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1.
In Arthrobacter oxydans, Klebsiella aerogenes and Sporosarcina ureae, growth with urea as a nitrogen source turned out to be more sensitive to inhibition by EDTA than that with ammonia. The inhibition was overcome by added nickel chloride, but not by other divalent metal ions tested. In A. oxydans the uptake of 63Ni was paralleled by an increase in urease (urea amidohydrolase, EC 3.5.1.5) activity under certain conditions. Following growth with radioactive nickel, urease from this strain was enriched by heat treatment and acetone fractionation. Copurification of 63Ni and urease was observed during subsequent Sephadex gel chromatography. Almost the entire labelling was detected together with the purified enzyme after focusing on polyacrylamide gel. The relative molecular mass of the purified urease was estimated to be 242,000. The pH optimum was 7.6, the K
m-value 12.5 mmol/l and the temperature optimum 40°C; heat stability was observed up to 65°C. In presence of 10 mmol/l EDTA the protein-nickel binding remained intact at pH 7; at pH 5 and below, nickel was irreversibly removed with concommitant loss of enzyme activity. The results demonstrated that nickel ions are required for active urease formation in the bacterial strains studied, and that urease from A. oxydans is a nickel-containing enzyme.Dedicated to Professor Dr. H.-G. Schlegel on the occasion of his 60th birthday 相似文献
2.
NAD-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.2) was purified to homogeneity from Sporosarcina ureae DSM 320; the native enzyme (M
r 250,000±25,000) is composed of subunits identical in molecular mass (M
r 42,000±3,000), suggesting a hexameric structure. In cell-free extracts and in its purified form, the enzyme was heat-stable, retaining 50% activity after 15 min incubation at temperatures up to 82°C. When exposed to low temperatures at pH values between 7.0 and 9.0. cell-free extracts and purified preparations lost enzyme activity rapidly and irreversibly. The addition of substrates, glycerol, or sodium chloride improved the stability of the enzyme with respect to cold lability and heat stability.Abbreviation NAD-GluDH
nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase 相似文献
3.
A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K
m and V
max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10−3 mM min−1, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM
L-Gln, 40 mM ethylamine, and 1.04 U His6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained. 相似文献
4.
Purification and partial characterization οf a new β-xylosidase from
Humicola grisea var. thermoidea
T. Iembo M.O. Azevedo C. Bloch Jr. E.X.F. Filho 《World journal of microbiology & biotechnology》2006,22(5):475-479
Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan
as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography.
Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified
enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h
of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol.
The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K
m and V
max values of 1.37 mM and 12.98 IU ml−1, respectively. 相似文献
5.
An N-acetylglucosaminidase produced by Streptomyces cerradoensis was partially purified giving, by SDS-PAGE analysis, two main protein bands with Mr of 58.9 and 56.4 kDa. The Km and Vmax values for the enzyme using p-nitrophenyl-β-N-acetylglucosaminide as substrate were of 0.13 mM and 1.95 U mg−1 protein, respectively. The enzyme was optimally activity at pH 5.5 and at 50 °C when assayed over 10 min. Enzyme activity
was strongly inhibited by Cu2+ and Hg2+ at 10 mM, and was specific to substrates containing acetamide groups such as p-nitrophenyl-β-N-acetylglucosaminide and p-nitrophenyl-β-D-N,N′-diacetylchitobiose. 相似文献
6.
Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420000; it consisted of subunits with molecular weights of 72400 (), 20400 (), and 13900 () in an estimated ()4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420000 to 100000. In the native enzyme, 4.09 (±0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.Abbreviation PAGE
polyacrylamide gel electrophoresis 相似文献
7.
The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography.
It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k
cat of 1.65 min−1 and a K
m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol−1). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C30 seems to be essential for enzyme activity. 相似文献
8.
For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular
mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme
was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a Km of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside. 相似文献
9.
Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu2+. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M
r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M
r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development. 相似文献
10.
λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization 总被引:1,自引:0,他引:1
λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension
cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular
mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg2+. With 0.2mM Cd2+ or Zn2+, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity
and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be
effective inhibitors of the plant enzyme too. The apparent Km values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione
(Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration
of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione
itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm.
Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48%
of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione
synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the
plant cell.
Dedicated to Professor A. Prison on the occasion of his 80th birthday 相似文献
11.
Z.T. Xing J.H. Cheng Q. Tan Y.J. Pan 《World journal of microbiology & biotechnology》2006,22(8):799-806
Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼
∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu2+. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M
r ∼
∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M
r␣∼
∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development. 相似文献
12.
Kamila Rzeznicka Sebastian Schätzle Dominique Böttcher Joachim Klein Uwe T. Bornscheuer 《Applied microbiology and biotechnology》2010,85(5):1417-1425
The nitrile hydratase (NHase, EC 4.2.1.84) genes (α and β subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (α subunit, Mr 23 kDa) and 218 amino acids (β subunit, Mr 24 kDa) and the NHase activator of 413 amino acids (Mr 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly
in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator
gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration
and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic
ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity
was observed for aromatic compounds only with E values ranging 5–17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30°C and showed the highest
stability at 4°C in thermal inactivation studies between 4°C and 50°C. 相似文献
13.
The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified
to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal
A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed.
The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C.
The kinetic parameters, K
m and V
max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min−1 ml−1. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase
activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the
capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of
40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of
glucose to cellotriose. 相似文献
14.
Marta E. Farías Ana M. Strasser de Saad Aída A. Pesce de Ruiz Holgado Dr. Guillermo Oliver 《Current microbiology》1991,22(4):205-211
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM
l-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent Km value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit. 相似文献
15.
A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg−1. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme
was unstable below pH 6.0: at pH 6 its half-life at 75°C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased
as p-nitrophenyl(pNP)-β-d-fucopyranoside (1067) > pNP-β-d-glucopyranoside (381) > pNP-β-d-galactopyranoside (18) > pNP-β-d-mannopyranoside (11 s−1 mM−1), indicating that the enzyme was a β-glycosidase. 相似文献
16.
γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195 000±10 000 in its dimeric form with an Mr of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme. 相似文献
17.
S. Singh 《Folia microbiologica》1995,40(5):529-533
Urease extracted from an alkaliphilic diazotrophic cyanobacteriumNostoc calcicola was partially purified and some of its properties were studied. Urease purified 39-fold from the crude enzyme extract showed
its optimum activity at pH 7.5 and at 40°C with aK
m value of 120 μmol/L. The enzyme was found to be sensitive to metal cations, particularly Hg2+, Ag+ and Cu2+. 4-Hydroxymercuribenzoate (a mercapto-group inhibitor) and acetohydroxamic acid (a chelating agent of nickel) inhibited,
the enzyme activity completely. These results suggest the involvement of an SH-group and Ni2+ in the activity of urease fromN. calcicola. 相似文献
18.
Elaine O’Connell Patrick Murray Charles Piggott Franck Hennequart Maria Tuohy 《Journal of applied phycology》2008,20(5):557-565
A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative
electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity
studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate
methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (Km value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH
5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg2+, Co2+ and Fe3+. 相似文献
19.
The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C
over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited
by bivalent metal ions (Ca++, Cu++, Cd++, and Hg++), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified
32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K
m
and Vmax values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first
time in Lactobacillus, and it proved to be a β-fructofuranosidase.
Received: 13 August 1999 / Accepted: 15 September 1999 相似文献
20.
Park SY Kim JT Kang SG Woo JH Lee JH Choi HT Kim SJ 《Applied microbiology and biotechnology》2007,77(1):107-115
Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result,
an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed
low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X1–S–X2–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together
with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C4) among various p-nitrophenyl esters (C2 to C18), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K
m (307 μM), k
cat (5.72 s−1), and k
cat/K
m (18.61 s−1 mM−1). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone
hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment. 相似文献