Measuring viscosity inside mesoporous silica using protein-bound molecular rotor probe.

Fluorescence spectroscopy of protein-bound molecular rotors Cy3 and Cy5 is used to monitor the effective viscosity inside the pores of two types of mesoporous silica (SBA-15 and MCF) with pore diameters between 8.9 and 33 nm. The ratio of the peak intensities is used to measure viscosity independently of solvent polarity, and the response of the lipase-bound dyes is calibrated using glycerol/water mixtures (no particles). The two dyes are either attached to the same protein or separate proteins in order to investigate potential effects of energy transfer (FRET) on the fluorescence properties, when using them as reporter dyes. The effective viscosity inside the pores at infinite protein dilution is one order of magnitude higher than in bulk water, and the effect of protein concentration on the measured viscosity indicates a stronger effect of protein-protein interactions in the pores than in similarly concentrated protein solutions without particles. In MCF-particles with octyl-groups covalently attached to the pore walls, a more efficient uptake of the lipase resulted in FRET between the protein-bound dyes even if the two dyes were attached to different proteins. In contrast to the unmodified particles the intensity-ratio method could therefore not be used to measure the viscosity, but the presence of FRET in itself indicates that octyl-protein interactions lead to a non-homogenous protein distribution in the pores. The dye labels also report a less polar pore environment as sensed by the proteins through a redshift in the dye emission. Both observations may help in understanding the higher efficiency of lipase immobilization in octyl-modified particles.

Immobilization of formaldehyde dehydrogenase in tailored siliceous mesostructured cellular foams and evaluation of its activity for conversion of formate to formaldehyde.

Formaldehyde dehydrogenase (FaldDH) is used as a catalyst to reduce formate to formaldehyde in a cascade reaction to convert CO2 to methanol. This enzyme, however, has low activity and is sensitive to substrate/product concentration and pH. To improve the performance of FaldDH, it can be immobilized through physical adsorption in siliceous mesostructured cellular foams (MCF), which physical properties are suitable for the immobilization of large molecules as FaldDH (molecular size of 8.6 × 8.6 × 19 nm). In this work two MCF materials were synthesized: MCF1 with a pore size of 26.8 nm and window size of 10.5 nm; and MCF2 with a pore size of 32.9 nm and window size of 13.0 nm. The surfaces of the materials were functionalized with octyl, mercaptopropyl or chloromethyl groups. FaldDH was successfully immobilized inside all the materials, yielding enzyme loadings of about 300 mg g-1 in MCF1 and more than 750 mg g-1 in MCF2. However, the enzyme was inactive upon immobilization on MCF1, whereas on MCF2 the enzyme retained its catalytic activity presumably owing to the larger pores of this material and the need for the enzyme to undergo configurational changes during the reaction. Using MCF2 functionalized with mercaptopropyl groups the activity of FaldDH was enhanced beyond that of the free enzyme. Additionally, low leakage of the enzyme from the MCF2 was observed during the reactions. Thus, tailored MCF is a highly attractive material for employment of the FaldDH enzyme.

Immobilisation on mesoporous silica and solvent rinsing improve the transesterification abilities of feruloyl esterases from Myceliophthora thermophila.

The immobilisation of four feruloyl esterases (FAEs) (FaeA1, FaeA2, FaeB1, FaeB2) from the thermophilic fungus Myceliophthora thermophila C1 was studied and optimised via physical adsorption onto various mesoporous silica particles with pore diameters varying from 6.6nm to 10.9nm. Using crude enzyme preparations, enrichment of immobilised FAEs was observed, depending on pore diameter and protein size. The immobilised enzymes were successfully used for the synthesis of butyl ferulate through transesterification of methyl ferulate with 1-butanol. Although the highest butyl ferulate yields were obtained with free enzyme, the synthesis-to-hydrolysis ratio was higher when using immobilised enzymes. Over 90% of the initial activity was observed in a reusability experiment after nine reaction cycles, each lasting 24h. Rinsing with solvent to remove water from the immobilised enzymes further improved their activity. This study demonstrates the suitability of immobilised crude enzyme preparations in the development of biocatalysts for esterification reactions.