The utilization of Triton X-100 for enhanced two-dimensional liquid-phase proteomics.

One of the main challenges in proteomics lies in obtaining a high level of reproducible fractionation of the protein samples. Automated two-dimensional liquid phase fractionation (PF2D) system manufactured by Beckman Coulter provides a process well suited for proteome studies. However, the protein recovery efficiency of such system is low when a protocol recommended by the manufacturer is used for metaproteome profiling of environmental sample. In search of an alternative method that can overcome existing limitations, this study replaced manufacturer's buffers with Triton X-100 during the PF2D evaluation of Escherichia coli K12. Three different Triton X-100 concentrations-0.1%, 0.15%, and 0.2%-were used for the first-dimension protein profiling. As the first-dimension result was at its best in the presence of 0.15% Triton X-100, second-dimension protein fractionation was performed using 0.15% Triton X-100 and the standard buffers. When 0.15% Triton X-100 was used, protein recovery increased as much as tenfold. The elution reliability of 0.15% Triton X-100 determined with ribonuclease A, insulin, α-lactalbumin, trypsin inhibitor, and cholecystokinin (CCK) affirmed Triton X-100 at 15% can outperform the standard buffers without having adverse effects on samples. This novel use of 0.15% Triton X-100 for PF2D can lead to greater research possibilities in the field of proteomics.

The antifouling potentiality of galactosamine characterized from Vibrio vulnificus exopolysaccharide.

To gain a better insight into biofilm composition, the exopolysaccharide (EPS) of the Gram-negative bacterium Vibrio vulnificus was studied. Monosaccharide composition analysis of the wild-type and mutant V. vulnificus EPS carried out with Bio-liquid chromatography revealed the presence of D-glucosamine, D-galactose, D-glucose and D-xylose in both strains. D-galactosamine was found only in the mutant that formed less biofilm compared to its wild-type. The influence of galactosamine on biofilm formation was then studied by adding this substance gradually to six different Gram-negative/positive bacteria associated with various autoinducers. Four bacterial species known to use the autoinducer type-2 signaling system produced less biofilm in the presence of galactosamine. No significant inhibition of biofilm formation was observed in bacteria that produce autoinducer type-1 signal molecules. Galactosamine was also immobilized on polymeric nanofibers to determine its re-usability for the study of biofilm inhibition. The immobilized galactosamine retained >65% of its initial antifouling activity after 10 repeated uses. The results of this study suggest the antifouling role of galactosamine for bacteria that produce AI-2.

Biochemical characterization and molecular docking analysis of novel esterases from Sphingobium chungbukense DJ77.

We identified three novel microbial esterase (Est1, Est2, and Est3) from Sphingobium chungbukense DJ77. Multiple sequence alignment showed the Est1 and Est3 have distinct motifs, such as tetrapeptide motif HGGG, a pentapeptide sequence motif GXSXG, and catalytic triad residues Ser-Asp-His, indicating that the identified enzymes belong to family IV esterases. Interestingly, Est1 exhibited strong activity toward classical esterase substrates, p-nitrophenyl ester of short-chain fatty acids and long-chain. However, Est3 did not exhibit any activity despite having high sequence similarity and sharing the identical catalytic active residues with Est1. Est3 only showed hydrolytic degradation activity to polycaprolactone (PCL). MOE-docking prediction also provided the parameters consisting of binding energy, molecular docking score, and molecular distance between substrate and catalytic nucleophilic residue, serine. The engineered mutEst3 has hydrolytic activity for a variety of esters ranging from p-nitrophenyl esters to PCL. In the present study, we demonstrated that MOE-docking simulation provides a valuable insight for facilitating biocatalytic performance.

Isolation and characterization of ethanol-producing Schizosaccharomyces pombe CHFY0201.

Ethanol-producing yeast strain, CHFY0201 was isolated from soil in South Korea using an enrichment technique in a yeast peptone dextrose medium supplemented with 5% (w/v) ethanol at 30 degrees C. The phenotypic and physiological characteristics, as well as molecular phylogenetic analysis based on the D1/D2 domains of the large subunit (26S) rDNA gene and the internally transcribed spacer (ITS) 1+2 regions suggested that the CHFY0201 was novel strain of Schizosaccharomyces pombe. During shaking flask cultivation, the highest ethanol productivity and theoretical yield of S. pombe CHFY0201 in YPD media containing 9.5% total sugars was 0.59 +/- 0.01 g/l/h and 88.4 +/- 0.91%, respectively. Simultaneous saccharification and fermentation for ethanol production was carried out using liquefied cassava (Manihot esculenta) powder in a 5 l lab-scale jar fermenter at 32 degrees C for 66 h with an agitation speed of 120 rpm. Under these conditions, S. pombe CHFY0201 yielded a final ethanol concentration of 72.1 +/- 0.27 g/l and a theoretical yield of 82.7 +/- 1.52% at a maximum ethanol productivity of 1.16 +/- 0.07 g/l/h. These results suggest that S. pombe CHFY0201 is a potential producer for industrial bioethanol production.

Immobilization of cross-linked lipase aggregates onto magnetic beads for enzymatic degradation of polycaprolactone.

Candida rugosa lipase was immobilized on amino-functionalized magnetic supports via cross-linked enzyme aggregates (CLEA) and used to enhance the enzymatic degradation of polycaprolactone (PCL). The maximum amounts of lipase immobilized on the magnetic beads using glutaraldehyde as a coupling agent were determined to be 33.7 mg/g of beads with an 81% recovery of activity after immobilization. Compared to the free enzyme, the immobilized lipase showed the optimum pH at 1 unit higher (pH 8.0) and also retained its enzymatic activity at higher temperatures. There was 62.9% retention of lipase activity after 30 consecutive reuses, indicating its stability and reusability in aqueous media. Moreover, the immobilized lipase maintained more than 80% of its initial activity during 30 days storage period, while the free lipase lost all under same condition. In addition, the immobilized lipase showed a more than 6-fold increase in biodegradability over the free lipase when the immobilized lipase was used to degrade PCL in a batch system. Higher thermal and storage stability, as well as good durability after repeated use of the immobilized lipase CLEA, highlights its potential applicability as large scale continuous systems for the enzymatic degradation of PCL.

Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift.

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.

Degradation of diphenyl phthalate by Sphingomonas chungbukensis.

More than 80% of diphenyl phthalate (DPP) at 100 mg l(-1) was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30 degrees C within 48 h. The maximum specific degradation rate was 5 mg DPP l(-1 )h(-1). It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.

Aptabody-aptatope interactions in aptablotting assays.

We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein-aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody-aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.

Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics.

Preventing the formation of positive transcription elongation factor b by human cyclin T1-binding RNA aptamer for anti-HIV transcription.

OBJECTIVE: Development of an innovative antitranscriptional technique for HIV. DESIGN: Systematic evolution of ligands by exponential enrichment (SELEX) technique that can characterize target-specific aptamer was employed to synthesize an aptamer that binds human cyclin T1 (CycT1). When CycT1-binding aptamer interferes the binding of cyclin-dependent kinase 9 (Cdk9) to CycT1, HIV transcription is likely to be discouraged. METHODS: Throughout SELEX steps, RNA aptamers having high specific affinity toward CycT1 were characterized. The binding interaction between selected aptamers and CycT1 was analyzed via various techniques. RESULTS: Both qualitative and quantitative analyses revealed Apt4 aptamer, among four candidates, has the highest specific affinity to CycT1. In the presence of Apt4, Cdk9 protein was unable to make interaction with CycT1. CONCLUSION: A specific RNA aptamer that identifies and binds to CycT1 with high affinity was successfully characterized. As CycT1 plays an important role in HIV transcription, this novel method that interferes and inhibits the transcription of HIV has the potential of being exploited in extended research fields, such as clinical therapy.