Vmh2 hydrophobin as a tool for the development of “self‐immobilizing” enzymes for biosensing

Abstract

Self-assembling proteins forming amyloid fibrils are promising candidates for the fabrication of biomaterials, due to the chemical and mechanical stability of their structures. Among potential applications, their use as platforms for enzyme immobilization is rapidly gathering attention. In this work, we demonstrate that the production of the enzyme glutathione-S-transferase (GST) fused to the class I hydrophobin Vmh2 from Pleurotus ostreatus represents an invaluable tool for the development of self-immobilizing enzymes useful for high throughput analyses. The proposed immobilization strategy is versatile since it can be applied, in principle, to every recombinant protein able to refold from Escherichia coli inclusion bodies. A GST based biosensor has been developed to quantify toxic compounds, such as the pesticides molinate and captan, in aqueous environmental samples. The main advantages of this sensor include simplicity and speed of preparation, high sensitivity, reusability, and accuracy. Biotechnol. Bioeng. 2017;114: 46-52. © 2016 Wiley Periodicals, Inc.

From Protein Features to Sensing Surfaces

“…Hydrophobins were used to drive enzymes to a hydrophobic polystyrene surface. Once fused at the gene level to glutathione-S-transferase (GST), a biosensor for the detection of pesticides molinate and captan was produced [ 122 ]. The hydrophobin-assisted immobilization of GST resulted in a higher affinity for the analytes and a higher catalytic activity, e.g., a lower K M and an almost double k cat [ 122 ].…”

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins

“…Upon functionalization of polystyrene multiwell plate with a combination of HFBs either lacking or exposing the α-factor, an inverted enzyme-linked immunosorbent assay (ELISA) approach was developed yielding a novel kind of biosensor with the lowest limit of detection reported at the time of publication. Vmh2 from P. ostreatus fused to the enzyme glutathione-S-transferase (Vmh2-GST) was exploited for the quantification of the pesticides molinate and captan, acting as inhibitors of the enzymatic activity [ 45 ]. The fused protein efficiently functionalized the polystyrene multiwell plate for the development of high throughput analyses ( Figure 3 ).…”

Paper-Based Electrochemical Devices for the Pharmaceutical Field: State of the Art and Perspectives

“…Moreover, the use of nanomaterials (e.g., metal nanoparticles and carbon nanomaterials) has shown a quantitative influence on the enhancement of electrochemical (bio)sensors and labon-a-chip performances, with clearly positive effects on the analyses. Indeed, such nanomaterials have demonstrated benefits for higher electrocatalytic properties and sensing response thanks to their large surface area, defect sites, high electrical conductivity, and good mechanical features (Farka et al, 2017;Piscitelli et al, 2017;Mazzaracchio et al, 2019). Owing to these astonishing features, nanomaterials are currently exploited for in vivo and in vitro medical applications in the form of robust and tuneable diagnostic and therapeutic platforms (Chen and Chatterjee, 2013).…”