Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over "Wider pH" Range.

Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a more comprehensive pH range, particularly in alkaline conditions over pH 8, has not been fully developed. On the other hand, enzyme immobilization following a suitable protocol is a long pending issue that determines the conformational stability, specificity, selectivity, enantioselectivity, and activity of the native enzymes at long-range pH. As a bridge between these two findings, in an attempt at a freezing temperature 273-278 K at an alkaline pH, the diazo-functionalized silica gel (SG) surface has been used to rapidly diazo couple pepsin through its inert center, the O-carbon of the phenolic -OH of surface-occupied Tyr residues in a multipoint mode: when all the various protein groups, viz., amino, thiol, phenol, imidazole, carboxy, etc., in the molecular sequence including those belonging to the active sites, remain intact, the inherent inbuilt interactions among themselves remain. Thereby, the macromolecule's global conformation and helicity preserve the status quo. The dimension of the SG-enzyme conjugate confirms as {Si(OSi)4 (H2O)1.03}n {-O-Si(CH3)2-O-C6H4-N═N+}4·{pepsin}·yH2O; where the values of n and y have been determined respectively as 347 and 188. The material performs the catalytic activity much better at 7-8.5 than at pH 2-3.5 and continues for up to six months without any appreciable change.

'Urease immobilized single-kit' for sensing of thiourea-glucose pair employing fluorescence 'Turn off - Turn on' and as an efficient sorbent for selective sample cleanup of thiourea.

The tenfold lowering in binding energy for TU-Tyrosine in immobilized urease (Kb: 4.7 × 103) with respect to the native enzyme (Kb: 6.5 × 104) begets easy desorption of thiourea (TU) by glucose (GL) with an eventual formation of a more strong TU- GL adduct; that rejuvenates the kit-material ready for the subsequent cycle(s). The sorption-desorption heeds fluorescence turn-off and turn-on in DCM for selective sensing of TU- GL pair at their respective linear range of concentration 2.5-26.1 ppm and 2.36-11.57 ppm. The process was found to be static (KSV ≥ 2.25 × 103 L mol-1), exothermic (ΔH: -0.08 kJ mol-1), spontaneous (ΔG: -21.1 kJ mol-1) and marginally entropy gaining (ΔS: 0.07 kJ mol-1 K-1). The 'bulk material' (200 ± 20 µm) brilliantly preconcentrates TU with an enrichment factor of 106.2 after its selective extraction at near-neutral pH from a large volume sample (800 mL) of low concentration (30 ppm). A very dilute solution (0.05 mmol L-1) of GL at minimum volume (6 mL) acts as a stripping agent and provides a longer life (200 cycles with good extraction efficiency) to the material. The method was found to be efficient in the analysis of fruit juice as a real sample.

Exuberant Immobilization of Urease on an Inorganic SiO2 Support Enhances the Enzymatic Activities by 3-fold for Perennial Utilization.

Urease has been covalently immobilized on a 3-D networking silica gel (SG) using dimethyldichlorosilane (DMDCS) as second generation silane coupling reagent and m-nitroaniline as linker component in a robust methodology and subsequently characterized as [{Si(OSi)4(H2O)0.05}205.2] n=4{OSi(CH3)2-NH-C6H4-N═N-urease}·282.5H2O (molecular mass 263 445 g or 263.4 kDa). Selective coupling of tyrosine residue with an identifiable m-nitroaniline modified SG unit prevents enzyme-enzyme cross-linking leading to enhancement of enzymatic activity. The material worked at room temperature and its activity (luminescent and ammonia releasing efficiency) was enhanced by 3-fold (for both synthetic and real sample) compared to native enzyme values at neutral pH. Up to 30 days and 30 cycles, this 3-fold activity remains as such but reduces gradually to native enzyme level after 60 days and 60 cycles of reuse.

Solid phase extraction, separation and preconcentration of rare elements thorium(IV), uranium(VI), zirconium(IV), cerium(IV) and chromium(III) amid several other foreign ions with eriochrome black T anchored to 3-D networking silica gel.

The present work reports the systematic studies on extraction, separation and preconcentration of Th(IV), U(VI), Zr(IV), Ce(IV) and Cr(III) amid several other foreign ions using EBT anchored {SiO2}n3-D microarray. The effect of various sorption parameters, such as pH, concentration, temperature, sample volume, flow-rate and co-existing foreign ions were investigated. Quantitative sorption was ensured at solution pH: 6.0-6.5 for Th(IV), Ce(IV), Cr(III) and pH: 2.75-3.0 for Zr(IV), U(VI) couple. Analysis on extracted species and extraction sites reveals that [Th4(µ(2)-OH)8(H2O)4](8+), [Ce6(µ(2)-OH)12(H2O)5](12+), [Cr3(µ(2)-OH)4(H2O)](5+), [(UO2)3(µ(2)-OH)5(H2O)3](+) and [Zr4(µ(2)-OH)8(H2O)0.5](8+) for the respective metal ions gets extracted at HOMO of the extractor. HOMO-{metal ion species} was found to be 1:1 complexation. Sorption was endothermic, entropy-gaining, instantaneous and spontaneous in nature. A density functional theory (DFT) calculation has been performed to analyze the 3-D structure and electronic distribution of the synthesized extractor.

Polypyrimidine tract binding protein (PTB) associates with intronic and exonic domains to squelch nuclear export of unspliced RNA.

Retention of unspliced pre-messenger RNA (pre-mRNA) in the nucleus is essential for cell survival. Available nuclear factors must recognize and discern between diverse export signals present in pre-mRNA to establish an export inhibitory complex. We found that polypyrimidine domains present in both intron and exon were important for export inhibition of a minigene transcript based on hepatitis B virus pregenomic RNA. Overexpression of PTB drastically reduced export and presence of RRM4 domain seemed critical. This inhibitory network overrode stimulation from an exonic export-facilitating element. We posit that binding of PTB to multiple loci on pre-mRNA regulates nuclear retention.

Immunofiltration assay for aflatoxin B1 based on the separation of pre-immune complexes.

A new approach for quantitative determination of AFB1 based on the separation of pre-immune complexes in the same immunoassay system has been developed. No additional step for the separation of pre-immune complexes is required. The method uses a test device for separation of pre-immune complexes from the free AFB1-enzyme conjugate by filtration through the membrane strips spotted with anti-AFB1 antibody. The bound enzyme conjugate was visualized by super-catalyzed reporter deposition (Super-CARD) signal amplification method. The measured signal intensity is directly proportional to the amount of AFB1 present in the sample. The detection limit obtained by the present method was 15 pg/ml. The data on the analytical parameters indicate that the new format of AFB1 detection in foodstuffs is reproducible, accurate and specific. The method is user friendly and does not require any costly equipment or a well-equipped laboratory.

Identification of a unique splicing regulatory cluster in hepatitis B virus pregenomic RNA.

HBV particles with genome derived from spliced mRNAs accumulate in patients with virus-derived severe liver necrosis and fibrosis. We investigated the role of an intronic element (intronic splicing silencer-long, ISS(L)) on splicing of HBV minigene transcripts. Removal of the entire ISS(L) showed two-fold increase in splicing while shorter deletions within ISS(L) indicated isolated clusters of activator and repressor domains. Activator domains stimulated splicing in presence of PRE, a long HBV 3' exon and even when present in a heterologous context. Mutations in the repressor domain unexpectedly augmented repression. The role of this intronic splicing regulatory element could be important for HBV pathogenesis.

Filtration-based staining of proteins on membranes.

A filtration-based staining method has been developed for sensitive estimation of proteins in a batch of samples. It is based on focused absorption of an applied protein standard (or sample) and dye solution on nitrocellulose membrane through an aqueous network of capillary channels formed between the membrane and a wetted filter paper. The method does not require any equipment for creating vacuum. Compared with the conventional pouring methods, the new approach reduces the consumption of staining solution and lowers the staining and destaining times (from 1.5 h to approximately 10 min) without compromising the sensitivity.

Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples.

Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B(1) (AFB(1)) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB(1) and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 microg kg(-1) for AFB(1) and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB(1) and OA in chili with accuracy and precision. The AFB(1) and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.

Filtration-based tyramide amplification technique--a new simple approach for rapid detection of aflatoxin B1.

The catalyzed reporter deposition (CARD) method of signal amplification, also called "tyramide signal amplification", has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B(1) (AFB(1)) in a variety of foodstuffs with a detection limit of 12.5 microg kg(-1). The assay procedure involves sequential addition of standards or sample, AFB(1)-horseradish peroxidase (HRP) conjugate, B-T, avidin-HRP, and substrate solution over anti-AFB(1) antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB(1) at concentrations from 25 to 100 mg kg(-1) were between 95 and 105%. AFB(1) results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.

In situ sample cleanup during immunoassay: a simple method for rapid detection of aflatoxin B, in food samples.

A strategy for rapid in situ elimination of interfering substances that are present in extracts of food samples during assay is described in this article. The novel feature of this method is that the sample purification is carried out as a part of the assay, and a separate sample cleanup step is not required. The assay procedure involves the sequential addition of standard or sample, cleaning solutions, and aflatoxin B1-horseradish peroxidase conjugate (AFB1-HRP) over antibody-spotted zones of a membrane, and 3,3'-diaminobenzidine was used as the substrate for visualization. We have determined that trifluoroacetic acid and propionic acids at concentrations of 100 mM are highly effective for cleaning groundnut, wheat, corn, and poultry feed samples and that NaHCO3 (100 mM) is successful in cleaning processed soybean. In all cases, subsequent washing was performed with phosphate-buffered saline solution to facilitate the removal of traces of adhering interfering substances. A batch of 12 samples can be analyzed within 8 min either by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards or, more precisely, by densitometry. The method was tested for the analysis of AFB1 in groundnut, wheat, corn, processed soybean, chili, and poultry feed. The detection limit obtained was 5 microg/kg, except for chili, where it was 10 microg/kg. The average recoveries from different noninfected food samples spiked with AFB1 at concentrations of 5 to 100 microg/kg were between 99 and 105%. The values obtained for infected corn and groundnut samples correlated well with the estimates obtained by high-pressure liquid chromatography. The absence of a sample extraction step reduces the cost and labor involved in the assay. The method may be potentially applicable to the assay of other mycotoxins and environmental pollutants.