Immobilization of Pseudomonas delafieldii with magnetic polyvinyl alcohol beads and its application in biodesulfurization

Pseudomonas delafieldii was immobilized in magnetic polyvinyl alcohol (PVA) beads using a hydrophilic magnetic fluid, which was prepared by a co-precipitation method. The beads had distinct super-paramagnetic properties and were compared with immobilized cells in non-magnetic PVA beads. Their desulfurizing activity was increased slightly from 8.7 to 9 mmol sulfur kg(-1) (dry cell) h(-1). The main advantages was that the magnetic immobilized cells maintain a high desulfurization activity and remain in good shape after 7 times of repeated use, while the non-magnetic immobilized cells could only be used for 5 times. Furthermore, the magnetic immobilized cells could be easily collected or separated magnetically from the biodesulfurization reactor.     

Rhamnolipid production by Pseudomonas aeruginosa immobilised in polyvinyl alcohol beads

A marine bacterium, Pseudomonas aeruginosa BYK-2 (KCTC 18012P), was immobilised by entrapment in 10% (w/v) polyvinyl alcohol beads and optimized for the continuous production of rhamnolipid. The relative activity of rhamnolipid production was maintained at 80 approximately 90% of the initial production during 15 cycles in a repeated batch culture. Continuous culture was performed in a 1.8 1 airlift bioreactor, yielding 0.1 g rhamnolipid h(-1) at a dilution rate of 0.0 18 h(-1), 25 degrees C, initial pH 7, and 0.5 vvm aeration rate with a 1.21 working volume.     

The use of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Biodegradation of high concentration of nitrobenzene by Pseudomonas corrugata embedded in peat-phosphate esterified polyvinyl alcohol

Efficiency on biodegradation of high concentration of nitrobenzene (NB) by peat-phosphate esterified polyvinyl alcohol-embedded NB-degrading bacteria Pseudomonas corrugata was conducted compared to free bacteria cells. Its biodegradation kinetics, reuse ability, degradation effect in the absence of the essential element needed for the growth of bacteria and degradation efficiency of the raw water from the contaminated site were also invested. Results show that the degradation rate when the concentration of NB was at 600, 750, and 900 mg/L reached 91.02, 83.23, and 55.9 %, which was higher than that observed in free bacteria at the same concentration levels. Biodegradation kinetics of the material could be well described by first- and zero-order kinetics when the concentration of NB was at 300, 450 mg/L and 600, 750, 900 mg/L, respectively. Stable degradation activity (stayed at a level of approximately 70 %) was displayed during the 11th repeat-batch experiment. The affect of absence of phosphorus in the medium can be abated ascribed to the addition of peat, which contributes with organic matter and other elements such as nitrogen and phosphorus necessary to maintain metabolically active the microorganisms. Effective biodegradation of the raw water from the experimental site revealed that the material can be a potential candidate for treating NB-contaminated wastewater in the practical setting.     

Modification of cellulosic fabric using polyvinyl alcohol, part-II: Colorfastness properties

A series of aqueous solutions of poly(vinyl alcohol) of various commercial products were prepared and applied onto the surfaces of cotton and blends of cotton/polyester fabrics. Fourier transform infrared spectrophotometer was used to confirm the molecular structure of the polyvinyl alcohol used. Performance tests such as colorfastness to rubbing (dry and wet) and colorfastness to washing were determined. The controlling variables affecting the performance properties of the finished substrate such as post-treatment with poly(vinyl alcohol) of various commercial trades, concentration and dilutions were studied. Crocking, washing and hue change of the treated dyed and printed fabrics is accompanied by the formation of semi-inter-penetrated network structure due to the presence of the hydroxyl (-OH) groups which make feasible to a number of grafting and physical cross linking reactions of polymer backbone.     

Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy

This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8 μm diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

Cr(VI) reduction by Pseudomonas aeruginosa immobilized in a polyvinyl alcohol/sodium alginate matrix containing multi-walled carbon nanotubes

Pseudomonas aeruginosa (P. aeruginosa) was immobilized with polyvinyl alcohol (PVA), sodium alginate and multiwalled carbon nanotubes (MCNTs). After immobilization, the beads were subjected to freeze-thawing to enhance mechanical strength. When exposed to 80 mg/L Cr(VI), the immobilized bacteria were able to reduce 50% of them in 84 h, however the free cells were deactivated at this concentration. The beads were used to reduce 50 mg/L Cr(VI) for nine times, with the reduction efficiency above 90% in the first five times and 65% in the end.     

S genotyping in Japanese plum and sweet cherry by allele-specific hybridization using streptavidin-coated magnetic beads

Key message

We report a rapid and reliable method for S genotyping of Rosaceae fruit trees, which would to be useful for successful planting of cross-compatible cultivars in orchards.

Abstract

Japanese plum (Prunus salicina) and sweet cherry (Prunus avium), belonging to the family Rosaceae, possess gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, S-RNase and SFB (S-haplotype-specific F-box gene). For successful planting of cross-compatible cultivars of Rosaceae fruit trees in commercial orchards, it is necessary to obtain information on S genotypes of cultivars. Recently, a method of dot-blot analysis utilizing allele-specific oligonucleotides having sequences of SFB-HVa region has been developed for identification of S haplotypes in Japanese plum and sweet cherry. However, dot-blot hybridization requires considerable time and skill for analysis even of a small number of plant samples. Thus, a quick and efficient method for S genotyping was developed in this study. In this method, instead of a nylon membrane used for dot-blot hybridization, streptavidin-coated magnetic beads are used to immobilize PCR products, which are hybridized with allele-specific oligonucleotide probes. Our improved method allowed us to identify 10 S haplotypes (S-a, S-b, S-c, S-d, S-e, S-f, S–h, S-k, S-7 and S-10) of 13 Japanese plum cultivars and 10 S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) of 13 sweet cherry cultivars utilizing SFB or S-RNase gene polymorphism. This method would be suitable for identification of S genotypes of a small number of plant samples.     

l-Dopa synthesis using tyrosinase immobilized on magnetic beads

Magnetic beads were prepared via suspension polymerization of glycidyl methacrylate (GMA) and methyl methacrylate (MMA) in the presence of ferric ions. Following polymerization, thermal co-precipitation of the Fe(III) ions in the beads with Fe(II) ions under alkaline condition resulted in encapsulation of Fe₃O₄ nano-crystals within the polymer matrix. The magnetic beads were activated with glutaraldehyde, and tyrosinase enzyme was covalently immobilized on the support via reaction of amino groups under mild conditions. The immobilized enzyme was used for the synthesis of l-Dopa (1-3,4-dihydroxy phenylalanine) which is a precursor of dopamine. The immobilized enzyme was characterized by temperature, pH, operational and storage stability experiments. Kinetic parameters, maximum velocity of the enzyme (V_max) and Michaelis–Menten constant (K_m) values were determined as 1.05 U/mg protein and 1.0 mM for 50–75 μm and 2.00 U/mg protein and 4.0 mM for 75–150 μm beads fractions, respectively. Efficiency factor and catalytic efficiency were found to be 1.39 and 0.91 for 75–150 μm beads and 0.73 and 0.75 for 50–75 μm beads fractions, respectively. The catalytic efficiency of the soluble tyrosinase was 0.37. The amounts of immobilized protein were on the 50–75 μm and 75–150 μm fractions were 2.7 and 2.8 mg protein/g magnetic beads, respectively.     

Ammonia removal from livestock wastewater by ammonia-assimilating microorganisms immobilized in polyvinyl alcohol

We isolated ammonia-assimilating microorganisms from the livestock manure treatment systems and evaluated their ammonia-assimilating ability. Many isolates utilized ammonia at high rates when they were purely cultivated in a nitrogen-limited medium to which sterilized lagoon extract had been added. Some isolates that were immobilized in polyvinyl alcohol (PVA) utilized ammonia present in the media containing viable lagoon microorganisms. Staining with 4′,6′-diamidino-2-phenylindole (DAPI) indicated that the immobilized high ammonia-assimilating isolates grew dominantly within the PVA beads. High ammonia-assimilating isolates in the mixed culture containing viable lagoon microorganisms were identified as Pseudomonas spp. and member of Rhizobiaceae species by partial sequencing of the 16S ribosomal DNA.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Repeated alcohol administration during adolescence causes changes in the mesolimbic dopaminergic and glutamatergic systems and promotes alcohol intake in the adult rat

Adolescence is a developmental period which the risk of drug and alcohol abuse increases. Since mesolimbic dopaminergic system undergoes developmental changes during adolescence, and this system is involved in rewarding effects of drugs of abuse, we addressed the hypothesis that ethanol exposure during juvenile/adolescent period over-activates mesolimbic dopaminergic system inducing adaptations which can trigger long-term enduring behavioural effects of alcohol abuse. We treated juvenile/adolescent or adult rats with ethanol (3 g/kg) for two-consecutive days at 48-h intervals over 14-day period. Here we show that intermittent ethanol treatment during the juvenile/adolescence period alters subsequent ethanol intake. In vivo microdialysis demonstrates that ethanol elicits a similar prolonged dopamine response in the nucleus accumbens of both adolescent and adult animals pre-treated with multiple doses of ethanol, although the basal dopamine levels were higher in ethanol-treated adolescents than in adult-treated animals. Repeated ethanol administration also down-regulates the expression of DRD2 and NMDAR2B phosphorylation in prefrontal cortex of adolescent animals, but not of adult rats. Finally, ethanol treatment during adolescence changes the acetylation of histones H3 and H4 in frontal cortex, nucleus accumbens and striatum, suggesting chromatin remodelling changes. In summary, our findings demonstrate the sensitivity of adolescent brain to ethanol effects on dopaminergic and glutamatergic neurotransmission, and suggest that abnormal plasticity in reward-related processes and epigenetic mechanisms could contribute to the vulnerability of adolescents to alcohol addiction.     

Allele-specific hybridization using streptavidin-coated magnetic beads for species identification, S genotyping, and SNP analysis in plants

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase (SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

A purification method of the diagnostic enzyme Bacillus uricase using magnetic beads and non-specific protease

A simple purification method of theBacillus uricase (Uao) was newly developed. The gene coding for Uao with a C-terminal 6-histidine tag (Uao-HT) was constructed and overexpressed. Using the non-specific proteases, such as proteinase K, the tag was easily removed because Uao-HT includes its C-terminal region to be specifically cleaved by them. Such treatment of Uao-HT with the proteases did not affect its enzymatic properties and enabled us to purify it from the crude extract with a single-step protocol; the cell lysate containing Uao-HT was mixed with the Ni ion-chelating magnetic beads and then the adsorbed enzyme was eluted with the proteinase K-containing buffer after untagged proteins were washed out. The isolated enzyme yielded a single band on SDS–PAGE and was fully active. This method is extremely useful for high-throughput purification of mutants because of compatibility with automation.     

Diagnostic model of saliva peptide finger print analysis of oral squamous cell carcinoma patients using weak cation exchange magnetic beads

Saliva diagnostics utilizing nanotechnology and molecular technologies to detect oral squamous cell carcinoma (OSCC) has become an attractive field of study. However, no specific methods have been established. To refine the diagnostic power of saliva peptide fingerprints for the early detection of OSCC, we screened the expression spectrum of salivary peptides in 40 T1 stage OSCC patients (and healthy controls) using MALDI-TOF-MS combined with magnetic beads. Fifty proteins showed significantly different expression levels in the OSCC samples (P<0.05). Potential biomarkers were also predicted. The novel diagnostic proteomic model with m/z peaks of 1285.6 Da and 1432.2 Da are of certain value for early diagnosis of OSCC.     

Short communication: Methylparathion degradation by Pseudomonas sp. A3 immobilized in sodium alginate beads

An organophosphate-degrading soil isolate of Pseudomonas sp. A3, immobilized at 5% (wet wt/v) cell mass in 3% (w/v) sodium alginate beads, detoxified 99% of 1 mm methylparathion in 48 h. The beads were re-usable for five batches, the sixth batch only giving 73% methylparathion removal.