SummaryThe presence of synthetic dyes in wastewaters generated by the textile industry constitutes a serious environmental and health problem that urges the scientific community on an appropriate action. As a proof‐of‐concept, we have developed a novel approach to design enzymatic bioreactors with the ability to decolorize dye solutions through the immobilization of the bacterial CueO laccase‐like multicopper oxidase from Escherichia coli on polyhydroxybutyrate (PHB) beads by making use of the BioF affinity tag. The decolorization efficiency of the system was characterized by a series of parameters, namely maximum enzyme adsorption capacity, pH profile, kinetic constants, substrate range, temperature and bioreactor recycling. Depending on the tested dye, immobilization increased the catalytic activity of CueO by up to 40‐fold with respect to the soluble enzyme, reaching decolorization efficiencies of 45–90%. Our results indicate that oxidase bioreactors based on polyhydroxyalkanoates are a promising alternative for the treatment of coloured industrial wastewaters.

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-β-galactosidase, containing the choline-binding module C-LytA and the β-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-β-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials. Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.