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D-tagatose, a type of rare sugar that has 92% sweetness when compared to sucrose, but with 1/3 of its energy value, is obtained from the isomerization of D-galactose by the enzyme L-arabinose isomerase EC 5.3.1.4. This high value-added enzyme obtained from Enterococcus faecium had its three-dimensional structure predicted from crystallographic studies and structural modeling, making it possible to design a recombinant enzyme with the insertion of a fusion label, LSLt (lectin Laetiporus sulphureus), which allows the enzyme to be efficiently, simply and inexpensively purified in a single affinity step, having carrier agarose and lactose as eluent. A TEV endoprotease recognition sequence (Tobacco Etch Virus) was also inserted, allowing cleavage of the fusion label following a purification process. The success in the construction resulted in an active enzyme and of easy purification, since the support does not require any type of activation. In the characterization of the enzyme, 50 ° C and 5.5 were obtained as the ideal temperature and pH, respectively. The studies also showed that the presence of the Mn2 + ion positively affects the catalytic activity of LSLt-LAI. As for immobilization, which occurred after 15 minutes, a yield of 95% was obtained, with activity recovered around 88%. The study of D-galactose bioconversion in D-tagatose showed promising results, 28%, high conversion rate when compared to studies already performed with the enzyme obtained from Enterococcus faecium in its native or recombinant form. A D-tagatose, um tipo de açúcar raro que apresenta 92% do poder de doçura quando comparada à sacarose, porém com 1/3 de seu valor energético, é obtida a partir da isomerização da D-galactose pela enzima L-arabinose isomerase EC 5.3.1.4. Esta enzima, de alto valor agregado, obtida a partir de Enterococcus faecium, teve sua estrutura tridimensional predita a partir de estudos cristalográficos e modelagem estrutural, possibilitando projetar-se uma enzima recombinante com a inserção de uma etiqueta de fusão, LSLt (lectina de Laetiporus sulphureus), que permite que a enzima seja purificada de modo eficaz, simples e barato em uma única etapa de afinidade, tendo agarose como suporte e lactose como eluente. Uma sequência de reconhecimento da endoprotease TEV (Tobacco Etch Virus), também foi inserida, permitindo clivar a etiqueta de fusão após um processo de purificação. O sucesso na construção resultou em uma enzima ativa e de fácil purificação, já que o suporte não exige nenhum tipo de ativação. Na caracterização da enzima, alcançou-se 50°C e 5,5 como temperatura e pH ideais, respectivamente. Os estudos também mostraram que a presença do íon Mn2+ afetou positivamente na atividade catalítica da LSLt-LAI. Quanto á imobilização, que ocorreu após 15 minutos, obteve-se um rendimento de 95%, com atividade recuperada em torno de 88%. O estudo da bioconversão de D-galactose em D-tagatose mostrou resultados promissores, 28%, alta taxa de conversão quando comparada a estudos já realizados com a enzima obtida de Enterococcus faecium em sua forma nativa ou recombinante.

A ciclodextrina glicosiltransferase (CGTase) é a única enzima capaz de catalisar a reação de ciclização a partir do amido e, assim, formar oligossacarídeos cíclicos conhecidos como ciclodextrinas (CDs). Através desta reação é produzida uma mistura de α-, β- e γ-CD que, respectivamente, contém 6, 7 e 8 resíduos de glicose. As CDs têm atraído enorme atenção devido ao seu grande potencial de aplicação em diversas áreas da indústria. Potencial este proporcionado por sua estrutura cônica, com interior hidrofóbico, capaz de encapsular sólidos, líquidos e gases, conferindo propriedades importantes e protegendo-os. Neste trabalho foi estudada a imobilização de uma CGTase em sílica mesoporosa de forma direcionada às cisteínas presentes em sua superfície, alterando a exposição do sítio ativo. A ligação via cisteínas nativas da proteína aumentou em quatro vezes a eficiência da imobilização, quando comparada a ligação via grupamento amino. Esta, no entanto, apresentou maior atividade enzimática em faixas mais amplas de temperatura e pH, além de maior estabilidade operacional, mantendo 100 % de sua atividade após 200 h de reação contínua a 60 °C e pH 4. Ainda que apresentando menor estabilidade da ligação, o derivado obtido por ligação dissulfeto manteve 40 % da atividade inicial durante 200 h e então, o suporte pôde ser recarregado e reutilizado por igual período. Os suportes desenvolvidos apresentaram estabilidade satisfatória, possibilitando o uso do derivado imobilizado em reator de leito fixo operado de forma contínua. Quando avaliado em relação a produção das três ciclodextrinas principais, o derivado cuja imobilização da enzima ocorreu via grupamento amino, evidenciou a possibilidade de modulação da produção apenas variando as condições de reação. α- e β-CD foram produzidas preferencialmente em pH 8,0 e 2 min (3,44 mg mL-1 e 3,51 mg mL-1, respectivamente), enquanto que pH mais ácido (4,0) e maior tempo de reação (141 min) favoreceram a formação de γ-CD (3,35 mg mL-1), com baixa formação α-CD (0,75 mg mL-1). Por fim, os resultados deste estudo evidenciam a importância da imobilização da CGTase para a estabilização de sua estrutura a fim de aplicá-la em sistemas contínuos de produção de CDs onde é possível modular o perfil dos produtos gerados em função das condições de reação, aumentando assim a produtividade do biocatalisador. Cyclodextrin glycosyltransferase (CGTase) is the only enzyme capable of catalyzing the cyclization reaction from the starch and thus forming cyclic oligosaccharides known as cyclodextrins (CDs). Through this reaction, is produced a mixture of α-, β- and γ-CD containing, 6, 7 and 8 glucose residues respectively. Cyclodextrins (CD) have been attracting considerable attention because of its great potential for application in various areas of industry. This potential is provided by its conical structure with hydrophobic interior, capable of encapsulating solids, liquids and gases, changing important features and protecting them. In this work, the immobilization of CGTase in mesoporous silica was studied in a way directed to cysteines present on its surface, altering the exposure of the active site. The connection via native cysteine of the protein increased by four times the efficiency of immobilization compared to amino groups connection. The binding of amino groups, however, showed greater enzymatic activity in wider ranges of temperature and pH, and higher operational stability, while maintaining 100 % of its activity after 200 h of continuous reaction at 60 °C and pH 4. Although showing less stable connection, the derivative obtained by disulfide bond retained 40 % of the initial activity for 200 h and then, the support could be reloaded and reused for the same period. Developed supports showed satisfactory stability, enabling the use of the derivative assets in a packed bed reactor and operated continuously. It was demonstrated the possibility of modulating the CDs production just varying the reaction conditions, using the derivative of which the enzyme immobilization occurred via amino group, to evaluate the production of three main cyclodextrins. α- and β-CD were produced preferentially at pH 8.0 and 2 min (3.44 mg mL-1 and 3.51 mg mL-1, respectively), whereas the more acid pH (4.0) and longer reaction (141 min) favored the formation of γ-CD (3.35 mg mL-1 and 0.75 mg mL-1 of α-CD). Finally, the results of this study show the importance of the immobilization of CGTase to the stabilization of its structure in order to apply it in continuous CD production systems, where it is possible to modulate the profile of the products generated as a function of the reaction conditions, thus increasing the productivity of the biocatalyst.

The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here, we present a unique method to attach a single DNA address to proteins in one step during the purification from the E. coli lysate by fusion to human O6-alkylguanine-DNA-alkyltransferase (SNAP-tagTM) and the Avitag. Use of the conjugates in converting a DNA chip into a protein chip by self assembly is demonstrated.

A novel SMART module, dubbed "DNA-SMART" (DNA substrate modification and replication by thermoforming) is reported, where polymer films are premodified with single-stranded DNA capture strands, microthermo formed into 3L) structures, and postmodified with complementary DNA-protein conjugates to realize complex biologically active surfaces within microfluidic devices. As a proof of feasibility, it is demonstrated that microchannels presenting three different proteins on their inner curvilinear surface can be used for selective capture of cells under flow conditions.

A three-enzyme cascade reaction was successfully realized in a continuous flow microreactor. The first enzyme (Candida antarctica lipase B, also known as Pseudozyma antarctica lipase B) and the third enzyme (horseradish peroxidase) of the cascade process were immobilized in a mild non-contact manner via ssDNA-ssDNA interaction in discrete zones on the capillary wall, whereas the second enzyme (glucose oxidase) was kept in the mobile phase. The unique combined feature of patterning, possibility of loading and stripping, and modularity in a fused silica microchannel is demonstrated. By changing the distance between the two enzyme patches, the reaction time available for glucose oxidase could be independently and modularly varied. The reusability of the enzymatic microfluidic system was shown by using the hybridization and dehybridization capabilities of DNA as a tool for subsequent enzyme immobilization and removal.

A novel approach is proposed to prepare a set of immobilized derivatives of a enzyme covalently rigidified through different regions of its surface. Six different variants of penicillin G acylase (PGA) from Escherichia coli (which lacks Cys) were prepared by introducing a unique Cys residue via site-directed mutagenesis in six different enzyme regions which were rich in Lys residues. All variants exhibited a similar activity and stability compared to those of the native enzyme. Each variant was immobilized on supports having a low concentration of reactive disulfide moieties and a high concentration of poorly reactive epoxy groups. After immobilization at pH 7.0 by site-directed thiol-disulfide intermolecular exchange, derivatives were further incubated at pH 10.0 for 48 h to promote an additional intramolecular reaction between Lys residues of enzyme and epoxy groups of the support. The establishment of at least three covalent attachments per PGA molecule was determined for all immobilized enzyme variants. The different derivatives exhibited diverse stability against several distorting agents and different selectivity in two interesting reactions. The derivative of the PGA variant obtained by replacement of GlnB380 by Cys was the most stable against heat and organic cosolvents: it preserved 90% of the initial activity and was 30-fold more stable than soluble PGA. This derivative also exhibited an improved enantioselectivity in the hydrolysis of chiral esters (E was improved from 8 to 16) and in kinetically controlled synthesis of amides (synthetic yields were increased from 31 to 49%)., The authors gratefully recognize the support from the Spanish CICYT (project. BIO-2005-8576). We gratefully recognize MEC for a fellowship for López-Gallego and Montes. We thank Antibióticos SA for the kind donation of reagents. The help and comments from Dr. Angel Berenguer (Universidad de Alicante) are gratefully recognized.

Nanografted monolayers (NAMs) of DNA, show novel physico-chemical properties, that make them ideally suited for advanced biosensing applications. In comparison with alternative solid-phase techniques for diagnostic DNA detection, NAMs can be distinguished because of their small size and by the high homogeneity of the DNA surface coverage. These two properties favor the extreme miniaturization and ultrasensitivity in high-throughput biosensing devices. The systematic use of NAMs for quantitative DNA (and protein) detection has so far suffered from the lack of a control on key fabrication parameters, such as the ss- or ds-DNA surface coverage. Here we report on a combined experimental-computational study that allows to estimate the surface density of the grafted DNA by analyzing the sample mechanical response, that is the DNA patch height vs. applied tip load curves. It is shown that the same analysis scheme can be used to detect the occurrence of hybridization with complementary strands in solution and estimate its efficiency. Thanks to these quantitative relationships it is possible to use a single AFM-based setup to: (i) fabricate a DNA NAM, (ii) control the DNA surface coverage, (iii) characterize its level of hybridization helping the design of NAMs with pre-determined fabrication parameters.