Biocatalytic Platform Research Articles

Nanobiohybrid Extracellular Vesicle Nanoreactor with Improving Metabolical Activity for Biocatalytic Therapy.

Extracellular vesicles (EVs) hosting enzymatic activities that function as independent metabolic units are attractive natural biocatalytic platforms. However, directly using these metabolically active nanoreactors for effective biocatalytic applications remains challenging, mainly due to their constrained catalytic capabilities. Here, we construct an EV-templated nanobiohybrid system by engineering an EV surface with a photoresponsive zeolitic imidazolate framework (ZIF). The deposition of ZIF nanostructures on EVs not only contributes to improved biocatalytic stability but also enables interfacial coupling between photoexcited electrons from the ZIF and the enzymatic reaction of metabolically active EVs. Nearly 300% of biomass conversion efficiency increment could be achieved by EVs derived from macrophages. This enhanced biocatalysis, high catalytic stability, and low cytotoxicity endowed the EV@ZIF nanosystem with robust biosynthesis and antimicrobial activity. When evaluated in a mouse periodontitis model, we show that the autologous biocatalytic EV@ZIF demonstrated efficient therapeutic capability by killing bacteria and inhibiting inflammation. This nanoengineering strategy will benefit the future optimization of metabolically active EV nanoreactors as biocatalysts for a broad range of therapeutics.

Engineering Neuroglobin for Synthesis of Chiral Organoborons via Carbene B-H Insertion.

Organoborons have recently received much attention, while a biocatalytic platform for the synthesis of chiral organoborons is limited only to Rma cytochrome c. In this study, we exploited the other heme protein, neuroglobin (Ngb), and engineered a quadruple mutant, A15C/H64G/V68F/F28M Ngb, by redesigning the heme active site using the structural information on A15C Ngb and molecular docking studies. The enzyme was shown to be efficient in catalyzing carbene transfer B-H insertion reactions between pyridine/quinoline boranes and benzyl 2-diazopropanoates and their derivatives (29 examples). The designed cavity in the heme distal site favors the binding of large volume substrates such as those containing a quinoline, naphthyl, or biphenyl group. As further determined by the X-ray crystallography of 6c, the chiral products are in the R-configuration, with up to 98:2 e.r. Furthermore, both the whole cell and cell lysate containing the enzyme are reactive toward the B-H insertion reactions. This study presents a convenient biocatalytic platform that may be generally applicable for the synthesis of functional chiral organoborons.

Chitin membrane for efficient laccase immobilization and synergistic removal of 17α-ethynylestradiol from water-based solutions

A unique chitin–laccase membrane was fabricated as an environmentally friendly biocatalytic platform, utilizing 1-butyl-3-methylimidazolium acetate as the solvent for chitin. Observations using scanning electron microscopy showed that the chitin-laccase membrane possessed a uniform and densely packed structure. Based on the presence of FT-IR signals at 1020 cm−1 and changes in the intensity of signals at 1540 cm−1 and 1645 cm−1, the effectiveness of laccase immobilization was confirmed. FT-IR mapping revealed that the enzyme is evenly distributed on the surface of the membrane. The catalytic activity of the native enzyme and laccase immobilized using the membrane was determined based on a model reaction, and the retention of high activity was confirmed using real solutions. Laccase immobilized using the chitinous membrane retained over 60 % of its initial activity after 30 days of storage at 4 °C. By contrast, the free enzyme retained <40 % of its initial activity. Moreover, the activity of chitin-laccase system remained at 85 % after 5 cycles. This novel chitin–laccase combination was tested in the 17α-ethynylestradiol (EE2) removal from water-based solutions. It was found that EE2 underwent synergistic degradation through concurrent adsorption and biocatalytic transformation, with enzymatic conversion as the dominant mechanism.

Stereoselective Synthesis of Oxetanes Catalyzed by an Engineered Halohydrin Dehalogenase.

Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In our work, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3-disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ-haloalcohols (via HheC M1-M3) and ring opening of oxetanes (via HheC M4-M5) to access both (R)- and (S)-configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49% with enantioselectivities exceeding 99% ee and E>200. The current strategy is broadly applicable as demonstrated by expansion of substrate scope to include up to 18 examples for dehalogenations and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH-catalyzed chiral oxetane ring constructions, offering efficient access to both enantiomers of chiral oxetanes and 1,3-disubstituted alcohols.

Stereoselective Synthesis of Oxetanes Catalyzed by an Engineered Halohydrin Dehalogenase

Although biocatalysis has garnered widespread attention in both industrial and academic realms, the enzymatic synthesis of chiral oxetanes remains an underdeveloped field. Halohydrin dehalogenases (HHDHs) are industrially relevant enzymes that have been engineered to accomplish the reversible transformation of epoxides. In our work, a biocatalytic platform was constructed for the stereoselective kinetic resolution of chiral oxetanes and formation of 1,3‐disubstituted alcohols. HheC from Agrobacterium radiobacter AD1 was engineered to identify key variants capable of catalyzing the dehalogenation of γ‐haloalcohols (via HheC M1‐M3) and ring opening of oxetanes (via HheC M4‐M5) to access both (R)‐ and (S)‐configured products with high stereoselectivity and remarkable catalytic activity, yielding up to 49% with enantioselectivities exceeding 99% ee and E&gt;200. The current strategy is broadly applicable as demonstrated by expansion of substrate scope to include up to 18 examples for dehalogenations and 16 examples for ring opening. Additionally, the functionalized products are versatile building blocks for pharmaceutical applications. To shed light on the molecular recognition mechanisms for the relevant variants, molecular dynamic (MD) simulations were performed. The current strategy expands the scope of HHDH‐catalyzed chiral oxetane ring constructions, offering efficient access to both enantiomers of chiral oxetanes and 1,3‐disubstituted alcohols.

N-Halogenation by Vanadium-Dependent Haloperoxidases Enables 1,2,4-Oxadiazole Synthesis.

Nitrogen-containing compounds are valuable synthetic intermediates and targets in nearly every chemical industry. While methods for nitrogen-carbon and nitrogen-heteroatom bond formation have primarily relied on nucleophilic nitrogen atom reactivity, molecules containing nitrogen-halogen bonds allow for electrophilic or radical reactivity modes at the nitrogen center. Despite the growing synthetic utility of nitrogen-halogen bond-containing compounds, selective catalytic strategies for their synthesis are largely underexplored. We recently discovered that the vanadium-dependent haloperoxidase (VHPO) class of enzymes are a suitable biocatalyst platform for nitrogen-halogen bond formation. Herein, we show that VHPOs perform selective halogenation of a range of substituted benzamidine hydrochlorides to produce the corresponding N'-halobenzimidamides. This biocatalytic platform is applied to the synthesis of 1,2,4-oxadiazoles from the corresponding N-acylbenzamidines in high yield and with excellent chemoselectivity. Finally, the synthetic applicability of this biotechnology is demonstrated in an extension to nitrogen-nitrogen bond formation and the chemoenzymatic synthesis of the Duchenne muscular dystrophy drug, ataluren.

N‐Halogenation by Vanadium‐Dependent Haloperoxidases Enables 1,2,4‐Oxadiazole Synthesis

Nitrogen‐containing compounds are valuable synthetic intermediates and targets in nearly every chemical industry. While methods for nitrogen‐carbon and nitrogen‐heteroatom bond formation have primarily relied on nucleophilic nitrogen atom reactivity, molecules containing nitrogen‐halogen bonds allow for electrophilic or radical reactivity modes at the nitrogen center. Despite the growing synthetic utility of nitrogen‐halogen bond‐containing compounds, selective catalytic strategies for their synthesis are largely underexplored. We recently discovered that the vanadium‐dependent haloperoxidase (VHPO) class of enzymes are a suitable biocatalyst platform for nitrogen‐halogen bond formation. Herein, we show that VHPOs perform selective halogenation of a range of substituted benzamidine hydrochlorides to produce the corresponding N’‐halobenzimidamides. This biocatalytic platform is applied to the synthesis of 1,2,4‐oxadiazoles from the corresponding N‐acylbenzamidines in high yield and with excellent chemoselectivity. Finally, the synthetic applicability of this biotechnology is demonstrated in an extension to nitrogen‐nitrogen bond formation and the chemoenzymatic synthesis of the Duchenne muscular dystrophy drug, ataluren.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.

Biocatalytic Regio‐ and Enantiocomplementary Synthesis of Chiral Aryloxazolidinones

AbstractWe described the protein engineering of the halohydrin dehalogenase HheG to enhance its R enantioselectivity for the synthesis of chiral 4‐aryloxazolidinones via α‐regioselective ring‐opening of aryl epoxides with cyanate. Additionally, we achieved the inversion of its regioselectivity and enantioselectivity, resulting in a HheG variant tailored for the synthesis of chiral 5‐aryloxazolidinones via β‐regioselective and S‐enantioselective ring‐opening of aryl epoxides with cyanate. Leveraging these engineered mutants, we developed a biocatalytic platform capable of synthesis of both chiral 4‐aryloxazolidinones (up to 47% yield, 98% ee, and 99% regioselectivity) and chiral 5‐aryloxazolidinones (up to 46% yield, &gt;99% ee, and 98% regioselectivity). Furthermore, a collaborative approach utilizing two regio‐ and enantioselective HheG mutants has been demonstrated to enable the simultaneous synthesis of chiral 4‐aryloxazolidinones and chiral 5‐aryloxazolidinones.

Enzyme-integrated metal-organic framework platform for cascade detection of α-amylase

As one of the most important industrial enzymes, α-amylase is widely used in food processing, such as starch sugar and fermentation, bringing high added value to industry of more than a trillion dollars. We developed a multi-enzyme system (Glu&Gox@Cu-MOF-74) prepared by embedding α-glucosidase (Glu) and glucose oxidase (Gox) into the biomimetic metal-organic framework Cu-MOF-74 using in situ encapsulation within 15 min at room temperature for efficient and sensitive detection of α-amylase activity. Benefitting from the remarkable peroxidase-mimicking property and rigid skeleton of Cu-MOF-74, the biocatalytic platform exhibited excellent cascade activity and tolerance in various extremely harsh environments compared to natural enzymes. On this basis, a cascade biocatalytic platform was constructed for the detection of α-amylase activity with wide linear range (5–100 U/L) and low limit of detection (1.45 U/L). The colorimetric cascade scheme is important for the sensitive and selective determination of α-amylase in complex fermentation samples, and the detection time is short (∼0.5 h). This work provides new ideas for the detection of α-amylase based on the cascade amplification method.

Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling

BackgroundThe global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD3 (cholecalciferol) in the human body, 25-hydroxy-VitD3 (calcifediol, 25OHVitD3), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD3 to value-added 25OHVitD3 are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates.ResultsIn this study, we used a whole-cell approach for biocatalytic 25OHVitD3 synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD3 hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD3 to 25OHVitD3 using either O2 via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD3 titer achieved was 1.85 g L–1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD3 solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity.ConclusionsThe established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD3 to 25OHVitD3 offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD3 synthesis.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

Lipase B from Candida antarctica immobilized on amphiphilic Janus halloysite nanosheet and application in biphasic interface conversion

Lipase B from Candida antarctica (CALB) plays a prominent role as a biocatalyst in several industries, especially for biphasic conversion of functional lipids. Herein, an amphiphilic Janus halloysite nanosheet (JHNS) was fabricated and employed simultaneously as a solid surfactant for stabilizing Pickering emulsion and as a carrier for immobilizing CALB, with the aim to realize highly efficient biphasic bioconversion. The obtained JHNS could stabilize Pickering emulsion for at least 7 days. Immobilization of CALB on JHNS improved the substrate affinity, catalytic efficiency, thermal stability, and alkaline tolerance of the enzyme. Moreover, JHNS-based immobilized CALB was exploited as a biocatalytic platform for the conversion of retinyl acetate, with almost twice increase in conversion efficiency. Taken together, the JHNS-based immobilized CALB paves the way for the design of efficient biphasic conversion system for the production of added-value lipids.

Bio-mitigation of organic pollutants using horseradish peroxidase as a promising biocatalytic platform for environmental sustainability

A wide array of environmental pollutants is often generated and released into the ecosystem from industrial and human activities. Antibiotics, phenolic compounds, hydroquinone, industrial dyes, and Endocrine-Disrupting Chemicals (EDCs) are prevalent pollutants in water matrices. To promote environmental sustainability and minimize the impact of these pollutants, it is essential to eliminate such contaminants. Although there are multiple methods for pollutants removal, many of them are inefficient and environmentally unfriendly. Horseradish peroxidase (HRP) has been widely explored for its ability to oxidize the aforementioned pollutants, both alone and in combination with other peroxidases, and in an immobilized way. Numerous positive attributes make HRP an excellent biocatalyst in the biodegradation of diverse environmentally hazardous pollutants. In the present review, we underlined the major advancements in the HRP for environmental research. Numerous immobilization and combinational studies have been reviewed and summarized to comprehend the degradability, fate, and biotransformation of pollutants. In addition, a possible deployment of emerging computational methodologies for improved catalysis has been highlighted, along with future outlook and concluding remarks.

A biocatalytic platform for asymmetric alkylation of α-keto acids by mining and engineering of methyltransferases

Catalytic asymmetric α-alkylation of carbonyl compounds represents a long-standing challenge in synthetic organic chemistry. Herein, we advance a dual biocatalytic platform for the efficient asymmetric alkylation of α-keto acids. First, guided by our recently obtained crystal structures, we develop SgvMVAV as a general biocatalyst for the enantioselective methylation, ethylation, allylation and propargylation of a range of α-keto acids with total turnover numbers (TTNs) up to 4,600. Second, we mine a family of bacterial HMTs from Pseudomonas species sharing less than 50% sequence identities with known HMTs and evaluated their activities in SAM regeneration. Our best performing HMT from P. aeruginosa, PaHMT, displays the highest SAM regeneration efficiencies (TTN up to 7,700) among HMTs characterized to date. Together, the synergistic use of SgvMVAV and PaHMT affords a fully biocatalytic protocol for asymmetric methylation featuring a record turnover efficiency, providing a solution to the notorious problem of asymmetric alkylation.

Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR

We demonstrate an atom-efficient and easy to use H2-driven biocatalytic platform for the enantioselective synthesis of isotopically labelled amino acids for protein NMR studies.

Efficient Production of Epoxy-Norbornane from Norbornene by an Engineered P450 Peroxygenase.

Epoxy-norbornane (EPO-NBE) is a crucial building block for the synthesis of various biologically active heterocyclic systems. To develop an efficient protocol for producing EPO-NBE using norbornene (NBE) as a substrate, cytochrome P450 enzyme from Pseudomonas putida (CYP238A1) was examined and its crystal structure (PDB code: 7X53) was resolved. Molecular mechanism analysis showed a high energy barrier related to iron-alkoxy radical complex formation. Therefore, a protein engineering strategy was developed and an optimal CYP238A1NPV variant containing a local hydrophobic "fence" at the active site was obtained, which increased the H2 O2 -dependent epoxidation activity by 7.5-fold compared with that of CYP238A1WT . Among the "fence", Glu255 participates in an efficient proton transfer system. Whole-cell transformation using CYP238A1NPV achieved an EPO-NBE yield of 77.6 g ⋅ L-1 in a 30-L reactor with 66.3 % conversion. These results demonstrate the potential of this system for industrial production of EPO-NBE and provides a new biocatalytic platform for epoxidation chemistry.

Development of a P450 Fusion Enzyme for Biaryl Coupling in Yeast.

Despite the diverse and potent bioactivities displayed by axially chiral biaryl natural products, their application in drug discovery is limited by restricted access to these complex molecular scaffolds. In particular, fundamental challenges remain in controlling the site- and atroposelectivity in biaryl coupling reactions. In contrast, Nature has a wealth of biosynthetic enzymes that catalyze biaryl coupling reactions with catalyst-controlled selectivity. In particular, a growing subset of fungal P450s have been identified to catalyze site- and atroposelective biaryl couplings. Herein, we optimize a whole-cell biocatalytic platform in Pichia pastoris to synthesize biaryl molecules through the recombinant production of the fungal P450 KtnC. Moreover, engineering redox self-sufficient fusion enzymes further improves the efficiency of the system. Altogether, this work provides a platform for biaryl coupling reactions in yeast that can be applied to engineering a currently underexplored pool of fungal P450s into selective biocatalysts for the synthesis of complex biaryl compounds.

Fluorescent sensor based on pyrroloquinoline quinone (PQQ)-glucose dehydrogenase for glucose detection with smartphone-adapted signal analysis.

A new nano-structured platform for fluorescent analysis using PQQ-dependent glucose dehydrogenase (PQQ-GDH) was developed, particularly using a smartphone for transduction and quantification of optical signals. The PQQ-GDH enzyme was immobilized on SiO2 nanoparticles deposited on glass microfiber filter paper, providing a high load of the biocatalytic enzyme. The platform was tested and optimized for glucose determination using a wild type of the PQQ-GDH enzyme. The analysis was based on the fluorescence generated by the reduced form of phenazine methosulfate produced stoichiometrically to the glucose concentration. The fluorescent signals were generated at separate analytical spots on the paper support under wavelength (365nm) UV excitation. The images of the analytical spots, dependent on the glucose concentration, were obtained using a photo camera of a standard smartphone. Then, the images were processed and quantified using software installed in a smartphone. The developed biocatalytic platform is the first step to assembling a large variety of biosensors using the same platform functionalized with artificial allosteric chimeric PQQ-dependent glucose dehydrogenase activated with different analytes. The future combination of the artificial enzymes, the presently developed analytical platform, and signal processing with a smartphone will lead to novel point-of-care and end-user biosensors applicableto virtually all possible analytes.

A Biocatalytic Platform for the Synthesis of Enantiopure Propargylic Alcohols and Amines.

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70–99% yield.