Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein

The analysis of Pleurotus ostreatus genome reveals the presence of automatically annotated 53 lipase and 34 carboxylesterase putative coding-genes. Since no biochemical or physiological data are available so far, a functional approach was applied to identify lipases from P. ostreatus. In the tested growth conditions, four lipases were found expressed, with different patterns depending on the used C source. Two of the four identified proteins (PleoLip241 and PleoLip369), expressed in both analysed conditions, were chosen for further studies, such as an in silico analysis and their molecular characterization. To overcome limits linked to native production, a recombinant expression approach in the yeast Pichia pastoris was applied. Different expression levels were obtained: PleoLip241 reached a maximum activity of 4000 U/L, whereas PleoLip369 reached a maximum activity of 700 U/L. Despite their sequence similarity, these enzymes exhibited different substrate specificity and diverse stability at pH, temperature, and presence of metals, detergents and organic solvents. The obtained data allowed classifying PleoLip241 as belonging to the “true lipase” family. Indeed, by phylogenetic analysis the two proteins fall in different clusters. PleoLip241 was used to remove the hydrophobic layer from wool surface in order to improve its dyeability. The encouraging results obtained with lipase treated wool led to forecast PleoLip241 applicability in this field.

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“…Both plasmids were linearized by Bsi WI and transformed into P . pastoris BG10 by electroporation, as already reported [ 25 ].…”

Section: Methodssupporting

confidence: 70%

New lipases by mining of Pleurotus ostreatus genome

et al. 2017

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“…Vmh2 adhesion ability was also combined with the fluorescence emission of the Green Fluorescent Protein (GFP) by genetic fusion [ 46 ]. Vmh2-GFP was proven to be a smart and effective tool for the study of Vmh2 self-assembling and was used as the active biological element in the realization of an ultrasensitive thrombin biosensor.…”

Section: Plastic Functionalizationmentioning

confidence: 99%

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

et al. 2017

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Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

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“…Only a few proteins belonging to Class I HFBs have been analyzed so far and they show quite different self‐assembling mechanisms (Gandier et al, ; Gravagnuolo et al, ). We have focused our work on the discovery of new Class I HFBs, (Armenante et al, ) and the characterization of their self‐assembling (Gravagnuolo et al, ; Longobardi et al, ) to enable modern biotechnological applications (Gravagnuolo et al, ,; Kaur et al, ; Longobardi et al, , ; Patel et al, ; Piscitelli et al, ,; Politi et al, , ). Additionally, because of their critical role in fungal biology, the knowledge of their mechanism of action may be useful for the discovery of novel antifungals (Pham et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…These structures, thanks to their unique properties and their nanoscale size, are suitable building blocks of advanced bio-nanomaterials (Amit et al, 2012;Bleem and Daggett, 2017;Cherny and Gazit, 2008;Knowles and Mezzenga, 2016;Kumar et al, 2014;Reynolds et al, 2014). They find applications in nanodevices, biosensors, (Hauser et al, 2014;Piscitelli et al, 2017a) nanocomposites, (Li et al, 2012(Li et al, , 2014 drug delivery, (Bolisetty et al, 2014;Seras-Franzoso et al, 2016;Unzueta et al, 2017), and catalysis Piscitelli et al, 2017b). Despite the fact that all amyloidogenic proteins are different in their native monomeric conformation, their polymerization leads to the same fibrillary structure with a cross b-sheet fold (Biancalana and Koide, 2010;Fowler et al, 2007;Hamley, 2007;Ruggeri et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Self‐assembly of two hydrophobins from marine fungi affected by interaction with surfaces

Cicatiello

Dardano

Pirozzi

et al. 2017

Biotech & Bioengineering

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Hydrophobins are amphiphilic fungal proteins endowed with peculiar characteristics, such as a high surface activity and an interface triggered self-assembly. Several applications of these proteins have been proposed in the food, cosmetics and biomedical fields. Moreover, their use as proteinaceous coatings can be effective for materials and nanomaterials applications. The discovery of novel hydrophobins with diverse properties may be advantageous from both the scientific and industrial points of view. Stressful environmental conditions of fungal growth may induce the production of proteins with peculiar features. Two Class I hydrophobins from fungi isolated from marine environment have been recently purified. Herein, their propensity to aggregate forming nanometric fibrillar structures has been compared, using different techniques, such as circular dichroism, dynamic light scattering and Thioflavin T fluorescence assay. Furthermore, TEM and AFM images indicate that the interaction of these proteins with specific surfaces, are crucial in the formation of amyloid fibrils and in the assembly morphologies. These self-assembling proteins show promising properties as bio-coating for different materials via a green process. Biotechnol. Bioeng. 2017;114: 2173-2186. © 2017 Wiley Periodicals, Inc.

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Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein

Cited by 30 publications

References 34 publications

New lipases by mining of Pleurotus ostreatus genome

New lipases by mining of Pleurotus ostreatus genome

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

Self‐assembly of two hydrophobins from marine fungi affected by interaction with surfaces

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