Coupling protein scaffold and biosilicification: A sustainable and recyclable approach for d-mannitol production via one-step purification and immobilization of multienzymes.

Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.

Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Catalytic Synthesis of (S)-CHBE by Directional Coupling and Immobilization of Carbonyl Reductase and Glucose Dehydrogenase.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.

Development of Direct Electron Transfer-Type Extended Gate Field Effect Transistor Enzymatic Sensors for Metabolite Detection.

In this work, direct electron transfer (DET)-type extended gate field effect transistor (EGFET) enzymatic sensors were developed by employing DET-type or quasi-DET-type enzymes to detect glucose or lactate in both 100 mM potassium phosphate buffer and artificial sweat. The system employed either a DET-type glucose dehydrogenase or a quasi-DET-type lactate oxidase, the latter of which was a mutant enzyme with suppressed oxidase activity and modified with amine-reactive phenazine ethosulfate. These enzymes were immobilized on the extended gate electrodes. Changes in the measured transistor drain current (ID) resulting from changes to the working electrode junction potential (φ) were observed as glucose and lactate concentrations were varied. Calibration curves were generated for both absolute measured ID and ΔID (normalized to a blank solution containing no substrate) to account for variations in enzyme immobilization and conjugation to the mediator and variations in reference electrode potential. This work resulted in a limit of detection of 53.9 µM (based on ID) for glucose and 2.12 mM (based on ID) for lactate, respectively. The DET-type and Quasi-DET-type EGFET enzymatic sensor was then modeled using the case of the lactate sensor as an equivalent circuit to validate the principle of sensor operation being driven through OCP changes caused by the substrate-enzyme interaction. The model showed slight deviation from collected empirical data with 7.3% error for the slope and 8.6% error for the y-intercept.

Enzymatic Galvanic Redox Potentiometry for In Vivo Biosensing.

Redox potentiometry has emerged as a new platform for in vivo sensing, with improved neuronal compatibility and strong tolerance against sensitivity variation caused by protein fouling. Although enzymes show great possibilities in the fabrication of selective redox potentiometry, the fabrication of an enzyme electrode to output open-circuit voltage (EOC) with fast response remains challenging. Herein, we report a concept of novel enzymatic galvanic redox potentiometry (GRP) with improved time response coupling the merits of the high selectivity of enzyme electrodes with the excellent biocompatibility and reliability of GRP sensors. With a glucose biosensor as an illustration, we use flavin adenine dinucleotide-dependent glucose dehydrogenase as the recognition element and carbon black as the potential relay station to improve the response time. We find that the enzymatic GRP biosensor rapidly responds to glucose with a good linear relationship between EOC and the logarithm of glucose concentration within a range from 100 µM to 2.65 mM. The GRP biosensor shows high selectivity over O2 and coexisting neurochemicals, good reversibility, and sensitivity and can in vivo monitor glucose dynamics in rat brain. We believe that this study will pave a new platform for the in vivo potentiometric biosensing of chemical events with high reliability.

Ketogluconate production by Gluconobacter strains: enzymes and biotechnological applications.

Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d-glucose to d-gluconate, which then diverges and splits into 2 pathways producing 5-keto-d-gluconate and 2-keto-d-gluconate and subsequently 2,5-diketo-d-gluconate. These transformations are facilitated by membrane-bound d-glucose dehydrogenase, glycerol dehydrogenase, d-gluconate dehydrogenase, and 2-keto-d-gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains.

Sustainable asymmetric synthesis of diltiazem precursor enabled by recombinant Escherichia coli whole cells co-expressing an engineered ketoreductase and glucose dehydrogenase.

As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)-3-(4-methoxyphenyl) glycidate ((2R,3S)-MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)-methyl 2-chloro-3-hydroxy-3-(4-methoxyphenyl)propanoate ((3S)-2). We report the efficient reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (3) to (3S)-2 using an engineered enzyme SSCRM2 possessing 4.5-fold improved specific activity, which was obtained through the structure-guided site-saturation mutagenesis of the ketoreductase SSCR by reliving steric hindrance and undesired interactions. With the combined use of the co-expression fine-tuning strategy, a recombinant E. coli (pET28a-RBS-SSCRM2 /pACYCDuet-GDH), co-expressing SSCRM2 and glucose dehydrogenase, was constructed and optimized for protein expression. After optimizing the reaction conditions, whole-cell-catalyzed complete reduction of industrially relevant 300 g L-1 of 3 was realized, affording (3S)-2 with 99% ee and a space-time yield of 519.1 g∙L-1 ∙d-1 , representing the highest record for the biocatalytic synthesis of (3S)-2 reported to date. The E-factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3S)-2 generated in this atom-economic synthesis was transformed to (2R,3S)-MPGM in 95% yield with 99% ee.

Characterization and Application of a Novel Glucose Dehydrogenase with Excellent Organic Solvent Tolerance for Cofactor Regeneration in Carbonyl Reduction.

An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of ßD-αE-αF-αG-ßG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40 °C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55 °C for 5 h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System.

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.

A New Digital Point-of-Care Tool With Advanced Blood Glucose Measuring Technology.

cobas® pulse is a point-of-care blood glucose (BG) measuring system for multiple-patient use in professional healthcare settings. The system provides advances in connectivity and BG measuring technology, and has multiple fail-safes to improve accuracy and reduce the risk of user error. Flavin adenine dinucleotide-dependent glucose dehydrogenase on the working electrode catalyzes oxidation of ß-D-glucose in the blood sample. A redox mediator/electron acceptor, on both the working and the counter electrode, facilitates diffusion of electrons in proportion to the glucose concentration and compensates for the effects of potential interfering agents. During development, >1 million test strip measurements were performed using >8000 test scenarios to refine the algorithm model. No clinically relevant interference was identified with extreme variations in blood properties and drugs in whole blood samples.

Wiring of bilirubin oxidases with redox polymers on gas diffusion electrodes for increased stability of self-powered biofuel cells-based glucose sensing.

A new redox polymer/bilirubin oxidase (BOD)-based gas diffusion electrode was designed to be implemented as the non-current and non-stability limiting biocathode in a glucose/O2 biofuel cell that acts as a self-powered glucose biosensor. For the proof-of-concept, a bioanode comprising the Os-complex modified redox polymer P(VI-co-AA)-[Os(bpy)2Cl]Cl and FAD-dependent glucose dehydrogenase to oxidize the analyte was used. In order to develop an optimal O2-reducing biocathode for the biofuel cell Mv-BOD as well as Bp-BOD and Mo-BOD have been tested in gas diffusion electrodes in direct electron transfer as well as in mediated electron transfer immobilized in the Os-complex modified redox polymer P(VI-co-AA)-[Os(diCl-bpy)2]Cl2. The resulting biofuel cell exhibits a glucose-dependent current and power output in the concentration region between 1 and 10 mM. To create a more realistic test environment, the performance and long-term stability of the biofuel cell-based self-powered glucose biosensor has been investigated in a flow-through cell design.

The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

Development of a new chemo-enzymatic catalytic route for synthesis of (S)- 2-chlorophenylglycine.

(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant EsLeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (BmGDH) for NADH regeneration, EsLeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C.

An artificial pathway for trans-4-hydroxy-L-pipecolic acid production from L-lysine in Escherichia coli.

Trans-4-hydroxy-L-pipecolic acid (trans-4-HyPip) is a hydroxylated product of L-pipecolic acid, which is widely used in the pharmaceutical and chemical industries. Here, a trans-4-HyPip biosynthesis module was designed and constructed in Escherichia coli by overexpressing lysine α-oxidase, Δ1-piperideine-2-carboxylase reductase, glucose dehydrogenase, lysine permease, catalase and L-pipecolic acid trans-4-hydroxylase for expanding the lysine catabolism pathway. A total of 4.89 g/L of trans-4-HyPip was generated in shake flasks from 8 g/L of L-pipecolic acid. By this approach, 14.86 g/L of trans-4-HyPip was produced from lysine after 48 h in a 5 L bioreactor. As far as we know, this is the first multi-enzyme cascade catalytic system for the production of trans-4-HyPip using E. coli from L-lysine. Therefore, it can be considered as a potential candidate for the industrial production of trans-4-HyPip in microorganisms.

G6PD Deficiency Is Crucial for Insulin Signaling Activation in Skeletal Muscle.

Glucose 6-P dehydrogenase (G6PD) is the first rate-limiting enzyme in pentose phosphate pathway (PPP), and it is proverbial that G6PD is absent in skeletal muscle. However, how and why G6PD is down-regulated during skeletal muscle development is unclear. In this study, we confirmed the expression of G6PD was down-regulated during myogenesis in vitro and in vivo. G6PD was absolutely silent in adult skeletal muscle. Histone H3 acetylation and DNA methylation act together on the expression of G6PD. Neither knock-down of G6PD nor over-expression of G6PD affects myogenic differentiation. Knock-down of G6PD significantly promotes the sensitivity and response of skeletal muscle cells to insulin; over-expression of G6PD significantly injures the sensitivity and response of skeletal muscle cells to insulin. High-fat diet treatment impairs insulin signaling by up-regulating G6PD, and knock-down of G6PD rescues the impaired insulin signaling and glucose uptake caused by high-fat diet treatment. Taken together, this study explored the importance of G6PD deficiency during myogenic differentiation, which provides new sight to treat insulin resistance and type-2 diabetes.

Asymmetric Hydrogenation of C = C Bonds in a SpinChem Reactor by Immobilized Old Yellow Enzyme and Glucose Dehydrogenase.

The application of immobilized enzymes in pharmaceutical and bulk chemical production has been shown to be economically viable. We demonstrate the exceptional performance of a method that immobilizes the old yellow enzyme YqjM and glucose dehydrogenase (GDH) on resin for the asymmetric hydrogenation (AH) of C = C bonds in a SpinChem reactor. When immobilized YqjM and GDH are reused 10 times, the conversion of 2-methylcyclopentenone could reach 78%. Which is because the rotor of the SpinChem reactor effectively reduces catalyst damage caused by shear force in the reaction system. When the substrate concentration is 175 mM, an 87% conversion of 2-methylcyclopentenone is obtained. The method is also observed to perform well for the AH of C = C bonds in other unsaturated carbonyl compounds with the SpinChem reactor. Thus, this method has great potential for application in the enzymatic production of chiral compounds.

Nanostructured Interface Loaded with Chimeric Enzymes for Fluorimetric Quantification of Cyclosporine A and FK506.

Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.

A membraneless starch/O2 biofuel cell based on bacterial surface regulable displayed sequential enzymes of glucoamylase and glucose dehydrogenase.

Enzymatic biofuel cells (EBFCs) provide a new strategy to enable direct biomass-to-electricity conversion, posing considerable demand on sequential enzymes. However, artificial blend of multi-enzyme systems often suffer biocatalytic inefficiency due to the rambling mixture of catalytic units. In an attempt to construct a high-performance starch/O2 EBFC, herein we prepared a starch-oxidizing bioanode based on displaying a sequential enzyme system of glucoamylase (GA) and glucose dehydrogenase (GDH) on E.coli cell surfaces in a precise way using cohesin-dockerin interactions. The enzyme stoichiometry was optimized, with GA&GDH (3:1)-E.coli exhibiting the highest catalytic reaction rate. The bioanode employed polymerized methylene blue (polyMB) to collect electrons from the oxidation of NADH into NAD+, which jointly oxidized starch together with co-displayed GA and GDH. The bioanode was oxygen-insensitive, which can be combined with a laccase based biocathode, resulting in a membranless starch/O2 EBFC in a non-compartmentalized configuration. The optimal EBFC exhibited an open-circuit voltage (OCV) of 0.74 V, a maximum power density of 30.1 ± 2.8 µW cm-2, and good operational stability.

One pot purification and co-immobilization of His-tagged old yellow enzyme and glucose dehydrogenase for asymmetric hydrogenation.

In this study, a novel kind of Ni-NTA modified monodispersed SiO2 nanoflowers (Ni-NTA@SiO2 nanoflowers) were successfully synthesized. The obtained Ni-NTA@SiO2 nanoflowers were used to specifically adsorb and purify His-tagged old yellow enzyme (OYE1) and glucose dehydrogenase (GDH), which allows access to optically pure (3 S)- 3-methyl-cyclohexanone through asymmetric hydrogenation reaction, and forms a cofactor regeneration system. The protein loading amount on Ni-NTA@SiO2 nanoflowers was 40.17 mg/g support and the activity recoveries of OYE1 and GDH were 81.53% and 79.68%, respectively. The effects of pH and temperature on the activity of free and co-immobilized enzymes were investigated, and the stability as well as reusability were also measured. Compared to free enzymes, the co-immobilized enzymes showed higher thermal and storage stability. The co-immobilized enzymes were applied to asymmetric reduction of CC bonds for the synthesis of a chiral center with excellent enantioselectivity (ee > 99%), and the conversion was 46.02% after 7 cycles. This work introduced a one-pot multi-enzyme purification and co-immobilization strategy to construct efficient cofactor regeneration system with high activity and stability.

A cytochrome b-glucose dehydrogenase chimeric enzyme capable of direct electron transfer.

The development of third generation biosensors depends on the availability of direct electron transfer (DET) capable enzymes. A successful strategy is to fuse a cytochrome domain to an enzyme to fulfil the function of a built-in redox mediator between the catalytic center and the electrode. In this study, we fused the cytochrome domain of Neurospora crassa CDH IIA (NcCYT) N-terminally to glucose dehydrogenase from Glomerella cingulata (GcGDH) to generate the chimeric enzyme NcCYT-GcGDH in a large amount for further studies. Heterologous expression in P. pastoris and chromatographic purification resulted in 1.8 g of homogeneous chimeric enzyme. Biochemical and electrochemical characterization confirmed that the chimeric enzyme is catalytically active, able to perform interdomain electron transfer (IET) and direct electron transfer (DET) via the fused cytochrome domain. The midpoint redox potential of the fused b-type cytochrome is 91 mV vs. SHE at pH 6.5 and the specific current obtained on a porous graphite electrode is 2.3 µA cm-2. The high current obtained on this simple, unmodified electrode at a rather low redox potential is a promising starting point for further optimization. The high yield of NcCYT-GcGDH and its high specific activity supports the application of the chimeric enzyme in bioelectrocatalytic applications.