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Hollow cross-linked enzyme aggregates of laccase (h-CLEA laccase) can be prepared by employing a millifluidic reactor carrying two coaxial laminar flows. In a confluence zone where acetonitrile and an aqueous solution of laccase meet, diffusion of acetonitrile into the aqueous solution gives rise to rapid precipitation of laccase aggregates at the water/acetonitrile interface, as is evidenced by fluorescence images. By controlling the flow rates carefully in the laminar flow regions, h-CLEA laccase around 220 ± 10 nm can be obtained, and the size of the h-CLEA laccase increases with increasing flow rates of both solutions. The h-CLEA laccase particles are distinctly different from CLEA laccase prepared in batch processes. The former only consist a crust of cross-linked enzymes (with a hollow core) whereas the latter has a highly porous structure. When the h-CLEA laccase is used as biocatalysts, their activity (0.26 U/mg) is comparable to that of free enzymes at neutral pH due to the hollow structure. Moreover, the activity of h-CLEA laccase is higher than that of free laccase at high pH. For example, trypan blue (a dye molecule) can be decolorized completely in the presence of h-CLEA laccase within 270 min even at pH 10.0, at which the free enzyme completely loses its activity. Because of their uniform sizes, h-CLEA laccase can be trapped in a membrane for continuous degradation of trypan blue up to 96 h without losing any activity. This study shows the superiority of h-CLEA laccase compared to other types of immobilized enzymes. [ABSTRACT FROM AUTHOR]

Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200 nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300 nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48 h and release reducing sugar concentrations up to 2.48 ± 0.08 g/l and 4.99 ± 0.09 g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity. [ABSTRACT FROM AUTHOR]

In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N , N -dimethyl- n -octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules. [ABSTRACT FROM AUTHOR]