共查询到20条相似文献,搜索用时 31 毫秒
1.
Hyunjun Choe Sumi Lee Hyojin Hwang Jeong Chan Joo Dae Haeng Cho Yong Hwan Kim 《Biotechnology and Bioprocess Engineering》2014,19(4):613-620
The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD+. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K m) of 5.87 μM, a turnover rate (k cat) of 0.24/sec, and a catalytic efficiency (k cat/K m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k cat/K m, 107.75/mM/sec). 相似文献
2.
Jan R. Andreesen Ebtisam El Ghazzawi Gerhard Gottschalk 《Archives of microbiology》1974,96(1):103-118
In a medium containing a trace element solution and 10-4 M ferrous ions the growth yield ofClostridium formicoaceticum on fructose was 5.5 g of weight per l; in the absence of metal ion solution it was 1 g per l. The specific activity of methyl viologen dependent formate dehydrogenase under both conditions was 0.28 and 0.03 units per mg of protein, respectively. It could be increased to 9.75 units when the growth medium contained 10-4 M tungstate and 10-5 M selenite in addition. Molybdate was only about 40% as effective as tungstate. Tungstate or molybdate could not be replaced by vanadate, selenite not by sulfide. The formate dehydrogenase catalyzed also the reduction of CO2 to formate. The highest rate of formate synthesis was observed when pyruvate served as the reductant. No pyruvate: formate exchange but rapid pyruvate: CO2 exchange could be observed with cell-free extracts ofC. formicoaceticum. Pyruvate is fermented byC. formicoaceticum to yield up to 1.16 mole acetate per mole of pyruvate. Resting cells accumulated some formate in addition to acetate. 相似文献
3.
- Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
- Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
- Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea 1 and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
- 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
- The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
- The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
- Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.
4.
The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe2+ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H2O2 was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H2O2 was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction 相似文献
5.
Eddy?Van der?Linden Tanja?Burgdorf Michael?Bernhard Boris?Bleijlevens B?rbel?Friedrich Simon?P.?J.?Albracht
Infrared spectra of 15N-enriched preparations of the soluble cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha are presented. These spectra, together with chemical analyses, show that the Ni-Fe active site contains four cyanide groups and one carbon monoxide molecule. It is proposed that the active site is a (RS)2(CN)Ni(-RS)2Fe(CN)3(CO) centre (R=Cys) and that H2 activation solely takes place on nickel. One of the two FMN groups (FMN-a) in the enzyme can be reversibly released upon reduction of the enzyme. It is now reported that at longer times also one of the cyanide groups, the one proposed to be bound to the nickel atom, could be removed from the enzyme. This process was irreversible and induced the inhibition of the enzyme activity by oxygen; the enzyme remained insensitive to carbon monoxide. The Ni-Fe active site was EPR undetectable under all conditions tested. It is concluded that the Ni-bound cyanide group is responsible for the oxygen insensitivity of the enzyme.Abbreviations BV benzyl viologen - DCIP 2,6-dichlorophenol-indophenol - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - MV methyl viologen - SH soluble NAD+-reducing hydrogenase - XAS X-ray absorption spectroscopy 相似文献
6.
Kazuo Iwai Noboru Suzuki Shusaku Mizoguchi 《Bioscience, biotechnology, and biochemistry》2013,77(3):267-274
Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was found in fresh spinach leaves and purified about 60-fold by treatments of ammonium sulfate, protamine sulfate, dialysis, and DEAE-cellulose column chromatography. Some properties of the enzyme were investigated. Optimum pH was found to be 7.5, and optimum temperature was observed to be at 37°C. In the enzyme reaction, FAH4 and formate were required specifically as the substrates, and Mg++ and ATP were essential components. The Michaelis constants for dl-FAH4, formate, ATP and magnesium chloride were 1.7×10?3 m, 1.7×10?2 m, 4.1×10?4 m and 3.3×10?3 m, respectively. The primary product formed in the reaction catalyzed by the enzyme was suggested as N10-formyl-FAH4 spectrophotometrically. It was observed that the enzyme also catalyzed the reverse reaction. The possible role of the enzyme in plants was discussed. 相似文献
7.
Cell-free extracts of Prochloron didemni were assayed for ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) and phosphoribulokinase (EC 2.7.1.19), two key enzymes in the reductive pentose-phosphate cycle. In an RuBP-dependent reaction, the production of two molecules of 3-phosphoglycerate per molecule of CO2 fixed was shown. Phosphoribulokinase activity was demonstrated by the production of ADP from ribulose 5-phosphate (Ru5P) and ATP and by measurement of ATP-, Ru5P-dependent 14CO2 fixation in the presence of excess spinach RuBP carboxylase. When Prochloron RuBP carboxylase was purified from cell-free extracts by isopycnic centrifugation in reoriented linear 0.2 to 0.8 M sucrose gradients, the enzyme sedimented to a position which corresponded to that for the 520,000-dalton spinach enzyme. After polyacrylamide gel electrophoresis (PAGE) of Prochloron enzyme, a major band of enzyme activity corresponded to that for the spinach enzyme. Considerably more additional carboxylase activity was found in a less mobile species than was the case for spinach RuBP carboxylase. Sodium dodecyl sulfate-PAGE of the Prochloron enzyme indicates that it is composed of both large (molecular weight, MW=57,500) and small (MW=18,800) subunits. 相似文献
8.
Lysosome-solubilized pig liver NADH-cytochrome b5 reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK 2=0.22 min?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[14C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b5 reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme. 相似文献
9.
The purified choline acetyltransferase from human striatal tissue was found to have aK m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH4)2SO4, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole. 相似文献
10.
Y. Okai 《Molecular biology reports》1982,8(3):167-172
Rat liver chromatin-bound RNA polymerase II could be differentially solubilized into two distinct populations, loosely and tightly bound enzymes, by a simple method. By this method the recovery of the solubilized enzyme from the chromatin fraction could be increased considerably as compared with the procedure of Yu (1). The two chromatin-bound enzymes had different properties:
- Loosely bound enzyme was easily extractable from chromatin with relatively mild ionic condition (0.5 M NaCl); the tightly bound enzyme had to be solubilized by more drastic conditions such as sonication or nuclease treatment.
- Loosely bound enzyme could not efficiently transcribe the chromatin template, but the tightly bound enzyme was active toward the same template. The latter enzyme is involved in the tight complex with the RNA synthesis activating factors.
- Cycloheximide treatment in vivo suggests that the two enzymes have different turn-over rates.
11.
The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO2 via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min-1 mg-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg2+ ions, NH 4 + or K+ ions, and SH-groups. Mn2+, but not Ca2+, could partially substitute for Mg2+; Na+ could not substitute for K+ or NH 4 + . The pH-optima,V max-values and the apparentK M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK M value for pyruvate (40 μM) being low and the apparentK M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO2 assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed. 相似文献
12.
J. C. Cox 《Archives of microbiology》1989,152(4):397-400
Four classes of Escherichia coli mutants deficient in either or both of their anaerobic selenium-containing formate dehydrogenases (FDH) were isolated. A class I mutant devoid of FDHH activity specifically linked to benzyl viologen (BV) produced a small amount of the FDHH 80,000 dalton selenopeptide. Three class II mutants were deficient in FDHN activity specifically linked to phenazine methosulfate (PMS) and exhibited a selenopeptide doublet rather than the FDHN 110,000 dalton selenosubunit. Three class III mutants were selenium incorporation deficient and did not exhibit either FDH activity or 75Selabeled selenopolymers. A class IV mutant was devoid of PMS-linked FDHN activity; neither its FDHN 110,000 dalton selenosubunit nor its BV-linked FDHH activity was fully regulated by nitrate.Abbreviations FDH
formate dehydrogenase
- BV
benzyl viologen
- MV
methyl viologen
- PMS
phenazine methosulfate
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
13.
- Cells of Vibrio succinogenes, treated with EDTA at pH 8, catalyze the phosphorylation of their endogenous ADP and AMP as a function of the electron transport from formate to fumarate. The P/fumarate ratio obtained from the initial velocity of the phosphorylation on initiation of the electron transport and from the activity of fumarate reduction in the steady state was 0.90. The phosphorylation was prevented by 10μmol/g protein carbonylcyanide-3-chlorophenylhydrazone.
- The esterification of external phosphate in the presence of ADP, hexokinase and glucose is catalysed by a membrane preparation of V. succinogenes in the steady state of fumarate reduction by H2. The phosphorylation was fully abolished by either 5μmol/g protein carbonylcyanide-4-trifluoromethoxyphenylhydrazone or 30μmol/g protein carbonylcyanide-3-chlorphenylhydrazone. Phosphorylation was blocked also by dicyclohexylcarbodiimide, an inhibitor of the Mg2+-dependent membrane bound ATP synthase, and by low concentrations of the inhibitors of electron transport 2-(n-nonyl)-4-hydroxyquinoline-N-oxide or 4-chloromercuriphenylsulfonate.
- The P/fumarate ratios, measured with the membrane preparation, were found to increase with progressive inhibition of the electron transport from hydrogen to fumarate by means of 4-chloromercuriphenylsulfonate. The extrapolated ratio at vanishing electron transport activity was 0.47.
- About 50% of the membrane preparation was found to consist of inverted vesicles with the hydrogenase and formate dehydrogenase oriented to the inside. The residual part is considered as being incapable of performing energy transduction. The extrapolated P/fumarate ratio valid for the inverted vesicles was 0.94.
14.
Silvia Yuko Eguchi Hiroyuki Nakata Naomichi Nishio Shiro Nagai 《Applied microbiology and biotechnology》1984,20(3):213-217
Summary The resting cells of methanogen strain HU could be used as biocatalyser for converting exoge nous NADP+ into NADPH, using either formate or hydrogen as electron donor. To enhance the conversion efficiency of NADPH from NADP+, several inhibitors of methylcoenzyme M reductase were used in order to avoid further oxidation of NADPH to CH4. When methyl viologen (7.5 mol ml-1) was added to the reaction mixture (17 mg of dry cells, 2 mg Triton X-100, 294 mol of Na-formate and 12 mol of NADP+ per ml reaction mixture), 9.6 mol ml-1 NADPH (80% yield) could be produced in a 2-h reaction, compared with 7.2 mol ml-1 NADPH (60% yield) in a 6-h reaction in the absence of methyl viologen. Molecular hydrogen istead of formate also served as electron donor to convert NADP+ into NADPH. A gas mixture of H2/N2 (75/25) yielded 9.8 mol ml-1 NADPH (82% yield) in a 3-h reaction in the absence of formate, suggesting that H2 might be a promising, inexpensive electron donor for this reaction system. 相似文献
15.
Yoshiteru Hashimoto Toshihide Sakashita Hiroshi Fukatsu Hiroyoshi Sato Michihiko Kobayashi 《Applied and environmental microbiology》2014,80(1):61-69
Previously, we isolated a new enzyme, N-substituted formamide deformylase, that catalyzes the hydrolysis of N-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726–13731, 2004, doi:10.1073/pnas.0405082101). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizing N-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C1 to C3), were active as acid substrates for the reverse reaction. Through this reaction, N-substituted carboxamides, such as NBFA, N-benzylacetamide, and N-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates. 相似文献
16.
Lijuan Wang Rihe Peng Yongsheng Tian Jing Han Wei Zhao Bo Wang Man Liu Quanhong Yao 《World journal of microbiology & biotechnology》2014,30(11):2967-2973
5-Enopyruvylshikimate-3-phosphate synthase (EPSP synthase) is an important enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants and microorganisms. A novel class II EPSP synthase AroA S. fredii from Sinorhizobium fredii NGR234 was overexpressed in Escherichia coli BL21. It was purified to homogeneity and its catalytic properties were studied. The enzyme exhibited optimum catalytic activity at pH 8.0 and 50 °C. It was stable below 40 °C, and over a broad range of pH 5.0–9.0. The EPSP synthase was increasingly activated by 100 mM of the chlorides of NH4 +, K+, Na+ and Li+. Kinetic analysis of AroA S. fredii suggested that the enzyme exhibited a high glyphosate tolerance and high level of affinity for phosphoenolpyruvate, which indicates the enzyme with a high potential for structural and functional studies and its potential usage for the generation of transgenic crops resistant to the herbicide. 相似文献
17.
α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff’s staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0–7.0. The enzyme was inhibited by EDTA, Hg2+, Ag2+, and Cu2+. The activity lost by EDTA was completely regained by addition of Zn2+. The purified enzyme was characterized in terms of the kinetic parameters K m (1.6 mM) and V max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn2+ as a metal ion suggested that the enzyme belongs to class II α-Mannosidase. 相似文献
18.
Kinetic characteristics of monoamine oxidase and serum cholinesterase in several related rat strains 总被引:1,自引:0,他引:1
This study was performed in order to delineate differences in kinetic enzyme characteristics of brain monoamine oxidase (MAO) and plasma cholinesterase (ChE) derived from the Walker-Walker (Fawn Hooded, FH) rat and from its putative ancestors, the Wistar (W) and Long-Evans (LE). As compared with the enzyme isolated from the other two strains, brain MAO from FH has both a higher V max and increased reaction rate at lower substrate concentrations. It may thus be described as a “more efficient” enzyme. This study confirms previous work which shows that plasma ChE activity of females is higher than that of males. Fluoride ion is a noncompetitive inhibitor of the Wistar ChE, is a competitive inhibitor of the FH enzyme, and has no effect on the LE enzyme. Dibucaine is a competitive inhibitor in all cases except one: ChE derived from the FH female is uncompetitively inhibited. A comparison of the inhibitor constants shows that FH ChE is more resistant to Dibucaine than is that of W, and that LE is the most sensitive. FH cholinesterase is twice as resistant to the action of fluoride as is the Wistar enzyme. 相似文献
19.
Antisera raised against the rare FD phenotype enzyme were exhaustively absorbed with SS and FF phenotype enzyme immobilized on agarose gels. When it was absorbed with the FF phenotype enzyme, the antiserum no longer reacted with the F-variant enzyme, but did with the S-, D-, and I-variants, as determined by electrophoretic retardation experiments and precipitation of antigen-antibody complexes using staphylococcal protein A. When the antiserum was absorbed with SS phenotype enzyme, it no longer reacted with S-, D-, or I-variant enzyme, but did have some reactivity with the F-variant, as seen in the protein A assay. Based upon the IgG concentration, which bound 40% of the appropriate enzyme, 1/20 of the antiserum preparation was specific for the S-, D-, and I-variant shared specificity, and 1/400 was specific for the F-variant alone. 相似文献