Response Surface Optimization for Decaffeination and Theophylline Production by Fusarium solani.

Coffee processing industries generate caffeine-containing waste that needs to be treated and decaffeinated before being disposed. Five fungal isolates obtained on caffeine-containing mineral media were tested for their ability to utilize caffeine at high concentrations. An isolate identified as Fusarium solani could utilize caffeine as a sole source of carbon and nitrogen up to 5 g/l and could degrade it to an extent of 30-53 % in 120 h. Sucrose that was added as an auxiliary substrate (5 g/l) enhanced the biodecaffeination of caffeine to 88 % in 96 h. The addition of co- substrate (sucrose) not only resulted in higher biodecaffeination efficiency, but also reduced the incubation period from the initial 120 to 96 h. Theophylline and 3-methyl xanthine were obtained as the major metabolites of decaffeination at 96 and 120 h, respectively. Response surface methodology used to optimize the process parameters for maximum biodecaffeination as well as theophylline production showed that a pH of 5.8, temperature of 24 °C and inoculum size of 4.8 × 10(5) spores/ml have resulted in a complete biodecaffeination of caffeine as well as the production of theophylline with a yield of 33 % (w/w). Results thus show that a viable and sustainable process can be developed for the detoxification of caffeine along with the recovery of theophylline, a commercially important chemical.

Methylcobalamin--a form of vitamin B12 identified and characterised in Chlorella vulgaris.

Vitamin B12 is among the most essential biomolecules required for crucial metabolic processes in humans. Vitamin B12 was extracted from Chlorella vulgaris biomass under aqueous conditions, partially purified by passing the extract through amberlite XAD-2, Sep-Pak columns, and further purified by HPLC. The target peak eluent was subjected to characterisation by tandem mass spectrometry (MS/MS), selected ion recording (SIR) and multiple reaction monitoring (MRM) and identified as methylcobalamin (Me-Cbl). Quantification of Me-Cbl was carried out by microbiological and chemiluminescence methods, and found to be 29.87±2 µg/100 g and 26.84±2 µg/100 g dry weight, respectively. The presence of Me-Cbl was further substantiated using gold nanoparticle (AuNPs) based aptamer analysis, and found to be 28.02±2 µg/100 g dry weight. Good similarity was observed among all the methods. Methylcobalamin, a form of vitamin B12 was identified in C. vulgaris and this finding enhances its use as a nutritional supplement.

Quantum dots as optical labels for ultrasensitive detection of polyphenols.

Considering the fact that polyphenols have versatile activity in-vivo, its detection and quantification is very much important for a healthy diet. Laccase enzyme can convert polyphenols to yield mono/polyquinones which can quench Quantum dots fluorescence. This phenomenon of charge transfer from quinones to QDs was exploited as optical labels to detect polyphenols. CdTe QD may undergo dipolar interaction with quinones as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, "turn-off" fluorescence method was applied for ultrasensitive detection of polyphenols by using laccase. We observed proportionate quenching of QDs fluorescence with respect to polyphenol concentration in the range of 100 µg to 1 ng/mL. Also, quenching of the photoluminescence was highly efficient and stable and could detect individual and total polyphenols with high sensitivity (LOD-1 ng/mL). Moreover, proposed method was highly efficient than any other reported methods in terms of sensitivity, specificity and selectivity. Therefore, a novel optical sensor was developed for the detection of polyphenols at a sensitive level based on the charge transfer mechanism.

Quantum dots as nano plug-in's for efficient NADH resonance energy routing.

The routing of fluorescent signals from NADH to quantum dots (QDs) has been a subject of extensive research for FRET based applications. In the present study, the spectral cross talk of NAD(+)/NADH with QDs was used to monitor the reaction of NAD(+)-dependent dehydrogenase enzyme. CdTe QD may undergo dipolar interaction with NADH as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, non-radiative energy transfer can take place from NADH to CdTe QD enhancing QDs fluorescence. Energy routing assay of NADH-QD was applied for detection of formaldehyde as a model analyte in the range 1000-0.01 ng/mL by the proposed technique. We observed proportionate quenching of CdTe QD fluorescence by NAD(+) and enhancement in the presence of NADH formed by various concentrations of enzyme (0.028-0.4 U). Hence, it was possible to detect formaldehyde in the range 1000-0.01 ng/mL with a limit of detection (LOD) at 0.01 ng/mL and regression coefficient R(2)=0.9982. Therefore, a unique optical sensor was developed for the detection of the formaldehyde in sensitive level based on the above mechanism. This method can be used to follow the activity of NAD(+)-dependent enzymes and detection of dehydrogenases in general.

A membrane protein based biosensor: use of a phosphate--H+ symporter membrane protein (Pho84) in the sensing of phosphate ions.

A label free biosensor for direct detection of inorganic phosphate based on potential-step capacitance measurements has been developed. The high-affinity Pho84 plasma membrane phosphate/proton symporter of Saccharomyces cerevisiae was used as a sensing element. Heterologously expressed and purified Pho84 protein was immobilized on a self-assembled monolayer (SAM) on a capacitance electrode. Changes in capacitance were recorded upon exposure to phosphate compared to the control substance, phosphate analogue methylphosphonate. Hence, even without the explicit use of lipid membranes, the Pho84 membrane protein could retain its capacity of selective substrate binding, with a phosphate detection limit in the range of the apparent in vivo K(m). A linear increase in capacitance was monitored in the phosphate concentration range of 5-25 µM. The analytical response of the capacitive biosensor is in agreement with that the transporter undergoes significant conformational changes upon exposure to inorganic phosphate, while exposure to the analogue only causes minor responses.

Purification, identification, and characterization of methylcobalamin from Spirulina platensis.

The present study reports methylcobalamin in Spirulina platensis using high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), microbiological assay, chemiluminescence assay, liquid chromatography-mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS). Extraction of vitamin B12 from S. platensis was carried out without using cyanide. Partial purification was achieved using Amberlite XAD-2 followed by elution with 80% (v/v) methanol. Activated charcoal facilitated removal of impurities in S. platensis extract and in further purification of vitamin B12. The purified fraction was identified to contain methylcobalamin as analyzed by HPLC and TLC. Authenticity of methylcobalamin was further confirmed by LC-MS and MS/MS. Quantitation of methylcobalamin in a test sample of S. platensis biomass was performed using microbiological assay and chemiluminescence assay and was found to be 38.5±2 and 35.7±2 µg/100 g of dry biomass, respectively.

Thiol-stabilized luminescent CdTe quantum dot as biological fluorescent probe for sensitive detection of methyl parathion by a fluoroimmunochromatographic technique.

Quantum dots (QDs) are preferred as high-resolution biological fluorescent probes because of their inherent optical properties compared with organic dyes. This intrinsic property of QDs has been made use of for sensitive detection of methylparathion (MP) at picogramme levels. The specificity of the assay was attributed to highly specific immunological reactions. Competitive binding between free MP and CdTe QD bioconjugated MP (MP-BSA-CdTe) with immobilized anti-MP IgY antibodies was monitored in a flow-injection system. The fluorescence intensity of MP-BSA-CdTe bioconjugate eluted from the column was found to be directly proportional to the free MP concentration. Hence, it was possible to detect MP in a linear range of 0.1-1 ng mL(-1) with a regression coefficient R(2) = 0.9905. In this investigation, IgY proved advantageous over IgG class immunoglobulins in terms of yield, stability, cost effectiveness, and enhancement of assay sensitivity. The photo-absorption spectrum of bioconjugated CdTe QD (lambda(max) = 310 nm) confirmed nano-biomolecular interactions. The results suggest the potential application of bioconjugation and nano-biomolecular interactions of QDs for biological labeling and target analyte detection with high sensitivity.

Enhancement of chemiluminescence for vitamin B12 analysis.

In the current article, chemiluminescence (CL) from the vitamin B(12) and luminol reaction was studied under alkaline conditions to develop a sensitive analytical method for vitamin B(12) using the carbonate enhancement effect. The method was successfully applied to the determination of vitamin B(12) in vitamin B(12) tablets, multivitamin capsules, and vitamin B(12) injections. Experimental parameters were optimized, including luminol concentration, urea-hydrogen peroxide (urea-H(2)O(2)) concentration, effect of pH, and sequence of addition of reactants for obtaining maximum CL, which was not explored previously. The limit of detection was 5 pg/ml, and the linear range was 10 pg/ml to 1 microg/ml with a regression coefficient of R(2)=0.9998. The importance of these experimental parameters and the carbonate enhancement effect is discussed based on the knowledge of the mechanism of oxidation of luminol and decomposition of urea-H2O2 in the presence of vitamin B(12). Extraction of vitamin B(12) was carried out, and the observed recovery was 97-99.2% with a relative standard deviation in the range of 0.30-1.09%. The results obtained were compared with those of the flame atomic absorption spectrometry method.

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives.

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.