Partial Characterization of Three Bacteriophages Isolated from Aquaculture Hatchery Water and Their Potential in the Biocontrol of Vibrio spp.

Bacteriophages are currently considered one of the most promising alternatives to antibiotics under the 'One Health' approach due to their ability to effectively combat bacterial infections. This study aimed to characterize Vibrio species in hatchery water samples collected from an aquaculture farm and investigate the biocontrol potential of their bacteriophages. Vibrio spp. (n = 32) isolates confirmed by LNA probe-based qPCR were used as hosts. Three Vibrio phages were isolated. IKEM_vK exhibited a broad host range, infecting V. harveyi (n = 8), V. alginolyticus (n = 2), V. azureus (n = 1), and V. ordalii (n = 1). IKEM_v5 showed lytic activity against V. anguillarum (n = 4) and V. ordalii (n = 1), while IKEM_v14 was specific to V. scophtalmi (n = 4). The morphological appearance of phages and their lytic effects on the host were visualized using scanning electron microscopy (SEM). All three phages remained relatively stable within the pH range of 6-11 and up to 60 °C. The lytic activities and biofilm inhibition capabilities of these phages against planktonic Vibrio cells support their potential applications in controlling vibriosis in aquaculture systems.

A review of aptamer-conjugated nanomaterials for analytical sample preparation: Classification according to the utilized nanomaterials.

BACKGROUND: Sample extraction before detection is a critical step in analysis. Since targets of interest are often found in complex matrices, the sample can not be directly introduced to the analytical instrument. Nanomaterials with unique physical-chemical properties are excellent supports for use in sorbent-based extraction. However, they lack selectivity and thus need to be functionalized with target-capturing molecules. Antibodies and molecularly imprinted polymers (MIPs) can be used for this purpose, but they have some problems that limit their practical applications. Hence, functionalization of nanomaterials for selectivity remains a problem. RESULTS: Nucleic acid aptamers are affinity reagents that can provide superiority to antibodies since they can be selected in vitro and at a lower cost. Moreover, aptamers can be chemically synthesized and easily modified with different functional groups. Hence, aptamers are good candidates to impart selectivity to the nanomaterials. Recent studies focus on the integration of aptamers with magnetic nanoparticles, carbon-based nanomaterials, metal-organic frameworks, gold nanoparticles, gold nanorods, silica nanomaterials, and nanofibers. The unique properties of nanomaterials and aptamers make the aptamer-conjugated nanomaterials attractive for use in sample preparation. Aptamer-functionalized nanomaterials have been successfully used for selective extraction of proteins, small molecules, and cells from different types of complex samples such as serum, urine, and milk. In particular, magnetic nanoparticles have a wider use due to the rapid extraction of the sample under magnetic field. SIGNIFICANCE: In this review, we aim to emphasize how beneficial features of nanomaterials and aptamers could be combined for extraction or enrichment of the analytes from complex samples. We aim to highlight that the benefits are twofold in terms of selectivity and efficiency when employing nanomaterials and aptamers together as a single platform.

Selection of DNA aptamers for the aptamer-assisted magnetic capture of the purified xylanase from Aspergillus niger.

Xylanases are a group of enzymes that catalyze the hydrolysis of xylan. Xylanases have wide industrial applications, and they can produced by various organisms. In this study, we aimed to develop aptamers for the capture of xylanase produced by a wild-type Aspergillus niger strain. Xylanase was produced by Aspergillus niger in a 5-liter stirred-tank bioreactor and then purified by column chromatography. Magnetic bead-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was performed to select DNA aptamers specific to the purified xylanase. After nine rounds of selection, next-generation sequencing (NGS) analysis was performed. Four aptamers, namely AXYL-1, AXYL-2, AXYL-3, and AXYL-4, were identified for further characterization. The binding properties of the selected aptamers were characterized by fluorescence quenching (FQ) analysis and an enzyme-linked aptamer assay (ELAA). The Kd values were found to be in the low µM range. Then, each aptamer was immobilized on streptavidin-coated magnetic particles, and the recovery ratio of xylanase was determined. Although AXYL-1 wasn't effective, AXYL-2, AXYL-3, and AXYL-4 were proven to capture the xylanase. The maximum recovery rate of xylanase was found to be approximately 54 %.

Recombinant Production and Biochemical Characterization of Thermostable Arabinofuranosidase from Acidothermophilic Alicyclobacillus Acidocaldarius.

The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius.

Deacetylation of Histones and Non-histone Proteins in Inflammatory Diseases and Cancer Therapeutic Potential of Histone Deacetylase Inhibitors.

Epigenetic changes play an important role in the pathophysiology of autoimmune diseases such as allergic asthma, multiple sclerosis, lung diseases, diabetes, cystic fibrosis, atherosclerosis, rheumatoid arthritis, and COVID-19. There are three main classes of epigenetic alterations: post-translational modifications of histone proteins, control by non-coding RNA and DNA methylation. Since histone modifications can directly affect chromatin structure and accessibility, they can regulate gene expression levels. Abnormal expression and activity of histone deacetylases (HDACs) have been reported in immune mediated diseases. Increased acetylated levels of lysine residues have been suggested to be related to the overexpression of inflammatory genes. This review focuses on the effect of HDAC modifications on histone and non-histone proteins in autoimmune diseases. Furthermore, we discuss the potential therapeutic effect of HDAC inhibitors (HDACi) used in these diseases.

Magnetic Nanoparticle-Based Electrochemical Sensing Platform Using Ferrocene-Labelled Peptide Nucleic Acid for the Early Diagnosis of Colorectal Cancer.

Diagnostic biomarkers based on epigenetic changes such as DNA methylation are promising tools for early cancer diagnosis. However, there are significant difficulties in directly and specifically detecting methylated DNA regions. Here, we report an electrochemical sensing system based on magnetic nanoparticles that enable a quantitative and selective analysis of the methylated septin9 (mSEPT9) gene, which is considered a diagnostic marker in early stage colorectal cancer (CRC). Methylation levels of SEPT9 in CRC samples were successfully followed by the selective recognition ability of a related peptide nucleic acid (PNA) after hybridization with DNA fragments in human patients' serums and plasma (n = 10). Moreover, this system was also adapted into a point-of-care (POC) device for a one-step detection platform. The detection of mSEPT9 demonstrated a limit of detection (LOD) value of 0.37% and interference-free measurement in the presence of branched-chain amino acid transaminase 1 (BCAT1) and SRY box transcription factor 21 antisense divergent transcript 1 (SOX21-AS1). The currently proposed functional platform has substantial prospects in translational applications of early CRC detection.

Indiscriminate SARS-CoV-2 multivariant detection using magnetic nanoparticle-based electrochemical immunosensing.

The increasing mutation frequency of the SARS-CoV-2 virus and the emergence of successive variants have made correct diagnosis hard to perform. Developing efficient and accurate methods to diagnose infected patients is crucial to effectively mitigate the pandemic. Here, we developed an electrochemical immunosensor based on SARS-CoV-2 antibody cocktail-conjugated magnetic nanoparticles for the sensitive and accurate detection of the SARS-CoV-2 virus and its variants in nasopharyngeal swabs. The application of the antibody cocktail was compared with commercially available anti-SARS-CoV-2 S1 (anti-S1) and anti-S2 monoclonal antibodies. After optimization and calibration, the limit of detection (LOD) determination demonstrated a LOD = 0.53-0.75 ng/mL for the antibody cocktail-based sensor compared with 0.93 ng/mL and 0.99 ng/mL for the platforms using anti-S1 and anti-S2, respectively. The platforms were tested with human nasopharyngeal swab samples pre-diagnosed with RT-PCR (10 negatives and 40 positive samples). The positive samples include the original, alpha, beta, and delta variants (n = 10, for each). The polyclonal antibody cocktail performed better than commercial anti-S1 and anti-S2 antibodies for all samples reaching 100% overall sensitivity, specificity, and accuracy. It also showed a wide range of variants detection compared to monoclonal antibody-based platforms. The present work proposes a versatile electrochemical biosensor for the indiscriminate detection of the different variants of SARS-CoV-2 using a polyclonal antibody cocktail. Such diagnostic tools allowing the detection of variants can be of great efficiency and economic value in the fight against the ever-changing SARS-CoV-2 virus.

Bimolecular fluorescence complementation assay to explore protein-protein interactions of the Yersinia virulence factor YopM.

Yersinia outer protein M (YopM) is one of the effector proteins and essential for virulence. YopM is delivered by the Yersinia type III secretion system (T3SS) into the host cell, where it shows immunosuppressive effect through interaction with host proteins. Therefore, protein-protein interactions of YopM is significant to understand its molecular mechanism. In this study, we aimed to explore protein-protein interactions of YopM with the two components of T3SS, namely LcrV and LcrG. We used bimolecular fluorescence complementation (BiFC) assay and monitored the reassembly of green fluorescence protein in Escherichia coli. As an indicator of the protein-protein interaction, we monitored the in vivo reconstitution of fluorescence by measuring fluorescence intensity and imaging the cells under fluorescence microscope. We showed, for the first time, that YopM interacts with LcrG, but not with LcrV. Here, we propose BiFC assay as a simple method to screen novel interaction partners of YopM.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

Dye-Loaded Polymersome-Based Lateral Flow Assay: Rational Design of a COVID-19 Testing Platform by Repurposing SARS-CoV-2 Antibody Cocktail and Antigens Obtained from Positive Human Samples.

The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified via magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 via commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol-b-polycaprolactone (mPEG-b-PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92-94%) and reproducibility (96-98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91-97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications.

Quantitative paper-based dot blot assay for spike protein detection using fuchsine dye-loaded polymersomes.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays are the gold standard for virus diagnosis. Point-of-care (POC) technologies have shown great progress during this period. Herein, we propose a novel fuchsine dye-loaded polymersome for a colorimetric paper-based dot blot spike protein diagnostic assay for COVID-19 via smartphone-assisted sensing. The prepared platform aimed to create an adaptable tool that competes with traditional nanoparticle-based assays employing gold and silver. Analytical characterization and application of the testing platform showed high sensitivity (10 times better than gold nanoparticles), stability, fast turnaround, and reproducibility. The potential and possibilities demonstrated by the current platform could be observed in its adaptability for different markers and pathologies. In addition, smartphone-assisted sensing emphasizes the ability to use the tool at home by common peoples which can lower the burden on the healthcare facilities and reach more underdeveloped regions.

Screen printed electrode-based biosensor functionalized with magnetic cobalt/single-chain antibody fragments for cocaine biosensing in different matrices.

On-site detection of substance abuse is an important approach in the preventive and intervention protocols implementations. It is known that the traditional methods are heavy, time-consuming, and need a high level of logistical requirements. As such, biosensors represent great potential to simplify and improve substance abuse detection. In this study, we have designed a functionalized screen-printed electrode (SPE) electrochemical biosensor with cobalt oxide nanoparticles and single-chain antibody fragments (scFvs) for cocaine detection. Different electrochemical techniques such as differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectrometry were used to examine the functionality of the designed biosensor. Furthermore, SEM observations were performed to observe the surface changes after functionalization. The results showed that the linearity ranged between 5.0 and 250 ng/mL and a detection limit of 3.6 ng/mL (n = 6). These results were compared to results obtained from Q-TOF/MS where four different matrices (serum, sweat, urine, and saliva) were spiked with 100 ng/mL cocaine and were analyzed by both methods (Biosensor and Q-TOF/MS). Results showed a higher performance of the biosensor compared to traditional methods. In addition, the selectivity of the biosensor was shown in the presence of different interferents where the designed platform showed a specific response to only cocaine. In conclusion, the designed biosensor proposes great potential for portable and on-site substance abuse detection in addition to boasting the capability of reuse of the SPE and thus, reducing the costs related to such applications.

The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene delivery vector.

Non-viral gene delivery systems have great potential for safe and efficient gene therapy, while inefficient cellular and nuclear uptake remain as the major hurdles. Novel approaches are needed to enhance the transfection efficiency of non-viral vectors. In accordance with this need, the objective of this study was to construct a non-viral vector that could achieve gene delivery without using additional lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by using the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding green fluorescent protein (GFP) was labeled with quantum dots (QDs) via peptide-nucleic acid (PNA) recognition site. Recombinant YopM protein was then attached to the conjugate via a second PNA recognition site. The YopM ̶ QDs ̶ pDNA conjugate was transfected into HeLa cells without using additional transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was successfully delivered to the nucleus. As control, naked pDNA was transfected into the cells by using a commercial transfection reagent. The Y. pseudotuberculosis YopM-functionalized conjugate achieved the highest GFP expression, compared to other two YopM proteins and the transfection reagent. To the best of our knowledge, YopM protein was used for the first time in a non-viral gene delivery vector.

Single-stranded DNA (ssDNA) Aptamer targeting SipA protein inhibits Salmonella Enteritidis invasion of intestinal epithelial cells.

Salmonella Enteritidis is an important pathogen that can invade the intestinal cells of its host causing salmonellosis. SipA protein, an effector protein secreted by T3SS, maintains invasion of host cells more efficient. Thus, inhibitory aptamers against SipA protein were developed using magnetic bead-based Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. The enriched sequences were obtained after 9 SELEX rounds. Among which, an aptamer namely Apt17 displayed Kd values equivalent to 114.9 and 63.4 nM at 27 °C and 37 °C, respectively. The effect of Apt17 on adhesion and invasion of Caco-2 cells by the tested strains was determined. While the adhesion and invasion of Salmonella Enteritidis TM 6 were inhibited by 70% and 37.7%, those of Salmonella Enteritidis TM 68 were inhibited by 45.71% and 39.5% respectively. These results represent a corner stone for future studies that could aim to develop putative inhibitors against Salmonellosis.

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics.

Aptamer-based point-of-care (POC) diagnostics platforms may be of substantial benefit in forensic analysis as they provide rapid, sensitive, user-friendly, and selective analysis tools for detection. Aptasensors have not yet been adapted commercially. However, the significance of the applications of aptasensors in the literature exceeded their potential. Herein, in this review, a bottom-up approach is followed to describe the aptasensor development and application procedure, starting from the synthesis of the corresponding aptamer sequence for the selected analyte to creating a smart surface for the sensitive detection of the molecule of interest. Optical and electrochemical biosensing platforms, which are designed with aptamers as recognition molecules, detecting abused drugs are critically reviewed, and existing and possible applications of different designs are discussed. Several potential disciplines in which aptamer-based biosensing technology can be of greatest value, including forensic drug analysis and biological evidence, are then highlighted to encourage researchers to focus on developing aptasensors in these specific areas.

MerR-fluorescent protein chimera biosensor for fast and sensitive detection of Hg2+ in drinking water.

Mercury ion (Hg2+ ) is a universal pollutant and its detection is crucial for public healthcare. In this study, we developed a novel fluorescent biosensor by construction of a protein fusion between the mercury-sensing transcription factor MerR and enhanced yellow fluorescent protein (EYFP). Hg2+ -induced conformational change of MerR was transduced into fluorescence signal. Fluorescence intensity of the biosensor protein decreased with increasing concentrations of Hg2+ and a linear response was obtained in the range of 0.5-40 nM. The limit of detection was 0.5 nM, which was much lower than the maximum allowed level in water. The biosensor specificity was highly dependent on type and concentration of metal ion. The biosensor exhibited high specificity in a mixture of metal ions at 0.5 nM concentration. However, the interference effect was more pronounced at 40 nM concentration of metal ions. The measurement was completed in less than 1 Min with no need for sample preparation or preincubation steps. The biosensor achieved accurate and reliable detection in the spiked drinking water sample, as validated by the inductively coupled plasma optical emission spectrometry.

A highly sensitive DNA aptamer-based fluorescence assay for sarcosine detection down to picomolar levels.

Sarcosine is an amino acid derivative, which is considered as a key metabolite in various metabolic processes. Therefore, simple and sensitive detection methods are needed for further understanding its metabolic role and diagnostic value. In this study, we developed a novel method that meets the need for practical and sensitive detection in a complex medium mimicking urine conditions. For this aim, we selected sarcosine-specific DNA aptamers using graphene oxide-assisted systemic evolution of ligands by exponential enrichment (GO-SELEX). The candidate aptamers were labeled with 6-carboxyfluorescein (6-FAM) at their 5' ends. Two aptamers, namely 9S and 13S produced a significant fluorescence signal upon sarcosine binding. Both aptamers enabled a sensitive analysis with a detection limit of 0.5 pM. The linear detection ranged between 5 pM and 50 µM for 9S aptamer, while 13S aptamer enabled a wider linear detection range between 5 pM and 500 µM. The aptamer-based assay allowed rapid detection with no need for chemical derivatization of sarcosine and sophisticated instruments. Moreover, the aptamer-based assay was free of interference from urea and human serum albumin.

Improving thermal and detergent stability of Bacillus stearothermophilus neopullulanase by rational enzyme design.

Neopullulanase, a glycosyl hydrolase from Bacillus stearothermophilus (bsNpl), is a potentially valuable enzyme for starch and detergent industries. However, as the protein is not active at elevated temperatures and high surfactant concentrations, we aimed to increase its stability by rational enzyme design. Nine potentially destabilizing cavities were identified in the crystal structure of the enzyme. Based on computational predictions, these cavities were filled by residues with bulkier side chains. The five Asp46Glu, Val239Leu, Val404Leu, Ser407Thr and Ala566Leu exchanges resulted in a drastic stabilization of bsNpl against inactivation by heat and detergents. The catalytic activity of the variants was identical to the wild-type enzyme.

The covalent bioconjugate of multiwalled carbon nanotube and amino-modified linearized plasmid DNA for gene delivery.

Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.

Development of genetically encoded fluorescent protein constructs of hyperthermophilic maltose-binding protein.

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.