Silver and copper nanoparticles were produced by chemical reduction of their respective nitrates by ascorbic acid in the presence of chitosan using microwave heating. Particle size was shown to increase by increasing the concentration of nitrate and reducing the chitosan concentration. Surface zeta potentials were positive for all nanoparticles produced and these varied from 27.8 to 33.8 mV. Antibacterial activities of Ag, Cu, mixtures of Ag and Cu, and Ag/Cu bimetallic nanoparticles were tested using Bacillus subtilis and Escherichia coli. Of the two, B. subtilis proved more susceptible under all conditions investigated. Silver nanoparticles displayed higher activity than copper nanoparticles and mixtures of nanoparticles of the same mean particle size. However when compared on an equal concentration basis Cu nanoparticles proved more lethal to the bacteria due to a higher surface area. The highest antibacterial activity was obtained with bimetallic Ag/Cu nanoparticles with minimum inhibitory concentrations (MIC) of 0.054 and 0.076 mg/L against B. subtilis and E. coli, respectively.
A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).
Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT-qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced.
Plasmodium knowlesi is the fifth Plasmodium species that can infect humans. The Plasmodium merozoite surface protein-1(42) (MSP-1(42)) is a potential candidate for malaria vaccine. However, limited studies have focused on P. knowlesi MSP-1(42).
In this article, gelatin/copper activated faujasites (CAF) composite scaffolds were fabricated by lyophilisation technique for promoting partial thickness wound healing. The optimised scaffold with 0.5% (w/w) of CAF, G (0.5%), demonstrated pore size in the range of 10-350 μm. Agar disc diffusion tests verified the antibacterial role of G (0.5%) and further supported that bacterial lysis was due to copper released from the core of CAF embedded in the gelatin matrix. The change in morphology of bacteria as a function of CAF content in gelatin scaffold was studied using SEM analysis. The confocal images revealed the increase in mortality rate of bacteria with increase in concentration of incorporated CAF in gelatin matrix. Proficient oxygen supply to needy cells is a continuing hurdle faced by tissue engineering scaffolds. The dissolved oxygen measurements revealed that CAF embedded in the scaffold were capable of increasing oxygen supply and thereby promote cell proliferation. Also, G (0.5%) exhibited highest cell viability on NIH 3T3 fibroblast cells which was mainly attributed to the highly porous architecture and its ability to enhance oxygen supply to cells. In vivo studies conducted on Sprague Dawley rats revealed the ability of G (0.5%) to promote skin regeneration in 20 days. Thus, the obtained data suggest that G (0.5%) is an ideal candidate for wound healing applications.
This study investigates the applicability of multivariate statistical techniques including cluster analysis (CA), discriminant analysis (DA), and factor analysis (FA) for the assessment of seasonal variations in the surface water quality of tropical pastures. The study was carried out in the TPU catchment, Kuala Lumpur, Malaysia. The dataset consisted of 1-year monitoring of 14 parameters at six sampling sites. The CA yielded two groups of similarity between the sampling sites, i.e., less polluted (LP) and moderately polluted (MP) at temporal scale. Fecal coliform (FC), NO3, DO, and pH were significantly related to the stream grouping in the dry season, whereas NH3, BOD, Escherichia coli, and FC were significantly related to the stream grouping in the rainy season. The best predictors for distinguishing clusters in temporal scale were FC, NH3, and E. coli, respectively. FC, E. coli, and BOD with strong positive loadings were introduced as the first varifactors in the dry season which indicates the biological source of variability. EC with a strong positive loading and DO with a strong negative loading were introduced as the first varifactors in the rainy season, which represents the physiochemical source of variability. Multivariate statistical techniques were effective analytical techniques for classification and processing of large datasets of water quality and the identification of major sources of water pollution in tropical pastures.
Matched MeSH terms: Escherichia coli/growth & development
The emergence of novel diseases caused by microbial pathogens and the undesirable side effects of certain antibiotics has been a recent dilemma in the medical arena. Consequently, it has stirred the discovery of many naturally occurring agents which could possibly provide important ramifications against various pharmacological targets and to combat various ailments. The main aim of the present study was to determine the antimicrobial activity of the crude methanolic extract of Piper (P.) sarmentosum against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, Vibrio cholera and Streptococcus pneumoniae.
Silver nanoparticles (Ag NPs) were synthesized by the chemical reducing method in the external and interlamellar space of montmorillonite (MMT) as a solid support at room temperature. AgNO(3) and NaBH(4) were used as a silver precursor and reducing agent, respectively. The most favorable experimental conditions for synthesizing Ag NPs in the MMT are described in terms of the initial concentration of AgNO(3). The interlamellar space limits changed little (d-spacing = 1.24-1.47 nm); therefore, Ag NPs formed on the MMT suspension with d-average = 4.19-8.53 nm diameter. The Ag/MMT nanocomposites (NCs), formed from AgNO(3)/MMT suspension, were characterizations with different instruments, for example UV-visible, PXRD, TEM, SEM, EDXRF, FT-IR, and ICP-OES analyzer. The antibacterial activity of different sizes of Ag NPs in MMT were investigated against Gram-positive, ie, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) and Gram-negative bacteria, ie, Escherichia coli, Escherichia coli O157:H7, and Klebsiella pneumoniae, by the disk diffusion method using Mueller-Hinton agar (MHA). The smaller Ag NPs were found to have significantly higher antibacterial activity. These results showed that Ag NPs can be used as effective growth inhibitors in different biological systems, making them applicable to medical applications.
Klebsiella pneumoniae causes neonatal sepsis and nosocomial infections. One of the strains, K. pneumoniae MGH 78578, shows high level of resistance to multiple microbial agents. In this study, domain family, amino acid sequence and topology analyses were performed on one of its hypothetical protein, YggG (KPN_03358). Structural bioinformatics approaches were used to predict the structure and functionality of YggG protein. The open reading frame (ORF) of yggG, which was a putative metalloprotease gene, was also cloned, expressed and characterized. The ORF was PCR amplified from K. pneumoniae MGH 78578 genomic DNA and cloned into a pET14-b vector for heterologous expression in Escherichia coli. The purified YggG protein was subsequently assayed for casein hydrolysis under different conditions. This protein was classified as peptidase M48 family and subclan gluzincin. It was predicted to contain one transmembrane domain by TMpred. Optimal protein expression was achieved by induction with 0.6 mM isopropyl thiogalactoside (IPTG) at 25 °C for six hours. YggG was purified as soluble protein and confirmed to be proteolytically active under the presence of 1.25 mM zinc acetate and showed optimum activity at 37 °C and pH 7.4. We confirmed for the first time that the yggG gene product is a zinc-dependent metalloprotease.
Using the chemical reduction method, silver nanoparticles (Ag NPs) were effectively synthesized into the zeolite framework in the absence of any heat treatment. Zeolite, silver nitrate, and sodium borohydride were used as an inorganic solid support, a silver precursor, and a chemical reduction agent, respectively. Silver ions were introduced into the porous zeolite lattice by an ion-exchange path. After the reduction process, Ag NPs formed in the zeolite framework, with a mean diameter of about 2.12-3.11 nm. The most favorable experimental condition for the synthesis of Ag/zeolite nanocomposites (NCs) is described in terms of the initial concentration of AgNO(3). The Ag/zeolite NCs were characterized by ultraviolet-visible spectroscopy, powder X-ray diffraction, transmission electron microscopy, scanning electron microscopy, energy dispersive X-ray fluorescence, and Fourier transform infrared. The results show that Ag NPs form a spherical shape with uniform homogeneity in the particle size. The antibacterial activity of Ag NPs in zeolites was investigated against Gram-negative bacteria (ie, Escherichia coli and Shigella dysentriae) and Gram-positive bacteria (ie, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus) by disk diffusion method using Mueller-Hinton agar at different sizes of Ag NPs. All of the synthesized Ag/zeolite NCs were found to have antibacterial activity. These results show that Ag NPs in the zeolite framework can be useful in different biological research and biomedical applications.
A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 microg AHL h(-1) per 10(9) CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif (106)HXDH-59 amino acids-H(169)-21 amino acids-D(191) for N-acylhomoserine lactone lactonases.
In this study, antibacterial characteristic of silver/poly (lactic acid) nanocomposite (Ag/PLA-NC) films was investigated, while silver nanoparticles (Ag-NPs) were synthesized into biodegradable PLA via chemical reduction method in diphase solvent. Silver nitrate and sodium borohydride were respectively used as a silver precursor and reducing agent in the PLA, which acted as a polymeric matrix and stabilizer. Meanwhile, the properties of Ag/PLA-NCs were studied as a function of the Ag-NP weight percentages (8, 16, and 32 wt% respectively), in relation to the use of PLA. The morphology of the Ag/PLA-NC films and the distribution of the Ag-NPs were also characterized. The silver ions released from the Ag/PLA-NC films and their antibacterial activities were scrutinized. The antibacterial activities of the Ag/PLA-NC films were examined against Gram-negative bacteria (Escherichia coli and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by diffusion method using Muller-Hinton agar. The results indicated that Ag/PLA-NC films possessed a strong antibacterial activity with the increase in the percentage of Ag-NPs in the PLA. Thus, Ag/PLA-NC films can be used as an antibacterial scaffold for tissue engineering and medical application.
This study was carried out to evaluate the antibacterial activity of aqueous and organic extracts of Thymus capitatus L. (Lamiaceae) leaves and stems. Dried ground powder leaves and stems were extracted with water (aqueous extracts), ethanol, dichloromethane and hexane (Soxhlet extracts). The antibacterial activity of these extracts was evaluated against bacteria using disc diffusion method. The result obtained showed that the leaves had stronger antibacterial activity than the stems extracts. The ethanolic extract had the highest yield products and the high antibacterial activity than all other solvents. The results suggest that essential oil as non-polar organic compounds could be the main active compounds in this plant. Therefore the antibacterial activity of leaves ethanol extracts (LEE) was compared with essential oils leaves extracts (LEO) of T. capitatus. The LEO showed greater antibacterial activity than LEE. The LEO showed a broad spectrum of antibacterial activity and the Pseudomonas aeruginosa was the most sensitive bacteria.
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
A minisequencing method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was developed for rapid identification of single nucleotide polymorphisms at bla(TEM) gene codons 104, 164 and 238 associated with extended-spectrum activity on TEM-type beta-lactamases. The method was validated by testing the Escherichia coli and Klebsiella pneumoniae strains possessing the known bla(TEM) gene sequences.
To assess the antibacterial and antifungal properties of human cerumen by studying its effect on the growth of Staphylococcus aureus, Esherichia coli, Pseudomonas aeruginosa and Candida albicans.
Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.
In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+)-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.