Development of a biosensing platform based on a laccase-hydrophobin chimera
A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.
KeywordsImmobilization Chimera proteins L-DOPA Caffeic acid Self-assembly
The authors would like to thank Dr. Manuela Rossi of the Department of Earth Sciences, Environment and Resources, Università degli studi di Napoli “Federico II”, for her assistance in acquisition of stereo microscope images.
This work was funded by grants from the University Federico II, (000023_ALTRI_DR_409_2017_Ricerca Ateneo_GIARDINA) “Immobilization of ENzymes on hydrophobin-functionalized NAnomaterials” (IENA). This project is funded by Ministry of Education, Universities and Research and co-funded by European Union’s Horizon 2020 research and innovation programme under the framework of ERA-NET Cofund MarTERA (Maritime and Marine Technologies for a new Era), “FLAshMoB, Functional Amyloid chimera for Marine Biosensing” (ID: 172).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with animals performed by any of the authors. Moreover, all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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