Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization

Masakari, Yosuke; Hara, Chiaki; Araki, Yasuko; Gomi, Keiko; Ito, Keisuke

doi:10.1016/j.enzmictec.2019.109387

Cited by 14 publications

(6 citation statements)

References 41 publications

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“…Thus, a method involving Nafion-assisted dispersion was utilized in this study. In order to optimize the concentration of S-MWNT/Nafion, ESI, a powerful tool for measuring the charge transfer resistance (Rct) value at the interface between the electrode and the electrolyte and analyzing the dynamics of electron transfer [ 28 ] was used to measure the electron transfer efficiency of S-MWNT. As shown in Figure 4 a, the Rct value and the straight-line angle in the low-frequency range varied for the electrodes modified with a series of concentration (0.1, 0.5, and 1 mg/mL) of S-MWNT.…”

Section: Resultsmentioning

confidence: 99%

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Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

Han

Gong

Zhang

et al. 2021

IJMS

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The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10−2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10−2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

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Section: Resultsmentioning

confidence: 99%

“…The method used to determine the GDH activity was modified from a previous report [ 28 ]. Briefly, the reaction mixture, consisting of 1.75 mL phosphate buffer (0.1 M, pH 6.0), 0.1 mL D-glucose (2 M), 50 μL PMS (24 mM), and 50 μL DCIP (2.4 mM) was mixed thoroughly and preheated at 37 °C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

Han

Gong

Zhang

et al. 2021

IJMS

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“…Dehydrogenase activity for l -arginine and l -citrulline was assayed in a reaction mixture containing 25 mM of each substrate, 1.8 mM of 2,6-dichlorophenolindophenol (DCIP), 15 mM of phenazine methosulfate (PMS), 200 mM of KPB (pH 6.0), and the enzyme, in a final volume of 1.5 mL at 37 ℃ according to a partially modified method from a previous study (Masakari et al 2020 ). The activity was calculated by monitoring the decrease in the absorbance of DCIP at 600 nm using a U-3900 spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Modification of substrate specificity of l-arginine oxidase for detection of l-citrulline

Yamamoto,

Masakari,

Araki

et al. 2023

AMB Expr

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Enzymatic detection of citrulline, a potential biomarker for various diseases, is beneficial. However, determining citrulline levels requires expensive instrumental analyses and complicated colorimetric assays. Although l-amino acid oxidase/dehydrogenase is widely used to detect l-amino acids, an l-citrulline-specific oxidase/dehydrogenase has not been reported. Therefore, in this study, we aimed to develop an l-citrulline-specific enzyme by introducing a mutation into l-arginine oxidase (ArgOX) derived from Pseudomonas sp. TPU 7192 to provide a simple enzymatic l-citrulline detection system. The ratio of the oxidase activity against l-arginine to that against l-citrulline (Cit/Arg) was 1.2%, indicating that ArgOX could recognize l-citrulline as a substrate. In the dehydrogenase assay, the specific dehydrogenase activity towards l-arginine was considerably lower than the specific oxidase activity. However, the specific dehydrogenase activity towards l-citrulline was only slightly lower than the oxidase activity, resulting in improved substrate specificity with a Cit/Arg ratio of 49.5%. To enhance the substrate specificity of ArgOX, we performed site-directed mutagenesis using structure-based engineering. The 3D model structure indicated that E486 interacted with the l-arginine side chain. By introducing the E486 mutation, the specific dehydrogenase activity of ArgOX/E486Q for l-citrulline was 3.25 ± 0.50 U/mg, which was 3.8-fold higher than that of ArgOX. The Cit/Arg ratio of ArgOX/E486Q was 150%, which was higher than that of ArgOX. Using ArgOX/E486Q, linear relationships were observed within the range of 10–500 μM l-citrulline, demonstrating its suitability for detecting citrulline in human blood. Consequently, ArgOX/E486Q can be adapted as an enzymatic sensor in the dehydrogenase system. Graphical Abstract

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“…Substitutions such as Lys → Arg and Ser → Ala have also been observed to be common when comparing mesophilic to thermophilic proteins [105]. Other core determinants of protein thermal stability include increased hydrogen bonding [106][107][108], the introduction of disulfide bonds [109][110][111], salt bridges [112,113], loop shortening [114,115], optimization of electrostatic surfaces [116,117], enhanced solvation in specific protein regions [118], and augmented intramolecular hydrophobic packing [119,120]. The factors mentioned previously largely share a similar basis, aiming to reduce the molecule's entropy; that is, enzyme rigidity is required for higher thermostability [108].…”

Section: Thermolability Of Sucrose Isomerasesmentioning

confidence: 99%

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases’ Thermostability

Sardiña-Peña,

Mesa-Ramos,

Iglesias-Figueroa

et al. 2023

IJMS

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Due to their ability to produce isomaltulose, sucrose isomerases are enzymes that have caught the attention of researchers and entrepreneurs since the 1950s. However, their low activity and stability at temperatures above 40 °C have been a bottleneck for their industrial application. Specifically, the instability of these enzymes has been a challenge when it comes to their use for the synthesis and manufacturing of chemicals on a practical scale. This is because industrial processes often require biocatalysts that can withstand harsh reaction conditions, like high temperatures. Since the 1980s, there have been significant advancements in the thermal stabilization engineering of enzymes. Based on the literature from the past few decades and the latest achievements in protein engineering, this article systematically describes the strategies used to enhance the thermal stability of sucrose isomerases. Additionally, from a theoretical perspective, we discuss other potential mechanisms that could be used for this purpose.

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Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization

Cited by 14 publications

References 41 publications

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay

Modification of substrate specificity of l-arginine oxidase for detection of l-citrulline

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases’ Thermostability

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