Weak protein-nanoparticle (NP) interactions are studied in a low binding regime as a model for the soft protein corona around nanoparticles in complex biological fluids. Noncovalent, reversible interactions between Subtilisin Carlsberg (SC) and silica NPs shows significant alteration in conformation and enzymatic activity in a NP-size dependent manner. Very weak interactions between SC and silica NPs were revealed by centrifugation-based separations and further supported by small-angle X-ray scattering, while bovine serum albumin was used as a strongly interacting reference. Secondary and tertiary structure changes of SC were studied via circular dichroism and correlated to enzymatic activity where the enzyme kinetics showed a critical role for nanoparticle size.

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The ionic liquids, [bmim]Br and [bmim][N(CN) 2 ] (where [bmim] = 1-butyl-3-methylimidazolium), stimulated laccase-catalysed oxidation of catechol when provided at concentrations between 10-20% and 50-60% (v/v) in water, respectively. However, activity was inhibited at higher and lower concentrations. [bmim][BF 4 ] was inhibitory at all concentrations tested, but residual activity was still retained in [bmim][BF 4 ] with ≤ 20% water.Laccases have attracted a great deal of attention as potential biocatalysts for manufacturing pharmaceutical intermediates and speciality chemicals, and for bioremediation. 1-3 Their catalytic versatility is a consequence of the unusual reaction mechanism in which the substrate (or mediator) is oxidised to a free radical intermediate using dioxygen as the electron acceptor. 4 This reactive intermediate can then undergo a wide range of reactions with other chemicals, and, most importantly, its mobility overcomes the requirement for close contact between the enzyme and its substrate. This means that laccases exhibit excellent catalytic activity even with water-insoluble substrates (e.g. lignin). 5 Further improvements in mass transport can be achieved by operating the reactions in conventional organic solvents, 6-8 but the range of solvents suitable for use with enzymes is extremely restricted, and there are serious problems with safety and environmental acceptability. For these reasons, we wished to explore the use of ionic liquids as alternative solvents. There are literally millions of ionic liquids, 9-11 offering new solvents with an unprecedented variety of chemical and physical properties. This offers a realistic prospect of tailoring the solvent to match the requirements of both the enzyme and the bioprocess with exquisite precision. Furthermore, most ionic liquids have no detectable vapour pressure at room temperature, which can provide new approaches to product work-up and separation (for example 12,13 ), and allows operation in non-flameproof areas. It is also possible to design environmentally benign, non-toxic ionic liquids, 9,14 and some can even be produced from renewable feedstocks. 15 In addition, ionic liquids are electrically conducting (unlike conventional solvents), 16 and this may be beneficial for exploitation of laccases in biosensors and biofuel cells. 17There is already some evidence that ionic liquids can be used with laccases, but the activity in 4-methyl-1butylpyridinium tetrafluoroborate ([ 4 mbpy][BF 4 ]) or 1-butyl-3methylimidazolium hexafluorophosphate, [bmim][PF 6 ], mixed

Long-range electron transfer (ET) in horseradish peroxidase (HRP) was studied with a wild-type recombinant form of HRP, rHRP, and recombinant forms containing histidine and cysteine tags at Gln1, Asn57, Asn189, or Ser309 amino acid residues of the protein. Chemisorption of the enzyme onto the Au electrodes through the tags introduced in different positions of the protein surface provided anisotropic orientations of the rHRPs on the Au surface, which allowed a restricted "rotation" of the rHRP molecules on the electrodes. Atomic force microscopy (AFM) studies revealed the monolayer coverage of the enzyme on gold surfaces and the specific orientations of different forms of rHRP, which may be characterized by different distances between the heme active site of rHRP and the gold electrode. The efficiency of long-range ET between the electrode and the heme of rHRP was estimated from direct non-catalytic electrochemistry of rHRPs differently orientated on Au and compared with the theoretically calculated values from the protein ET model (C. C. Page, C. C. Moser, X. Chen, P. L. Dutton, Nature, 1999, 402, 47-51), under the assumption that ET occurs within the protein structure between the heme and the tag-modified amino acid residue of the protein. Comparative analysis of the long-range ET through the rHRP showed that the highest ET rates, obtained for the rHRP forms containing the tags at C- or N-termini of the enzyme, did not correlate with the shortest ET distance, but were instead consistent with the directional ET along the most favourable ET pathway within the protein matrix.