scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

scholarly journals d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

1992 ◽  
Vol 282 (3) ◽  
pp. 747-752 ◽  
Author(s):  
O A M al-Bar ◽  
C D O'Connor ◽  
I G Giles ◽  
M Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Phosphinic Acid ◽  
Mature Protein ◽  
Bamhi Fragment ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals A new method for the extraction and purification of K99 pili from enterotoxigenic Escherichia coli and their characterization

1982 ◽  
Vol 201 (3) ◽  
pp. 505-513 ◽  
Author(s):  
K Altmann ◽  
N A Pyliotis ◽  
T K S Mukkur
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Average Length ◽  
Bacterial Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterotoxigenic Escherichia Coli ◽  
Total Amino Acid ◽  
Apparent Homogeneity

It was found that K99 pili from enterotoxigenic Escherichia coli (of bovine origin) could be extracted by treatment with 3M-KSCN solution. The K99 pili were purified by preparative isoelectric focusing to apparent homogeneity as judged by the presence of a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the molecular weight of this component was calculated to be 12 600 +/- 300. This indicated that the K99 pili were composed of a single subunit. On analytical ultracentrifugation, a single boundary with an s20,w of 12.2 S at a concentration of 0.42 mg/ml was observed. The average length of purified pili at zero concentration was approx. 160 nm and the diameter was 7.4 +/- 0.6 nm. Amino acid analysis of the purified K99 pili revealed that sulphur-containing amino acids, cysteine and methionine, were absent. Aromatic amino acids, phenylalanine and tyrosine, previously reported to be absent [Isaacson (1977) Infect. Immun. 15. 272-279], constituted 7.14% of the total amino acid residues present. On immunoelectrophoresis, purified K99 pili migrated towards the cathode and caused mannose-resistant haemagglutination of horse, but not of sheep or guinea-pig, red blood cells. Pili from enterotoxigenic E. coli of porcine and human origin and from another bacterial species, namely Fusiformis nodosus, could also be extracted by the treatment of respective micro-organisms with 3 M-KSCN.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12

1981 ◽  
Vol 193 (3) ◽  
pp. 861-867 ◽  
Author(s):  
R H Jackson ◽  
A Cornish-Bowden ◽  
J A Cole
Keyword(s):  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soret Band ◽  
Horse Heart Cytochrome ◽  
E Coli ◽  
Highly Active ◽  
Covalently Bound

A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480–490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite.

Survey, purification, and properties of sugar phosphate phosphohydrolase among microorganisms

1983 ◽  
Vol 61 (12) ◽  
pp. 1292-1303 ◽  
Author(s):  
Francis Y. M. Choy ◽  
Ya-Pin Lee
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Physiological Role ◽  
Lactobacillus Bulgaricus ◽  
Sugar Phosphate ◽  
Phosphotransferase System ◽  
Enzyme Preparations ◽  
E Coli ◽  
Apparent Homogeneity

Sugar phosphate phosphohydrolase was purified approximately 500- to 600-fold to apparent homogeneity from Escherichia coli B, Escherichia coli C, Escherichia coli var. communior, Escherichia acidilactici, Enterobacter aerogenes, Neisseria meningitidis, and Saccharomyces cereviseae. The molecular weights of the enzyme as estimated by gel filtration ranged from 97 × 103 to 101 × 103. The enzyme was composed of two subunits with the same molecular weight which ranged from 50 × 103 to 52 × 103, as determined by sodium dodecyl sulfate gel electrophoresis. Homogeneous enzyme preparations hydrolyse all the tested α-D-aldohexose 1-phosphate, D-(keto or aldo)hexose 6-phosphate, and pentose phosphate substrates significantly. When the microorganisms were transferred from growth medium with 1% glucose to that without glucose, there were dramatic increases in both the specific and total enzyme activities. At least three isozymes appeared to be present in S. cereviseae, and two appeared to be present in E. coli B, E. coli var. communior, and N. meningitidis. Rabbit antiserum immunized against sugar phosphate phosphohydrolase purified from E. coli B cross-reacted with both the crude extracts and purified preparations of the enzyme from the other microorganisms. The presence of neither sugar phosphate phosphohydrolase activity nor immunocross-reacting material was detected in the following microorganisms: Aspergillus niger, Azotobacter chroococcum, Bacillus subtilis, Bacillus pumilis, Citrobacter freundii, Clostridium butyricum, Corynebacterium xerosis, Flavobacterium aquatile, Flavobacterium synxanthum, Lactobacillus bulgaricus, Micrococcus coralinus, Neisseria perflava, Neurospora crassa, Penicilium expansum, Penicilium notatum, Proteus mirabilis, Proteus vulgaris, Pseudomonas fluorescens, Saccharomyces fermenti, Sarcina lutea, and Streptomyces antibioticus. At present, no conclusive relationship can be established between the phosphoenolpyruvate phosphotransferase system and the enzyme sugar phosphate phosphohydrolase among microorganisms. The physiological role of sugar phosphate phosphohydrolase as a transferase and regulatory enzyme is discussed.

scholarly journals Expression of the Corynebacterium glutamicum panD Gene Encoding l-Aspartate-α-Decarboxylase Leads to Pantothenate Overproduction in Escherichia coli

1999 ◽  
Vol 65 (4) ◽  
pp. 1530-1539 ◽  
Author(s):  
Nicole Dusch ◽  
Alfred Pühler ◽  
Jörn Kalinowski
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Specific Productivity ◽  
Decarboxylase Activity ◽  
Coding Region ◽  
E Coli ◽  
Enhanced Expression

ABSTRACT The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panDmutant strain. Sequence analysis revealed that the coding region ofpanD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A definedC. glutamicum panD mutant completely lackedl-aspartate-α-decarboxylase activity and exhibited β-alanine auxotrophy. The C. glutamicum panD(panDC.g. ) as well as the E. coli panD (panDE.c. ) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of l-aspartate-α-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed inE. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of thepanDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of thepanDC.g. gene was sufficient β-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external β-alanine supplementation. The enhanced expression ofpanDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)−1h−1.

scholarly journals Expression of the Methanobacterium thermoautotrophicum hpt Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in Escherichia coli

1999 ◽  
Vol 181 (6) ◽  
pp. 1958-1962 ◽  
Author(s):  
Jørgen Sauer ◽  
Per Nygaard
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Methanobacterium Thermoautotrophicum ◽  
Recombinant Enzyme ◽  
Functional Complementation ◽  
Gene Encoding ◽  
Apparent Homogeneity ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT The hpt gene from the archaeon Methanobacterium thermoautotrophicum, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into Escherichia coli. The hpt-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine. The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the hpt gene. The recombinant enzyme was purified to apparent homogeneity.

scholarly journals Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli

1992 ◽  
Vol 288 (3) ◽  
pp. 1037-1044 ◽  
Author(s):  
R E Milner ◽  
J Busaan ◽  
M Michalak
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Fusion Proteins ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Soluble Form ◽  
E Coli ◽  
Terminal Domain ◽  
Isolation And Characterization

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

Sign in / Sign up

Export Citation Format

Share Document