scholarly journals Engineering Cofactor Specificity of a Thermostable Phosphite Dehydrogenase for a Highly Efficient and Robust NADPH Regeneration System

2021 ◽  
Vol 9 ◽  
Author(s):  
Gamal Nasser Abdel-Hady ◽  
Takeshi Ikeda ◽  
Takenori Ishida ◽  
Hisakage Funabashi ◽  
Akio Kuroda ◽  
...  
Keyword(s):  
Shikimic Acid ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Site Directed Mutagenesis ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Promising Alternative ◽  
Practical Applications ◽  
Cofactor Specificity ◽  
Highly Efficient ◽  
Phosphite Dehydrogenase

Nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases catalyze a range of chemical reactions useful for practical applications. However, their dependence on the costly cofactor, NAD(P)H remains a challenge which must be addressed. Here, we engineered a thermotolerant phosphite dehydrogenase from Ralstonia sp. 4506 (RsPtxD) by relaxing the cofactor specificity for a highly efficient and robust NADPH regeneration system. The five amino acid residues, Cys174–Pro178, located at the C-terminus of β7-strand region in the Rossmann-fold domain of RsPtxD, were changed by site-directed mutagenesis, resulting in four mutants with a significantly increased preference for NADP. The catalytic efficiency of mutant RsPtxDHARRA for NADP (Kcat/KM)NADP was 44.1 μM–1 min–1, which was the highest among the previously reported phosphite dehydrogenases. Moreover, the RsPtxDHARRA mutant exhibited high thermostability at 45°C for up to 6 h and high tolerance to organic solvents, when bound with NADP. We also demonstrated the applicability of RsPtxDHARRA as an NADPH regeneration system in the coupled reaction of chiral conversion of 3-dehydroshikimate to shikimic acid by the thermophilic shikimate dehydrogenase of Thermus thermophilus HB8 at 45°C, which could not be supported by the parent RsPtxD enzyme. Therefore, the RsPtxDHARRA mutant might be a promising alternative NADPH regeneration system for practical applications.

scholarly journals Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinghui Xiong ◽  
Hefeng Chen ◽  
Ran Liu ◽  
Hao Yu ◽  
Min Zhuo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Whole Cell ◽  
Cyclohexanone Monooxygenase ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Whole Cell Biocatalyst ◽  
Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

scholarly journals Exploration of the cofactor specificity of wild-type phosphite dehydrogenase and its mutant using molecular dynamics simulations

RSC Advances ◽  
2021 ◽  
Vol 11 (24) ◽  
pp. 14527-14533
Author(s):  
Kunlu Liu ◽  
Min Wang ◽  
Yubo Zhou ◽  
Hongxiang Wang ◽  
Yudong Liu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Wild Type ◽  
Cofactor Specificity ◽  
Phosphite Dehydrogenase ◽  
Dynamics Simulations

Phosphite dehydrogenase (Pdh) catalyzes the NAD-dependent oxidation of phosphite to phosphate with the formation of NADH.

A Promising Alternative for Sustainable and Highly Efficient Solar‐Driven Deuterium Evolution at Room Temperature by Photocatalytic D 2 O Splitting

ChemSusChem ◽  
2020 ◽  
Vol 13 (11) ◽  
pp. 2935-2939 ◽  
Author(s):  
Gongchang Zeng ◽  
Heping Zeng ◽  
Lishan Niu ◽  
Jiayi Chen ◽  
Ting Song ◽  
...  
Keyword(s):  
Room Temperature ◽  
Promising Alternative ◽  
Highly Efficient

scholarly journals Self-standing heterostructured NiC x -NiFe-NC/biochar as a highly efficient cathode for lithium–oxygen batteries

2020 ◽  
Vol 11 ◽  
pp. 1809-1821
Author(s):  
Shengyu Jing ◽  
Xu Gong ◽  
Shan Ji ◽  
Linhui Jia ◽  
Bruno G Pollet ◽  
...  
Keyword(s):  
Low Cost ◽  
Discharge Voltage ◽  
Research Attention ◽  
Practical Applications ◽  
Highly Efficient ◽  
Electrochemical Processes ◽  
Nickel Carbide ◽  
Specific Discharge ◽  
High Theoretical Capacity ◽  
Lithium Oxygen Batteries

Lithium–oxygen batteries have attracted research attention due to their low cost and high theoretical capacity. Developing inexpensive and highly efficient cathode materials without using noble metal-based catalysts is highly desirable for practical applications in lithium–oxygen batteries. Herein, a heterostructure of NiFe and NiC x inside of N-doped carbon (NiC x -NiFe-NC) derived from bimetallic Prussian blue supported on biochar was developed as a novel self-standing cathode for lithium–oxygen batteries. The specific discharge capacity of the best sample was 27.14 mAh·cm−2 at a stable discharge voltage of 2.75 V. The hybridization between the d-orbital of Ni and s and p-orbitals of carbon in NiC x , formed at 900 °C, enhanced the electrocatalytic performance due to the synergistic effect between these components. The structure of NiC x -NiFe-NC efficiently improved the electron and ion transfer between the cathode and the electrolyte during the electrochemical processes, resulting in superior electrocatalytic properties in lithium–oxygen batteries. This study indicates that nickel carbide supported on N-doped carbon is a promising cathode material for lithium–oxygen batteries.

scholarly journals The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine

2020 ◽  
Vol 295 (9) ◽  
pp. 2822-2838 ◽  
Author(s):  
Alexandra A. Richter ◽  
Stefanie Kobus ◽  
Laura Czech ◽  
Astrid Hoeppner ◽  
Jan Zarzycki ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Catalytic Core ◽  
Chemical Chaperone ◽  
Practical Applications ◽  
Acetyl Group ◽  
Function Relationship ◽  
Thermotolerant Bacterium ◽  
Stress Protectant ◽  
Relationship Of

Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2–2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

scholarly journals Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

2019 ◽  
Vol 20 (4) ◽  
pp. 916 ◽  
Author(s):  
Diana Humer ◽  
Oliver Spadiut
Keyword(s):  
Catalytic Activity ◽  
Horseradish Peroxidase ◽  
Signal Sequence ◽  
Enzyme Engineering ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Promising Alternative ◽  
Wide Range ◽  
Quadruple Mutant ◽  
Commercial Applications

Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme.

Highly efficient near-infrared and semitransparent polymer solar cells based on an ultra-narrow bandgap nonfullerene acceptor

2019 ◽  
Vol 7 (8) ◽  
pp. 3745-3751 ◽  
Author(s):  
Juan Chen ◽  
Guangda Li ◽  
Qinglian Zhu ◽  
Xia Guo ◽  
Qunping Fan ◽  
...  
Keyword(s):  
Solar Cells ◽  
Near Infrared ◽  
Polymer Solar Cells ◽  
Practical Applications ◽  
Low Bandgap ◽  
Highly Efficient ◽  
Narrow Bandgap ◽  
Nonfullerene Acceptor

Non-fullerene polymer solar cells based on a low bandgap polymer PTB7-Th and an ultra-narrow bandgap acceptor ACS8 exhibited an optimal PCE of 13.2%, indicating that the blend of PTB7-Th/ACS8 has potential for the practical applications of PSCs.

FORECASTING FOREIGN EXCHANGE RATES WITH ARTIFICIAL NEURAL NETWORKS: A REVIEW

2004 ◽  
Vol 03 (01) ◽  
pp. 145-165 ◽  
Author(s):  
WEI HUANG ◽  
K. K. LAI ◽  
Y. NAKAMORI ◽  
SHOUYANG WANG
Keyword(s):  
Neural Networks ◽  
Artificial Neural Networks ◽  
Exchange Rates ◽  
Network Architecture ◽  
Future Research ◽  
Promising Alternative ◽  
Practical Applications ◽  
Future Research Directions ◽  
Artificial Neural ◽  
Input Variables

Forecasting exchange rates is an important financial problem that is receiving increasing attention especially because of its difficulty and practical applications. Artificial neural networks (ANNs) have been widely used as a promising alternative approach for a forecasting task because of several distinguished features. Research efforts on ANNs for forecasting exchange rates are considerable. In this paper, we attempt to provide a survey of research in this area. Several design factors significantly impact the accuracy of neural network forecasts. These factors include the selection of input variables, preparing data, and network architecture. There is no consensus about the factors. In different cases, various decisions have their own effectiveness. We also describe the integration of ANNs with other methods and report the comparison between performances of ANNs and those of other forecasting methods, and finding mixed results. Finally, the future research directions in this area are discussed.

scholarly journals Purification and cDNA Cloning of NADPH-Dependent Aldoketoreductase, Involved in Asymmetric Reduction of Methyl 4-Bromo-3-Oxobutyrate, from Penicillium citrinum IFO4631

2005 ◽  
Vol 71 (2) ◽  
pp. 1101-1104 ◽  
Author(s):  
Hiroyuki Asako ◽  
Ryuhei Wakita ◽  
Kenji Matsumura ◽  
Masatoshi Shimizu ◽  
Jun Sakai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cdna Cloning ◽  
Cdna Clone ◽  
Asymmetric Reduction ◽  
Optical Purity ◽  
Penicillium Citrinum ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
High Optical Purity

ABSTRACT Penicillium citrinum was found to catalyze the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate [(S)-BHBM] with high optical purity. From the strain, a cDNA clone encoding a novel NADPH-dependent alkyl 4-halo-3-oxobutyrate reductase (KER) was isolated. Escherichia coli cells overexpressing KER produced (S)-BHBM in the presence of an NADPH-regeneration system.

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