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Journal for Biophysical Chemistry

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Multitechnique study on a recombinantly produced Bacillus halodurans laccase and an S-layer/laccase fusion protein

Abstract

Methods for organizing functional materials at the nanometer scale are essential for the development of novel fabrication techniques. One of the most relevant areas of research in nanobiotechnology concerns technological utilization of self-assembly systems, wherein molecules spontaneously associate into reproducible supramolecular structures. For this purpose, the laccase of Bacillus halodurans C-125 was immobilized on the S-layer lattice formed by SbpA of Lysinibacillus sphaericus CCM 2177 either by (i) covalent linkage of the enzyme to the natural protein self-assembly system or (ii) by construction of a fusion protein comprising the S-layer protein and the laccase. The laccase and the S-layer fusion protein were produced heterologously in Escherichia coli. After isolation and purification, the properties of the proteins, as well as the specific activity of the enzyme moiety, were investigated. Interestingly, the S-layer part confers a much higher solubility on the laccase as observed for the sole enzyme. Comparative spectrophotometric measurements of the enzyme activity revealed similar but significantly higher values for rLac and rSbpA/Lac in solution compared to the immobilized state. However, rLac covalently linked to the SbpA monolayer yielded a four to five time higher enzymatic activity than rSbpA/Lac immobilized on a solid support. Combined quartz crystal microbalance with dissipation monitoring (QCM-D) and electrochemical measurements (performed in an electrochemical QCM-D cell) revealed that rLac immobilized on the SbpA lattice had an approximately twofold higher enzymatic activity compared to that obtained with the fusion protein. nt]mis|Authors have contributed equally to this work. nt]mis|Author to whom correspondence should be addressed

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Ferner-Ortner-Bleckmann, J., Schrems, A., Ilk, N. et al. Multitechnique study on a recombinantly produced Bacillus halodurans laccase and an S-layer/laccase fusion protein. Biointerphases 6, 63–72 (2011). https://doi.org/10.1116/1.3589284

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