Design of a structure-based fluorescent biosensor from bioengineered arginine deiminase for rapid determination of L-arginine

doi:10.1016/j.ijbiomac.2020.09.134

International Journal of Biological Macromolecules

Volume 165, Part A, 15 December 2020, Pages 472-482

https://doi.org/10.1016/j.ijbiomac.2020.09.134 Get rights and content

Highlights

•
L-arginine was rapidly detected by fluorescent biosensor with one-enzyme system.
•
L-arginine level detected by the biosensor was comparable with mass spectrometry.
•
Determination of L-arginine level in complexed-matrix samples could be performed.

Abstract

Rationally designed mutations on recombinant arginine deiminase (ADI) could act as a ‘turn-off’ L-arginine (L-Arg) fluorescent biosensor and provide an alternative method for rapid determination of L-Arg. Double mutations were introduced on the Cys²⁵¹➔Ser²⁵¹ and Thr²⁶⁵➔Cys²⁶⁵ of recombinant ADI, rendering a single cysteine present on the protein surface for the site-specific attachment of a fluorophore, fluorescein-5-maleimide. The double mutations on ADI (265C) and its fluorescein-labelled form (265Cf) conserved the catalytic efficiency of wild-type ADI. Upon binding to L-Arg, 265Cf induced structural conformational changes and rendered the fluorescein moiety to move closer to Trp²⁶⁴, resulting in fluorescence quenching. The duration of fluorescence quenching was dependant on the L-Arg concentration. A linear relationship between the time at the maximum rate of fluorescence change and L-Arg concentrations, which ranged from 2.5 to 100 μM, was found with R² = 0.9988. The measurement time was within 0.15–4 min. Determination of L-Arg concentration in fetal bovine serum could be achieved by the standard addition method and without sample pre-treatment. The result showed a good agreement with the one determined by mass spectrometry, suggesting our biosensor as a promising tool for the detection of L-Arg in biological samples.

Introduction

L-Arginine (L-Arg) is recognized as one of the miracle molecules due to the significant roles it plays in acting as precursor and anti-aggregating agent of a lot of proteins and molecules for metabolism [1,2]. Its concentrations can be indicators of the degree of healthiness of people and the quality of food [3]. Generally, the normal L-Arg concentration in humans is about 100–120 μM [4]. This range can be a kind of references of physiological conditions since elevated plasma L-Arg concentration has been observed in some arginase deficiency persons while declined L-Arg concentration has been found in L-Arg auxotrophic cancer patients [3,[5], [6], [7], [8], [9], [10], [11], [12]]. On the other hand, measurement of plasma L-Arg concentrations is one of the key parameters that reflects the potency of arginine-depleting drugs for the treatment of cancers [[13], [14], [15]]. In the area of food processing, L-Arg is an index for monitoring the safety of beverages, especially in wine production [[16], [17], [18], [19]]. During the fermentation process, L-Arg is converted into urea, which is accumulated and sequentially reacts with ethanol to produce a hazardous and carcinogenic compound, ethyl carbamate [[16], [17], [18], [19]]. Therefore, the monitoring of L-Arg concentrations in biological and food samples is fundamentally important.

There are numerous methods to detect L-Arg concentrations, such as ionization mass spectrometry and high-performance liquid chromatography to provide accurate quantitative analyses [[20], [21], [22]], but they involve high operating cost and long measurement time. Biosensors, on the contrary, can provide fast response and high specificity for determination, and thus, they are commonly used in drug discovery, diagnosis, and food safety and processing [23,24]. Different types of L-Arg biosensors have been developed and they exhibit wide linear detection ranges with rapid response time. Details of their characteristics are summarized in Table 1. However, these biosensors have the following drawbacks: (a) The specificity of biosensor is reduced due to using ammonium (NH₃) as an analyte. Additional measurement of NH₃ concentration is thus required for samples that contain NH₃ [[25], [26], [27]], (b) Two enzymes system (Arginase/Urease) might introduce more variations than one enzyme system into the detection of L-Arg [26,[28], [29], [30], [31], [32], [33]].

Targeting the problems mentioned above, in the present study, we developed a fluorescent biosensor with a single enzyme, arginine deiminase (ADI), to directly measure L-Arg concentration. This enzyme was bio-engineered for the site-specific attachment of the fluorophore, fluorescein-5-maleimide (F5M). The incorporation of F5M into ADI would allow the generation of fluorescence intensity changes upon the binding of L-Arg with ADI, showing a high specificity of the biosensor. Our biosensor could successfully detect the L-Arg concentration in fetal bovine serum, a result that was comparable with the results determined by Mass-spectrometry. Therefore, it provided an alternative method for rapid and accurate determination of L-Arg concentration in biological samples.

Section snippets

Materials and instruments

The DNA sequence of wild-type arginine deiminase (WT-ADI) inserted into pET3a vector, pET3a/WT-ADI, was purchased from GeneScript. Fluorescein-5-maleimide (F5M) was purchased from Invitrogen. L-arginine (L-Arg) and fetal bovine serum (HyClone™) were purchased from ThermoFisher. Fluorescence measurements were performed on an Aglient Cary Eclipse Fluorescence Spectrophotometer (Agilent). Electrospray ionization mass spectrometry (ESI–MS) experiments were conducted using an Agilent 6540 QTOF mass

Protein expression and purification

Wild-type arginine deiminase (WT-ADI) and two corresponding mutants (44C and 265C) were expressed and purified. They were highly expressed as inclusion bodies and were purified by a single-step column with high purity (Fig. S1). High purity of WT-ADI was observed after the re-folding process (Fig. S1, lane 4). The purity of WT-ADI was further enhanced using the Q column in 20% elution buffer (Fig. S1, lane 7). The purified WT-ADI had a yield of about 16 mg/l cell, which accounted for about 88%

Discussion

In the present study, we developed a fluorescent biosensor (265Cf) that provided an alternative method for the rapid and specific determination of L-Arginine (L-Arg). One of the main features of our biosensor is the high specificity. Existing developed L-Arg biosensors utilize immobilized enzymes, such as arginases, ureases, and arginine deiminases to generate ammonium for analysis [3,[25], [26], [27]]. The ability of ammonium to generate potential and pH differences renders it to become

Conclusion

A fluorescent L-arginine (L-Arg) biosensor (265Cf) was developed by site-specific attachment of fluorescein-5-maldeimide (F5M) to generate fluorescence quenching, which were induced by the structural conformational changes of the bioengineered ADI as a result of L-Arg binding. A linear relationship between the time at the maximum rate of the fluorescence change and the concentrations of L-Arg was revealed. The linear detection range was 2.5–100 μM with good linearity of R² = 0.9988. The assay

CRediT authorship contribution statement

Suet-Ying Tam: Data Curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - Original Daft

Sai-Fung Chung: Data Curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – Review & Editing

Yu Wai Chen: Software, Writing – Review & Editing

Yik-Hing So: Investigation

Pui-Kin So: Investigation

Wing-Lam Cheong: Formal analysis, Writing – Review & Editing

Kwok-Yin Wong: Conceptualization, Funding acquisition, Supervision

Yun-Chung Leung:

Funding

This work was supported by University Supporting Fund (1-BBAE), Project of Strategic Importance (1-ZE18 & 1-ZE21), the Lo Ka Chung Charitable Foundation Limited (847E), Research in Chirosciences and Chemical Biology Funding Scheme (BBX8), and PolyU Strategic Development Special Project (ZVH9).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

We would like to thank the University Research Facility in Life Sciences (ULS) for providing instruments for doing the studies.

References (42)

S. Haghighi-Poodeh et al.
Characterization of arginine preventive effect on heat-induced aggregation of insulin
Int. J. Biol. Macromol.
(2020)
N. Verma et al.
L-arginine biosensors: a comprehensive review
Biochem. Biophys. Rep.
(2017)
P.N. Cheng et al.
Remission of hepatocellular carcinoma with arginine depletion induced by systemic release of endogenous hepatic arginase due to transhepatic arterial embolisation, augmented by high-dose insulin: arginase as a potential drug candidate for hepatocellular carcinoma
Cancer Lett.
(2005)
Y.L. Vissers et al.
Plasma arginine concentrations are reduced in cancer patients: evidence for arginine deficiency?
Am. J. Clin. Nutr.
(2005)
C.A. Uthurry et al.
Ethyl carbamate production induced by selected yeasts and lactic acid bacteria in red wine (vol 94, pg 262-270, 2006)
Food Chem.
(2007)
X. Lai et al.
Development, validation, and comparison of four methods to simultaneously quantify l-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2015)
D.M. Ivnitskii et al.
Biosensor based on direct-detection of membrane-potential induced by immobilized hydrolytic enzymes
Anal. Chim. Acta
(1993)
O.Y. Saiapina et al.
Development and optimization of a novel conductometric bi-enzyme biosensor for L-arginine determination
Talanta
(2012)
N. Stasyuk et al.
Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode
Biosens. Bioelectron.
(2012)
O.V. Soldatkina et al.
Conductometric biosensor for arginine determination in pharmaceutics
Bioelectrochemistry
(2018)

N.Y. Stasyuk et al.

L-arginine-selective microbial amperometric sensor based on recombinant yeast cells over-producing human liver arginase I

Sensors Actuators B Chem.

(2014)

M. Knipp et al.

A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline

Anal. Biochem.

(2000)

K. Das et al.

Crystal structures of arginine deiminase with covalent reaction intermediates; implications for catalytic mechanism

Structure

(2004)

A. Galkin et al.

Structural insight into arginine degradation by arginine deiminase, an antibacterial and parasite drug target

J. Biol. Chem.

(2004)

D.S. Lind

Arginine and cancer

J. Nutr.

(2004)

L. Hu et al.

Association of plasma arginine with breast cancer molecular subtypes in women of Liaoning province

IUBMB Life

(2016)

L.C. Burrage et al.

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency

Hum. Mol. Genet.

(2015)

W.T. Shen et al.

A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor

Cell Death Dis.

(2017)

S. Xu et al.

Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer

Cancer Sci.

(2018)

S.M. Tsui et al.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Cancer Cell Int.

(2009)

P.E. Hall et al.

A phase I study of pegylated arginine deiminase (Pegargiminase), cisplatin, and pemetrexed in argininosuccinate synthetase 1-deficient recurrent high-grade glioma

Clin. Cancer Res.

(2019)

Cited by (13)

Photo-responsive oxidase-like nanozyme based on a vanadium-docked porphyrinic covalent organic framework for colorimetric L-Arginine sensing
2023, Analytica Chimica Acta
This study reports the development of a vanadium-docked porphyrinic covalent organic framework as a novel class of highly polar photoactive materials. Thanks to its extended π-electron conjugation and high chemical stabilities, this framework can serve as an oxidase-Like photo-nanozyme for photocatalytic oxidation of o-phenylenediamine (o-PDA) and a colorimetric substrate for the production of the yellow-colored oxidized o-PDA (o-PDAox). The physicochemical properties of the as-prepared photo-nanozyme were characterized by several analytical techniques. Its enhanced light harvesting and charge separation and transfer were also verified by electrochemical and spectroscopic analysis. This photo-nonenzymatic colorimetric assay was applied for the sensitive L-Arginine (L-Arg) detection as a typical amino acid in the linear range of 8.1 nM–330 μM with a limit of detection (LOD) of 3.5 nM. The findings of this research confirmed the safety and feasibility of the proposed photo-nonenzymatic colorimetric sensing strategy for the detection of L-Arg and other similar biomolecules in food samples. Kinetic investigation revealed that the photo-responsive oxidase mimic exhibits satisfactory K_m (0.47 mM) and V_max (42.0 μM/s) values. This work broadened our insight into the development of modified porphyrinic–COF–based visible light-responsive oxidase-like photo-nanozyme for environmentally friendly colorimetric biosensing.
Carboxyl porphyrin as signal molecule for sensitive fluorescent detection of aflatoxin B<inf>1</inf> via ARGET-ATRP
2022, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
Citation Excerpt :
During the construction process of fluorescent biosensors, the selection of fluorescent molecules was also an important factor which affected the sensitivity of the biosensor. In the past decade, various fluorophores have been introduced as the signal-reporting units for fluorescent biosensors, such as cyanine dyes [26], coumarin [27], anthracene [28], naphthalimide [29], fluorescein [30], rhodamine [31] and porphyrins [32], etc. Among them, porphyrins are macrocyclic compounds consisting of four pyrroles linked by four methyl carbons at its α-positions in a macrocyclic manner.
In this work, a novel fluorescent biosensor for sensitive detecting of aflatoxin B₁ (AFB₁) was constructed through activators regenerated by electron transfer for atom transfer radical polymerization (ARGET-ATRP) for the first time. The AFB₁ antigen was immobilized on the carboxy magnetic beads (MBs) by forming a sandwich-type “aptamer-antigen–antibody” immune system. Then, acrylamid (AM) was introduced through ARGET-ATRP to provide binding sites for the signaling molecules. Finally, carboxy porphyrins (TPP*) were connected with monomers through an amide bond and fixed on the MBs. Under the optimal experimental conditions, the fluorescence intensity and the logarithm of the concentration of AFB₁ showed a good relationship from 100 fg mL⁻¹ to 100 ng mL⁻¹, with the limit of detection (LOD) as low as 8.38 fg mL⁻¹. In addition, the method shows good selectivity and excellent reproducibility. More importantly, the biosensor has applied to the quantitative analysis of AFB₁ in four Chinese medicines, and this strategy could potentially serve as a novel means for sensitive detecting of AFB₁ in complex matrices.
Arginine-hydrolyzing enzymes for electrochemical biosensors
2022, Current Opinion in Electrochemistry
Citation Excerpt :
The highly selective enzymes involved in Arg metabolism, especially recombinant ones, are promising tools for biosensors’ construction [2,23,40]. For Arg analysis, arginine deiminase (ADI) [22,24,26–32], arginase (ARG) in combination with urease [22,23,33–35], arginine oxidase (ArgO) and natural or artificial peroxidase (PO) [36–40] as well as arginine decarboxylase (ADC) can be applied (Figure 1c). This review focuses on the peculiarities of electrochemical enzyme-based biosensors for Arg analysis and their advantages over other approaches.
Since l-Arginine (Arg) is a semi-essential amino acid for humans, its adequate amount must be consumed in the diet to prevent certain negative consequences related to insufficient synthesis of this amino acid under specific physiological conditions. Arg metabolism results in the production of a biochemically diverse range of such products as urea, some amino acids, creatine, polyamines, nitric oxide, etc. Arg, an important biomarker in clinical diagnostics, is also used for prevention/treatment of different diseases, including cancer and COVID-19. Furthermore, it serves as an indicator of food and beverages quality.
A variety of optic and electrochemical methods for Arg determination have already been suggested. The biosensor systems based on the enzymes of Arg metabolism were shown to be the most promising tools for Arg assay. This review focuses on the peculiarities of electrochemical biosensors for Arg assay based on the use of Arg-degrading enzymes and on the analysis of their advantages as compared to other approaches.
Amperometric biosensors for L-arginine and creatinine assay based on recombinant deiminases and ammonium-sensitive Cu/Zn(Hg)S nanoparticles
2022, Talanta
Citation Excerpt :
The highly selective enzymes involved in Arg and Crn metabolism, including recombinant ones, are the promising tools for biosensor construction. For Arg analysis, arginine decarboxylase, arginase with urease [27–30], arginine oxidase [28,31], and arginine deiminase (ADI) may be utilized [27,28,32–34]. For assay of Crn, creatinine deiminase (CDI) or multienzyme complexes are usually employed [5,6,22,35–38].
There are limited data on amperometric biosensors (ABSs) based on deiminases that produce ammonium as a byproduct of enzymatic reaction. The most frequently proposed biosensors utilizing such a mode are based on potentiometric transducers, which contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Thus, the construction of a one-enzyme ABS is a practical problem. In our manuscript ABSs for the direct measurement of creatinine (Crn) and l-arginine (Arg), based on the recombinant bacterial creatinine deiminase (CDI) and arginine deiminase (ADI), are described. To choose the best chemosensor on ammonium ions, a number of nanoparticles (NPs) were synthesized and characterized using cyclic voltammetry. Hybrid Cu/Zn(Hg)S-NPs, having a good selectivity and an extremely high sensitivities towards ammonium ions (5660 A M⁻¹ m⁻² at +170 mV and 1870 A M⁻¹ m⁻² at −300 mV, respectively), was selected for the development of deiminase-based ABSs. The novel biosensors exhibited very high sensitivities (2660 A M⁻¹ m⁻² to Crn for CDI-ABS; 1570 A M⁻¹ m⁻² to Arg for ADI-ABS), broad linear ranges, low limits of detection, satisfactory storage stabilities and good selectivities towards natural substrates. The constructed CDI-ABS and ADI-ABS were tested on real samples of biological fluids and juices for Crn and Arg assay, respectively. High correlations of the obtained results with the reference methods were demonstrated for the target analytes.
Microbial arginine deiminase: A multifaceted green catalyst in biomedical sciences
2022, International Journal of Biological Macromolecules
Arginine deiminase is a well-recognized guanidino-modifying hydrolase that catalyzes the conversion of L-arginine to citrulline and ammonia. Their biopotential to regress tumors via amino acid deprivation therapy (AADT) has been well established. PEGylated formulation of recombinant Mycoplasma ADI is in the last-phase clinical trials against various arginine-auxotrophic cancers like hepatocellular carcinoma, melanoma, and mesothelioma. Recently, ADIs have attained immense importance in several other biomedical applications, namely treatment of Alzheimer's, as an antiviral drug, bioproduction of nutraceutical L-citrulline and bio-analytics involving L-arginine detection. Considering the wide applications of this biodrug, the demand for ADI is expected to escalate several-fold in the coming years. However, the sustainable production aspects of the enzyme with improved pharmacokinetics is still limited, creating bottlenecks for effective biopharmaceutical development. To circumvent the lacunae in enzyme production with appropriate paradigms of ‘quality-by-design’ an explicit overview of its properties with ‘biobetter’ formulations strategies are required. Present review provides an insight into all the potential biomedical applications of ADI along with the improvements required for its reach to clinics. Recent research advances with special emphasis on the development of ADI as a ‘biobetter’ enzyme have also been comprehensively elaborated.
Biosensing technology in food production and processing
2022, Advanced Sensor Technology: Biomedical, Environmental, and Construction Applications
The emergence of nanomaterial-based signal transduction technologies and the combination of different types of biosensors have resulted in a promising revolution in designing and developing next-generation sensing platforms in the food industry. In this chapter, the most recent biosensor-based studies in food production and processing are reviewed. The applicability of different biosensors in the food quality sector was evaluated by determining polyphenols and antioxidant capacities, screening of food-grade ingredients and additives, assessing the food authenticity, recognizing the food freshness, and monitoring the quality of fermented beverages. In the food safety sector, the efficiency of biosensors was appraised to detect allergens, antibiotics, pathogenic microorganisms, fungal and bacterial toxins, heavy metals, and other chemical contaminants such as insecticide and pesticide residues in food products. The industrialized biosensing platforms with their interesting limits of detection will open new avenues for improved quality and safety of food products with better consumer perception.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Design of a structure-based fluorescent biosensor from bioengineered arginine deiminase for rapid determination of L-arginine

Highlights

Abstract

Introduction

Section snippets

Materials and instruments

Protein expression and purification

Discussion

Conclusion

CRediT authorship contribution statement

Funding

Declaration of competing interest

Acknowledgements

Int. J. Biol. Macromol.

Biochem. Biophys. Rep.

Cancer Lett.

Am. J. Clin. Nutr.

Food Chem.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Anal. Chim. Acta

Talanta

Biosens. Bioelectron.

Bioelectrochemistry

Sensors Actuators B Chem.

Anal. Biochem.

Structure

J. Biol. Chem.

Arginine and cancer

J. Nutr.

Association of plasma arginine with breast cancer molecular subtypes in women of Liaoning province

IUBMB Life

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency

Hum. Mol. Genet.

A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor

Cell Death Dis.

Recombinant human arginase induces apoptosis through oxidative stress and cell cycle arrest in small cell lung cancer

Cancer Sci.

Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)

Cancer Cell Int.

A phase I study of pegylated arginine deiminase (Pegargiminase), cisplatin, and pemetrexed in argininosuccinate synthetase 1-deficient recurrent high-grade glioma

Clin. Cancer Res.