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1.
Y. Tsuji H. Yamanaka T. Fukui T. Kawamoto A. Tanaka 《Applied microbiology and biotechnology》1997,47(2):114-119
In this paper we report on the enzymatic preparation of d-p-trimethylsilylphenylalanine (d-TMS-Phe). First, dl-5-(p-trimethylsilylphenylmethyl)hydantoin␣(dl-TMS-Phe-Hyd) was synthesized chemically and subjected to bacterial hydrolysis to obtain N-carbamoyl-d-p-trimethylsilylphenylalanine (C-d-TMS-Phe), but no strains examined showed sufficient hydantoinase activity on this compound. However, Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-d-amino acid amidohydrolase (DCase), was found to be able to hydrolyze C-dl-TMS-Phe prepared chemically from the hydantoin. When C-dl-TMS-Phe was hydrolyzed with cells of Blastobacter sp. A17p-4, its optical purity was low because N-carbamoyl-l-amino acid amidohydrolase (LCase) coexisted in the cells. DCase and LCase in the cell-free extract of Blastobacter sp. A17p-4 could be separated by DEAE-Sephacel column chromatography. The optimum pH for the hydrolysis of C-dl-TMS-Phe by the partially purified DCase was 8.0 and addition of 2.5 % N,N-dimethylformamide was effective in raising the substrate concentration without inactivation of DCase. Under the optimized
conditions, highly optically pure (98 % enantiomeric excess) d-TMS-Phe could be obtained from C-dl-TMS-Phe with partially purified DCase.
Received: 12 July 1996 / Received revision: 11 September 1996 / Accepted: 2 November 1996 相似文献
2.
3.
Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific
isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing
was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the
three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal
inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized
proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation
with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori.
Received: 21 February 2002 / Accepted: 28 March 2002 相似文献
4.
α-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml−1; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h. 相似文献
5.
Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60
Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M
r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K
m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co2+, Mn2+ and Fe2+ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH2-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%. 相似文献
6.
Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system 总被引:1,自引:0,他引:1
T. Mustafa C. Agnisola J. K. Hansen 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1997,167(2):98-104
The effects of l-arginine, and its analogues N
ω-nitro-l-arginine methyl ester and N
ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation.
l-Arginine, at 10–8 mol · l–1induced a slight vasodilatory effect (max 10%). N
ω-nitro-l-arginine methyl ester and N
ω-Nitro-l-arginine in the range 10–8–10–4 mol · l–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10–4 mol · l–1
N
ω-nitro-l-arginine or N
ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced
a strong vasoconstriction (already significant at 10–5 mol · l–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and
serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine
induced a biphasic response with vasodilation at low concentration (max 15% at 10–8 mol · l–1). Serotonin displayed a dose-response vasodilation in the range 10–8–10–4 mol · l–1 (max 20%). These vasodilative effects were reduced or abolished by 10–4 mol · l–1
l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system.
Accepted: 1 July 1996 相似文献
7.
N5-(l-1-Carboxyethyl)-l-ornithine: NADP+ oxidoreductase [N5-(CE)ornithine synthase] catalyzes the NADPH-dependent reductive condensation between pyruvic acid and the terminal amino group ofl-ornithine andl-lysine to yield N5-(l-1-carboxyethyl)-l-ornithine and N6-(l-1-carboxyethyl)-l-lysine respectively. Polyclonal antibodies against N5-(CE)ornithine synthase purified fromStreptococcus lactis K1 have been used for the immunochemical (Western blot) detection and sizing of this enzyme in various lactic acid bacteria. The enzyme was confined to about one-half of the strains ofS. lactis examined. N5-(CE)ornithine synthase is constitutive, and in strains K1, 6F3, and (plasmid-free)H1-4125 the native enzyme is a tetramer composed of identical subunits of Mr=38,000. However, in other strains, including 133 (ATCC 11454), C10, and ML8, the molecular weight of the native enzyme is approximately 130,000 and the corresponding subunit Mr=35,000. Analyses of the amino acid pool components maintained byS. lactis K1 during growth in medium containing [14C] labeled and unlabeled arginine have revealed that (i) exogenous arginine is the precursor of intracellular ornithine, citrulline, and N5-(CE)ornithine, and (ii) the rates of turnover of ornithine and citrulline were considerably faster than that of N5-(CE)ornithine. These data account for the biosynthesis and accumulation of N5-(CE)ornithine byS. lactis. 相似文献
8.
9.
V. Andreoni G. Baggi S. Bernasconi C. Foglieni F. Pelizzoni 《Applied microbiology and biotechnology》1990,32(6):633-636
Summary
N-(Benzyloxycarbonyl)-l-asparty-l-phenylalanine methyl ester, the precursor of the synthetic sweetener aspartame, was continuously synthesized in an immobilized thermolysin plug-flow type reactor at 25° C with the substrates (N-benzyloxycarbonyl-l-aspartic acid and l-phenylalanine methyl ester) dissolved in ethyl acetate. The immobilized enzyme was quite stable in ethyl acetate containing 2.5% 0.01 M 2-(N-morpholino)ethanesulphonic acid-NaOH buffer, pH 6.0, and 20 mM CaCl2 with or without the substrate at 25° C. By periodically washing the column, we could conduct a continuous reaction for over 500 h with an average yield of 95% and a space velocity of 1.85 h –1.Offprint requests to: K. Nakanishi 相似文献
10.
J. Hlaváček J. Pícha V. Vaněk J. Jiráček J. Slaninová V. Fučík M. Buděšínský D. Gilner R. C. Holz 《Amino acids》2010,38(4):1155-1164
A series of N
α-acyl (alkyl)- and N
α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N
α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N
α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the
compounds tested provided IC50 values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N
α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC50 value of 85 μM while N
α-trifluoroacetyl-l-ornithine (1f), N
α-ethoxycarbonyl-l-ornithine (2b), and N
α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC50 values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N
α-acetyl-d-ornithine (1a) and N
α-fluoro- (1f), N
α-chloro- (1g), N
α-dichloro- (1h), and N
α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC50 values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that
ArgE is likely the bacterial enzymatic target. 相似文献
11.
H. Kneifel Kerstin Elmendorff Eberhard Hegewald Carl J. Soeder 《Archives of microbiology》1997,167(1):32-37
Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic
acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and
by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic
acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl β-d-glucopyranoside.
Received: 27 March 1996 / Revision received: 18 October 1996 / Accepted: 30 October 1996 相似文献
12.
We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector. This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose. The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted. Sequences required for regulated expression
of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene. The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U. maydis.
Received: 16 March 1996 / Accepted: 22 August 1996 相似文献
13.
J. M. Crous I. S. Pretorius W. H. van Zyl 《Applied microbiology and biotechnology》1996,46(3):256-260
First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1
P
) and terminator (PGK1
T
) sequences on a multicopy episomal plasmid. The resulting construct PGK1
P
-abf B-PGK1
T
was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.
Received: 29 January 1996/Received revision: 3 May 1996/Accepted: 9 May 1996 相似文献
14.
Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant
to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type
or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was
much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells. 相似文献
15.
N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co2+ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co2+ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn2+ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h. 相似文献
16.
Erhard Bieberich Bettina Freischütz Shyh-Shyurng Liour Robert K. Yu 《Journal of neurochemistry》1998,71(3):972-979
Abstract: Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP-NeuAc:GM1 α2-3-sialyltransferase (ST-IV) and UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (Gal-NAc-T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108-15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST-IV activity to 45% of that of control cells with t1/2 = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST-IV due to the inhibition of a serine/threonine-specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST-IV, the activity of GalNAc-T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc-T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up-regulation of GalNAc-T in conjunction with down-regulation of ST-IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a-series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108-15 cells can be specifically regulated by both protein kinase A- and protein kinase C-related phosphorylation systems. 相似文献
17.
S. Nakamori S. Kobayashi T. Nishimura H. Takagi 《Applied microbiology and biotechnology》1999,52(2):179-185
We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately
910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate.
The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution
(wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered
that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes.
Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999 相似文献
18.
Gérard Strecker Jean-Michel Wieruszeski Claude Martel Jean Montreuil 《Glycoconjugate journal》1987,4(4):329-337
Alkaline borohydride reductive cleavage of hen ovomucin resulted in the release of a series of neutral and acidic oligosaccharide-alditols.1H-NMR spectroscopy in combination with fast ion bombardment-mass spectrometry in negative ion mode were used for investigation of the structures of three oligosaccharide-alditols. The following structures were established:
Abbreviations NeuAc
N-acetyl-d-neuraminic acid
- Gal
d-galactose
- GlcNAc
N-acetyl-d-glucosamine
- Gal-NAc-ol
N-acetyl-d-galactosaminitol
- NMR
nuclear magnetic resonance
- FAB-MS
fast atom bombardmentmass spectrometry 相似文献
19.
Primary structure of the majorO-glycosidically linked carbohydrate unit of human von Willebrand factor 总被引:5,自引:0,他引:5
Bruno Samor Jean-Claude Michalski Claudine Mazurier Maurice Goudemand Pieter De Waard Johannes F G Vliegenthart Gérard Strecker Jean Montreuil 《Glycoconjugate journal》1989,6(3):263-270
A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz1H-NMR spectroscopy to be: NeuAc(2-3)Gal(1-3)[NeuAc(2-6)]GalNAc-ol.Abbreviations ConA
concanavalin A
- LCA
Lens culinaris agglutinin
- vWF
von Willebrand factor
- NeuAc
N-acetylneuraminic acid
- Gal
d-galactose
- GalNAc-ol
N-acetyl-d-galactosaminitol
- HMW
high molecular weight
- LMW
low molecular weight 相似文献
20.
Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with
xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were
found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro.
Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996 相似文献