共查询到20条相似文献,搜索用时 109 毫秒
1.
B. Repič Lampret Jurka Kidrič Bogdan Kralj Ljubinka Vitale Miroslav Pokorny Metka Renko 《Archives of microbiology》1999,171(6):397-404
Lapstatin, a low-molecular-weight aminopeptidase inhibitor, was purified to homogeneity from Streptomyces rimosus culture filtrates. The purification procedure included extraction with methanol, followed by chromatography on Dowex 50WX4,
AG50WX4, and HPLC RP C18 columns. By amino acid analysis, mass spectrometry, and NMR spectroscopy, the structure of lapstatin was shown to be 3-amino-2-hydroxy-4-methylpentanoylvaline.
Lapstatin inhibited the extracellular leucine aminopeptidases from Streptomyces rimosus, Streptomyces griseus, and Aeromonas proteolytica with an IC50 in the range of 0.3–2.4 μM. IC50 values for other enzymes tested were at least tenfold higher. Leucine aminopeptidase from Streptomyces griseus was inhibited in a competitive manner, with an inhibition constant of 5 × 10–7 M. Lapstatin is the first low-molecular-weight compound isolated from streptomycetes shown to inhibit an autogenous aminopeptidase.
Received: 7 December 1998 / Accepted: 29 March 1999 相似文献
2.
Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme 总被引:9,自引:0,他引:9
C. G. van Ginkel G. B. Rikken A. G. M. Kroon S. W. M. Kengen 《Archives of microbiology》1996,166(5):321-326
A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative
bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose
chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min–1 (mg protein)–1. The dismutase obeyed Michaelis-Menten kinetics. The V
max and K
m calculated for chlorite were 2,200 U (mg protein)–1 and 170 μM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole.
Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had
a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase
displayed maxima for activity at pH 6.0 and 30° C.
Received: 9 April 1996 / Accepted: 12 August 1996 相似文献
3.
Macedo ML Diz Filho EB Freire MG Oliva ML Sumikawa JT Toyama MH Marangoni S 《The protein journal》2011,30(1):9-19
The present paper describes the purification, characterization and determination of the partial primary structure of the first
trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity.
SDS–PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15
and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is
a competitive inhibitor with an equilibrium dissociation constant of 10−9 M for trypsin. The partial NH2- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different
sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity
against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis. 相似文献
4.
Two extracellular isoenzymes of polygalacturonase, isolated from the brown-rot fungus Postia placenta, were purified 342-fold by Mono S cation-exchange chromatography. The temperature optimum ranged from 25 °C to 37 °C, and the pH optimum ranged from 3.2 to 3.9. Apparent pI values of the isoenzymes (3.2 and 3.4) were lower than any previously reported. The estimated molecular mass from a single
band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (PAGE) was 34 kDa. Isoenzymes of polygalacturonase in native
PAGE and isoelectric focusing gels were identified by substrate/ agar overlays (zymograms). Comparison of viscosity reduction
rates with release of reducing sugars indicated that the enzyme from P. placenta is endo-acting. The objective of this study was to isolate polygalacturonase from the brown-rot fungus P. placenta and characterize the properties of the enzyme.
Received: 31 October 1995/Received revision: 12 February 1996/Accepted: 4 March 1996 相似文献
5.
H. Sztajer W. Wang H. Lünsdorf A. Stocker R. D. Schmid 《Applied microbiology and biotechnology》1996,45(5):600-606
Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified
lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission
electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic
studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and
did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at
45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability.
Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996 相似文献
6.
Purification and characterization of tubulin from ginkgo pollen 总被引:2,自引:0,他引:2
Tubulin was purified by a combination of acetone powder preparation, DEAE Sephadex A-50 chromatography, Sephacryl S-300 gel
filtration, and Mono Q anion exchange chromatography from the pollen of ginkgo (Ginkgo biloba L.), a typical gymnosperm. The average yield of tubulin is 2 mg per 100 g of pollen grain. The purified tubulin is electrophoretically
homogeneous. It seems to be composed of two subunits on SDS-PAGE and is resolved as two major spots on two-dimensional electrophoresis,
preliminarily indicating that there are no obvious tubulin isotypes in ginkgo pollen. The apparent molecular weights of the
two subunits are about 54 kDa and 52 kDa respectively, estimated from the SDS-PAGE. It was also demonstrated that tubulin
from ginkgo pollen is immunochemically related to animal brain tubulin, and the purified tubulin was polymerized to microtubular
aggregates in the presence of taxol and GTP in vitro.
Received: 13 April 1996 / Revision accepted: 24 March 1997 相似文献
7.
Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing
up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme
has a molecular mass of 108000 (gel filtration) or 112000 (native electrophoresis), and consists of four identical subunits
with a molecular mass of 27 000 (SDS-electrophoresis).
The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates
to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity
were not cleaved. Therefore, the described enzyme was designated “insulin-cleaving proteinase” (ICP). 相似文献
8.
Hollow-fibre modules containing microporous membrane material were evaluated as bioreactors for waste gas treatment. The
reactors were inoculated with the propene-utilizing strain Xanthobacter Py2, which formed a biofilm on the inner side of the fibres. The removal of the poorly soluble volatile propene from synthetic
waste gas was monitored for up to 170 days. The maximum removal rates were 70–110 g propene per m3 reactor per hour. A gas residence time of 80 s was required to remove 95% of an initial propene concentration of 0.84 g/m3. The presence of ammonium in the liquid medium resulted in the development of an additional population of nitrifying organisms.
Therefore, nitrate was used as the source of nitrogen in later experiments. During long-term operation, the propene removal
rates gradually decreased. At low liquid velocities (1–5 cm/s) clogging of individual fibres with excess biomass was observed.
Elevation of the liquid velocity in the fibres to 90 cm/s resulted in the formation of a dense biofilm and prevented clogging
of the fibres. However, also at this high liquid velocity a gradual decrease in propene removal rate was observed. These results
suggest that aging of biofilms is a very important factor in long-term operation of hollow-fibre bioreactors.
Received: 24 November 1995 / Received revision: 14 February 1996 / Accepted: 20 February 1996 相似文献
9.
A. M. Moilanen T. Lundell T. Vares A. Hatakka 《Applied microbiology and biotechnology》1996,45(6):792-799
The effects of high manganese [180 μM Mn(II)] concentration and addition of malonate (10 mM) were studied in nitrogen-limited
cultures of the white-rot fungus, Phlebia radiata. High levels of manganese alone showed no systematic influence on the production of lignin peroxidase (LiP), manganese peroxidase
(MnP) or laccase. In contrast, high-manganese containing cultures of P. radiata showed lower efficiency in the mineralization of 14C-ring-labelled synthetic lignin ([14C]DHP). The highest rates of mineralization, up to 30% in 18 days, were reached in low- manganese(2 μM)-containing cultures
when malonate was omitted. Degradation of [14C]DHP was substantially restricted by the addition of malonate. The combination of high manganese and malonate resulted in
increased levels of MnP and laccase production, whereas LiP production was repressed. Also, the profiles of expression of
the MnP and LiP isozymes were affected. A new P. radiata MnP isozyme of pI 3.6 (MnP3) was found in the high-manganese cultures. Addition of malonate alone caused some repression but also stimulating
effects on distinctive MnP and LiP isozymes. The results indicate that manganese and malonate are individual regulators of
MnP and LiP expression and have different roles in the degradation of lignin by P. radiata.
Received: 30 August 1995/Received revision: 10 January 1996/Accepted: 12 February 1996 相似文献
10.
The synthesis of poly(3-hydroxyalkanoates) (PHA) by Pseudomonas putida KT2442 growing on long-chain fatty acids was studied in continuous cultures. The effects of the growth rate on the biomass
and polymer concentration were determined and it was found that the PHA concentrations decreased with increasing growth rates.
The highest volumetric productivity was 0.13 g PHA l-1 h-1 at a specific growth rate (μ) of 0.1 h-1. The molecular mass of the polymer remained constant at all growth rates but changes in the monomeric composition of the
PHA synthesized were observed. Variation of the carbon to nitrogen (C/N) ratio of the substrate feed at μ=0.1 h-1 revealed optimal PHA formation at C/N=20 mol/mol. In order to optimize PHA production P. putida KT2442 was cultivated to high cell densities in oxygen-limited continuous cultures. In this way a maximum biomass concentration
of 30 g/l containing approximately 23% PHA was achieved. This corresponds to a volumetric productivity of 0.69 g l-1 h-1.
Received: 14 December 1995 / Received revision: 18 April 1996 / Accepted: 22 April 1996 相似文献
11.
Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater 总被引:6,自引:0,他引:6
Denitrification of a high-strength synthetic wastewater (150 g NO-
3 l-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand
l-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other
was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of
9.54 g NO-
3 l-1 day-1 (2.19 g N as NO-
3 l-1 day-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO
x
g volatile suspended solids h-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium
did not occur, even in the one-step process.
Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996 相似文献
12.
T. Kobayashi Y. Hakamada J. Hitomi K. Koike S. Ito 《Applied microbiology and biotechnology》1996,45(1-2):63-71
Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease,
designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two
minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease
had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein
was observed at pH 11.0 and at 55°C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE,
although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and
at 60°C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease.
The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values
and at 5°C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease
at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.
Received: 12 December 1994/Received last revision: 9 June 1995/Accepted: 31 July 1995 相似文献
13.
A. Ragout F. Siñeriz R. Kaul D. Guoqiang B. Mattiasson 《Applied microbiology and biotechnology》1996,46(2):126-131
Streptococcus salivarius subsp. thermophilus was cultivated in a chemostat in order to obtain an adhesive phenotype of this strain. When the system was operated at low
dilution rates (D<0.2 h-1) for about 4 weeks, the strain formed a visible film on the surface of the culture vessel. The biofilm cells were not washed
out even when dilution rates were increased (D=6.9 h-1), and this resulted in a high biomass productivity (P=4.1 g l-1h-1). On the other hand, when the culture was grown at dilution rates faster than 0.2 h-1, only the free suspended cells were present in the culture broth, and were washed out at velocities of about 1.0 h-1. The biomass productivity was consequently lower (P=1.33 g l-1h-1) than in the previous case. The selected adhesive phenotype was grown on different glass beads and the possibility of lactate
fermentation in a continuous and semicontinuous mode was demonstrated.
Received: 16 August 1995/Received revision: 18 March 1996/Accepted: 25 March 1996 相似文献
14.
Degradation of pyrene by Mycobacterium flavescens 总被引:1,自引:0,他引:1
A strain of Mycobacterium flavescens was isolated from polluted sediments. It was capable of utilizing pyrene as a sole source of carbon and energy. When pyrene
was supplied as a suspension at 50 μg/ml, the generation time was 9.6 h and the rate of pyrene utilization was 0.56 μg ml-1 day-1. In addition to pyrene, the strain could mineralize phenanthrene (17.7%) and fluoranthene (17.9%), but failed to mineralize
naphthalene, chrysene, anthracene, fluorene, acenaphthene and benzo[a]pyrene, as determined by recovery of radiolabeled CO2 in incubations conducted for 2 weeks under growth conditions. Metabolites produced during growth on pyrene were detected
and characterized by HPLC and GC-MS. The product of initial ring oxidation, 4,5-dihydroxy-4,5-dihydropyrene was identified,
as well as ring-fission products including 4-phenanthroic acid, phthalic acid, and 4,5-phenanthrenedioic acid.
Received: 3 October 1995/Received last revision: 1 April 1996/Accepted: 15 April 1996 相似文献
15.
K. Katoh M. Ohbo M. Wakui 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1996,166(6):369-374
In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty
acids, changes in intracellular calcium concentration ([Ca2+]i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine
raised [Ca2+]i in acinar cells in a concentration-dependent manner. The rise in [Ca2+]i in response to the stimulation with octanoate (10 mmol ⋅ l-1) was reduced in a medium without CaCl2, but was markedly enhanced by reintroduction of CaCl2 into the medium up to 2.56 mmol ⋅ l-1. Perfusion of the cells with a medium containing octanoate (5 mmol ⋅ l-1) or acetylcholine (0.5 μmol ⋅ l-1) immediately raised inward current across the cell membrane at a holding-membrane potential of −30 mV. The inward current
became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate
and acetylcholine, respectively, being consistent with that for Cl-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes
in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other
cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses
immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised
amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin
inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol ⋅ l-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate
and acetylcholine (5.5 μmol ⋅ l-1). We conclude that fatty acids and acetylcholine increase [Ca2+]i, which consequently evokes a rise in transmembrane ion (Cl-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes
activated by stimulation with fatty acids in ovine pancreatic acinar cells.
Accepted: 14 May 1996 相似文献
16.
Overwintering populations of Mesodinium rubrum (Ciliophora: Haptorida) in lakes of the Vestfold Hills, East Antarctica 总被引:1,自引:0,他引:1
J. A. E. Gibson Kerrie M. Swadling Tracey M. Pitman Harry R. Burton 《Polar Biology》1997,17(2):175-179
The autotrophic ciliate Mesodinium rubrum Lohmann was observed during winter and spring in saline lakes ranging in salinity from 2 to 78‰ in the Vestfold Hills, Antarctica.
The ciliate remained active during winter, and contained chlorophyll even though the level of light available for photosynthesis
was minimal. No evidence of encystment as a means of survival during winter was observed. A seasonal study in one of the lakes,
Ace Lake, revealed that M. rubrum was present throughout the year at abundances ranging from 1×104 to 3.5×105 cells l-1. During the winter period, when little light penetrated the lake’s ice cover, cells were most common immediately under the
ice at 2 m, where cell numbers were typically 8×104 cells l-1.
Received: 3 January 1996/Accepted: 21 April 1996 相似文献
17.
J. V. Pothuluri F. E. Evans T. M. Heinze C. E. Cerniglia 《Applied microbiology and biotechnology》1996,45(5):677-683
Benzo[e]pyrene is a pentacyclic aromatic hydrocarbon, which, unlike its structural isomer benzo[a]pyrene, is not a potent carcinogen or mutagen. The metabolism of benzo[e]pyrene was studied using the filamentous fungus Cunninghamella elegans ATCC 36112. C. elegans metabolized 65% of the [9, 10, 11, 12-3H]benzo[e]pyrene and unlabeled benzo[e]pyrene added to Sabouraud dextrose broth cultures after 120 h of incubation. Three major metabolites of benzo[e]pyrene were separated by reversed-phase high-performance liquid chromatography. These metabolites were identified by 1H and 13C NMR, UV-visible, and mass spectral analyses as 3-benzo[e]pyrenylsulfate, 10-hydroxy-3-benzo[e]pyrenyl sulfate, and benzo[e]pyrene 3-O-β-glucopyranoside.
Received: 7 September 1995/Received revision: 14 November 1995/Accepted: 11 December 1995 相似文献
18.
19.
Previously it was demonstrated that bacteria are capable of transforming soluble uranyl ion, U(VI), to insoluble uraninite,
U(IV); however, the rate for this transformation has not been determined. We report the kinetic coefficients for Desulfovibrio desulfuricans DSM 1924 grown in a continuous-flow chemostat where pyruvate was the electron donor and sulfate was the electron acceptor.
The medium was supplemented with 1 mM uranyl nitrate, and the chemostat flow rate ranged from 1.12 ml/h to 4.75 ml/h with
incubation at 28°C. The maximum rate of pyruvate utilization (k) was determined to be 4.7 days-1, while the half-velocity constant (K
s) was 127 mg/l. The yield coefficient (Y) of cells per mole of pyruvate oxidized was calculated to be 0.021 g, while the endogenous decay coefficient (k
d) was determined to be 0.072 days-1. More than 90% of U(VI) was transformed to U(VI) in the chemostat under the conditions employed.
Received: 7 September 1995/Received last revision: 10 January 1996/Accepted: 5 February 1996 相似文献
20.
Chandan Shee Ashwani K. Sharma 《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):115-120
A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10? 9 M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata. 相似文献