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1.
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
2.
M. J. Pettinari G. J. Vazquez N. Krüger P. S. Vary A. Steinbüchel B. S. Méndez 《Applied microbiology and biotechnology》1998,49(6):737-742
A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a
protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties
by recombinant E. coli strains.
Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998 相似文献
3.
Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli 总被引:2,自引:0,他引:2
Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder
as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l−1 and 50 g l−1 respectively in 47 h. When concentrated whey solution containing 210 g l−1 lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l−1 and 69 g l−1 respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry
weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate.
Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998 相似文献
4.
Hayashi H Takehara M Hattori T Kimura T Karita S Sakka K Ohmiya K 《Applied microbiology and biotechnology》1999,51(3):348-357
A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da,
and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da.
XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11
xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high
overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested
that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was
689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome.
Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998 相似文献
5.
A. Blanco P. Díaz J. Martínez T. Vidal A. L. Torres F. I. J. Pastor 《Applied microbiology and biotechnology》1998,50(1):48-54
The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced
amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases.
The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the
presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres
showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced
the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished.
Electron-microscope analysis showed that the surface of straw fibres was modified by CelA.
Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998 相似文献
6.
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
7.
The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal
DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334
amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology
to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic
activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the
cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K
m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61.
Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998 相似文献
8.
M. Kataoka K. Yamamoto H. Kawabata M. Wada K. Kita H. Yanase S. Shimizu 《Applied microbiology and biotechnology》1999,51(4):486-490
The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7%
enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of
the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.
Received: 5 October 1998 / Received revision: 16 November 1998 / Accepted: 5 December 1998 相似文献
9.
R. N. Z. A. Rahman S. Fujiwara M. Takagi T. Imanaka 《Molecular & general genetics : MGG》1998,257(3):338-347
The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH,
and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric
GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both
enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino
acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native
KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar
to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h
for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production.
KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs.
Received: 6 May 1997 / Accepted: 23 September 1997 相似文献
10.
A. Arai S. Masuda A. Matsuyama S. Murakami M. Nakajima 《Applied microbiology and biotechnology》1998,49(3):272-276
The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular
mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino
acid sequence of the purified pyruvate kinase from M. thermodiastatica.
Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997 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.
High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium 总被引:1,自引:0,他引:1
G. Miksch R. Neitzel K. Friehs E. Flaschel 《Applied microbiology and biotechnology》1999,51(5):627-632
The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome
of Klebsiella planticola with high transposition frequency. A Bacillus hybrid β-glucanase located on an RSF1010-derived plasmid was mobilized from E.coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments
it was shown that the production of β-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter
used for the expression of the kil gene the secretion of β-glucanase into the medium started at the transition from the exponential to the stationary phase,
as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.
Received: 22 July 1998 / Received revision: 25 November 1998 / Accepted: 29 November 1998 相似文献
13.
P. J. Punt I. A. van Gemeren J. Drint-Kuijvenhoven J. G. M. Hessing G. M. van Muijlwijk-Harteveld A. Beijersbergen C. T. Verrips C. A. M. J. J. van den Hondel 《Applied microbiology and biotechnology》1998,50(4):447-454
The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous
fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount
of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed
fusion protein were detected in the total protein extracts of these strains.
Received: 9 February 1998 / Received last revision: 26 May 1998 / Accepted: 14 June 1998 相似文献
14.
Kacena MA Merrell GA Manfredi B Smith EE Klaus DM Todd P 《Applied microbiology and biotechnology》1999,51(2):229-234
Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space
flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions.
This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary
phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic
bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase
concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space.
Received: 27 February 1998 / Received revision: 21 August 1998 / Accepted: 3 September 1998 相似文献
15.
M. E. Gustafson R. A. Clayton P. B. Lavrik G. V. Johnson R. M. Leimgruber S. R. Sims D. E. Bartnicki 《Applied microbiology and biotechnology》1997,47(3):255-261
Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration
of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this
truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified
by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography
steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating
full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity
and suitability for use in safety-assessment studies.
Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996 相似文献
16.
A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in
the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated
molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted
amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active
at pH 7 and a temperature of 40° C.
Received: 6 February 1998 / Accepted: 6 November 1998 相似文献
17.
The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining
the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published
findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than
control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates.
Received: 3 December 1998 / Received revision: 23 February 1999 / Accepted: 14 March 1999 相似文献
18.
Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the
molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average
molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent
on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated
in E. coli cells during the accumulation of PHB.
Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996 相似文献
19.
S. Nakotte S. Schaffer M. Böhringer P. Dürre 《Applied microbiology and biotechnology》1998,50(5):564-567
Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 × 102 transformants/μg DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 μF, and 600 Ω. The method also allowed the
taxonomic group IV strain NI-4082 to be transformed (101 transformants/μg DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis
method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be
easily preserved as spore suspensions; under all conditions tested plasmids were maintained.
Received: 17 March 1998 / Received revision: 17 August 1998 / Accepted: 26 August 1998 相似文献
20.
A. E. Cazemier J. C. Verdoes H. J. M. Op den Camp J. H. P. Hackstein A. J. J. van Ooyen 《Applied microbiology and biotechnology》1999,52(2):232-239
A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA
fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A
had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase
Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding
domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473,
which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.
Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999 相似文献