Construction of a novel bioanode for amino acid powered fuel cells through an artificial enzyme cascade pathway

The glyceraldehyde dehydrogenase fromThermoplasma acidophilum(TaAlDH) is a microbial enzyme that catalyzes the oxidation of D-glyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol. Various mutants ofTaAlDH were constructed by a random approach followed by site-directed and saturation mutagenesis in order to improve the properties of the enzyme that are essential for its functioning within the cascade. Two enzyme variants, wild-typeTaAlDH (TaAlDHwt) and an F34M+S405N variant (TaAlDH F34M+S405N), were successfully crystallized. Crystals ofTaAlDHwt belonged to the monoclinic space groupP1211 with eight molecules per asymmetric unit and diffracted to a resolution of 1.95 Å.TaAlDH F34M+S405N crystallized in two different space groups: triclinicP1 with 16 molecules per asymmetric unit and monoclinicC121 with four molecules per asymmetric unit. These crystals diffracted to resolutions of 2.14 and 2.10 Å for theP1 andC121 crystals, respectively.

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Comparative Activity of Adenosine Deaminase Acting on RNA (ADARs) Isoforms for Correction of Genetic Code in Gene Therapy

Current Gene Therapy ◽

10.2174/1566523218666181114122116 ◽

2019 ◽

Vol 19 (1) ◽

pp. 31-39 ◽

Cited By ~ 3

Author(s):

Md. Thoufic A. Azad ◽

Umme Qulsum ◽

Toshifumi Tsukahara

Keyword(s):

Amino Acid ◽

Adenosine Deaminase ◽

Rna Binding ◽

Stop Codon ◽

Single Amino Acid ◽

Fluorescence Signal ◽

Artificial Enzyme ◽

Western Blots ◽

Stem Loop ◽

Hek 293

Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD. Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into HEK 293 cells, we observed fluorescence signals of various intensities. Results: ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots and sequencing confirmed that ADAR1-DD was more active than any other DD. Conclusion: This study provides information that should facilitate the rational use of ADAR variants for genetic restoration and treatment of genetic diseases.

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Development and Characterization of Cellulolytic Enzymatic Fuel Cells with DNA-Organized Multi-Enzyme Cascade

ECS Meeting Abstracts ◽

10.1149/ma2016-02/44/3229 ◽

2016 ◽

Keyword(s):

Fuel Cells ◽

Enzyme Cascade

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High-efficiency artificial enzyme cascade bio-platform based on MOF-derived bimetal nanocomposite for biosensing

Talanta ◽

10.1016/j.talanta.2020.121374 ◽

2020 ◽

Vol 220 ◽

pp. 121374 ◽

Cited By ~ 1

Author(s):

Min Liu ◽

Junsong Mou ◽

Xiaohan Xu ◽

Feifei Zhang ◽

Jianfei Xia ◽

...

Keyword(s):

High Efficiency ◽

Artificial Enzyme ◽

Enzyme Cascade

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A Four-Step Enzymatic Cascade for Efficient Production of L-Phenylglycine from Biobased L-Phenylalanine

10.1101/2022.01.14.476296 ◽

2022 ◽

Author(s):

Yuling Zhu ◽

Jifeng Yuan

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Industrial Applications ◽

Efficient Production ◽

E Coli ◽

Amycolatopsis Orientalis ◽

Enzyme Cascade ◽

Pharmaceutical Industries ◽

Rate Limiting

Enantiopure amino acids are of particular interest in the agrochemical and pharmaceutical industries. Here, we reported a multi-enzyme cascade for efficient production of L-phenylglycine (L-Phg) from biobased L-phenylalanine (L-Phe). We first attempted to engineer Escherichia coli for expressing L-amino acid deaminase (LAAD) from Proteus mirabilis, hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis, (S)-mandelate dehydrogenase (SMDH) from Pseudomonas putida, the endogenous aminotransferase (AT) encoded by ilvE and L-glutamate dehydrogenase (GluDH) from E. coli. However, 10 mM L-Phe only afforded the synthesis of 7.21 mM L-Phg. The accumulation of benzoylformic acid suggested that the transamination step might be rate-limiting. We next used leucine dehydrogenase (LeuDH) from Bacillus cereus to bypass the use of L-glutamate as amine donor, and 40 mM L-Phe gave 39.97 mM (6.04 g/L) L-Phg, reaching 99.9% conversion. In summary, this work demonstrated a concise four-step enzymatic cascade for the L-Phg synthesis from biobased L-Phe, with a potential for future industrial applications.

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Tandem Friedel-Crafts-Alkylation-Enantioselective-Protonation by Artificial Enzyme Iminium Catalysis

10.33774/chemrxiv-2021-09qr1 ◽

2021 ◽

Author(s):

Reuben Leveson-Gower ◽

Ruben de Boer ◽

Gerard Roelfes

Keyword(s):

Amino Acid ◽

Small Molecule ◽

Bond Formation ◽

Side Chain ◽

Artificial Enzymes ◽

Protein Scaffolds ◽

Artificial Enzyme ◽

Protein Scaffold ◽

Tertiary Carbon ◽

Canonical Amino Acid

The incorporation of organocatalysts into protein scaffolds, i.e. the production of organocatalytic artificial enzymes, holds the promise of overcoming some of the limitations of this powerful catalytic approach. In particular, transformations for which good reactivity or selectivity is challenging for organocatalysts may find particular benefit from translation into a protein scaffold so that its chiral microenvironment can be utilised in catalysis. Previously, we showed that incorporation of the non-canonical amino acid para-aminophenylalanine into the non-enzymatic protein scaffold LmrR forms a proficient and enantioselective artificial enzyme (LmrR_pAF) for the Friedel-Crafts alkylation of indoles with enals. The unnatural aniline side-chain is directly involved in catalysis, operating via a well-known organocatalytic iminium-based mechanism. In this study, we show that LmrR_pAF can enantioselectively form tertiary carbon centres not only during C-C bond formation, but also by enantioselective protonation. Control over this process is an ongoing challenge for small-molecule catalysts for which general solutions do not exist. LmrR_pAF can selectively deliver a proton to one face of a prochiral enamine intermediate delivering product enantiomeric excesses and yields that rival the best organocatalyst for this transformation. The importance of various side-chains in the pocket of LmrR is distinct from the Friedel-Crafts reaction without enantioselective protonation, and two particularly important residues were probed by exhaustive mutagenesis. This study shows how organocatalytic artificial enzymes can provide solutions to transformations which otherwise require empirical optimisation and design of multifunctional small molecule catalysts.

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Production of (R)-mandelic acid from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00374-6 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Benedict Ryan Lukito ◽

Zilong Wang ◽

Balaji Sundara Sekar ◽

Zhi Li

Keyword(s):

Mandelic Acid ◽

Kinetic Resolution ◽

Direct Synthesis ◽

Maximum Yield ◽

Direct Conversion ◽

Artificial Enzyme ◽

E Coli ◽

Racemic Form ◽

Enzyme Cascade ◽

Renewable Feedstocks

Abstract(R)-mandelic acid is an industrially important chemical, especially used for producing antibiotics. Its chemical synthesis often uses highly toxic cyanide to produce its racemic form, followed by kinetic resolution with 50% maximum yield. Here we report a green and sustainable biocatalytic method for producing (R)-mandelic acid from easily available styrene, biobased L-phenylalanine, and renewable feedstocks such as glycerol and glucose, respectively. An epoxidation-hydrolysis-double oxidation artificial enzyme cascade was developed to produce (R)-mandelic acid at 1.52 g/L from styrene with > 99% ee. Incorporation of deamination and decarboxylation into the above cascade enables direct conversion of L-phenylalanine to (R)-mandelic acid at 913 mg/L and > 99% ee. Expressing the five-enzyme cascade in an L-phenylalanine-overproducing E. coli NST74 strain led to the direct synthesis of (R)-mandelic acid from glycerol or glucose, affording 228 or 152 mg/L product via fermentation. Moreover, coupling of E. coli cells expressing L-phenylalanine biosynthesis pathway with E. coli cells expressing the artificial enzyme cascade enabled the production of 760 or 455 mg/L (R)-mandelic acid from glycerol or glucose. These simple, safe, and green methods show great potential in producing (R)-mandelic acid from renewable feedstocks.

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