Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

Maenpuen, Somchart; Pongsupasa, Vinutsada; Pensook, Wiranee; Anuwan, Piyanuch; Kraivisitkul, Napatsorn; Pinthong, Chatchadaporn; Phonbuppha, Jittima; Luanloet, Thikumporn; Wijma, Hein J.; Fraaije, Marco W.; Lawan, Narin; Chaiyen, Pimchai; Wongnate, Thanyaporn

doi:10.1002/cbic.201900737

Cited by 19 publications

(17 citation statements)

References 77 publications

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“…We replaced the formerly used glucose 6‐ phosphate (G6P) dehydrogenase (G6PD), which uses expensive G6P substrate, with formate dehydrogenase (FDH), which uses inexpensive formate to generate NADH. The formerly used flavin reductase (C 1 ‐WT), which transfers electrons from NADH to FAD to generate reduced FAD as a substrate for HadA, was replaced by the more thermostable C 1 ‐A166L variant [20] (Figure S2).…”

Section: Resultsmentioning

confidence: 99%

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

et al. 2022

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D-Luciferin (D-LH 2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH 2 analogues with � 51 % yield. The method can synthesize the novel 5'-methyl-D-LH 2 and 4',5'-dimethyl-D-LH 2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH 2 emits brighter and longer wavelength light than the D-LH 2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

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Section: Resultsmentioning

confidence: 99%

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

et al. 2022

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“…A double reciprocal plot of initial rates was used to determine competitive inhibition using Equation ( 2), where v and V represent the initial and maximum velocities, A and I are the substrate and inhibitor concentrations, K A is the Michaelis constant for substrate, and the secondary plot of slope versus inhibitor concentration was used to determine K i , the inhibition constant. 2018) [24] and Maenpuen et al (2020), [25] respectively.…”

Section: Determination Of the K I Value Of Azide Ion Inhibitionmentioning

confidence: 95%

A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex

Boonkumkrong,

Chunthaboon,

Munkajohnpong

et al. 2024

Biotechnology Journal

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NAD+‐dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s−1 at 25°C compared to 7.7 ± 1.0 s−1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6‐fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2O2 inactivation, showing a 200‐fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4‐nitrophenol by the reaction of HadA, which produced a H2O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.

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“…However, as the two-component flavin-dependent monooxygenase such as C 2 requires a cognate partner, a C 1 flavin reductase, to supply reduced flavin, both enzymes need to be robust enough for the prolonged bioconversion process. Therefore, C 1 was engineered using computational approaches to obtain two variants, A166L and A58P, which have greater thermostability than the wild-type enzyme . Moreover, these two variants, especially A166L exhibits solvent-tolerant activity toward several solvents including dimethyl sulfoxide (DMSO), methanol, and ethanol.…”

Section: Production Of Lignocellulosic Biomass-derived Productsmentioning

confidence: 99%

“…Therefore, C 1 was engineered using computational approaches to obtain two variants, A166L and A58P, which have greater thermostability than the wild-type enzyme. 395 Moreover, these two variants, especially A166L exhibits solvent-tolerant activity toward several solvents including dimethyl sulfoxide (DMSO), methanol, and ethanol. Further optimization of the C 2 system with the engineered C 1 , or by replacing C 1 with a more thermostable flavin reductase (e.g., PheA2 from Bacillus thermoglucosidasius A7 396 or Th-Fre from Streptomyces violaceusniger strain SPC6 397 ) should be useful for further improvement of hydroxylation of PA derivatives to synthesize valuable chemicals in the future.…”

Section: Phenolic Acid-derived Productsmentioning

confidence: 99%

“…This can be done through target engineering of residues with high B-factor values 811 or using computer assisted methodology via several softwares such as FireProt, 812 FRESCO, 813 or disulfide by designed. 814 For example, the thermostability of flavin reductase 395 and dehalogenating monooxygenase 815 can be increased by rational engineering, allowing their reactions to be used in converting low-value toxic waste into high-value compounds such as Dluciferin. 816 Enzyme engineering also allows incorporation of genes encoding for enzymes with improved properties into cells so that new and efficient metabolic pathways can be directly obtained.…”

Section: Concluding Remarks and Futurementioning

confidence: 99%

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Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability

et al. 2021

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Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO 2 and CH 4 ) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.

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Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

Cited by 19 publications

References 77 publications

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex

Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability

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