
Chiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium-chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2-hydroxyacetophenone (2-HAP) to (R)-(-)-1-phenyl-1,2-ethanediol ((R)-PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg−1 and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme-coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two-phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l−1). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.  相似文献   

2.
NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2–C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.Abbreviations HPLC high performance liquid chromatography - DEAE diethyl amino ethyl - IEF isoelectrofocusing - NTG nitrosoguanidine - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - pI isoelectric point  相似文献   

3.
The optimal concentrations of diaphorase, methyl viologen (MV2+) and NAD+ in the mediated electrocatalytic reduction of NAD+ were decided by applying cyclic voltammetry. The steady-state catalytic current was achieved under the conditions of 1.5 U diaphorase ml–1, 0.2 mM MV2+, and 4.8 mM NAD+ at the scan rate of 2 mV s–1, suggesting that the rate of the electrocatalytic reaction is the highest under the former conditions. However, NAD+ was effective at 0.3 mM as it was at 4.8 mM when the electrocatalysis is coupled with an enzymatic synthesis requiring NADH. In effect, the electrochemical procedure under the conditions of 1.5 U diaphorase ml–1, 0.2 mM MV2+, and 0.3 mM NAD+ worked satisfactorily to drive an enzymatic reduction of pyruvate to d-lactate in the presence of d-lactate dehydrogenase.  相似文献   

4.
Summary l-Phenylalanine dehydrogenase [l-phenylalanine: NAD+-oxidoreductase (deaminating)] of Rhodococcus sp. strain M4 was studied emphasizing its application for the production of l-phenylalanine. A high enzyme level (30,000 U·l-1, 25–30 U·mg-1 in the crude extract) could be reached during aerob degradation of l-phenylalanine (10 g·l-1) under optimized growth coditions. A partial purification of the intracellular enzyme by liquid-liquid extraction, and DEAE-cellulose led to a specific activity of more than 1300 U·mg-1. The continuous production of l-phenylalanine in an enzyme-membrane-reactor for 350h resulted in a space-time yield of 456 g·l-1·d-1 with a mean substrate conversion of 95%. Consumption of phenylalanine dehydrogenase was 1,500 U·kg Phe-1.Abbreviations BSA bovine serum albumine - pheDH l-phenylalanine dehydrogenase - phepyr phenylpyruvate - OD optical density - FDH formate dehydrogenase  相似文献   

5.
Cell-free extracts of crotonate-grown cells of the syntrophic butyrate-oxidizing bacteriumSyntrophospora bryantii contained high hydrogenase activities (8.5–75.8 µmol · min–1 mg–1 protein) and relatively low formate dehydrogenase activities (0.04–0.07 µmol · min–1 mg–1 protein). The K M value and threshold value of the hydrogenase for H2 were 0.21 mM and 18 µM, respectively, whereas the K M value and threshold value of the formate dehydrogenase for formate were 0.22 mM and 10 µM, respectively. Hydrogenase, butyryl-CoA dehydrogenase and 3-OH-butyryl-CoA dehydrogenase were detected in the cytoplasmic fraction. Formate dehydrogenase and CO2 reductase were membrane-bound, likely located at the outer aspect of the cytoplasmic membrane. Results suggest that during syntrophic butyrate oxidation H2 is formed intracellularly while formate is formed at the outside of the cell.  相似文献   

6.
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.  相似文献   

7.
A new immobilisate of alcohol dehydrogenase (ADH) is described in which all components for the reaction, i.e. enzyme, the coenzyme NADP+, the buffer and other cofactors (trace elements), are immobilized together. It is an all-inclusive catalyst. The support is a cheap, commercially-available, superabsorbent polymer. The immobilisation is easy to achieve. The superabsorbed ADH is, even when dried, a stable and storable catalyst for at least five weeks at −18°C. Asymmetric reductions of the prochiral ketones, acetophenone, 4-acetylpyridine and ethyl acetoacetate, with a superabsorbed ADH from Lactobacillus brevis (ADH 002) and a superabsorbed ADH from Thermoanaerobicum sp. (ADH 005) in 2-propanol as both the organic solvent and the cofactor-regenerating substrate are given. Yields of chiral (R) and (S)-alcohols from 97–100% were achieved within 18 to 48 h with enantiomeric excesses of >99%. The superabsorbed ADH was easily separated by filtration and could be reused at least four times.  相似文献   

8.
This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF6]: Butyl-methyl-imidazoliumhexafluorophosphate  相似文献   

9.
A new NADH oxidase, useful for the regeneration of NAD+, was isolated and characterized from Lactobacillus brevis. In crude extracts the activity was from 10–15 U mg–1. After purification by four chromatographic steps, an activity of 116 U mg–1 was obtained with 14% yield. Highest activity was from pH 5.5–7 and at 40°C. The enzyme requires dithiothreitol to prevent oxidative deactivation. The K m value for NADH was 24 M.  相似文献   

10.
A hollow fiber module was used as a reactor for conversion of ethanol to acetaldehyde in the presence of horse liver alcohol dehydrogenase as catalyst. Mass transport rates for NAD+, the overall acetaldehyde generation rate, catalyst effectiveness factors, and the overall order of the reaction with respect to NAD+ concentration were measured. A coupled-substrate reactor with continuous in situ regeneration of cofactor was also examined. Two substrates of opposite redox state were added simultaneously to the feed stream. NADH and acetaldehyde concentrations were monitored in the effluent stream. The cofactor recycle number, or ratio of moles of product to moles of NADH produced, exceeded 10,000 under certain conditions. While decreasing the NAD+ concentration in the feed stream decreased reactor productivity somewhat, it greatly enhanced the ratio of product formed per mole of NAD+ fed to the reactor. It is suggested that high cofactor costs in dehydrogenase reactors may be overcome with efficient in situ regeneration and secondary recovery and recycling of cofactor from the process stream.  相似文献   

11.
Alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH)-catalyzed one-pot cascade conversion of an alcohol to an amine provides a simple preparation of chiral amines. To enhance the cofactor recycling in this reaction, we report a new concept of coupling whole-cells with the cell-free system to enable separated intracellular and extracellular cofactor regeneration and recycling. This was demonstrated by the respective biotransformation of racemic 4-phenyl-2-butanol 1a and 1-phenyl-2-propanol 1b to (R)-4-phenylbutan-2-amine 3a and (R)-1-phenylpropan-2-amine 3b . Escherichia coli cells expressing S-enantioselective CpsADH, R-enantioselective PfODH, and NADH oxidase (NOX) was developed to oxidize racemic alcohols 1a–b to ketones 2a–b with full conversion via intracellular NAD+ recycling. AmDH and glucose dehydrogenase (GDH) were used to convert ketones 2a–b to amines (R)- 3a–b with 89–94% conversion and 891–943 times recycling of NADH. Combining the cells and enzymes for the cascade transformation of racemic alcohols 1a–b gave 70% and 48% conversion to the amines (R)- 3a and (R)-3 b in 99% ee, with a total turnover number (TTN) of 350 and 240 for NADH recycling, respectively. Improved results were obtained by using the E. coli cells with immobilized AmDH and GDH: (R)- 3a was produced in 99% ee with 71–84% conversion and a TTN of 1410-1260 for NADH recycling, the highest value so far for the ADH–AmDH-catalyzed cascade conversion of alcohols to amines. The concept might be generally applicable to this type of reactions.  相似文献   

12.
The effect of gene knockout on metabolism in the pflA, pflB, pflC, and pflD mutants of Escherichia coli was investigated. Batch cultivations of the pfl mutants and their parent strain were conducted using glucose as a carbon source. It was found that pflA and pflB mutants, but not pflC and pflD mutants, produced large amounts of d-lactate from glucose under the microaerobic condition, and the maximum yield was 73%. In order to investigate the metabolic regulation mechanism, we measured enzyme activities for the following eight enzymes: glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase, lactate dehydrogenase (LDH), phosphoenolpyruvate carboxylase, acetate kinase, and alcohol dehydrogenase. Intracellular metabolite concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate, pyruvate, acetyl coenzyme A as well as ATP, ADP, AMP, NADH, and NAD+ were also measured. It was shown that the GAPDH and LDH activities were considerably higher in pflA and pflB mutants, which implies coupling between NADH production and consumption between the two corresponding reactions. The urgent energy requirement was shown by the lower ATP/AMP level due to both oxygen limitation and pfl gene knockout, which promoted significant stepping-up of glycolysis when using glucose as a carbon source. It was shown that the demand for energy is more important than intracellular redox balance, thus excess NADH produced through GAPDH resulted in a significantly higher intracellular NADH/NAD+ ratio in pfl mutants. Consequently, the homolactate production was achieved to meet the requirements of the redox balance and the energy production through glycolysis. The effect of using different carbon sources such as gluconate, pyruvate, fructose, and glycerol was investigated.  相似文献   

13.
NAD+-dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD+ or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile (MtFDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli, as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that MtFDH exhibits higher catalytic efficiency on NaHCO3 compared to formate. Notable, recombinant MtFDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with Tm 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that MtFDH possess a huge potential for CO2 reduction or NADH generation and under extreme alkaline conditions.  相似文献   

14.
【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。  相似文献   

15.
Recombinant pyridine nucleotide transhydrogenase (PNT) from Escherichia coli has been used to regenerate NAD+ and NADPH. The pnta and pntb genes encoding for the - and -subunits were cloned and co-expressed with NADP+-dependent alcohol dehydrogenase (ADH) from Lactobacillus kefir and NAD+-dependent formate dehydrogenase (FDH) from Candida boidinii. Using this whole-cell biocatalyst, efficient conversion of prochiral ketones to chiral alcohols was achieved: 66% acetophenone was reduced to (R)-phenylethanol over 12h, whereas only 19% (R)-phenylethanol was formed under the same conditions with cells containing ADH and FDH genes but without PNT genes. Cells that were permeabilized with toluene showed ketone reduction only if both cofactors were present.  相似文献   

16.
A new nicotinamide cofactor-dependent alcohol dehydrogenase from Pseudomonas strain SBD6 (PADH) was isolated and purified 150-fold to homogeneity using a combination of salt precipitation, anion-exchange chromatography, gel filtration chromatography, and dye matrix chromatography. Approximately 10 mg of pure enzyme can be obtained from 10 g of wet cells. The enzyme has four subunits with a total molecular weight of 162,000. Incubation with the metal chelators 1,10-phenanthroline, 2-aminoethanethiol, hydroxyquinolinesulfonic acid, N-ethylmaleimide, and potassium cyanide result in complete loss of activity. The enzyme is very stable (t1/2 7 days at pH 7 and 25°C in the absence of 2-propanol and 18 days in the presence of 10% 2-propanol, v/v) and possesses a broad substrate specificity with transfer of the pro-(R) hydride from NADH to the si face of carbonyl substrates to give (R)-alcohols in high enantiomeric excess, a stereochemical process different from that of other known alcohol dehydrogenases. Synthetic scale reductions are facilitated with 2-propanol as a hydride source for the regeneration of NADH. The kinetic mechanism is ordered bi-bi with the cofactor binding first. Based on NAD and 2-propanol, the kinetic parameters of the enzyme were determined to be Vmax = 29.9 Units mg−1 at 25°C and pH 8.5, KmNAD = 0.36 m and Km2-propanol = 0.19 m .  相似文献   

17.

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis.  相似文献   

18.
Active site substituted Cd(II) horse liver alcohol dehydrogenase has been studied by Perturbed Angular Correlation of Gamma rays Spectroscopy during turnover conditions for benzaldehyde and 4-trans-(N,N-dimethylamino)cinnamaldehyde. The ternary complex between alcohol dehydrogenase NAD+ and Cl, and the binary complex between alcohol dehydrogenase and orthophenanthroline have also been studied. The Nuclear Quadrupole Interaction parameters have been interpreted in terms of different coordination geometries for Cd(II) in the catalytic zinc site of the enzyme. Calculation of the nuclear quadrupole interaction for cadmium in the catalytic site of the enzyme with and without coenzyme, based upon the four coordinated geometries determined from X-ray diffraction, agrees with the experimentally determined values. The ternary complexes between enzyme, NAD+ and either Cl or trifluoroethanol and the binary complex between enzyme and orthophenanthroline have almost identical spectral parameters which are not consistent with a four coordinated geometry, but are consistent with a five coordinated geometry. The nonprotein ligands for the ternary complex with trifluoroethanol are suggested to be an alkoxide group and a water molecule. The Nuclear Quadrupole Interaction parameters for the productive ternary complex between enzyme, NADH and an aldehyde is consistent with the four coordinated geometry predicted from X-ray diffraction data having the carbonyl group of the aldehyde substituting the water molecule as ligand to the metal.Abbreviations LADH Horse liver alcohol dehydrogenase - H4Zn2LADH derivative of LADH free of zinc in the catalytic site - 111CdZn2LADH derivative of LADH with 111Cd (carrier free) in the catalytic site - Cd2Zn2LADH derivative of LDH with 2 mole of Cd(II) per mole LADH in the catalytic site - PAC pertubed angular correlation of gamma rays - NQI Nuclear quadrupole interaction - AOM Angular overlap model - trifluoroethanol 2,2,2-trifluoroethanol - DACA trans-4-(N,N-dimethylamino)cinnamaldehyde - NAD+ and NADH oxidized and reduced nicotinamide adenine dinucleotide - NADH2 reduced 1,4,5,6-tetrahydronicotinamide adenine dinucleotide The experimental work was carried out at the Niels Bohr Institute Risø, 4000 Roskilde and Blegdamsvej 19, 2100 Copenhagen, Denmark Offprint requests to: R. Bauer  相似文献   

19.
A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L−1 d−1, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.  相似文献   

20.
Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD+, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault  相似文献   

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