共查询到20条相似文献,搜索用时 46 毫秒
1.
Liu JQ Odani M Yasuoka T Dairi T Itoh N Kataoka M Shimizu S Yamada H 《Applied microbiology and biotechnology》2000,54(1):44-51
The dtaAX gene encoding a pyridoxal 5′-phosphate (pyridoxal-P)-dependent low-specificity d-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The
predicted amino acid sequence displayed 54% identity with that of d-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which
was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography
steps. The recombinant low-specificity d-threonine aldolase was shown to be an efficient biocatalyst for resolution of l-β-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.
Received: 9 September 1999 / Received revision: 1 November 1999 / Accepted: 12 November 1999 相似文献
2.
Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M
r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal
lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was
shown to be stable over a broad range of pH and temperature.
Received: 1 April 1999 / Received last revision: 11 June 1999 / Accepted: 11 June 1999 相似文献
3.
Koibuchi K Nagasaki H Yuasa A Kataoka J Kitamoto K 《Applied microbiology and biotechnology》2000,54(1):59-68
A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight
mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported
previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have
isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and
a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding
sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.
Received: 23 August 1999 / Received last revision: 7 January 2000 / Accepted: 14 January 2000 相似文献
4.
J. Geistlinger K. Weising W. J. Kaiser G. Kahl 《Molecular & general genetics : MGG》1997,256(3):298-305
The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated
amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA
fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the
Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat
units (CATTT)n, (CATTA)n, (CATATCATTT)n and (TATTT)n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus- specific microsatellite amplification
and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational
mechanisms have contributed to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic
F1 progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the
parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length
allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)53 to (TATTT)65], while the remainder of the sequence was unchanged.
Received: 11 March 1997 / Accepted: 21 June 1997 相似文献
5.
Hong KW Iwatsuki H Takenaka O Hayasaka I Murayama Y Ito S Inoue-Murayama M 《Primates; journal of primatology》2007,48(2):151-155
Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding
regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans
and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)7–9 and (CA)4–6. Gorillas had the (TA)9–10 repeat tracts and orangutans had monomorphic (TA)7 repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)2, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of
ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA)
n
, (GA)2(TA)8(CA)4(TA). In apes that region included {(TA)
n
(CA)
n
}
n
. Gibbon sequences included (TATG)
n
and (TATC)
n
and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan
lineage. In addition, a great diversity of allele length was detected in each gibbon species. 相似文献
6.
Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO3. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO3, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced
in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA
was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass
was not changed. Optimal temperatures were 24–28 °C.
Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999 相似文献
7.
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
8.
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
9.
Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources 总被引:1,自引:0,他引:1
van Zyl WH Eliasson A Hobley T Hahn-Hägerdal B 《Applied microbiology and biotechnology》1999,52(6):829-833
Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l−1
d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose
consumption rate increased a further threefold when 20 g l−1
d-glucose or raffinose was used as co-substrate together with 50 g l−1
d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after
82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose
as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and
much less acetic acid formation.
Received: 12 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999 相似文献
10.
S. Venditti M. A. Vega-Palas G. Di Stefano E. Di Mauro 《Molecular & general genetics : MGG》1999,262(2):367-377
Telomeric heterochromatin plays an essential role in telomere function, including the regulation of telomere length. We observe
that in Saccharomyces cerevisiae an imbalance in the dosage of genes for two protein components of heterochromatin (namely Sir3p and histone H4) causes modifications
in telomere length and telomere sequence organization. The effects of Sir3p/H4 imbalance were analyzed in yeast strains in
which the wild-type SIR3 gene (normally a single-copy gene) was either absent or present in 20–30 copies, and both histone H4 genes (HHF1 and HHF2) were present or HHF1 was deleted, thus covering a wide range of viable gene-dosage combinations. Modifications of telomeres and of subtelomeric
regions were identified by analyzing both the overall telomere population and by focusing on two single telomeric regions:
the left telomere of chromosome III (LIII) and the right telomere of chromosome XI (RXI). The modifications induced by alteration
of the Sir3p/H4 ratio consist of a reduction in the length and an increase in the instability of the terminal block of (C1–3A)n repeats and in susceptibility to insertion of Y′ elements into this repeat element. Restoration of the wild-type gene ratio
(by removal of the extra copies of SIR3 or by complementation with the missing second copy of HHF) restored the original telomere organization, both with respect to the length of the (C1–3A)n repeat stretch and the absence of Y′ elements. This behavior shows that the stability of the wild-type sequence organization
requires maintenance of the normal structure of telomeric heterochromatin.
Received: 23 March 1999 / Accepted: 10 June 1999 相似文献
11.
Using high-cell-density culture of Escherichia coli under the control of an l-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The
inducer, l-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected
the partition of interferon-α (IFN-α) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation
of IFN-α on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration
time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol
(N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation.
The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion
bodies in the total expressed IFN-α.
Received: 21 May 1999 / Received last revision: 16 August 1999 / Accepted: 2 September 1999 相似文献
12.
S M Kotwal M M Gote M I Khan J M Khire 《Journal of industrial microbiology & biotechnology》1999,23(1):661-667
The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg−1 protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source
and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation,
DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular
weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis.
The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K
m
S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly
inhibited by Ag+ and Hg2+. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K
i) for galactose was 11 mM.
Received 28 January 1999/ Accepted in revised form 07 April 1999 相似文献
13.
Ye K Shibasaki S Ueda M Murai T Kamasawa N Osumi M Shimizu K Tanaka A 《Applied microbiology and biotechnology》2000,54(1):90-96
An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied
to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The
GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase
precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence
derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by
immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine
Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of
GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell
fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface,
selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including
nutrient concentrations in the media, through the emission of fluorescence.
Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999 相似文献
14.
Soluble carbonic anhydrase (CA, EC 4.2.1.1) inducible by low levels of CO2 was purified from the unicellular green alga Chlorella sorokiniana grown at alkaline pH. The purified CA had a specific activity of 2,300 units (mg protein)−1. The molecular mass of the CA was found to be 100 kDa by non-dissociating (native)-polyacrylamide gel electrophoresis and
50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 50-kDa subunit was recognized by concanavalin A.
These results suggest that the protein has a dimeric form with two 50-kDa subunits that are glycosylated in an asparagine-linked
manner. The native CA was revealed by isoelectric focusing to be a very acidic protein with an isoelectric point of 4.2. About
60% of the CA activity was inhibited by 0.5 M NaCl. The enzyme was inactivated over 95% by preincubation with 50 mM dithiothreitol
but not with 1 mM dithiothreitol. After partial amino acid sequence analysis, a cDNA clone of the CA was isolated and characterized.
The cloned cDNA fragment encoded a 348-amino-acid polypeptide (36,709 Da) including an NH2-terminal hydrophobic signal peptide composed of 35 amino acids (3,725 Da). Conserved regions of sequences found in animal
CAs, in the periplasmic (pCA) and the intracellular CAs of Chlamydomonas, and in the plasma-membrane-bound CA of Dunaliella (Dca) were also found in this Chlorella CA. The signal sequence was significantly homologous to the pCA and the Dca. The internal signal sequence between the large
and the small subunits reported for pCA was not found in this Chlorella CA. The soluble CA of this alga was an α-type CA with salt-sensitive, periplasm-locating and acidic properties and very different
from pCA and Dca with their salt-sensitive/neutral and salt-resistant/acidic properties, respectively.
Received: 25 May 1998 / Accepted: 9 July 1998 相似文献
15.
Matsusaki H Abe H Taguchi K Fukui T Doi Y 《Applied microbiology and biotechnology》2000,53(4):401-409
Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate
(3HA) units of 4–12 carbon atoms. The genes encoding β-ketothiolase (PhbARe) and NADPH-dependent acetoacetyl-CoA reductase (PhbBRe) from Ralstoniaeutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1
Ps gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB−4 to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6–12 carbon atoms. The introduction of the three genes into
GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated
at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1
Ps gene only. In addition, recombinant strains of R. eutropha PHB−4 produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant
strains, R. eutropha PHB−4/pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that
the copolyesters obtained here were random copolymers of 3HB and 3HA units.
Received: 12 July 1999 / Received revision: 1 October 1999 / Accepted: 2 October 1999 相似文献
16.
Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli 总被引:1,自引:0,他引:1
K. Saito H. Yamazaki Y. Ohnishi S. Fujimoto E. Takahashi S. Horinouchi 《Applied microbiology and biotechnology》1998,50(2):193-198
The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with
the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag
at the NH2-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that
the equilibrium lay far in the direction of trehalose synthesis.
Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998 相似文献
17.
M. J. Artolozaga E. Kubátová J. Volc H. M. Kalisz 《Applied microbiology and biotechnology》1997,47(5):508-514
Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass
of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of
isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and
up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate
specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at
pH 8.0, with a catalytic constant (k
cat) of 111.0 s−1 and an affinity constant (K
m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at
pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k
cat/K
m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k
cat decreased to 60.9 s−1 and the K
m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl2, AgNO3 and ZnCl2, and 50% by lead acetate, CuCl2 and MnCl2.
Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996 相似文献
18.
J. F. Martín 《Applied microbiology and biotechnology》1998,50(1):1-15
Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase1 and isopenicillin N (IPN) synthase1 respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its
active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin
N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular
mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms,
biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic
acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association
of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes
involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P7 and P8) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative
to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase
encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin
clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling
precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have
been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new
molecules.
Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998 相似文献
19.
C. J. Park J. H. Lee S.-S. Hong H.-S. Lee S. C. Kim 《Applied microbiology and biotechnology》1998,50(1):71-76
To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino
acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers
of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units
in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers
by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly
as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were
purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was
obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1
in molecular mass, amino acid sequence and ACE-inhibiting activity.
Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998 相似文献
20.
The report describes a system for somatic embryogenesis and direct plant regeneration from the embryos of Manihot glaziovii. Somatic embryos were obtained by culturing young leaf lobes (3–6 mm long) adjacent to the apex in Murashige and Skoog medium
containing 18 μm 2,4-dichlorophenoxy acetic acid for 20 days and then transferring them to a maturation medium with 0.5 μm 6-benzylaminopurine. Secondary embryogenesis was induced from cotyledonary segments of somatic embryos by using the same
protocol as that for primary embryogenesis. For regeneration, somatic embryos were cultured in medium supplemented with 10−4 m kinetin and 53.4% of them developed into plantlets. Linamarin and linamarase were not detected in calli or in somatic embryos.
Linamarin content was found to be highest in leaves of regenerated plantlets, followed by stem and root tissues. Levels of
linamarase activity were almost the same in leaves and stem tissues and very low in roots.
Received: 19 April 1999 / Revision received: 11 August 1999 / Accepted: 17 August 1999 相似文献