Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Abstract

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.

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…Hooks et al (2013) also pointed out that displaying N-acetylneuraminic acid aldolase (NanA) from E. coli on PHA particles through N-and C-terminal fusion of PhaC resulted in varying catalytic performance . Moreover, similar findings were reported for phasins where fusion of different foreign polypeptides to the BioF tag (PHA-binding domain of PhaF) might have contributed to inconsistency of the physical adsorption function of the BioFtagged enzyme to the PHA particle surface (Bello-Gil et al, 2018a). A brief comparison of the PHA particle technology with other biological assemblies, detailing their advantages and limitations, is provided in Table 2.…”

An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase‐like multicopper oxidase on poly‐3‐hydroxybutyrate

“…Figure shows that BioF–CueO‐funcionalized PHB can be reused at least in the short term (five 1 h cycles). This is in accordance with the ability of a BioF–β‐galactosidase fusion adsorbed on PHB to maintain its enzymatic function after several activity + washing cycles (Bello‐Gil et al ., ). To evaluate reusability at longer incubation times and with a different dye, we analysed the activity of the minibioreactor on IC after several 20 h of cycles.…”

“…S6) yielded a dissociation constant of 0.7 ± 0.1 mg ml −1 and a maximum adsorption of 40 ± 2 μg of protein per mg of support. The affinity is similar to that displayed by the PhaF phasin in this system ( K d = 0.5 mg ml −1 ) although the binding capacity for BioF–CueO is appreciably higher (only 20 μg of protein per mg of support for PhaF) (Bello‐Gil et al ., ). Calculation of these features was essential to design the enzymatic experiments using immobilized protein, since they indicate which should be the initial soluble protein concentration to incubate with PHB for a given defined amount of actual immobilized enzyme per mg of support.…”

“…Commercial PHB (Sigma-Aldrich) was used in our study. A thorough biophysical analysis of this material has been recently reported (Bello-Gil et al, 2018). Ten milligrams of PHB particles were first washed with 20 mM Tris buffer pH 7.0 and incubated with increasing concentrations of the protein (0.1-2 mg ml À1 ) in the same buffer (600 ll).…”

“…Immobilization of CueO oxidase on PHB was achieved as a chimeric protein fused to the BioF affinity tag, the PHA binding domain of the PhaF phasin from Pseudomonas putida KT2442 (Prieto et al ., ; Moldes et al ., , ). Although very interesting in vivo procedures have been described using fusions with the PhaC synthase from Ralstonia eutropha leading to covalent attachment to PHA granules in vivo (Rehm et al ., ), we have recently demonstrated the feasibility of the non‐covalent interaction between PHB particles and the BioF tag for the construction of protein‐based biomaterials that demand, as in the present case, a good control of the amount of immobilized protein, versatility of available supports and easy matrix regeneration once the protein is inactivated (Bello‐Gil et al ., ). Here, the degradation capacity of PHB minibioreactors containing the CueO enzyme was evaluated on different industrial dyes and in the absence of usual laccase mediators (Bourbonnais et al ., ; Moldes and Sanroman, ; Ibarra et al ., ) with a view on decreasing process costs upon future scaleup.…”