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Fulltext Green Synthesized Silver Nanoparticles Immobilized on Activated Carbon Nanoparticles: Antibacterial Activity Enhancement Study and Its Application on Textiles Fabrics

Wibawa PJ, Nur M, Asy'ari M, Wijanarka W, Susanto H, Sutanto H, et al.

Molecules, 2021 Jun 22;26(13).
PMID: 34206375 DOI: 10.3390/molecules26133790

This research aimed to enhance the antibacterial activity of silver nanoparticles (AgNPs) synthesized from silver nitrate (AgNO3) using aloe vera extract. It was performed by means of incorporating AgNPs on an activated carbon nanoparticle (ACNPs) under ultrasonic agitation (40 kHz, 2 × 50 watt) for 30 min in an aqueous colloidal medium. The successful AgNPs synthesis was clarified with both Ultraviolet-Visible (UV-Vis) and Fourier Transform Infrared (FTIR) spectrophotometers. The successful AgNPs-ACNPs incorporation and its particle size analysis was performed using Transmission Electron Microscope (TEM). The brown color suspension generation and UV-Vis's spectra maximum wavelength at around 480 nm confirmed the existence of AgNPs. The particle sizes of the produced AgNPs were about 5 to 10 nm in the majority number, which collectively surrounded the aloe vera extract secondary metabolites formed core-shell like nanostructure of 8.20 ± 2.05 nm in average size, while ACNPs themselves were about 20.10 ± 1.52 nm in average size formed particles cluster, and 48.00 ± 8.37 nm in average size as stacking of other particles. The antibacterial activity of the synthesized AgNPs and AgNPs-immobilized ACNPs was 57.58% and 63.64%, respectively (for E. coli); 61.25%, and 93.49%, respectively (for S. aureus). In addition, when the AgNPs-immobilized ACNPs material was coated on the cotton and polyester fabrics, the antibacterial activity of the materials changed, becoming 19.23% (cotton; E. coli), 31.73% (polyester; E. coli), 13.36% (cotton; S. aureus), 21.15% (polyester; S. aureus).

Matched MeSH terms: Escherichia coli/growth & development*
Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library

Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J

Malar J, 2018 Oct 24;17(1):383.
PMID: 30355309 DOI: 10.1186/s12936-018-2531-y

BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

Matched MeSH terms: Escherichia coli/metabolism
Fulltext Optimisation of preparation conditions for Ti nanowires and suitability as an antibacterial material

Munisparan T, Yang ECY, Paramasivam R, Dahlan NA, Pushpamalar J

IET Nanobiotechnol, 2018 Jun;12(4):429-435.
PMID: 29768225 DOI: 10.1049/iet-nbt.2017.0186

Ultrafine titanium dioxide (TiO2) nanowires were synthesised using a hydrothermal method with different volumes of ethylene glycol (EG) and annealing temperatures. It shows that sodium titanate nanowires synthesised using 5 and 10 ml EG, which annealed at 400°C produced TiO2 nanowires that correspond to a photochemically active phase, which is anatase. The influences of annealing temperatures (400-600°C) on the morphological arrangement of TiO2 nanowires were evident in the field emission scanning electron microscopy. The annealing temperature of 500°C led to agglomeration, which formed a mixture of TiO2 nanoparticles and nanowires. High thermal stability of TiO2 nanowires revealed by thermogravimetric analysis and Fourier transform infrared spectroscopy spectrum showed the presence of the Ti-O-Ti vibrations as evidenced due to TiO2 lattices. An antibacterial study using TiO2 nanowires toward Escherichia coli and Klebsiella pneumoniae showed large zones of inhibition that indicated susceptibility of the microbe toward TiO2. Growth kinetic analysis shows that addition of TiO2 has reduced optical density (OD) suggesting an inhibition of the growth of bacteria. These results indicate TiO2 nanowires can be effectively used as an antimicrobial agent against gram-bacteria. The TiO2 nanowires could be exploited in the medical, packaging and detergent formulation industries and wastewater treatment.

Matched MeSH terms: Escherichia coli/drug effects
Prevalence of diarrheagenic Escherichia coli in finns with or without diarrhea during a round-the-world trip

Keskimäki M, Mattila L, Peltola H, Siitonen A

J Clin Microbiol, 2000 Dec;38(12):4425-9.
PMID: 11101575

The incidence of diarrhea and the prevalence of bacterial enteropathogens, viruses, and parasites in feces of subjects with and without diarrhea were evaluated in 204 Finns traveling round the world (from Finland to China, Malaysia, Australia, Fiji, Chile, and Brazil and back to Finland). Special emphasis was placed on the finding of diarrheagenic Escherichia coli (enterotoxigenic, enteropathogenic, Shiga toxin-producing, and enteroaggregative strains) by PCR from growth on primary culture plates. From the PCR-positive samples, corresponding strains were isolated, confirmed as E. coli, and O serotyped. Of all the subjects, 37% experienced a total of 90 episodes of diarrhea. No adenoviruses or rotaviruses were detected, and findings of parasites were insignificant. In contrast, enteropathogenic bacteria were present in 62% of the 65 diarrheal and in 33% of the 127 nondiarrheal samples (P < 0.001); diarrheagenic E. coli strains were found in 35 and 26% of these, respectively (not statistically significant). As a single pathogen, E. coli was found in 20 and 24% of samples (not significant). Of all diarrheagenic E. coli strains, enteropathogenic strains were the most commonly found independently of the clinical picture of the subjects, whereas Salmonella enterica as a single pathogen was the most common non-E. coli organism found in diarrheal samples. Multiple bacterial pathogens were found 10 times more commonly in diarrheal than in nondiarrheal samples (20 versus 2%; P < 0.001).

Matched MeSH terms: Escherichia coli/isolation & purification*
Purification of recombinant nucleocapsid protein of Newcastle disease virus from unclarified feedstock using expanded bed adsorption chromatography

Tan YP, Ling TC, Tan WS, Yusoff K, Tey BT

Protein Expr Purif, 2006 Mar;46(1):114-21.
PMID: 16139513

In the present work, a single-step purification of recombinant nucleocapsid protein (NP) of the Newcastle disease virus (NDV) directly from unclarified feedstock using an expanded bed adsorption chromatography (EBAC) was developed. Streamline 25 column (ID = 25 mm) was used as a contactor and Streamline chelating adsorbent immobilized with Ni2+ ion was used as affinity adsorbent. The dynamic binding capacity of Ni2+ -loaded Streamline chelating adsorbent for the NP protein in unclarified feedstock was found to be 2.94 mg ml(-1) adsorbent at a superficial velocity of 200 cm h(-1). The direct purification of NP protein from unclarified feedstock using expanded bed adsorption has resulted in a 31% adsorption and 9.6% recovery of NP protein. The purity of the NP protein recovered was about 70% and the volume of processing fluid was reduced by a factor of 10. The results of the present study show that the IMA-EBAC developed could be used to combine the clarification, concentration and initial purification steps into a single-step operation.

Matched MeSH terms: Escherichia coli/genetics
Fulltext Porcine placenta hydrolysate as an alternate functional food ingredient: In vitro antioxidant and antibacterial assessments

Laosam P, Panpipat W, Yusakul G, Cheong LZ, Chaijan M

PLoS One, 2021;16(10):e0258445.
PMID: 34695136 DOI: 10.1371/journal.pone.0258445

The production of bioactive peptides from animal-based raw materials highly depends on enzymatic hydrolysis. Porcine placenta is an underutilized biomass in Thailand's pig farms, yet it is still a source of proteins and beneficial compounds. Porcine placenta could be used as a protein substrate for the production of enzymatic hydrolysate, which could be employed as a functional food ingredient in the future. The goal of this study was to enzymatically produce porcine placenta hydrolysates (PPH) using three commercial enzymes (Alcalase, Flavouzyme, and papain) and evaluate their in vitro antioxidant and antibacterial activity. The degree of hydrolysis (DH) increased as the enzyme load and hydrolysis time increased, but the DH was governed by the enzyme class. The maximum DH was found after using 10% enzyme for 20 min of hydrolysis (36.60%, 31.40%, and 29.81% for Alcalase, Flavouzyme, and papain). Depending on the enzyme type and DH, peptides of various sizes (0.40-323.56 kDa) were detected in all PPH. PPH created with Alcalase had an excellent reducing capacity and metal chelating ability (p < 0.05), whereas PPH made with Flavourzyme and Papain had higher DPPH• and ABTS•+ inhibitory activities (p < 0.05). Papain-derived PPH also had a strong antibacterial effect against Staphylococcus aureus and Escherichia coli, with clear zone values of 17.20 mm and 14.00 mm, respectively (p < 0.05). When PPH was transported via a gastrointestinal tract model system, its antioxidative characteristics were altered. PPH's properties and bioactivities were thus influenced by the enzyme type, enzyme concentration, and hydrolysis time used. Therefore, PPH produced from porcine placenta can be categorized as an antioxidant and antibacterial alternative.

Matched MeSH terms: Escherichia coli/drug effects
Fulltext Efficient Synthesis, Structural Characterization, Antibacterial Assessment, ADME-Tox Analysis, Molecular Docking and Molecular Dynamics Simulations of New Functionalized Isoxazoles

Arzine A, Hadni H, Boujdi K, Chebbac K, Barghady N, Rhazi Y, et al.

Molecules, 2024 Jul 17;29(14).
PMID: 39064944 DOI: 10.3390/molecules29143366

This work describes the synthesis, characterization, and in vitro and in silico evaluation of the biological activity of new functionalized isoxazole derivatives. The structures of all new compounds were analyzed by IR and NMR spectroscopy. The structures of 4c and 4f were further confirmed by single crystal X-ray and their compositions unambiguously determined by mass spectrometry (MS). The antibacterial effect of the isoxazoles was assessed in vitro against Escherichia coli, Bacillus subtilis, and Staphylococcusaureus bacterial strains. Isoxazole 4a showed significant activity against E. coli and B. subtilis compared to the reference antibiotic drugs while 4d and 4f also exhibited some antibacterial effects. The molecular docking results indicate that the synthesized compounds exhibit strong interactions with the target proteins. Specifically, 4a displayed a better affinity for E. coli, S. aureus, and B. subtilis in comparison to the reference drugs. The molecular dynamics simulations performed on 4a strongly support the stability of the ligand-receptor complex when interacting with the active sites of proteins from E. coli, S. aureus, and B. subtilis. Lastly, the results of the Absorption, Distribution, Metabolism, Excretion and Toxicity Analysis (ADME-Tox) reveal that the molecules have promising pharmacokinetic properties, suggesting favorable druglike properties and potential therapeutic agents.

Matched MeSH terms: Escherichia coli/drug effects
Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria

Goh ZH, Tan SG, Bhassu S, Tan WS

J Virol Methods, 2011 Jul;175(1):74-9.
PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021

Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.

Matched MeSH terms: Escherichia coli/virology*
Identification and Characterization of Entamoeba histolytica Choline Kinase

Chang CH, See Too WC, Lim BH, Few LL

Acta Parasitol, 2024 Mar;69(1):426-438.
PMID: 38172465 DOI: 10.1007/s11686-023-00763-1

PURPOSE: Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future.
METHODOLOGY: E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes.
RESULTS: EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1 µmol/min/mg. Its Km for choline and ATP was 203 ± 26 µM and 3.1 ± 0.4 mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting.
CONCLUSIONS: With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

Matched MeSH terms: Escherichia coli/genetics
Enhancing refrigerated chicken breasts preservation: Novel composite hydrogels incorporated with antimicrobial peptides, bacterial cellulose, and polyvinyl alcohol

Si S, Huang X, Wang Q, Manickam S, Zhao D, Liu Y

Int J Biol Macromol, 2024 Nov;281(Pt 4):136505.
PMID: 39395516 DOI: 10.1016/j.ijbiomac.2024.136505

Microbial contamination annually leads to substantial food resource loss. Effective food packaging can mitigate food contamination and waste, yet conventional materials such as plastics often lack bacteriostatic activity. This study aimed to synthesise FengycinA-M3@bacterial cellulose@polyvinyl alcohol composite hydrogels via dual cross-linking with hydrogen and borate bonding, with the goal of enhancing antibacterial properties and prolonging the preservation period of refrigerated chicken breast. The composite hydrogel was subjected to comprehensive characterisation for structural, mechanical, water absorption, slow peptide release, antimicrobial capacity, biocompatibility, and chicken breast freshness preservation. The results showed that the composite hydrogel had a porous network structure and excellent gel elasticity and biocompatibility. It was effective in inhibiting Staphylococcus aureus and Escherichia coli, and prolonged the storage time of frozen chicken breast for up to 12 days. These findings emphasise the potential of hydrogel food packaging to prolong storage periods and its suitability for food industry applications due to ease of manufacture.

Matched MeSH terms: Escherichia coli/drug effects
Fulltext Antibacterial Mode of Action of Cinnamomum verum Bark Essential Oil, Alone and in Combination with Piperacillin, Against a Multi-Drug-Resistant Escherichia coli Strain

Yap PS, Krishnan T, Chan KG, Lim SH

J Microbiol Biotechnol, 2015 Aug;25(8):1299-306.
PMID: 25381741 DOI: 10.4014/jmb.1407.07054

This study aimed to investigate the mechanism of action of the cinnamon bark essential oil (CB), when used singly and also in combination with piperacillin, for its antimicrobial and synergistic activity against beta-lactamase TEM-1 plasmid-conferred Escherichia coli J53 R1. Viable count of this combination showed a complete killing profile at 20 h and further confirmed its synergistic effect by reducing the bacteria cell numbers. Analysis on the stability of treated cultures for cell membrane permeability by CB when tested against sodium dodecyl sulfate revealed that the bacterial cell membrane was disrupted by the essential oils. Scanning electron microscopy observation and bacterial surface charge measurement also revealed that CB causes irreversible membrane damage and reduces the bacterial surface charge. In addition, bioluminescence expression of Escherichia coli [pSB1075] and E. coli [pSB401] by CB showed reduction, indicating the possibility of the presence of quorum sensing (QS) inhibitors. Gas-chromatography and mass spectrometry of the essential oil of Cinnamomum verum showed that trans-cinnamaldehyde (72.81%), benzyl alcohol (12.5%), and eugenol (6.57%) were the major components in the essential oil. From this study, CB has the potential to reverse E. coli J53 R1 resistance to piperacillin through two pathways; modification in the permeability of the outer membrane or bacterial QS inhibition.

Matched MeSH terms: Escherichia coli/drug effects*; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/ultrastructure
Fulltext Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

Chang CY, Krishnan T, Wang H, Chen Y, Yin WF, Chong YM, et al.

Sci Rep, 2014;4:7245.
PMID: 25430794 DOI: 10.1038/srep07245

N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.

Matched MeSH terms: Escherichia coli/drug effects; Escherichia coli/metabolism
Recombineering linear BACs

Chen Q, Narayanan K

Methods Mol Biol, 2015;1227:27-54.
PMID: 25239740 DOI: 10.1007/978-1-4939-1652-8_2

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/metabolism
Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) by recombinant Escherichia coli expressing leucine metabolism-related enzymes derived from Clostridium difficile

Saika A, Watanabe Y, Sudesh K, Tsuge T

J Biosci Bioeng, 2014 Jun;117(6):670-5.
PMID: 24484910 DOI: 10.1016/j.jbiosc.2013.12.006

An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.

Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/metabolism
Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3)

Chew FN, Tan WS, Boo HC, Tey BT

Prep Biochem Biotechnol, 2012;42(6):535-50.
PMID: 23030465 DOI: 10.1080/10826068.2012.660903

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.

Matched MeSH terms: Escherichia coli/growth & development; Escherichia coli/chemistry*
Fulltext Engineering the production of major catechins by Escherichia coli carrying metabolite genes of Camellia sinensis

Umar KM, Abdulkarim SM, Radu S, Abdul Hamid A, Saari N

ScientificWorldJournal, 2012;2012:529031.
PMID: 22645428 DOI: 10.1100/2012/529031

A mimicked biosynthetic pathway of catechin metabolite genes from C. sinensis, consisting of flavanone 3 hydroxylase (F3H), dihydroflavonol reductase (DFR), and leucoanthocyanidin reductase (LCR), was designed and arranged in two sets of constructs: (a) single promoter in front of F3H and ribosome-binding sequences both in front of DFR and LCR; (b) three different promoters with each in the front of the three genes and ribosome-binding sequences at appropriate positions. Recombinant E. coli BL (DE3) harbouring the constructs were cultivated for 65 h at 26 °C in M9 medium consisting of 40 g/L glucose, 1 mM IPTG, and 3 mM eriodictyol. Compounds produced were extracted in ethyl acetate in alkaline conditions after 1 h at room temperature and identified by HPLC. Two of the four major catechins, namely, (-)-epicatechin (0.01) and (-)-epicatechin gallate (0.36 mg/L), and two other types ((+)-catechin hydrate (0.13 mg/L) and (-)-catechin gallate (0.04 mg/L)) were successfully produced.

Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/metabolism*
Fulltext Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109

Yee LN, Chuah JA, Chong ML, Phang LY, Raha AR, Sudesh K, et al.

Microbiol Res, 2012 Oct 12;167(9):550-7.
PMID: 22281521 DOI: 10.1016/j.micres.2011.12.006

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182 bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738 bp) and PHA synthase, class I (phaC(Co) gene, 1694 bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.

Matched MeSH terms: Escherichia coli/genetics*; Escherichia coli/metabolism
Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli

Zakaria II, Rahman RN, Salleh AB, Basri M

Appl Biochem Biotechnol, 2011 Sep;165(2):737-47.
PMID: 21633820 DOI: 10.1007/s12010-011-9292-1

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.

Matched MeSH terms: Escherichia coli/enzymology; Escherichia coli/genetics*
Organic-solvent stability of elastase strain K overexpressed in an Escherichia-Pseudomonas expression system

Wong CF, Salleh AB, Basri M, Abd Rahman RN

Biotechnol Appl Biochem, 2010 Sep;57(1):1-7.
PMID: 20726840 DOI: 10.1042/BA20100224

The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.

Matched MeSH terms: Escherichia coli/genetics; Escherichia coli/metabolism*
Feeding Artemia franciscana (Kellogg) larvae with bacterial heat shock protein, protects from Vibrio campbellii infection

Sung YY, Ashame MF, Chen S, Macrae TH, Sorgeloos P, Bossier P

J Fish Dis, 2009 Aug;32(8):675-85.
PMID: 19515074 DOI: 10.1111/j.1365-2761.2009.01046.x

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

Matched MeSH terms: Escherichia coli; Escherichia coli Proteins/metabolism

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