scholarly journals Improved expression of Catechol 1,2-dioxygenase gene from Burkholderia cepacia in Escherichia coli

2020 ◽  
Vol 129 (1D) ◽  
pp. 61-65
Author(s):  
Van Thanh Dang ◽  
Anh Thi Hoang ◽  
Gia Cat Tuong Tran ◽  
Thi Huyen Tran Pham ◽  
Le Thi Ha Thanh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Burkholderia Cepacia ◽  
Culture Conditions ◽  
Environmental Pollutant ◽  
Gene Encoding ◽  
Key Enzyme ◽  
Catechol 1,2 Dioxygenase ◽  
Dioxygenase Gene ◽  
Temperature Induction

Catechol 1,2-dioxygenase (CAT1) is a key enzyme for the ortho-cleavage pathway involved in the degradation of dibenzofuran, a dioxin derivative, which is a highly toxic environmental pollutant. The present study aims to investigate appropriate culture conditions for enhancing the expression of the cat1 gene encoding CAT1 enzyme from Burkholderia cepacia DF4 in Escherichia coli M15. The optimized culture conditions for gene expression are cell density at the time of induction, shaking speed, induction temperature, induction time, and inducer concentration. The highest level for CAT1 was obtained at the IPTG concentration of 1.2 mM, 10 hours after induction at 35 °C, shaking speed 200 rpm with cell density at OD600 0.7.

scholarly journals Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100

2003 ◽  
Vol 185 (9) ◽  
pp. 2786-2792 ◽  
Author(s):  
Michelle R. Gisi ◽  
Luying Xun
Keyword(s):  
Burkholderia Cepacia ◽  
Active Oxygen Species ◽  
Environmental Pollutant ◽  
Flavin Mononucleotide ◽  
Binding Property ◽  
Aromatic Substrates ◽  
Key Enzyme ◽  
Trichlorophenoxyacetic Acid ◽  
New Discovery

ABSTRACT Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH2)-utilizing monooxygenase, and FADH2 was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH2 and retarded its rapid oxidation by O2. A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea.

scholarly journals Proteomic Profiling of Recombinant Escherichia coli in High-Cell-Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical

2005 ◽  
Vol 71 (4) ◽  
pp. 1717-1728 ◽  
Author(s):  
Ilana S. Aldor ◽  
Denise C. Krawitz ◽  
William Forrest ◽  
Christina Chen ◽  
Julie C. Nishihara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Cell Density ◽  
High Cell Density ◽  
Antibody Fragment ◽  
Culture Conditions ◽  
Test Method ◽  
Differentially Expressed ◽  
Soluble Form ◽  
High Cell

ABSTRACT By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limited Escherichia coli fermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm. E. coli protein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using a t-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Dorra Ayadi ◽  
Radhouane Kammoun ◽  
Sonia Jemli ◽  
Samir Bejar
Keyword(s):  
Escherichia Coli ◽  
Bacillus Stearothermophilus ◽  
Active Form ◽  
Culture Conditions ◽  
Cyclodextrin Glucanotransferase ◽  
Gene Encoding ◽  
Box Behnken Design ◽  
Post Induction ◽  
Optimized Conditions ◽  
Maximum Expression

AbstractThe gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

scholarly journals Expression of Alcaligenes eutrophusFlavohemoprotein and Engineered VitreoscillaHemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth ofEscherichia coli

2000 ◽  
Vol 66 (1) ◽  
pp. 98-104 ◽  
Author(s):  
Alexander D. Frey ◽  
James E. Bailey ◽  
Pauli T. Kallio
Keyword(s):  
Escherichia Coli ◽  
Cell Density ◽  
Product Formation ◽  
Gene Encoding ◽  
Final Cell Density ◽  
E Coli ◽  
Amino Terminal ◽  
Positive Effects ◽  
Cell Densities ◽  
Carboxy Terminal

ABSTRACT Expression of the vhb gene encoding hemoglobin fromVitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant β-lactamase relative to the VHb-expressing control.

scholarly journals ADP-Dependent Phosphofructokinases in Mesophilic and Thermophilic Methanogenic Archaea

2001 ◽  
Vol 183 (24) ◽  
pp. 7145-7153 ◽  
Author(s):  
Corné H. Verhees ◽  
Judith E. Tuininga ◽  
Servé W. M. Kengen ◽  
Alfons J. M. Stams ◽  
John van der Oost ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phylogenetic Analysis ◽  
Methanogenic Archaea ◽  
Hyperthermophilic Archaea ◽  
Acetyl Phosphate ◽  
Gene Encoding ◽  
Substrate Specificities ◽  
Key Enzyme ◽  
Domains Of Life ◽  
Modified Embden Meyerhof Pathway

ABSTRACT Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PPi-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus andThermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcalesand Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK fromMethanococcus jannaschii was functionally expressed inEscherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order Thermococcales, the M. jannaschii ADP-PFK has an extremely lowKm for fructose 6-phosphate (9.6 μM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.

scholarly journals Inactivation of the ampDE Operon Increases Transcription of algD and Affects Morphology and Encystment of Azotobacter vinelandii

2000 ◽  
Vol 182 (17) ◽  
pp. 4829-4835 ◽  
Author(s):  
Cinthia Núñez ◽  
Soledad Moreno ◽  
Luis Cárdenas ◽  
Gloria Soberón-Chávez ◽  
Guadalupe Espín
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Azotobacter Vinelandii ◽  
Transmembrane Protein ◽  
Nucleotide Sequencing ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Alginate Production ◽  
Key Enzyme

ABSTRACT Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of analgD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDEoperon. Tn5 was located within the ampD gene, encoding a cytosolicN-acetyl-anhydromuramyl-l-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carryingampD or ampE mutations and one with anampDE deletion. The strain with a deletion of theampDE operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

scholarly journals SUB-CLONING OF GENES ENCODINGCYTHOCHROME P450 MONOOXYGENASE (CYP71AVI) INTOEXPRESSION VECTOR IN ESCHERICHIA COLI

2017 ◽  
Vol 10 (14) ◽  
pp. 69
Author(s):  
Imam Adi Wicaksono ◽  
Tresna Lestari ◽  
Evi Umayah Ulfa ◽  
Catur Riani ◽  
Elfahmi Elfahmi
Keyword(s):  
Escherichia Coli ◽  
Restriction Site ◽  
Restriction Analysis ◽  
Sequencing Analysis ◽  
Gene Encoding ◽  
P450 Monooxygenase ◽  
Recombinant Vector ◽  
Sds Page ◽  
Key Enzyme ◽  
Artemisinin Biosynthesis

Objective: Cytochrome P450 monooxygenase (CYP71AVI) is a key enzyme involved in the artemisinin biosynthesis pathway.In this research, sub-cloning gene encoding CYP71AVI into pETDUET1 vector in Escherichia coli has been done and then the expression products characterized with SDS-PAGE.Methods: Gene construction started with sub-cloning of cyp71avi gene from pJexpress401_cyp into pETDUET1 through restriction site NdeI and XhoI to get pETDUET1_cyp. Overproduction of CYP71AVI at temperature 37 °C has conducted by IPTG induction.Results: Confirmation of the recombinant vector pETDUET1_cyp was done by migration, restriction site and sequencing analysis. The result of pETDUET1_cyp restriction analysis with XhoI restriction enzyme showed one DNA band with experimental size 6585 bp.The CYP71AVI protein has been produced and characterized with SDS-PAGE method. Based on experimental calculation from SDS-PAGE analysis obtained molecular weight of CYP71AVI band was 57.55 kDa.Conclusion: Construction of gene encoding CYP71AVI into pETDUET1 as the co-expression vector in Escherichia colihas been succesfully and confirmed by migration, restriction site and sequencing analysis. The result of overproduction showed protein bands on SDS-PAGE analysis indicated as CYP71AVI. 

scholarly journals Requirement of Purine and Pyrimidine Synthesis for Colonization of the Mouse Intestine by Escherichia coli

2010 ◽  
Vol 76 (15) ◽  
pp. 5181-5187 ◽  
Author(s):  
Jacqueline Vogel-Scheel ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Gunnar Loh ◽  
Michael Blaut
Keyword(s):  
Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Gene Encoding ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
Key Enzyme ◽  
Mouse Intestine ◽  
Gnotobiotic Mice

ABSTRACT To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5′-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

scholarly journals Kloning Gen β-1,4 Glukanase dari Burkholderia cepaciake dalam Escherichia coli dan Karakterisasi Sekuennya

2014 ◽  
Vol 1 (3) ◽  
pp. 116-125
Author(s):  
Fitriani Winangsih ◽  
Maria Bintang ◽  
Tri Puji Priyatno
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Burkholderia Cepacia ◽  
Endophytic Bacteria ◽  
Expression System ◽  
Alpha Helix ◽  
Data Bank ◽  
Plant Production ◽  
Gene Encoding ◽  
Dna Fragment

The increasing of rice plant production has to deal with some constraints caused by pathogen infection such as by bacteria, viruses or fungi. Endophytic bacteria have antagonistic capacity against fungi and was used to prevent the invasion of the pathogen. Burkholderia cepacia is one of the endophytic bacteria carrying genes expressed in defense system against fungi by producing glucanase enzyme. The aim of this research was to clone a gene encoding β-1,4-glucanase from B. cepacia into the expression system in Escherichia coli. The clone of glucanase gene was isolated by PCR technique using DNA fragment of B. cepacia from rice plants. The Glu 1320 primer pairs were designed based on the glucanase gene nucleotide sequence on online database, with the length of the amplicon DNA of 1300 bp. Results from BlastN and BlastX analysis showed that the DNA fragment which was cloned into pGEM-T Easy vector had similarity with Endo-1,4-D-glucanase gene of Burkholderia mallei and Burkholderia pseudomallei. The identity of the cloned DNA fragment was 99% and E-value 0.0. Proteomic analysis of the amino acid sequence was done using Server Expasy Proteomic and the total of amino acid was 451 with, molecular weight of 48.363 kDa and isoelectric point (pI) of 5.87. The signal peptide had cleavage sites on position 23 and 24 in amino acid AAAAE. Recombinant protein clone was obtained from Protein Data Bank (PDB) database with the code of 4q2b.2.A. The protein consist of 349 residu which formed the secondary structure like of 7 betahairpin pairs, 20 turn, 3 helix-3/10, and 17 alpha-helix.

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