Using Spectroscopy to Guide the Adaptation of Aptamers into Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.

Immobilization of Candida antarctica lipase B onto Purolite® MN102 and its application in solvent-free and organic media esterification.

The aim of this study was to develop simple and efficient method for immobilization of Candida antarctica lipase B onto hydrophobic anion exchange resin Purolite® MN102 and to apply immobilized catalyst for the enzymatic synthesis of two valuable esters-isoamyl acetate and L-ascorbyl oleate. At optimized conditions (1 M phosphate buffer pH = 7, 7 h at 25 °C, and 18.75 mg of offered proteins g-1 of support), immobilized lipase with hydrolytic activity of 888.4 p-nitrophenyl butyrate units g-1 was obtained. Afterwards, preparation was applied for the solvent-free synthesis of isoamyl acetate from triacetin and isoamyl alcohol. At 75 °C, 1 M of isoamyl alcohol, and 6 mg ml-1 of enzyme 100 % yield was achieved in 6 h, while at prolonged reaction times, complete conversion was enabled even at lower temperatures, lower lipase loadings, and higher substrate concentrations. After 15 consecutive reuses (60 h), activity of catalyst dropped to 50 % of its initial value and total amount of 1.31 mol (170.55 g) of ester with 1 g of biocatalyst was produced. Even higher operational stability of lipase (25 % loss of activity in 200 h) was observed in the synthesis of L-ascorbyl oleate performed in organic solvent (t-butanol). Multiple use of one batch of immobilized biocatalyst in both cases led to a significant process cost reduction and substantial increment of corresponding productivities.

Design of biocompatible immobilized Candida rugosa lipase with potential application in food industry.

BACKGROUND: Biocatalysts are a promising alternative for the production of natural flavor compounds. Candida rugosa lipase (CRL) is a particularly important biocatalyst owing to its remarkable efficiency in both hydrolysis and synthesis. However, additional stabilization is necessary for successful industrial implementation. This study presents an easy and time-saving method for immobilizing this valuable enzyme on hydroxyapatite (HAP), a biomaterial with high protein-binding capacity. RESULTS: Targeted immobilized CRL was obtained in high yield of ≥98%. Significant lipase stabilization was observed upon immobilization: at 60 °C, immobilized lipase (HAP-CRL) retained almost unchanged activity after 3 h, while free CRL lost 50% of its initial activity after only 30 min. The same trend was observed with tested organic solvents. Methanol and hexane had the most pronounced effect: after 3 h, only HAP-CRL was stable and active, while CRL was completely inactivated. The practical value of the prepared catalyst was tested in the synthesis of the aroma ester methyl acetate in hexane. Reaction yields were 2.6 and 52.5% for CRL and HAP-CRL respectively. CONCLUSION: This research has successfully combined an industrially prominent biocatalyst, CRL, and a biocompatible, environmentally suitable carrier, HAP, into an immobilized preparation with improved catalytic properties. The obtained CRL preparation has excellent potential for the food and flavor industries, major consumers in the global enzyme market. © 2016 Society of Chemical Industry.

Influence of fatty acid on lipase-catalyzed synthesis of ascorbyl esters and their free radical scavenging capacity.

Fatty acid (FA) ascorbyl esters are recently emerging food, cosmetic, and pharmaceutical additives, which can be prepared in an eco-friendly way by using lipases as catalysts. Because they are amphiphilic molecules, which possess high free radical scavenging capacity, they can be applied as liposoluble antioxidants as well as emulsifiers and biosurfactants. In this study, the influence of a wide range of acyl donors on ester yield in lipase-catalyzed synthesis and ester antioxidant activity was examined. Among saturated acyl donors, higher yields and antioxidant activities of esters were achieved when short-chain FAs were used. Oleic acid gave the highest yield overall and its ester exhibited a high antioxidant activity. Optimization of experimental factors showed that the highest conversion (60.5%) in acetone was achieved with 5 g L(-1) of lipase, 50 mM of vitamin C, 10-fold molar excess of oleic acid, and 0.7 mL L(-1) of initial water. Obtained results showed that even short- and medium-chain ascorbyl esters could be synthesized with high yields and retained (or even exceeded) free radical scavenging capacity of l-ascorbic acid, indicating prospects of broadening their application in emulsions and liposomes.

Amplification of Signal on Cell Surfaces in Molecular Cascades.

We can formulate mixtures of oligonucleotide-antibody conjugates to act as molecular cascade-based automata that analyze pairs of cell surface markers (CD markers) on individual cells in a manner consistent with the implementation of Boolean logic-for example, by producing a fluorescent label only if two markers are present. While traditional methods to characterize cells are based on transducing signals from individual cell surface markers, these cascades can be used to combine into a single signal the presence of two or even more CDs. In our original design, oligonucleotide components irreversibly flowed from one antibody to another, driven by increased hybridizations, leading to the magnitude of the final signal on each cell being determined by the surface marker that was the least abundant. This is a significant limitation to the precise labeling of narrow subpopulations, and, in order to overcome it, we changed our design to accomplish signal amplification to a more abundant cell surface marker. We show the AMPLIFY function on two examples: (1) we amplify the fluorescent label from the CD19 marker onto a fivefold more abundant CD45, and (2) we amplify broadly distributed CD45RA to a more constant marker, CD3. We expect this new function to enable the increasingly complex Boolean analysis of cell surfaces.

Real-Time, Seconds-Resolved Measurements of Plasma Methotrexate In Situ in the Living Body.

Dose-limiting toxicity and significant patient-to-patient pharmacokinetic variability often render it difficult to achieve the safe and effective dosing of drugs. This is further compounded by the slow, cumbersome nature of the analytical methods used to monitor patient-specific pharmacokinetics, which inevitably rely on blood draws followed by post-facto laboratory analysis. Motivated by the pressing need for improved "therapeutic drug monitoring", we are developing electrochemical aptamer-based (EAB) sensors, a minimally invasive biosensor architecture that can provide real-time, seconds-resolved measurements of drug levels in situ in the living body. A key advantage of EAB sensors is that they are generalizable to the detection of a wide range of therapeutic agents because they are independent of the chemical or enzymatic reactivity of their targets. Three of the four therapeutic drug classes that have, to date, been shown measurable using in vivo EAB sensors, however, bind to nucleic acids as part of their mode of action, leaving open questions regarding the extent to which the approach can be generalized to therapeutics that do not. Here, we demonstrate real-time, in vivo measurements of plasma methotrexate, an antimetabolite (a mode of action not reliant on DNA binding) chemotherapeutic, following human-relevant dosing in a live rat animal model. By providing hundreds of drug concentration values, the resulting seconds-resolved measurements succeed in defining key pharmacokinetic parameters, including the drug's elimination rate, peak plasma concentration, and exposure (area under the curve), with unprecedented 5 to 10% precision. With this level of precision, we easily identify significant (>2-fold) differences in drug exposure occurring between even healthy rats given the same mass-adjusted methotrexate dose. By providing a real-time, seconds-resolved window into methotrexate pharmacokinetics, such measurements can be used to precisely "individualize" the dosing of this significantly toxic yet vitally important chemotherapeutic.

Novel glycoside of vanillyl alcohol, 4-hydroxy-3-methoxybenzyl-α-D-glucopyranoside: study of enzymatic synthesis, in vitro digestion and antioxidant activity.

Novel glucoside of physiological active vanillyl alcohol was synthesized for the first time using maltase from Saccharomyces cerevisiae as catalyst, and established its structure as 4-hydroxy-3-methoxybenzyl-α-D: -glucopyranoside. The key reaction factors for this transglucosylation reaction were optimized using response surface methodology and the highest yield so far in maltase catalyzed transglucosylation reaction was obtained. It was found out that optimum temperature of reaction was 37 °C, optimal maltose concentration was 60% (w/v), optimal pH was 6.6, and optimal concentration of vanillyl alcohol was 158 mM. Under these conditions, yield of glucoside was 90 mM with no by product formation. It was shown that this compound posses good antioxidant activity as well as stability in gastrointestinal tract. It was demonstrated that it is hydrolyzed on brush border membrane of enterocytes, so it can serve in protecting gastrointestinal system from oxidation, as well as source of anticonvulsive drug after the hydrolysis of glucoside on brush border membrane of small intestine.

A novel optical tracer for VMAT2 applied to live cell measurements of vesicle maturation in cultured human ß-cells.

The islet ß-cells integrate external signals to modulate insulin secretion to better regulate blood glucose levels during periods of changing metabolic demand. The vesicular monoamine transporter type 2 (VMAT2), an important regulator of CNS neurotransmission, has an analogous role in the endocrine pancreas as a key control point of insulin secretion, with additional roles in regulating ß-cell differentiation and proliferation. Here we report on the synthesis and biological characterisation of a fluorescent ligand for VMAT2 suitable for live cell imaging. Staining for VMAT2 and dopamine in live ß-cell cultures show colocalisation in specific vesicles and reveal a heterogeneous population with respect to cell size, shape, vesicle number, size, and contents. Staining for VMAT2 and zinc ion, as a surrogate for insulin, reveals a wide range of vesicle sizes. Immunohistochemistry shows larger ß-cell vesicles enriched for proinsulin, whereas smaller vesicles predominantly contain the processed mature insulin. In ß-cell cultures obtained from nondiabetic donors, incubation at non-stimulatory glucose concentrations promotes a shift in vesicle diameter towards the more mature insulin vesicles at the expense of the larger immature insulin secretory vesicle population. We anticipate that this probe will be a useful reagent to identify living ß-cells within complex mixtures for further manipulation and characterisation.

Macroporous Poly(GMA-co-EGDMA) for Enzyme Stabilization.

One of the most used procedures for enzyme stabilization is immobilization. Although immobilization on solid supports has been pursued since the 1950s, there are no general rules for selecting the best support for a giving application. A macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate (poly (GMA-co-EGDMA)) is a carrier consisting of macroporous beads for immobilizing enzymes of industrial potential for the production of fine chemicals and pharmaceuticals.

Synthesis of medium-chain length capsinoids from coconut oil catalyzed by Candida rugosa lipases.

A commercial preparation of Candida rugosa lipases (CRL) was tested for the production of capsinoids by esterification of vanillyl alcohol (VA) with free fatty acids (FA) and coconut oil (CO) as acyl donors. Screening of FA chain length indicated that C8-C12 FA (the most common FA found in CO triglycerides) are the best acyl-donors, yielding 80-85% of their specific capsinoids. Hence, when CO, which is rich in these FA, was used as the substrate, a mixture of capsinoids (vanillyl caprylate, vanillyl decanoate and vanillyl laurate) was obtained. The findings presented here suggest that our experimental method can be applied for the enrichment of CO with capsinoids, thus giving it additional health promoting properties.

Dual effect of benzyl alcohol on α-glucosidase activity: efficient substrate for high yield transglucosylation and non-competitive inhibitor of its hydrolytic activity.

Benzyl alcohol, a potent anesthetic and bacteriostatic, can be efficiently glucosylated by α-glucosidase from Saccharomyces cerevisiae to produce benzyl alcohol α-glucoside with a 75% yield. However, while studying the transglucosylation reaction conditions, it was found out that benzyl alcohol is a non-competitive inhibitor of α-glucosidase's hydrolytic activity (Ki=18mM, toward maltose). Due to its interesting ability to be glycosylated by the enzyme and to inhibit its hydrolytic activity, we proposed a plausible mechanism for the phenolic α-glucosydase inhibitor's binding, since the mechanism of inhibition has not yet been elucidated.

Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation.

Specificity of maltase to maltose in three different directions of reaction: hydrolytic, vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis.

Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that maltase has interestingly high K(m) value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate.

One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite.

Lipase A from Candida antarctica (CAL A) was purified to apparent homogeneity in a single step using hydroxyapatite (HAP) chromatography. CAL A bound to HAP was eluted with 10mM Na-phosphate buffer, pH 7.0 containing 0.5% Triton X-100. The protocol resulted in a 3.74-fold purification with 94.7% final recovery and 400.83 U/mg specific activity. Silver staining after SDS-PAGE revealed the presence a single band of 45 kDa. The enzyme exhibited a temperature optimum of 60°C, was unaffected by monovalent metal ions, but was destabilized by divalent metal ions (Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+)) and stimulated by 50mM Fe(2+). Detergents at 0.1% concentrations did not affect lipase activity. Except for Triton X-100, detergent concentrations of 1% had a destabilizing effect.

Macroporous poly(GMA-co-EGDMA) for enzyme stabilization.

One of the most used procedures for enzyme stabilization is immobilization. Although immobilization on solid supports has been pursued since the 1950s, there are no general rules for selecting the best support for a giving application. A macroporous copolymer of ethylene glycol dimethacrylate and glycidyl methacrylate is a carrier consisting of macroporous beads for immobilizing enzymes of industrial potential for the production of fine chemicals and pharmaceuticals.

Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.

Synthesis of hydroquinone-alpha-glucoside by alpha-glucosidase from baker's yeast.

Hydroquinone-alpha-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with alpha-glucosidase from baker's yeast. Only one phenolic -OH group was alpha-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 degrees C for 20 h with 50 mM: hydroquinone and 1.5 M: maltose in 100 mM: sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.