Technological developments enable the discovery of novel enzymes, the advancement of enzyme cascade designs and pathway engineering, moving biocatalysis into an era of technology integration, intelligent manufacturing and enzymatic total synthesis.

Abstract:We have successfully established a simple continuous colorimetric assay for the sensitive and reliable quantification of cytosine-5'-monophosphate-acetylneuraminate synthetase (CMP-sialate synthetase, CSS) activity based on the pH change of a released proton equivalent upon nucleotide activation of Neu5Ac or related analogues. Using this method, steady-state kinetic data of Neisseria meningitidis CSS were determined for cytosine-5'-triphosphate (CTP), N-acetylneuraminic acid (Neu5Ac) and eleven structural variations of the sialic acid, including backbone truncation, deamination, epimerization, and several N-acyl modifications. These data further demonstrate the unusual versatility of the N. meningitidis CSS for a general access to sialoconjugates containing non-natural sialic acid analogues. Remarkably, the assay allows covering a broad range of substrate parameters that span over more than three orders of magnitude for K M and k cat measurements. With the aid of a structural model built from X-ray crystal structure data, the kinetic data could be used to interpret potential protein contributions in substrate binding of Neu5Ac and its analogues. The Neu5Ac analogues were used for the preparation of neo-sialoconjugate analogues of 2,6-sialyllactose in a one-pot two-enzyme cascade, using N. meningitidis CSS together with the novel a2,6-sialyltransferase (a2,6SiaT) from Photobacterium leiognathi, which proved to be highly effective because of its optimum activity at alkaline pH, appropriately matching the requirements from the overall reaction system.

A pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.

Directed evolution of the thermostable transketolase from Geobacillus stearothermophilus based on a pH-based colorimetric screening of smart libraries yielded several mutants with up to 16-fold higher activity for aliphatic aldehydes and high enantioselectivity (>95% ee) in the asymmetric carboligation step.

Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst ) retained 100% activity for one week at 50 8C and for 3 days at 65 8C, and has an optimum temperature range around 60-70 8C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTK gst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 8C for 45 min, directly yielding 132 mg of pure mTK gst from 1 L of culture. The reaction rate of mTK gst with glycolaldehyde was 14 times higher at 70 8C than at 20 8C, and 4 times higher at 50 8C when compared to E. coli TK under identical conditions. When tested at 50 8C with other aldehydes as acceptors, mTK gst activity was approximately 3 times higher than those obtained at 20 8C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors -glycolaldehyde, d-glyceraldehyde and butyraldehyde -from which the corresponding products l-erythrulose 1, dxylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTK gst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.

Transketolase variants were engineered to utilize arylalkanals and benzaldehyde as substrates with up to 28-fold rate acceleration for C–C bond formation with good yields (50–73%) and virtually complete (3S)-stereoselectivity (>99% ee).

We report on the use of PDMS multichannels for affinity studies of DNA aptamer-human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5'-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3') has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I-IgE complex was found to be 2.7 x 10(-7) M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format.

Ultrasensitive detection of proteins is of great importance to proteomics studies. We report here a method to enhance detection sensitivity in surface plasmon resonance (SPR) spectroscopy by coupling a polymerization initiator to a biospecific interaction and inducing inline atom transfer radical polymerization (ATRP) for amplifying SPR response. Bacterial cholera toxin (CT) is chosen as the model protein that has been covalently immobilized on the surface for demonstrating the principle. The specific recognition is achieved by use of biotinylated anti-CT, which allows initiators with a biotin tag to be fixed at the protein binding site through a neutravidin bridge and triggers the localized growth of polymer brushes of poly(hydroxyl-ethyl methacrylate) (PHEMA) via an ATRP mechanism. To further enhance the signal, a second ATRP reaction is conducted that takes advantage of the hydroxyl groups of PHEMA brushes from the first step to form hyperbranched polymers onto the sensing surface. The two consecutive ATRP steps significantly improve SPR detection, allowing low amounts of CT that yield no direct measurement to be quantified with large signals. The resulting polymer film has been characterized by optical and atomic force microscopy. Ascorbic acid (AA) is employed as deoxygen reagent in the catalyst mixture that effectively suppresses oxygen interference, shortening the reaction time and making it possible for applying this ATRP approach to flow injection based SPR detection. A calibration curve of PHEMA amplification for CT detection based on surface coverage has been obtained that displays a correlation in a range from 8.23 x 10(-15) to 3.61 x 10(-12) mol/cm(2) with a limit of detection of 6.27 x 10(-15) mol/cm(2). The versatile biotin-neutravidin interaction used here should allow adaptation of ATRP enhancement to many other systems that include DNA, RNA, peptides, and carbohydrates, opening new avenues for ultrasensitive analysis of biomolecules with flow-injection assay and SPR spectroscopy.