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1.
Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties 总被引:2,自引:0,他引:2
Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension
cultures to a specific activity of 4.68 μmol (mg protein)−1 min−1. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of
5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus
sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD+) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations.
The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation
products.
Received: 1 April 1998 / Accepted: 18 November 1998 相似文献
2.
Emile Bol Nicolette J. Broers Wilfred R. Hagen 《Journal of biological inorganic chemistry》2008,13(1):75-84
Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have
been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring
of the relatively weak (ε ≈ 2 mM−1 cm−1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism
for three substrates (formaldehyde plus two ferredoxin molecules). The K
M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value
measured when benzyl viologen is used as an acceptor. The K
M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy
may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference
spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds
of the reaction. Two fast processes (k
obs1 = 4.7 s−1, k
obs2 = 1.9 s−1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes
could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling
over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k
obs3 = 6.10 × 10−2 s−1, k
obs4 = 2.18 × 10−2 s−1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation
route plus catalytic redox cycle is proposed. 相似文献
3.
Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis> 总被引:1,自引:0,他引:1
Hu GS Jia JM Hur YJ Chung YS Lee JH Yun DJ Chung WS Yi GH Kim TH Kim DH 《Molecular biology reports》2011,38(6):3741-3750
We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes
from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein
exhibited Michaelis–Menten kinetics with a K
m of 0.1013 mM, V
max of 4.858 μmol min−1, K
cat of 3.36 S−1, and K
cat/K
m is 33,168 M−1 S−1. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol−1 when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results
showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min
resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl
aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid
(tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K
i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K
i = 0.056 μM. 相似文献
4.
Characterization of malate dehydrogenase from the hyperthermophilic archaeon <Emphasis Type="Italic">Pyrobaculum islandicum</Emphasis> 总被引:1,自引:0,他引:1
Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic
archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins
are the same. The apparent K
m
values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with
specific activity as high as 470 U mg−1. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific
activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD+ were used as substrates and was not detected with NADP+. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines
suggesting minimal lateral mdh transfer between Archaea. 相似文献
5.
Uchida H Hojyo M Fujii Y Maeda Y Kajimura R Yamanaka H Sakurai A Sakakibara M Aisaka K 《Applied microbiology and biotechnology》2007,74(4):805-812
Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to
a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration,
was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively.
These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits
with apparently similar MM. The preparation acted on formate with K
m and V
max values of 11.7 mM and 262 μmol min−1 mg−1, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature
were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0
and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml−1 of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml−1 of a microbial aldehyde oxidase and 100 U ml−1 of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative
conversion of formaldehyde to carbon dioxide. 相似文献
6.
Laila Oukhattar Tarik Baibai Adnane Moutaouakkil Omar Assobhei Abdelaziz Soukri 《Reviews in Fish Biology and Fisheries》2008,18(3):263-271
The NAD+ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification
method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold
increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native
enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately
36 kDa. The Michaelis constants Km for both NAD+ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity Vmax of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions
of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against
the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose. 相似文献
7.
B B Nepple J Kessi R Bachofen 《Journal of industrial microbiology & biotechnology》2000,25(4):198-203
Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and
anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic
cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic
interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration.
The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations
of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K
m of 15±1.3 μM CrO4
2− and a V
max of 420±50 μmol min−1 mg protein−1 was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO4
2− and 100±9.6 μmol CrO4
2− min−1 mg protein−1 for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203.
Received 05 January 2000/ Accepted in revised form 27 May 2000 相似文献
8.
During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete
copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock),
and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible
form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution
in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The
relative molecular mass of native CP was in the range of 118–136 kDa, as estimated by gel filtration analysis on size exclusion
matrices, whereas SDS-PAGE analysis yielded a size of ∼37 kDa for the polypeptide. Substrate saturation kinetics studies were
conducted using ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H2O2 as substrates: K
m, V
max, and K
cat values for H2O2 were ∼22 μM, ∼447 nmol mg−1, and 0.33 s−1, respectively, and those for ABTS were ∼55 μM, ∼453 nmol mg−1, and 0.3 s−1, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme,
stable at temperatures up to 58°C.
Received: August 5, 2002 / Accepted: January 22, 2003
Acknowledgments This work was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada
(to M.K.). The financial support provided to A. M. in the form of a graduate studentship award by the AHFMR (Alberta Heritage
Foundation for Medical Research) and of a graduate teaching assistantship to A. S. by the Department of Biological Sciences,
University of Calgary, is gratefully acknowledged.
Correspondence to:M. Kapoor 相似文献
9.
Yoshifumi Maeda Atsuhide Yagyu Akihiko Sakurai Yutaka Fujii Hiroyuki Uchida 《World journal of microbiology & biotechnology》2008,24(6):797-804
Bacterium MEY43, an isolate from soil, produced aldehyde oxidase when it was cultivated in a medium containing methanol as
a sole source of carbon and energy. The methylotrophic bacterium was identified as Brevibacillus sp. Its cultivation in media containing other substrates, such as ethanol and glucose, resulted in little production of the
enzyme. Aldehyde oxidase purified from a cell-free extract of the bacterium was a hetero-trimeric protein comprised of large,
medium, and small subunits with molecular masses of 87, 35, and 19 kDa, respectively. Its UV/visible spectrum and the presence
of molybdenum, 5′-CMP, flavin adenine dinucleotide, iron, and acid-labile sulfur suggested that the enzyme belonged to the
xanthine oxidase family. The enzyme acted on a wide range of aliphatic and aromatic aldehydes. The K
m value for formaldehyde was 32 mM, whereas those for the other aldehydes tested were below 0.2 mM. When 10 mM glutaraldehyde
was treated with 2.0 units of the enzyme ml−1 in the presence of 100 units ml−1 catalase for 120 min, the concentration of the aldehyde decreased to below a detectable level. 相似文献
10.
Renata Matraszek 《Acta Physiologiae Plantarum》2008,30(3):361-370
The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity
of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO3
−–N, NH4
+–N, NH4NO3). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in
lettuce plants supplied with nitrate nitrogen (NO3
−–N) or mixed (NH4NO3) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction
of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity
in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants.
At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants
treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of
significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed
nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically
significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with
NO3
−–N or NH4NO3. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the
enzyme activity in the roots of NO3
−-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution
contained ammonium nitrogen (NH4
+–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs,
especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with
ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated
with NH4
+–N the enzyme activity in roots was even higher than in those supplied with NO3
−–N. 相似文献
11.
N. Borghol L. Mora T. Jouenne N. Jaffézic-Renault N. Sakly A. C. Duncan Y. Chevalier P. Lejeune A. Othmane 《Biotechnology and Bioprocess Engineering》2010,15(2):220-228
Electrochemical impedance spectroscopy (EIS) technique has proved to be an effective method for monitoring the immobilization
of various bioactive species such as enzymes, DNA, whole cells, and so forth. In this work we describe the development of
an electrochemical whole cell based biosensor. Biotinylated fluorescent E. coli are immobilized onto a cysteamine, Sulfo-NHS-LC-biotin, and avidin modified gold electrodes. Immobilized bacteria are clearly
observed using confocal microscopy. Electrochemical measurements are based on the charge-transfer kinetics of [Fe (CN)6]3−/4− redox couple. The experimental impedance data were modelised with a computer. SAM assembly and the subsequent immobilization
of bacteria on the gold bare electrodes greatly increased the electrontransfer resistance (R
et
) and reduced the constant phase element (CPE). It’s interesting to note, the hard immobilization of bacteria on the surface of electrode and do not remove during measurements.
The effect of glucose addition was studied in the range of 10−7 μM to 10 μM. The relation between the evolution of R
et
and D-glucose concentration was found to be linear for values ranging from 10−5 μM to 10−1 μM and reached saturation for higher concentrations. Such biosensor could be applied to a more fundamental study of cell
metabolism and drugs effect. 相似文献
12.
Mahdi Mohammadian Mehrnoosh Fathi-Roudsari Nasrin Mollania Arastoo Badoei-Dalfard Khosro Khajeh 《Journal of industrial microbiology & biotechnology》2010,37(8):863-869
Laccases (benzenediol oxygen oxidoreductase; EC 1.10.3.2) have many biotechnological applications because of their oxidation
ability towards a wide range of phenolic compounds. Within recent years, researchers have been highly interested in the identification
and characterization of laccases from bacterial sources. In this study, we have isolated and cloned a gene encoding laccase
(CotA) from Bacillus sp. HR03 and then expressed it under microaerobic conditions and decreased temperature in order to obtain high amounts of
soluble protein. The laccase was purified and its biochemical properties were investigated using three common laccase substrates,
2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). K
M and k
cat were calculated 535 μM and 127 s−1 for ABTS, 53 μM and 3 s−1 for 2, 6-DMP and 5 μM and 20 s−1 for SGZ when the whole reactions were carried out at room temperature. Laccase activity was also studied when the enzyme
was preincubated at 70 and 80°C. With SGZ as the substrate, the activity was increased three-fold after 50 min preincubation
at 70°C and 2.4-fold after 10 min preincubation at 80°C. Preincubation of the enzyme in 70°C for 30 min raised the activity
four-fold with ABTS as the substrate. Also, l-dopa was used as a substrate. The enzyme was able to oxidize l-dopa with the K
M and k
cat of 1,493 μM and 194 s−1, respectively. 相似文献
13.
Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which
had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids
had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots
of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear.
The K
m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures
was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence
of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by
NADH was linear competitive with respect to acetyl-CoA (K
is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K
is = 56 ± 3.8 μM and K
ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD+ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when
the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment
of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely
to be relieved by NAD+ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle.
Received: 14 July 1999 / Accepted: 20 September 1999 相似文献
14.
Beris FS De Smet L Karaoglu H Canakci S Van Beeumen J Belduz AO 《Journal of microbiology (Seoul, Korea)》2011,49(4):641-650
The G2ALT gene was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2. The gene is 666 bp long and encodes a protein 221 amino acids in length. The gene was overexpressed in E. coli and purified to homogeneity and biochemically characterized. The enzyme has a molecular mass of 24.5 kDa and it could be
classified as a member of the family of bacterial aluminium resistance proteins based on homology searches. When this fragment
was expressed in E. coli, it endowed E. coli with Al tolerance to 500 μM. The purified G2ALT protein is active at a broad pH range (pH 4.0–10.0) and temperature range
(25°C–80°C) with optima of 6.0 and the apparent optimal temperature of 73°C respectively. Under optimal conditions, G2ALT
exhibited a low ATPase activity with K
m
− and V
max
− values of 10±0.55 μM and 26.81±0.13 mg Pi released/min/mg enzyme, respectively. The ATPase activity of G2ALT requires Mg2+ and Na+ ions, while Zn2+ and Al3+ stimulate the activity. Cd2+ and Ag+ reduced the activity and Li+, Cu2+, and Co2+ inhibited the activity. Known inhibitors of most ATPases, like such as β-mercaptoethanol and ouabain, also inhibited the
activity of the G2ALT. These biochemical characterizations suggested that G2ALT belongs to the PP-loop ATPase superfamily
and it can be responsible for aluminium tolerance in A. gonensis G2. 相似文献
15.
Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic
voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance
Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type
monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the
electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated
into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only
for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms
of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)
diammonium salt (ABTS2−), K
Mapp, and the maximal current, I
max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal
current density and the efficiency of the catalysis, I
max/K
Mapp, were found to be 73 μA cm−2 and 208.2 μA cm−2 mM−1, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS2− in the cubic-phase environment. Rate constants were calculated to be 7.5 × 104 and 3.6 × 104 M−1 s−1 for native and hydrophobic laccase, respectively. 相似文献
16.
Irena Romanowska Ewa Kwapisz Magdalena Mitka Stanisław Bielecki 《Journal of industrial microbiology & biotechnology》2010,37(6):625-629
Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors.
The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration,
and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three
subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K
m values for NO3
− (110 μM) and for ClO3
− (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor
to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from
Gordonia species. 相似文献
17.
D. Licht S. S. Johansen E. Arvin B. K. Ahring 《Applied microbiology and biotechnology》1997,47(2):167-172
Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the
2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The
kinetic parameters for indole were an apparent maximum specific transformation rate (V
Amax) of 263 μmol mg total protein−1 day−1 and an apparent half-saturation constant (K
Am) of 139 μM. The V
Amax for quinoline was 170 μmol mg total protein−1 day−1 and K
Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation
period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline.
Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol
inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol
had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted
cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline.
Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996 相似文献
18.
Debabrata Sircar Chiranjit Mukherjee Till Beuerle Ludger Beerhues Adinpunya Mitra 《Acta Physiologiae Plantarum》2011,33(5):2019-2024
Hairy root cultures of Daucus carota respond to methyl-jasmonate treatment with enhanced accumulation of p-hydroxybenzoic acid. The final C1-side chain of this compound is shaped by p-hydroxybenzaldehyde dehydrogenase (HBD) that catalyzes the formation of p-hydroxybenzoic acid from p-hydroxybenzaldehyde in the presence of NAD+. HBD was biochemically characterized from cell-free hairy root extracts of D. carota. The preferred substrate for HBD was p-hydroxybenzaldehyde. The apparent K
m values were 54.8 and 74.4 μM for p-hydroxybenzaldehyde and NAD+, respectively. Divalent metal cations did not significantly affect enzyme activity. 相似文献
19.
Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their
substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO-
and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent K
m and V
max values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and
38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol
(C20 substrate), which functionally resembles the natural substrate lycopene. 相似文献
20.
Hemant Lata Suman Chandra Ikhlas Khan Mahmoud A. ElSohly 《In vitro cellular & developmental biology. Plant》2009,45(1):12-19
Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants
of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants
were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into
a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength
MS medium supplemented with 500 mg l−1 activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized
in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different
light levels (0, 500, 1,000, 1,500, or 2,000 μmol m−2 s−1). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m−2 s−1 and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced
at lower light intensities below 500 μmol m−2 s−1. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m−2 s−1) tested. Intercellular CO2 concentration (C
i) and the ratio of intercellular CO2 concentration to ambient CO2 (C
i/C
a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study. 相似文献