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1.
Chimeric α-amylase, produced by recombinant yeast cells, was purified by immunoaffinity chromatography by use of an anti-peptide
antibody and an eluent containing an antigen peptide. Chimeric α-amylase was adsorbed by the antibody against the peptide
corresponding to the C-terminal region of target α-amylase, and specifically eluted by the eluent containing the antigen peptide
used for immunization. A low concentration of the peptide could competitively elute adsorbed α-amylase, and the rate-limiting
step of the elution was mass transfer of desorbed α-amylase. With this specific method, target proteins can be effectively
eluted, and highly purified under mild conditions, from the antibody ligand showing a high-affinity for the adsorption step.
Received: 14 November 1996 / Received revising: 16 December 1996 / Accepted: 17 January 1997 相似文献
2.
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
3.
The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation,
acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da
by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein,
bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature
optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation
after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain
dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin
(4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400
μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5%
v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma)
produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.
Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997 相似文献
4.
Glycine-rich peptide toxin of cyanobacterium Scytonema MKU 106 was purified. UV spectral analysis showed an absorption maximum at 228 nm and the molecular mass was less than 12
kDa. The mortality rate of American boll worms (Helicoverpa armigera) was about 80% and 40% 84 h after treatment with 0.001% crude and purified peptide toxins respectively; 100% mortality was
observed after 108 h treatment with both purified and crude peptide toxins. The LC50 (lethal concentration to 50% of the population) for Heliothis larvae after 96 h was 8.3 μg/ml purified peptide toxin and 6.2 μg/ml crude peptide toxin. Observations also show that the
peptide toxin at 0.01% concentration acts as a biopesticide and at high (0.1%) concentrations it will act as an anti-feeding
compound for Stylepta derogata (leaf-roller) larvae of the cotton crop.
Received: 22 May 1996 / Accepted: 8 July 1996 相似文献
5.
Kiley Prilliman Mark Lindsey Y. Zuo Ken W. Jackson Ying Zhang W. Hildebrand 《Immunogenetics》1997,45(6):379-385
A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8+ T-cell response. Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide
that stimulates one individual’s CD8+ cytotoxic T lymphocytes will be presented by a second individual’s different class I molecules. Therefore, to facilitate
precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry
to assign precise peptide binding signatures to individual class I HLA molecules. In applying this strategy to HLA-B*1501, we isolated milligram quantities of B*1501-bound peptides and mapped them using mass spectrometry. Repeated analyses consistently assign the same peptide binding
signature to B*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of
their peptide signatures.
Received: 3 October 1996 / Revised: 20 November 1996 相似文献
6.
Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization 总被引:1,自引:0,他引:1
M. Sojo R. Bru D. Lopez-Molina F. Garcia-Carmona J.-C. Argüelles 《Applied microbiology and biotechnology》1997,47(5):583-589
Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol
oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum
within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant
levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time
of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis.
Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was
quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western
blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea
that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular
mass of 55 kDa.
Received: 26 September 1996 / Received revision: 30 December 1996 / Accepted: 4 January 1997 相似文献
7.
H. H. J. Cox F. J. Magielsen H. J. Doddema W. Harder 《Applied microbiology and biotechnology》1996,45(6):851-856
The performance at low water availability of styrene-degrading biofilters with the fungus Exophiala jeanselmei growing on perlite, the inert support, was investigated. E. jeanselmei degrades styrene at a water activity of 0.91–1. In biofilters, the styrene elimination capacity at a water activity of 0.91
is 5% of the maximal elimination capacity of 79 g m-3 h-1 (water activity 1). Application of dry air results in a rapid loss of styrene degradation activity, even at 40%–60% (w/w)
water in the filter bed and at a water activity of 1. Humidification of the gas and an additional supply of water to the filter
bed are necessary to maintain a high and stable styrene elimination capacity.
Received: 7 August 1995 / Received revision: 29 January 1996 / Accepted: 5 February 1996 相似文献
8.
V. V. Fokina A. V. Karpov I. A. Sidorov V. A. Andrjushina A. Y. Arinbasarova 《Applied microbiology and biotechnology》1997,47(6):645-649
β-Cyclodextrin changes the kinetic peculiarities of microbial 1-en-dehydrogenation of 6α-methylhydrocortisone: the reaction
rate, degree of conversion and respiratory-chain activity are increased. Respiratory-chain activity in the presence of β-cyclodextrin
is increased both in the presence and in the absence of 6α-methylhydrocortisone. A mathematical model is proposed to describe
the kinetics of the process. This model suggests the formation of different multi-component complexes consisting of inclusion
complexes and the functional unit involving the enzyme and the respiratory chain. All the parameters of the model were estimated
by data fitting. In the model framework the formation of multi-component complexes leads to an increase of the maximal reaction
rate and to a decrease of substrate inhibition. An approach is suggested for optimisation of 6α-methylhydrocortisone 1-en-dehydrogenation
in the presence of β-cyclodextrin. The optimal concentrations of 6α-methylhydrocortisone and β-cyclodextrin have been calculated.
Received: 26 September 1996 / Received revision: 16 January 1997 / Accepted: 17 January 1997 相似文献
9.
The biodegradation of an oily sludge is facilitated by a microbial tensio-active agent isolated from Pseudomonas aeruginosa USB-CS1. The optimal oil-in-water dispersion conditions are as follows: pH 6.5, temperature 30 °C, agitation 150 rev/min.
The total hydrocarbon content shows that the biodegradation of the oily substrate mediated by the biosurfactant or by the
biosurfactant–P. aeruginosa USB-CS1 complex is significantly higher after 30 days of incubation than that in other experimental conditions, by a mean
of 70%. Substrate fractionation by column chromatography reveals that, if biosurfactant is present, saturated and aromatic
compounds are more susceptible to microbial degradation than they are in other biodegradation systems by an average of 55%
and 40% respectively. These results suggest that the stimulatory effects of the biosurfactant on the biodegradation of the
oily substrate are limited over time by the loss of surface activity of the biosurfactant after 30 days of incubation.
Received : 7 August 1996 / Received revision : 6 December 1996 / Accepted : 4 January 1997 相似文献
10.
The mean maximum dive depth from 49 foraging bouts by northern rockhopper penguins, measured using capillary-tube depth gauges,
was 66±4 m (12–168 m). There were no differences in the maximum dive depths between male and female penguins. Northern rockhopper
penguins dived deeper in early than in late creche stages (83±7 vs 57±4 m), and this was associated with probable dietary
changes, squid dominating the diet by mass (44%) in November, and fish (64%) in December 1994 at Amsterdam Island.
Received: 10 January 1996/Accepted: 31 March 1996 相似文献
11.
N. Sriubolmas W. Panbangred S. Sriurairatana V. Meevootisom 《Applied microbiology and biotechnology》1997,47(4):373-378
Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG
concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations
(up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results
from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and
inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme
(i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation
of proenzyme (i.e., precursor polypeptide lacking a signal peptide).
Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996 相似文献
12.
C. Suresh A. K. Dubey S. Srikanta S. Umesh Kumar N. G. Karanth 《Applied microbiology and biotechnology》1999,51(5):673-675
A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases
of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then
saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and
maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries.
Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999 相似文献
13.
The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45°C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration
of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined.
Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses
concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the
molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production
rate was determined using the Nelder and Mead (Computer J 7:308–313, 1965) simplex optimisation method. The optimum concentrations
of the supplements were 0.576 g l-1 magnesium sulphate, 0.288 g l-1 potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect
the ethanol production rate.
Received: 29 January 1996/Received revision: 23 April 1996/Accepted: 29 April 1996 相似文献
14.
K. M. J. Van Laere G. Beldman A. G. J. Voragen 《Applied microbiology and biotechnology》1997,47(3):231-235
An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum
activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues
established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl
residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed
no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides.
Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996 相似文献
15.
A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene
(5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0.
Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein,
collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity
in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa
protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation
product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer
selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range
of potential natural protein substrates.
Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996 相似文献
16.
An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt.
approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM
o-phenanthroline; the activity was restored upon addition of 0.05 mM Co2+, Zn2+, or Ni2+. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides
and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM–1 s–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude
lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P1 and/or P1′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme
increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified
as an Arg(Lys)-preferring metalloaminopeptidase.
Received: 18 January 1996 / Accepted: 19 March 1996 相似文献
17.
The purpose of this commentary is to stress that in mixed-valence systems, electron localization and electron delocalization
are competitive, the first being most often the stronger. In iron-sulfur clusters where a delocalized Fe(II)Fe(III) pair of
maximal spin exists, a minimum value of 1200 cm–1 is proposed for the resonance integral β parametrizing the electron delocalization.
Received: 12 January 1996 / Accepted: 23 January 1996 相似文献
18.
M. Tschierske H. Drechsel G. Jung H. Zähner 《Applied microbiology and biotechnology》1996,45(5):664-670
The Zygomycete Cunninghamella elegans produces the polycarboxylate siderophore rhizoferrin. Production depends mainly on iron concentration in the medium. With
an optimized production medium the yield of rhizoferrin in a bioreactor could be increased to more than 4 g/l. Supplementation
of the basic production medium with different precursors led to the formation of nine new rhizoferrins. Both the diaminobutane
backbone and the citric acid side-chains of rhizoferrin could be substituted by appropriate analogues. These substitutions
led to new siderophores either with a variable length of diamine bridge or with fewer or different functional groups. The
proportion of the new diamine analogues relative to the total rhizoferrin could be markedly increased by the use of α-difluoromethylornithine,
an inhibitor of the ornithine decarboxylase.
Received: 11 September 1995/Received revision: 15 January 1996/Accepted: 22 January 1996 相似文献
19.
Hiroya Kobayashi Taku Kokubo Yusuke Abe Keisuke Sato Shoji Kimura Naoyuki Miyokawa Makoto Katagiri 《Immunogenetics》1996,44(5):366-371
The peptide motif of the HLA-DR53 (DRB4*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by
peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide
is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1*0901/DRB4*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated
peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of
biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides,
indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered
to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue
(I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position
10. This predicted motif is different from the binding motifs of other HLA-DR molecules.
Received: 29 April 1996 / Revised: 16 June 1996 相似文献
20.
H. Okamoto T. Fujiwara E. Nakamura T. Katoh H. Iwamoto H. Tsuzuki 《Applied microbiology and biotechnology》1997,48(1):27-33
Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23 894 Da,
completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease [J Bacteriol (1990) 172:1019–1023
and 1024–1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease
and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider
BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a
highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore,
Ca2+ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very
stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein
by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool
for selective cleavage.
Received: 24 December 1996 / Received revision: 3 February 1997 / Accepted: 22 February 1997 相似文献