scholarly journals Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection

2021 ◽  
Vol 22 (24) ◽  
pp. 13570
Author(s):  
Takenori Satomura ◽  
Kohei Uno ◽  
Norio Kurosawa ◽  
Haruhiko Sakuraba ◽  
Toshihisa Ohshima ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Lactate Dehydrogenase ◽  
Direct Electron Transfer ◽  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Electrochemical Devices ◽  
Amine Groups ◽  
Phenazine Ethosulfate ◽  
Excellent Stability

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

scholarly journals Identification and characterization of YLR328W, theSaccharomyces cerevisiaestructural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme

FEBS Letters ◽  
1999 ◽  
Vol 455 (1-2) ◽  
pp. 13-17 ◽  
Author(s):  
Monica Emanuelli ◽  
Francesco Carnevali ◽  
Maria Lorenzi ◽  
Nadia Raffaelli ◽  
Adolfo Amici ◽  
...  
Keyword(s):  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Identification And Characterization

scholarly journals The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (8) ◽  
pp. 2323-2329 ◽  
Author(s):  
Miguel Prudêncio ◽  
Robert R. Eady ◽  
Gary Sawers
Keyword(s):  
Amino Acid ◽  
Nitrite Reductase ◽  
Recombinant Enzyme ◽  
Leader Peptide ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Characterization of Mercaptoethylamine-Modified Gold Electrode Surface and Analyses of Direct Electron Transfer to Putidaredoxin

Langmuir ◽  
1995 ◽  
Vol 11 (12) ◽  
pp. 4818-4822 ◽  
Author(s):  
Ling Sang Wong ◽  
Vincent L. Vilker ◽  
William T. Yap ◽  
Vytas Reipa
Keyword(s):  
Electron Transfer ◽  
Electrode Surface ◽  
Gold Electrode ◽  
Direct Electron Transfer

scholarly journals Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Dominic W. S. Wong ◽  
Victor J. Chan ◽  
Amanda A. McCormack ◽  
Ján Hirsch ◽  
Peter Biely
Keyword(s):  
Glucuronic Acid ◽  
Active Form ◽  
Schizophyllum Commune ◽  
Constitutive Expression ◽  
Recombinant Enzyme ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Glucuronoyl Esterase ◽  
Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

scholarly journals Engineered Glucose Oxidase Capable of Quasi-Direct Electron Transfer after a Quick-and-Easy Modification with a Mediator

2020 ◽  
Vol 21 (3) ◽  
pp. 1137 ◽  
Author(s):  
Nanami Suzuki ◽  
Jinhee Lee ◽  
Noya Loew ◽  
Yuka Takahashi-Inose ◽  
Junko Okuda-Shimazaki ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Glucose Oxidase ◽  
Glucose Sensor ◽  
Direct Electron Transfer ◽  
Glucose Monitoring ◽  
Single Step ◽  
Redox Mediator ◽  
Electrochemically Active ◽  
Phenazine Ethosulfate ◽  
The Impact

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.

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