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.

Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction.

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.

Electrochemical and biosensing properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens.

We investigated the bioelectrochemical properties of an FAD-dependent glucose dehydrogenase from Trichoderma virens (TvGDH) and its electrochemical behaviour when immobilized on a graphite electrode. TvGDH was recently shown to have an unusual substrate spectrum and to prefer maltose over glucose as substrate, and hence could be of interest as recognition element in a maltose sensor. In this study, we determined the redox potential of TvGDH, which is -0.268 ± 0.007 V vs. SHE, and advantageously low to be used with many redox mediators or redox polymers. The enzyme was entrapped in, and wired by an osmium redox polymer (poly(1-vinylimidazole-co-allylamine)-{[Os(2,2'-bipyridine)2Cl]Cl}) with formal redox potential of +0.275 V vs. Ag|AgCl via poly(ethylene glycol) diglycidyl ether crosslinking onto a graphite electrode. When the TvGDH-based biosensor was tested with maltose it showed a sensitivity of 1.7 µA mM-1cm-2, a linear range of 0.5-15 mM, and a detection limit of 0.45 mM. Furthermore, it gave the lowest apparent Michaelis-Menten constant (KM app) of 19.2 ± 1.5 mM towards maltose when compared to other sugars. The biosensor is also able to detect other saccharides including glucose, maltotriose and galactose, these however also interfere with maltose sensing.

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10-2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10-2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

Designing a cross-linked redox network for a mediated enzyme-based electrode.

A bio-conjugated redox network matrix based on glucose dehydrogenase, thionine (diamine-containing mediator), and poly(ethylene glycol) diglycidyl ether (crosslinker) is developed on a glassy carbon electrode through covalent bonding with one-pot crosslinking. Electrons from the enzyme diffuse through the network producing 400 µA cm-2 of glucose oxidation current at 25 °C.

Co-immobilized Alcohol Dehydrogenase and Glucose Dehydrogenase with Resin Extraction for Continuous Production of Chiral Diaryl Alcohol.

Ni2+-functionalized porous ceramic/agarose composite beads (Ni-NTA Cerose) can be used as carrier materials to immobilize enzymes harboring a metal affinity tag. Here, a 6×His-tag fusion alcohol dehydrogenase Mu-S5 and glucose dehydrogenase from Bacillus megaterium (BmGDH) were co-immobilized on Ni-NTA Cerose to construct a packed bed reactor (PBR) for the continuous synthesis of the chiral intermediate (S)-(4-chlorophenyl)-(pyridin-2-yl) methanol ((S)-CPMA) NADPH recycling, and in situ product adsorption was achieved simultaneously by assembling a D101 macroporous resin column after the PBR. Using an optimum enzyme activity ratio of 2:1 (Mu-S5: BmGDH) and hydroxypropyl-ß-cyclodextrin as co-solvent, a space-time yield of 1560 g/(L·d) could be achieved in the first three days at a flow rate of 5 mL/min and substrate concentration of 10 mM. With simplified selective adsorption and extraction procedures, (S)-CPMA was obtained in 84% isolated yield.

Development of a Sensitive Self-Powered Glucose Biosensor Based on an Enzymatic Biofuel Cell.

Biofuel cells allow for constructing sensors that leverage the specificity of enzymes without the need for an external power source. In this work, we design a self-powered glucose sensor based on a biofuel cell. The redox enzymes glucose dehydrogenase (NAD-GDH), glucose oxidase (GOx), and horseradish peroxidase (HRP) were immobilized as biocatalysts on the electrodes, which were previously engineered using carbon nanostructures, including multi-wall carbon nanotubes (MWCNTs) and reduced graphene oxide (rGO). Additional polymers were also introduced to improve biocatalyst immobilization. The reported design offers three main advantages: (i) by using glucose as the substrate for the both anode and cathode, a more compact and robust design is enabled, (ii) the system operates under air-saturating conditions, with no need for gas purge, and (iii) the combination of carbon nanostructures and a multi-enzyme cascade maximizes the sensitivity of the biosensor. Our design allows the reliable detection of glucose in the range of 0.1-7.0 mM, which is perfectly suited for common biofluids and industrial food samples.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Co-evolution of activity and thermostability of an aldo-keto reductase KmAKR for asymmetric synthesis of statin precursor dichiral diols.

Aldo-keto reductase KmAKR-catalyzed asymmetric reduction offers a green approach to produce dichiral diol tert-butyl 6-substituted-(3R,5R/S)-dihydroxyhexanoates, which are important building blocks of statins. In our previous work, we cloned a novel gene of NADPH-specific aldo-keto reductase KmAKR (WT) from a thermotolerant yeast Kluyveromyces marxianus ZJB14056 and a mutant KmAKR-W297H/Y296W/K29H (Variant III) has been constructed and displayed strict diastereoselectivity towards tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1) but moderate activity and stability. Herein, to further co-evolve its activity and thermostability, we performed semi-rational engineering of Variant III by using a combinational screening strategy, consisting of tertiary structure analysis, loop engineering, and alanine scanning. As results, the "best" variant KmAKR-W297H/Y296W/K29H/Y28A/T63M (Variant VI) was acquired, whose Km, kcat/Km towards (5R)-1 was 0.66 mM and 210.77 s-1 mM-1, respectively, with improved thermostability (half-life of 14.13 h at 40 °C). Combined with 1.5 g dry cell weight (DCW) L-1Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for NADPH regeneration, 4.5 g DCW L-1Variant VI completely reduced (5R)-1 of up to 450 g L-1 within 7.0 h at 40 °C, yielding the corresponding optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-3, >99.5% d.e.p) with a space-time yield (STY) of 1.24 kg L-1 day-1, and this was the highest level documented in literatures so far on substrate loading and STY of producing (3R,5R)-3. Besides (5R)-1, Variant VI displayed strong activity on tert-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate ((5S)-2). 4.5 g DCW L-1Variant VI completely reduced 400 g L-1 (5S)-2, within 5.0 h at 40 °C, yielding optically pure tert-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate ((3R,5S)-4, >99.5% d.e.p) with a STY of 1.34 kg L-1 day-1. In summary, Variant VI displayed industrial application potential in statins biomanufacturing.

Mimic of the Cellular Antioxidant Defense System for a Sustainable Regeneration of Nicotinamide Adenine Dinucleotide (NAD).

The prolonged use of enzymes under oxidative stress is a major challenge in enabling effective enzymatic reaction pathways. Herein, we report a biomimetic antioxidant defensive strategy capable of providing adequate protection of enzymes against superoxide-mediated oxidation. Superoxide dismutase (SOD) and catalase (CAT) were chosen as scavengers and covalently encapsulated into silica nanoreactors, together with glucose dehydrogenase (GDH), which simultaneously should produce the coenzyme nicotinamide adenine dinucleotide (NADH, reduced form). By the enzymatic reactions of SOD and CAT, the interior of silica nanoreactors becomes a "ROS safe zone" to protect the glucose-dependent NADH production of coencapsulated GDH. We further combined this protected NADH-producing module with photocatalytic nanoparticles that enable the light-triggered oxidation of NADH back to NAD+ (oxidized form). In combination, these two modules allow interconversion between NAD+ and NADH by the addition of glucose or by light irradiation (LED lamp or sunlight). This protection and regeneration strategy is a versatile tool for enzyme applications for biological reactors, catalysis, or prototypes of artificial organelles or building blocks that contains fragile biomolecules and rely on the coenzyme NAD+/NADH.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.

Light-controlled imaging of biocatalytic reactions via scanning photoelectrochemical microscopy for multiplexed sensing.

A light-controlled multiplexing platform has been developed on the basis of a quantum dot-sensitized inverse opal TiO2 electrode with integrated biocatalytic reactions. Spatially resolved illumination enables multiplexed sensing and imaging of enzymatic oxidation reactions at relatively negative applied potentials.

A bio-inspired highly selective enzymatic glucose sensor using a red blood cell membrane.

In the development of enzymatic glucose sensors, accurate glucose sensing has been a challenging task because of the existence of numerous interfering molecules in the blood. Meanwhile, red blood cells (RBCs) selectively uptake glucose via a membrane protein called glucose transporter-1. In this study, we developed the RBC membrane (RBCM)-coated enzymatic glucose sensors that mimic the glucose uptake. The RBCM-coated sensors were examined via scanning electron microscopy, atomic force microscopy, and ATR-FTIR. We optimized the glucose permeability of the RBCM filter by controlling the thickness of the filter. The sensing range of the optimized sensor was 1-15 mM, the detection limit was 0.66 mM, and the sensitivity was 2.978 µA mM-1. Intriguingly, the RBCM-coated sensor was highly accurate and precise, despite the coexistence of glucose and interfering molecules (e.g., mannose, galactose, ascorbic acid, uric acid, and cysteine). For each interfering molecule, the errors of our sensor were 0.8 to 2.3%, which was 4.8-14.2 times more accurate than the uncoated one. A similar result was verified for a human serum sample containing countless interfering molecules. Also, the sensing performance of the sensor was consistent after 4 weeks of storage. The results suggest that applying RBCM may improve the selectivity of various types of glucose sensors including the continuous monitoring system.

Improved operational stability of mediated glucose enzyme electrodes for operation in human physiological solutions.

Stability of glucose-oxidising enzyme electrodes is affected by substances in physiological solutions, hampering deployment as long-term implantable biosensors or fuel cells. The performance of Nafion over-coated enzyme electrodes, consisting of multiwalled carbon nanotubes and flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) or glucose oxidase (GOx) crosslinked with osmium-complex based redox polymer, was compared to uncoated electrodes in presence of uric acid and artificial plasma. Nafion over-coating resulted in lower glucose oxidation current densities compared to no over-coating. The highest initial current density for Nafion over-coated electrodes in artificial plasma in 100 mM glucose was 8.0 ± 2.0 mA cm-2 for GOx electrodes with 0.5% w/v Nafion coating. These electrodes retained 83% of initial current after 12 h continuous operation in artificial plasma while similarly prepared FADGDH electrodes retained 58% signal. This is compared to retention of only 73% or 31% observed for GOx or FADGDH electrodes in artificial plasma with no Nafion membrane. Enzyme electrodes over-coated with Nafion maintain improved signal stability when tested continuously in the presence of uric acid, identified as being the main contributing substance to FADGDH enzyme electrode instability, showing promise for application to continuous use glucose-oxidising enzyme electrodes.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters.

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm-2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

pH-Neutralization, Redox-Balanced Process with Coupled Formate Dehydrogenase and Glucose Dehydrogenase Supports Efficient Xylitol Production in Pure Water.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization.

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144-297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144-297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144-297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

Lab on a tip: Applications of functional atomic force microscopy for the study of electrical properties in biology.

Electrical properties, such as charge propagation, dielectrics, surface potentials, conductivity, and piezoelectricity, play crucial roles in biomolecules, biomembranes, cells, tissues, and other biological samples. However, characterizing these electrical properties in delicate biosamples is challenging. Atomic Force Microscopy (AFM), the so called "Lab on a Tip" is a powerful and multifunctional approach to quantitatively study the electrical properties of biological samples at the nanometer level. Herein, the principles, theories, and achievements of various modes of AFM in this area have been reviewed and summarized. STATEMENT OF SIGNIFICANCE: Electrical properties such as dielectric and piezoelectric forces, charge propagation behaviors play important structural and functional roles in biosystems from the single molecule level, to cells and tissues. Atomic force microscopy (AFM) has emerged as an ideal toolkit to study electrical property of biology. Herein, the basic principles of AFM are described. We then discuss the multiple modes of AFM to study the electrical properties of biological samples, including Electrostatic Force Microscopy (EFM), Kelvin Probe Force Microscopy (KPFM), Conductive Atomic Force Microscopy (CAFM), Piezoresponse Force Microscopy (PFM) and Scanning ElectroChemical Microscopy (SECM). Finally, the outlook, prospects, and challenges of the various AFM modes when studying the electrical behaviour of the samples are discussed.

Wearable high-powered biofuel cells using enzyme/carbon nanotube composite fibers on textile cloth.

Wearable biofuel cells with flexible enzyme/carbon nanotube (CNT) fibers were designed on a cotton textile cloth by integrating two components: bioanode fibers for glucose oxidation and O2-diffusion biocathode fibers for oxygen reduction. The anode and cathode fibers were prepared through modification with glucose dehydrogenase and bilirubin oxidase, respectively, on multi-walled carbon nanotube-coated carbon fibers. Both biofibers woven on the cloth generated a power density of 48 µW/cm2 at 0.24 V from 0.1 mM glucose (human sweat amount), and of 216 µW/cm2 at 0.36 V, when glucose was supplied from a hydrogel tank containing 200 mM glucose. Our fiber-based biofuel cell deformed to an S-shape without a significant loss in cell performance. Furthermore, we demonstrated a series-connection involving the tying of biofibers on a cloth with batik-based ionic isolation. The booster four cells generate power at 1.9 V that illuminated an LED on the cloth.

Improving the catalytic efficiency of aldo-keto reductase KmAKR towards t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate via semi-rational design.

t-Butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-2) is an important chiral diol synthon of atorvastatin calcium. Previously, we constructed a variant KmAKR-W297H (M1) of Kluyveromyces marxianus aldo-keto reductase (KmAKR, designated as M0), possessing excellent diastereoselectivity but moderate activity towards t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1). In this work, KmAKR-W297H/Y296W/K29H (M3) was developed via semi-rational design. It exhibited much improved catalytic efficiency towards (5R)-1. The Km values of M3 for NADPH and (5R)-1 were 0.15 mmol/L and 1.41 mmol/L, and the maximal reaction rate vmax was 55.56 µmol/min/mg. Compared with M1, the catalytic efficiency kcat/Km of M3 was increased 2.64-fold. Coupled with Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, M3 took 3.5 h to completely reduce (5R)-1 at up to 100.0 g/L, producing 237.4 mmol/L (3R,5R)-2 in d.e.P value above 99.5%. The space-time yield (STY) of M3-catalyzed (3R,5R)-2 synthesis was 372.8 g/L/d.