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d‐Serine plays vital physiological roles in the functional regulation of the mammalian brain, where it is produced from l‐serine by serine racemase and degraded by d‐amino acid oxidase. In the present study, we identified a new d‐serine metabolizing activity of serine hydroxymethyltransferase (SHMT) in bacteria as well as mammals. SHMT is known to catalyze the conversion of l‐serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate, respectively. In addition, we found that human and Escherichia coli SHMTs have d‐serine dehydratase activity, which degrades d‐serine to pyruvate and ammonia. We characterized this enzymatic activity along with canonical SHMT activity. Intriguingly, SHMT required THF to catalyze d‐serine dehydration and did not exhibit dehydratase activity toward l‐serine. Furthermore, SHMT did not use d‐serine as a substrate in the canonical hydroxymethyltransferase reaction. The d‐serine dehydratase activities of two isozymes of human SHMT were inhibited in the presence of a high concentration of THF, whereas that of E. coli SHMT was increased. The pH and temperature profiles of d‐serine dehydratase and serine hydroxymethyltransferase activities of these three SHMTs were partially distinct. The catalytic efficiency (kcat/Km) of dehydratase activity was lower than that of hydroxymethyltransferase activity. Nevertheless, the d‐serine dehydratase activity of SHMT was physiologically important because d‐serine inhibited the growth of an SHMT deletion mutant of E. coli, ∆glyA, more than that of the wild‐type strain. Collectively, these results suggest that SHMT is involved not only in l‐ but also in d‐serine metabolism through the degradation of d‐serine. [ABSTRACT FROM AUTHOR]

The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.

A miniaturized trypsin reactor was prepared by coating a trypsin-containing gel on a porous silica monolith. The trypsin-encapsulated gel was prepared by the sol-gel method. The sol-gel reaction was optimized so that the sol solution containing trypsin forms a thin film on the sol-gel monolith. The trypsin was encapsulated into the gel matrix without losing its activity. The silica monolith was fabricated to fit into a 96-well microtiter plate well and could then be easily removed. The trypsin-immobilized monolith was reacted in the 96-well microtiter plate. After the reaction, the monolith was removed, and the enzymatic activity was measured. The large surface area of the monolith enabled the immobilized trypsin to achieve a high catalytic turnover rate. Furthermore, the kinetic parameter of the immobilized trypsin indicates the absence of diffusional limitations. The durability and repeatability of the fabricated trypsin-coated monolith was tested and found to be satisfactory. The encapsulated trypsin exhibits an increased stability even after continuous use compared with that in free solution. Furthermore, this on-plate bioreactor was applicable to the digestion of protein with multiple cleavage sites.

Plastic microchips are very promising analytical devices because they are less fragile and are suitable for mass production. However, due to their hydrophobicity, the surface strongly interacts with nonpolar analytes or species containing hydrophobic domains, resulting in significant uncontrolled adsorption on channel walls. This paper describes the poly(methyl methacrylate) surface treatment by dynamic coating additives that considerably decreases adsorption of analytes to channel walls. Among the additives studied, quaternary ammonium starch derivatives suppressed the adsorption of fluorescently labeled amino acids and peptides most effectively. The effect was valid over the wide pH range from 2.5 to 8.0. Using a 10 mM phosphate buffer (pH 7.0) with 3% (w/v) quaternary ammonium starch as the running buffer, Asp and Glu, respectively, migrated at 54.6 and 57.6 s with efficiencies of 380 000 and 370 000 plates/m. In addition, this cationic starch derivative was found to possess good solubility and low viscosity.

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 [micro]m) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a solgel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain [beta] and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain [beta] amino acid sequence and 73% of the lysozyme amino acid sequence.

Trypsin-encapsulated sol--gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol--gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed by electrophoretic separation on the same chip. The plastic microchip, which is made from poly(methyl methacrylate), generated enough electroosmotic flow that substrates and products could be satisfactorily separated. The sol--gel in the SR did not alter the separation efficiency of each peak. With the present device, the analytical time was significantly shortened compared to conventional tryptic reaction schemes. This on-chip microreactor was applicable to the digestion of protein with multiple cleavage sites and separation of digest fragments. Furthermore, the encapsulated trypsin exhibits increased stability, even after continuous use, compared with that in free solution.

A novel trypsin-encapsulation technique using the sol-gel method was developed for the preparation of an online enzyme reactor integrated into capillary electrophoresis. Trypsin was encapsulated in tetramethoxysilane-based hydrogel, and its enzymatic activity was evaluated using [alpha]-N-benzoyl-L-arginine ethyl ester and two peptides (bradykinin and [[Tyr.sup.8]]-bradykinin). The enzyme encapsulation was carried out in a single step under mild conditions within a capillary, and 1.5-cm gel was formed at the inlet of the capillary. The resultant monolithic reactor showed excellent enzymatic activity, which was ~700 times higher than that in free solution, without stopping the flow. Separation of the unreacted substrates and products in the same capillary also showed high selectivity, and sample size in this system decreased 3 orders of magnitude from conventional tryptic reaction schemes. The encapsulated trypsin maintains its substrate specificity even in a sol-gel matrix. Furthermore, the encapsulated trypsin exhibits increased stability even after continuous use compared to that in free solution.

A novel protein-encapsulation technique using sol-gels was developed for the preparation of monolithic capillary columns for capillary electrochromatography. Two chiral compounds, bovine serum albumin (BSA) and ovomucoid (OVM) from chicken egg white, were encapsulated in tetramethoxysilane-based hydrogel and their chiral selectivity was evaluated for the separation of some selected enantiomers (tryptophan, benzoin, eperisone, chlorpheniramine). The protein encapsulation was carried out within a capillary in a single step under mild conditions. The resultant monolithic columns showed adequate chromatographic performance, including mechanical strength, penetration of pressurized flow, and chiral separation. Two different proteins, BSA and OVM, were successfully encapsulated into the gel matrixes by changing the alkoxysilane compositions of the gel. Run-to-run repeatability was quite satisfactory. The consecutive analysis of the neutral compound, benzoin, by the OVM-encapsulated column showed good repeatability in the retention time (RSD = 1.23% for the first peak, N = 10). Under optimized conditions, the theoretical plate number for the first peak of benzoin reached 72 000 plates/m.