An injectable alginate-based hydrogel for microfluidic applications.

The objective of this study was to develop an injectable alginate based formulation for immobilizing enzymes into microfluidic systems. The gelation was induced upon lowering the pH by addition of d-glucono-δ-lactone (GDL) and release of Ca+ ions from solid CaCO3. The effects of GDL concentration on enzymatic activity and gelation time were investigated. The results indicated that increasing the GDL concentration increased both surface area and enzymatic activity. Also, chitosan was added to the formulation at different ratios to enhance the stability of enzyme during immobilization. For microfluidic application, 100µl spiral coil single channel microchip was fabricated and alginate GDL mixture containing ß-glucosidase was injected to the microchannel prior to gelation. Enzymatic conversion was performed by pumping substrate (pNPG) through the microchannel. The results indicated that the entire substrate was converted continuously during 24h without any leakage or deactivation of immobilized enzyme.

Formulation of organic and inorganic hydrogel matrices for immobilization of ß-glucosidase in microfluidic platform.

The aim of this study was to formulate silica and alginate hydrogels for immobilization of ß-glucosidase. For this purpose, enzyme kinetics in hydrogels were determined, activity of immobilized enzymes was compared with that of free enzyme, and structures of silica and alginate hydrogels were characterized in terms of surface area and pore size. The addition of polyethylene oxide improved the mechanical strength of the silica gels and 68% of the initial activity of the enzyme was preserved after immobilizing into tetraethyl orthosilicate-polyethylene oxide matrix where the relative activity in alginate beads was 87%. The immobilized ß-glucosidase was loaded into glass-silicon-glass microreactors and catalysis of 4-nitrophenyl ß-d-glucopyranoside was carried out at various retention times (5, 10, and 15 min) to compare the performance of silica and alginate hydrogels as immobilization matrices. The results indicated that alginate hydrogels exhibited slightly better properties than silica, which can be utilized for biocatalysis in microfluidic platforms.

Determining sites of interaction between prenisin and its modification enzymes NisB and NisC.

Although nisin is a model lantibiotic, our knowledge of the specific interactions of prenisin with its modification enzymes remains fragmentary. Here, we demonstrate that the nisin modification enzymes NisB and NisC can be pulled down in vitro from Lactococcus lactis by an engineered His-tagged prenisin. This approach enables us to determine important intermolecular interactions of prenisin with its modification machinery within L. lactis. We demonstrate that (i) NisB has stronger interactions with precursor nisin than NisC has, (ii) deletion of the propeptide part keeping the nisin leader intact leads to a lack of binding, (iii) NisB point mutants of highly conserved residues W616, F342A, Y346F and P639A are still able to dehydrate prenisin, (iv) NisB Δ(77-79)Y80F mutant decreased the levels of NisB-prenisin interactions and resulted in unmodified prenisin, (v) substitution of an active site residue H331A in NisC leads to higher amounts of the co-purified complex, (vi) NisB is present in the form of a dimer, and (vii) the region FNLD (-18 to -15) of the leader is an important site for binding not only to NisB, but also to NisC.