Successful synthesis of a glial-specific blood-brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin.

Successful manual synthesis of the TD2.2 peptide acting as a blood-brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.

Overview of Immobilized Enzymes' Applications in Pharmaceutical, Chemical, and Food Industry.

The use of immobilized enzymes in industry is becoming a routine process for the manufacture of many key compounds in the pharmaceutical, chemical, and food industry. Some enzymes like lipases are naturally robust and efficient, can be used for the production of many different molecules, and have found broad industrial applications. Some more specific enzymes, like transaminases, have required protein engineering to become suitable for applications in industrial manufacture. For all enzymes, the possibility to be immobilized and used in a heterogeneous form brings important industrial and environmental advantages such as simplified downstream processing or continuous process operations. Here, we present a series of large-scale applications of immobilized enzymes with benefits for the food, chemical, pharmaceutical, cosmetics, and medical device industries, some of them hardly reported before.

Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems.

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 Å of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100-200 Å of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 °C, fully preserving the activity after 20 days. In anhydrous oil at 40 °C, PEGylated TLL-Purolite C-18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e.g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium.

BACKGROUND: Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions. RESULTS: Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 µmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone. CONCLUSIONS: Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.

Layer-by-layer hydroxymethyl ferrocene modified sensor for one-step flow/stop-flow injection amperometric immunoassay of alpha-fetoprotein.

A rapid one-step flow/stop-flow injection amperometric immunoassay for alpha-fetoprotein (AFP) using a novel home-produced electrochemical sensor was proposed. The sensor was prepared using layer-by-layer adsorption of positively charged poly(allylamine) (PAA) and negatively charged hydroxymethyl ferrocene on a screen-printed electrode (SPE). The electrochemistry of the immobilized ferrocene moieties showed a surface-controlled electrode process. Based on an electrochemical enzyme-linked immunoassay with the immobilized ferrocene moieties as an electron transfer mediator between the electrode and the horseradish peroxidase (HRP)-labeled anti-AFP antibody, a calibration curve with two linear ranges from 5 to 20 and 20 to 150 ng ml-1 and a detection limit of 2 ng ml-1 for AFP determination was obtained under the optimized conditions of 0.891 ml min-1 flow rate, 20 microl injection volume and +25 mV applied potential. The sensor showed good repeatability and reproducibility and retained more than 95% of its original signal after 15 days of storage. The proposed method eliminated the need for washing and addition of any substrate or mediator. The complete assay could be handled in less than 25 min with a one-step injection of a 40 microl sample solution. The proposed method would be valuable for the diagnosis and monitoring of carcinoma and its metastasis.

Rapid and sensitive automated method for glucose monitoring in wine processing.

A rapid and sensitive automated method for glucose monitoring that might be employed during wine fermentation and processing was developed. A flow injection (FI) system coupled with an automated dilutor and the "redox-versatile" modified electrode were used to directly measure glucose in wine. To avoid interferences during wine analysis, different formulations of enzymatically modified carbon paste electrodes (CPE) were used and evaluated in oxidation and reduction mode. The best selectivity and sensitivity for glucose monitoring in real samples was obtained in cathodic mode at a fixed potential of 0 V versus Ag/AgCl using a CPE modified with glucose oxidase, horseradish peroxidase, and ferrocene as redox mediator. A total linear range of 0.02-50 g/L glucose was covered using this automated system and allowed the measurement of glucose in dry, medium, and sweet white or red wines without any sample pretreatment. The results showed a good correlation with the standard method, and the proposed method is very rapid, simple, and reliable and does not need skilled operators.

Synergetic effect for NADH oxidation of ferrocene and zeolite in modified carbon paste electrodes. New approach for dehydrogenase based biosensors.

The development of electrochemical biosensors using dehydrogenases associated with the corresponding cofactor is strongly related to the better understanding of NADH oxidation at the electrode surface. The aim is to lower the necessary overvoltage and consequently to escape interferences and electrode fouling. In this paper, we show that carbon paste electrode (CPE) modified with NaY zeolite fulfils this requirements thanks to its hydrophilic surface. Oxidation of NADH at ferrocene (FcH) modified carbon paste electrode exhibits a rather slow electrocatalytic effect. We demonstrated the existence of synergetic effect on the electrocatalytic oxidation of NADH when the CPE is doped with zeolite (NaY) and FcH mediator or with the zeolite exchanged beforehand with the mediator (Y-Ferricinium, YFcH). This cumulative effect permits to reach high sensitivity for NADH detection and offers new way for the development of enzymatic biosensors using dehydrogenases depending on NADH as cofactor.