Functionalization of micropipette tips with hydrophobin-laccase chimera and application to the electrochemical determination of caffeic acid in tea samples

This work is part of the I + D + I project PID2020-118376RA-I00 funded by MCIN/AEI/10.13039/501100011033 (...); E.C.R. thanks for the support of the grant “Beatriz Galindo” (BG20/00027) funded by the Ministry of Universities of the Spanish Government

FUNGAL SELF-ASSEMBLING PROTEIN LAYERS: NEW BIOTECH-TOOLS FOR BIO/NON-BIO HYBRID DEVICES

During recent years the demand of valuable scaffolds to biofunctionalize nanomaterials has been rapidly increasing. Self-assembling proteins, such as amyloid fibrils, are promising candidates for the functionalization of nanomaterials due to their chemical and mechanical stability. These fibrillar structures are typically associated to neurodegenerative diseases. However, it has been recently observed that many organisms, such as fungi and some bacteria, take advantage of the ability of polypeptides to form amyloids. Hydrophobins represent an example of functional amyloid fibrils produced at different growth stages by filamentous fungi. They are a large family of very active surface proteins and can be grouped into two distinct classes based on the stability of the amphipathic layers that they form. In particular, layers formed by class I share many structural properties with amyloid fibrils, solubilized only after harsh acid treatments Immobilization of active proteins on the hydrophobin layer has been proven as a simple approach for the functionalization of different surfaces useful in biotechnological applications. For most of these applications, a critical aspect concerns protein orientation with respect to the surface. To overcome this problem, design and recombinant production of fused engineered proteins combining the adhesive moiety of hydrophobins to target proteins can be a valuable choice. This PhD project has been focused on recombinant production in different hosts (Pichia pastoris and Escherichia coli) of chimera proteins built by selected target proteins (laccase, antimicrobial peptides and antibody) fused to an adhesive self-assembling class I hydrophobin, and on their exploitation in some application fields. The selected class I hydrophobin was Vmh2, extracted from the mycelium of the fungus Pleurotus ostreatus, one of the most hydrophobic hydrophobin able to spontaneously form stable and homogeneous layers on hydrophilic or hydrophobic surfaces, changing their wettability. As a first goal, the hydrophobin Vmh2 was fused to the laccase POXA1b from P. ostreatus, a high redox potential oxidase endowed with a remarkable stability at high temperature and at alkaline pH. The resulting fusion enzyme was secreted into the culture medium by the heterologous yeast P. pastoris and directly used for coating different surfaces, i.e. graphene and polystyrene, without additional purification steps. The immobilized enzyme was exploited to develop innovative optical and electrochemical biosensing platform for the detection of phenolic compounds. Subsequently, two chimeric proteins were recombinantly expressed in E. coli: in the first case, Vmh2 was fused to the antimicrobial peptide LL37, a small cationic amphiphilic peptide belonging to the family of cathelicidins which has an effective activity against a wide range of bacteria and fungi. The LL37-Vmh2 chimeric protein was successfully produced and its adhesive and antibiofilm capabilities, against Staphylococcus epidermidis, tested by coating polystyrene surfaces. The chimera HN1-Vmh2, the fusion between the hydrophobin and the anti-mesothelin ScFv antibody, was produced in the soluble fraction of E. coli whilst its purification and exploiation needs to be optimized. In conclusion, chimeric proteins obtained by the genetic fusion of the hydrophobin Vmh2 to target proteins represent a proof of concept of versatile and straightforward solutions of surface functionalizations that could be explored in several fields. Moreover, during the six months stay at the CNRS of Grenoble, the work was focused on carbon nanotubes (CNT) functionalization using the native laccase POXC from P. ostreatus. POXC is a promising biocathode for enzymatic biofuel cells (EBFCs), since it shows efficient ORR (Oxygen Reduction Reaction) performances and it is capable to operate in a conventional H2/air proton-exchange membrane fuel cell

Biosensors and Bioprocesses to address Environmental Pollution

Pollution caused by heavy metals is a serious threat for the environment and human health, because of their toxicity and persistency. Among heavy metals, arsenic represents a harsh pollutant able to contaminate air, water and soil; it is a component of the Earth crust and is present in many geothermal environments, but it is also released into the environment by the consumption of arsenic-containing products such as insecticides, pesticides, and chemotherapeutic drugs. Most of the current systems for monitoring and restoring the environment from heavy metals require several expensive and hard instrumentation; modern biotechnologies can be addressed to exploit metal bio-transformations for the set-up of easier and possibly cheaper devices and eco-sustainable processes. In this context thermophiles stand out, they are microorganisms adapted to live in harsh conditions, which often include high concentrations of heavy metals. Therefore, knowledges of their resistance mechanisms can lead to the development of new strategies to face the heavy metals pollution. Moreover, thermophilic microorganisms have been associated to sources of thermostable proteins and enzymes useful for several applications; in fact, since the last 30 years they have been deeply studied for their high potential in industrial biotechnologies. This PhD thesis is aimed at the exploitation of thermophilic microorganisms for environmental applications. In particular we here report the state of the art on the most common thermophilic resistance mechanisms to heavy metals (Chapter [2]); we also describe the set-up of electrochemical and optical biosensors for the monitoring of arsenic based on thermophilic enzymes (Chapter [3]), as well as the isolation and physiological, molecular and genetic characterization of new metal-tolerant thermophiles from extreme environments (Chapter [4]) as source of novel biomolecules and/or bioprocesses. Furthermore, we report the emerging technologies for genetic engineering of thermophilic microorganisms, and their employment against, not only heavy metals, but also organic pollutants and in the lignocellulose degradation (Chapter [5])

A New Strategy for As(V) Biosensing Based on the Inhibition of the Phosphatase Activity of the Arsenate Reductase from Thermus thermophilus

Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are noncompetitive inhibitors affecting the Vmax but not the KM of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 µm for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a sensitivity of 0.53 ± 0.03 mU/mg/µm and a limit of detection (LOD) of 0.28 ± 0.02 µm. The good sensitivity and specificity for As(V) point to consider inhibition of TtArsC phosphatase activity for the setup of a novel biosensor for the detection of As(V)

Microparticle-Based Biosensors for Anthropogenic Analytes

Anthropogenic pollution of water resources and the environment by various hazardous compounds and classes of substances raises concerns about public health impacts and environmental damage. Commercially available, portable and easy-to-use devices to detect and quantify these compounds are rather sparse, but would contribute to comprehensive monitoring and reliable risk assessment. The Soft Colloidal Probe (SCP) assay is a promising platform for the development of portable analytical devices and thus has a great potential for a transfer to industry. This assay is based on the differential deformation of an elastic particle, i.e., the SCP, as a function of analyte concentration, which affects the extent of interfacial interactions between the SCP and a biochip surface. The objective of this work was to adapt this assay for the detection of anthropogenic pollutants. Biomimetic molecular recognition approaches were used based on naturally occurring target proteins that specifically bind the anthropogenic pollutants of interest. This adaptation included the elaboration of strategies for site-specific immobilization of the respective proteins and functionalization of SCPs. In this work, it is demonstrated that the SCP method can be employed for the highly specific and sensitive detection of the critically discussed pesticide glyphosate by using the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Furthermore, a specific detection scheme for estrogens and compounds with estrogenic and antiestrogenic activity was developed by harnessing estrogen sulfotransferase as the biomimetic recognition element. In the second part of the thesis, improvements of the SCP sensing methodology are described. These improvements were achieved by accelerating data analysis and developing a novel synthesis method for SCPs that ensures monodisperse particles with superior reproducibility. Rapid extraction of interaction energies is achieved by using a pattern matching algorithm that reduces the time required for data analysis to a fraction. The microfluidics-assisted synthesis of SCPs enables the production of highly monodisperse SCPs with adjustable size and mechanical properties. Various functionalization approaches have been developed that allow easy and modular introduction of functional groups and biomolecules for SCP-based sensing approaches

A Novel SCP-RICM Assay Application: Indirect Detection of Analytes by Modulation of Protein-Protein Interactions

The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area. An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection. The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1. It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components. The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces. Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT Table of Content I List of Figures VII List of Tables IX List of Abbreviations XI 1 Introduction 1 1.1 Biosensors 1 1.2 Analytical Detection Methods: Copper 2 1.3 SCP-RICM Assay 3 1.3.1 Sensor Chip Surface 4 1.3.2 Soft Colloidal Probes 5 1.3.3 Reflection Interference Contrast Microscopy 6 1.4 Hydrophobins 9 1.4.1 Structure and Functions of Hydrophobins 9 1.4.2 Ex vivo Applications of Hydrophobins 11 1.4.3 Class I Hydrophobin: Ccg-2 12 1.5 Mitochondrial Respiratory Chain 14 1.5.1 Copper Transport in Yeast 14 1.5.2 S. cerevisiae Sco1 protein 18 1.5.3 S. cerevisiae Cox17 protein 21 1.6 SCP-RICM Assay for Copper Detection 23 1.7 Aim of the Study 24 2 Materials and Methods 25 2.1 Laboratory Equipment 25 2.1.1 Devices 25 2.1.2 Chemicals 26 2.1.3 Consumables 28 2.1.4 Antibodies 29 2.1.5 Enzymes 30 2.1.6 Molecular Weight Standards 30 2.1.7 DNA Oligonucleotides 31 2.1.8 Plasmids and Vectors 32 2.2 Microorganisms 33 2.2.1 Strains 33 2.2.2 Cultivation of Microorganisms 34 2.2.3 Preparation of Electrocompetent E. coli Cells 36 2.2.4 Preparation of E. coli Glycerol Stocks 36 2.3 Protein Design 37 2.4 Molecular Cloning Methods 38 2.4.1 Vector Template Preparation 38 2.4.2 Agarose Gel Electrophoresis 40 2.4.3 DNA Extraction from Agarose Gels 41 2.4.4 Polymerase Chain Reaction 41 2.4.5 DNA Restriction Digest 42 2.4.6 DNA Dialysis 43 2.4.7 Ligation of DNA Fragments 43 2.4.8 Isolation of DNA from E. coli 44 2.4.9 DNA Sequencing 45 2.4.10 Transformation of E. coli via Electroporation 45 2.5 Protein Detection and Quantification 46 2.5.1 SDS PAGE 46 2.5.2 Coomassie Staining 50 2.5.3 Western Blot Analysis 51 2.5.4 Immunological Detection 51 2.5.5 Protein Quantification: Lowry Assay 52 2.5.6 Protein Quantification: Bradford Assay 53 2.5.7 Protein Quantification: NanoDrop Measurement 53 2.6 Protein Purification and Storage 54 2.6.1 Expression Analysis of Recombinant Proteins 54 2.6.2 Solubility Analysis 54 2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55 2.6.4 Quantification of Purified Proteins 64 2.6.5 Dialysis of Purified Proteins 65 2.7 Glass Surface Functionalization 65 2.7.1 Glass Surface Preparation 66 2.7.2 Hydrophobin and Fusion Protein-Based Coating 66 2.7.3 Contact Angle Measurement 67 2.7.4 DRoPS Test 67 2.7.5 Atomic Force Microscopy 67 2.8 SCP Functionalization 68 2.8.1 Functionalization of SCPs with Proteins 68 2.8.2 Validation of SCP Functionalization with FITC Staining 69 2.9 SCP-RICM Assay and Its Analysis 69 3 Results 73 3.1 Generation of Recombinant Fusion Proteins 73 3.1.1 Sco1 and Sco1∆95 73 3.1.2 Cox17 84 3.1.3 Ccg-2 88 3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90 3.2 His-Tag Cleavage 92 3.3 Chip Surface Functionalization 94 3.3.1 Optimization of the Glass Chip Preparation 94 3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95 3.3.3 AFM Measurements 102 3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103 3.4 SCP Functionalization 104 3.4.1 SCP Functionalization and FITC Staining 104 3.4.2 Theoretical Package of Proteins on SCPs 106 3.5 SCP-RICM Assay 107 4 Discussion and Further Prospectives 113 4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113 4.2 Outlook and Further Prospects 119 4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119 4.2.2 Further Analysis of Chip Surface Functionalization 124 4.2.3 Alternative Chip Surface Functionalization Methods 126 4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128 4.2.5 SCP-RICM Assay: Copper Detection 130 4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131 4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133 5 Summary 137 6 Bibliography 141 7 Appendix 165 7.1 Sequences of Protein Constructs 165 7.1.1 Sequences of the Protein Construct Cox17_a 165 7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165 7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166 7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167 7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169 7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171 7.1.7 Sequences of the Hydrophobin Ccg-2 173 7.2 pET-28b(+): Plasmid Map 173 7.3 Nickel Removal During Dialysis 175 7.4 DGR Assay 176 7.5 SCP diameter 179 Acknowledgements 181 Declaration of Authorship 18

Applications and immobilization strategies of the copper-centred laccase enzyme : a review

DATA AVAILABILITY STATEMENT: No data was used for the research described in the article.Laccase is a multi-copper enzyme widely expressed in fungi, higher plants, and bacteria which facilitates the direct reduction of molecular oxygen to water (without hydrogen peroxide production) accompanied by the oxidation of an electron donor. Laccase has attracted attention in biotechnological applications due to its non-specificity and use of molecular oxygen as secondary substrate. This review discusses different applications of laccase in various sectors of food, paper and pulp, waste water treatment, pharmaceuticals, sensors, and fuel cells. Despite the many advantages of laccase, challenges such as high cost due to its non-reusability, instability in harsh environmental conditions, and proteolysis are often encountered in its application. One of the approaches used to minimize these challenges is immobilization. The various methods used to immobilize laccase and the different supports used are further extensively discussed in this review.The National Research Foundation (NRF) of South Africa.https://www.cell.com/heliyonChemical Engineerin

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