Biocatalytic Reactions Research Articles

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Interfacing Whole Cell Biocatalysis with a Biocompatible Pictet-Spengler Reaction for One-Pot Syntheses of Tetrahydroisoquinolines and Tryptolines.

Biocatalytic processes are highly selective and specific. However, their utility is limited by the comparatively narrow scope of enzyme-catalysed transformations. To expand product scope, we are developing biocompatible processes that combine biocatalytic reactions with chemo-catalysis in single-flask processes. Here, we show that a chemocatalysed Pictet-Spengler annulation can be interfaced with biocatalysed alcohol oxidation. This two-step, one-pot cascade reaction converts tyramine and aliphatic alcohols to tetrahydroisoquinoline alkaloids in aqueous buffer at mild pH. Tryptamine derivatives are also efficiently converted to tryptolines. Optimization of stoichiometry, pH, reaction time, and whole-cell catalyst deliver the tetrahydroisouinolines and tryptolines in >90 % and >40 % isolated yield, respectively, with excellent regioselectivity.

Photobiocatalytic conversion of solar energy to NH3 from N2 and H2O under ambient condition

NH3 is an important chemical fertilizer and expecting as H2 carrier. Several methods have been investigated for eco-friendly NH3 production under mild conditions instead of Haber-Bosch process using 400 °C, 20 MPa. Here, cyanobacterial Anabaena variabilis was utilized as a nitrogenase-producing biocatalyst that converts N2/H2O to NH3 under ambient conditions. Biocatalytic reactions revealed that MV•+ can penetrate cell membrane and transfer electrons generated in inorganic photocatalyst. We first reported photobiocatalytic NH3 production of cyanobacteria and TiO2. Comparing with natural system, NH3 formation rate of the hybrid system increased 81.3 times with an initial rate of 2031.7 nmol·h−1 and turnover number of 216.8.

A Simple Access to γ- and ε-Keto Arenes via Enzymatic Divergent C─H Bond Oxyfunctionalization.

Performing divergent C─H bond functionalization on molecules with multiple reaction sites is a significant challenge in organic chemistry. Biocatalytic oxyfunctionalization reactions of these compounds to the corresponding ketones/aldehydes are typically hindered by selectivity issues. To address these challenges, the catalytic performance of oxidoreductases is explored. The results show that combining the peroxygenase-catalyzed propargylic C─H bond oxidation with the Old Yellow Enzyme-catalyzed reduction of conjugated C─C triple bonds in one-pot enables the regio- and chemoselective oxyfunctionalization of sp3 C─H bonds that are distant from benzylic sites. This enzymatic approach yielded a variety of γ-keto arenes with diverse structural and electronic properties in yields of up to 99% and regioselectivity of 100%, which are difficult to achieve using other chemocatalysis and enzymes. By adjusting the C─C triple bond, the carbonyl group's position can be further tuned to yield ε-keto arenes. This enzymatic approach can be combined with other biocatalysts to establish new synthetic pathways for accessing various challenging divergent C─H bond functionalization reactions.

High hydrolytic activity of the metagenomic lipase LipC12 in deep eutectic solvents

Deep eutectic solvents (DESs) have been shown to increase the activity of lipases in biocatalytic processes, especially when added at intermediate concentrations. We evaluated the effect of DESs on the hydrolysis of p-nitrophenyl palmitate (p-NPP) by a metagenomic lipase, LipC12. The hydrogen bond acceptor was choline chloride (ChCl), while sorbitol (Sor), xylitol (Xyl), Glycerol (Gly), ethylene glycol (EG), triethylene glycol (TEG) and urea (U) were tested as hydrogen bond donors. The best results were obtained at 25% (v v−1) DES, with the increases in hydrolytic activity, compared to activity in buffer, being 18-fold for ChCl:Sor (2:1), and 10- to 12-fold for ChCl:Gly (1:2), ChCl:EG (1:2) and ChCl:Xyl (2:1). Dynamic Light Scattering analysis suggests that protein disaggregation played a key role in this activation: the hydrodynamic radius of LipC12 was 157 nm in buffer, but ranged from about 1 to 7 nm in DESs at different concentrations. The addition of 100 mM NaCl to the DES-containing media (at 25% v v−1 DES) slightly improved LipC12 activity. Addition of the hydrogen-bond donor alone also increased the p-NPP hydrolyzing activity with 30% (w v−1) sorbitol or xylitol giving 4.5-fold higher activities, compared to activity in buffer. Our fold-improvements in activity are the highest yet reported for the p-NPP-hydrolyzing activity of lipases and suggest that more studies are required, especially with other biocatalytic reactions such as esterification and transesterification.

Photo-enzyme-polymerized hydrogel platform exhibits photo-switchable redox reversibility for diabetic wound healing

Redox homeostasis catalyzed by series oxidoreductases is important to maintain normal physiological functions, including mutually contradictory reactive oxygen species generation or reduction by separated enzyme regulation during cellular metabolism. The unidirectional enzyme regulation of redox homeostasis by biocatalytic reaction is an emerging catalytic medicine approach to disease biomimetic treatments. Bioinspired by photosynthesis, an efficient photo-enzyme-coupled hydrogel platform (Gel) with a spatio-temporally controlled hydrogel network was constructed through photoenzymatic-initiated radical polymerization in the absence of the corresponding enzyme substrate. Then, the coupling mechanism of light-induced electron transfer-enzyme activity center allosteric was explored through free radical analysis and theoretical calculations. The photo-switchable redox performance has been demonstrated based on the special anaerobic dehydrogenases, the flavin adenine dinucleotide (FAD)-centered dihydrolipoamide dehydrogenase (DLD) platform. At last, both in vitro and in vivo biological effects have been verified to evaluate the bidirectional oxidation/antioxidant modulation for the complicated wound healing in diabetic mice and wound-infected animals. The photoenzymatic coupling dehydrogenase-laden hydrogel platform proposed in this work provides ideas for the engineering design of enzymes, and the physical-biochemical regulatory mechanisms also offers a theoretical basis for controllable activation of enzyme activity, allowing for potential biomedical applications in metabolic processes.

Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems

AbstractAdenosine‐5’‐triphosphate (ATP) plays a crucial role in many biocatalytic reactions and its regeneration can influence the performance of a related enzymatic reaction significantly. Here, we established electrochemically coupled ATP regeneration by pyruvate oxidase and acetate kinase (ACK) for the phosphorylation of mevalonate catalyzed by mevalonate kinase. A yield of 84 % for the product mevalonate phosphate was reached and a total turnover number for ADP of 68. These metrics are promising for the development of an economic feasible bioprocess and surpass many other enzymatic ATP regeneration systems. A comparison was made to polyphosphate kinases (PPKs), ACK, pyruvate kinase, and creatine kinase in terms of the phosphate donor properties and biocatalytic metrics of exemplary reactions. Furthermore, our system was expanded by a PPK that enables the phosphorylation of AMP, which can broaden the spectrum of applications even further.

Comparative Evaluation of the Asymmetric Synthesis of (S)-Norlaudanosoline in a Two-Step Biocatalytic Reaction with Whole Escherichia coli Cells in Batch and Continuous Flow Catalysis

Opioids are important analgesics, and their pharmaceutical application is increasing worldwide. Many opioids are based on benzylisoquinoline alkaloids (BIA) and are still industrially produced from Papaver somniferum (opium poppy). (S)-norlaudanosoline ((S)-NLS) is a complex BIA and an advanced intermediate for diverse pharmaceuticals. The efficient synthesis of this scaffold could pave the way for a plant-independent synthesis platform. Although a promising biocatalytic route to (S)-NLS using norcoclaurine synthase (NCS) and ω-transaminase (TAm) has already been explored, the cost-effectiveness of this process still needs much improvement. Therefore, we investigated whether the synthesis could also be performed using whole cells to avoid the use of (partially) purified enzymes. With an optimized mixing ratio of TAm- and NCS-containing cells in batch biotransformations, 50 mM substrate was converted within 3 h with more than 90% yield and a high enantiomeric excess of the product (95%). To further increase the space–time yield, the cells were immobilized to enable their retainment in fixed-bed reactors. A comparison of glass beads, Diaion HP-2MG and alginate revealed that the addition of Diaion during bacterial growth led to the most active immobilisates. To facilitate sustained production of (S)-NLS, a fixed-bed setup was constructed based on lithographically printed columns from biocompatible PRO-BLK 10 plastic. The continuous production at two scales (5 mL and 50 mL columns) revealed insufficient system stability originating from biocatalyst leaching and inactivation. Thus, while the use of whole cells in batch biotransformations represents an immediate process improvement, the transfer to flow catalysis needs further optimization.

Photoelectrochemical immunoassay of carcinoembryonic antigen based on hollow In2O3/In2S3 nanocolumn

AbstractThis work presents a split‐type photoelectrochemical (PEC) immunoassay platform for ultrasensitive determination carcinoembryonic antigen (CEA) using In2O3/In2S3 nanocolumn as the photoactive matrix. Initially, In2O3/In2S3 nanocolumn with high photocurrent response was obtained by vulcanizing metal‐organic framework‐derived hollow In2O3 nanomaterials for surface modification. Subsequently, sandwich immunoreactions were conducted utilizing an alkaline phosphatase‐labeled secondary antibody that served as a signal pointer. A large amount of ascorbic acid was produced by the biocatalytic reaction, which enhanced the anodic photocurrent of the In2O3/In2S3 nanocolumn‐modified electrode. Under optimized conditions, the In2O3/In2S3 nanocolumn‐based PEC immunoassay exhibited a promising photocurrent response to the target CEA in the operational scope of 0.02 ng/mL to 10 ng/mL, with a limit of detection of 8.4 pg/mL. Benefiting from the excellent photoelectrochemical response of In2O3/In2S3 nanocolumn to ascorbic acid and the separated detection operation, the constructed PEC immunosensing platform has the advantages of great precision and strong anti‐jamming capability, and the detection results are comparable to those of commercial ELISA kits. Collectively, this study not only offered an efficacious semiconducting surface modification strategy to enhance photocurrent response activity, but also offered a novel thought for the sensitive measurement of a marker of cancer.

Immobilization protects enzymes from plasma-mediated inactivation.

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications.

Impact of Deep Eutectic Solvents on Kinetics and Folding Stability of Formate Dehydrogenase

Specifically designed co-solvent mixtures are an efficient way to enhance the kinetics of enzyme-catalyzed reactions without compromising enzyme stability; among them, several deep eutectic solvents have emerged as exciting co-solvent mixtures for biocatalytic reactions. DESs nature allows one to tailor the enzyme-co-solvent interactions by using DESs constituents of diverse functional groups. In this work, the influence of co-solvents (betaine, glycerol, and sorbitol) and two DESs (betaine:glycerol and betaine:sorbitol) on the kinetics of candida boidinii Formate dehydrogenase was evaluated. The results showed a 30% increase in catalytic efficiency by adding 15 wt.-% of betaine to the buffered aqueous reaction media. Further, cbFDH folded-state stability was evaluated using differential scanning fluorimetry to finally obtain the binding affinity, unfolding curves, and thermodynamic unfolding parameters. The addition of glycerol, sorbitol, and DESs increased cbFDH protection against thermal stress, and this effect could be improved by increasing co-solvent concentrations. Moreover, DESs showed the ability to reduce the irreversibility of the unfolding process. Betaine was the only co-solvent that had a negative stability effect, which was offset by using betaine-based DESs. The latter was a result of the additivity of certain individual co-solvent effects on thermal stability. Non-monotonous stability effects were obtained by adding sorbitol to the buffer solutions, probably because hydrogen bond dynamics between cbFDH/co-solvent/water change dramatically with the amount of water present. Finally, DESs improved NAD+ binding affinity with cbFDH interestingly without direct correlation with the results obtained for kinetics.

A comprehensive analysis of key factors influencing methane production from CO2 using microbial methanogenesis cells

With increasing attention on carbon capture and utilization (CCU) technologies for the conversion of CO2 into chemical products, microbial methanogenesis cells (MMCs) have been extensively studied over the past few decades for biomethane production. Using rapidly accumulating data for MMCs with varying configurations and operating conditions, a comprehensive analysis was conducted here to investigate the critical factors that influence methane production rates (MPR) in these systems. A comparison of MPR and set potentials or current densities showed weak linear relationships (R2 < 0.6, p < 0.05), indicating the significant contributions of other important factors impacting methane production. A non-quantitative analysis of these additional parameters indicated the potential importance of using metal catalysts for anode materials where oxygen evolution reaction occurs, while most previous MMC research focused more on cathode materials where the biocatalytic reaction occurs. The use of undefined mixed anaerobic cultures as inocula was found to be sufficient for producing high MPRs, as the electrochemical environment at the cathode provides a strong selective pressure to converge on desirable methanogenic cultures. Other operational parameters, such as catholyte pH control and CO2 supply methods, were also important factors impacting MPR in MMCs, indicating the cumulative impact of these various factors will require careful consideration in future research.

Multienzymatic Synthesis of γ‐Lactam Building Blocks from Unsaturated Esters and Hydroxylamine

AbstractThe assembly of enzymatic cascades and multi‐step reaction sequences represents an attractive alternative to traditional synthetic‐organic approaches. The biocatalytic reaction mediators offer not only mild conditions and permit the use of environmentally benign reagents, but the high compatibility of different enzymes promises more streamlined reaction setups. In this study, a triple‐enzymatic strategy was developed that enables the direct conversion of γ,δ‐unsaturated esters to N‐hydroxy‐γ‐lactam building blocks. Hereby, a lipase‐catalyzed hydroxylaminolysis generates hydroxamic acid intermediates that are subsequently aerobically activated by horseradish peroxidase and glucose oxidase to cyclize in an intramolecular nitroso ene reaction. Utilizing the hydroxylaminolysis/ene‐cyclization sequence for the preparation of an aza‐spirocyclic lactam, the multi‐enzymatic methodology was successfully employed in the synthesis of key intermediates en route to alkaloids of the Cephalotaxus family.

Engineered Biocatalytic Synthesis of β-N-Substituted-α-Amino Acids.

Non-canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids. In this study, we used directed evolution to engineer the β-subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re-entry into the catalytic cycle and subsequent decomposition. Additional equivalents of l-serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β-substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

Chemo-enzymatic Synthesis of Rivastigmine Intermediate by Locally Isolated Soil Fungus Fusarium graminearum

Background: Biocatalytic reactions are known as a green technology, capable of delivering highly stereo-, chemo- and regioselective transformations that usually reduce the number of steps in a synthetic route Objective: This work describes a process for the production of a key chiral intermediate N-ethyl-Nmethyl-carbamic acid-3-(1S-hydroxy-ethyl)-phenyl ester converted into chiral alcohol which can be further converted into rivastigmine Methods: The fungi isolated from different soil samples by the soil dilution plate technique were screened for their ability to convert the selected intermediate. The selected organism is further grown under fermentation for gram scale conversion. Results: Among the different fungi isolated Fusarium graminearum selected for further study for reaction optimization and gram scale conversion of the ketone substrate Conclusion: The reaction under fermentative condition for 48 hours at 30°C, pH 6 with agitation speed of 200rpm found to be optimal condition for the overall bioreduction process of the ketone employed in the study.

Elucidating the Self-cleavage Dynamics of Hairpin Ribozyme by Mode-decomposed Infrared Spectroscopy.

While catalytic reactions of biomolecular processes play an indispensable role in life, extracting the underlying molecular picture often remains challenging. Based on ab initio simulations of the self-cleavage reaction of hairpin ribozyme, mode-decomposed infrared spectra, and cosine similarity analysis to correlate the product with reactant IR spectra, we demonstrate a strategy to extract molecular details from characteristic spectral changes. Our results are in almost quantitative agreement with the experimental IR band library of nucleic acids and suggest that the spectral range of 800-1200 cm-1 is particularly valuable to monitor self-cleavage. Importantly, the cosine similarities also disclose that IR peaks subject to slight shifts due to self-cleavage might be unrelated, while strongly shifting resonances can correspond to the same structural dynamics. This framework of correlating complex IR spectra at the molecular level along biocatalytic reaction pathways is broadly applicable.

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

A synthetic cell-free 36-enzyme reaction system for vitamin B12 production

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

RetroBioCat Database: A Platform for Collaborative Curation and Automated Meta-Analysis of Biocatalysis Data.

Despite the increasing use of biocatalysis for organic synthesis, there are currently no databases that adequately capture synthetic biotransformations. The lack of a biocatalysis database prevents accelerating biocatalyst characterization efforts from being leveraged to quickly identify candidate enzymes for reactions or cascades, slowing their development. The RetroBioCat Database (available at retrobiocat.com) addresses this gap by capturing information on synthetic biotransformations and providing an analysis platform that allows biocatalysis data to be searched and explored through a range of highly interactive data visualization tools. This database makes it simple to explore available enzymes, their substrate scopes, and how characterized enzymes are related to each other and the wider sequence space. Data entry is facilitated through an openly accessible curation platform, featuring automated tools to accelerate the process. The RetroBioCat Database democratizes biocatalysis knowledge and has the potential to accelerate biocatalytic reaction development, making it a valuable resource for the community.

Insights into Substrate Recognition by the Unusual Nitrating Enzyme RufO.

Nitration reactions are crucial for many industrial syntheses; however, current protocols lack site specificity and employ hazardous chemicals. The noncanonical cytochrome P450 enzymes RufO and TxtE catalyze the only known direct aromatic nitration reactions in nature, making them attractive model systems for the development of analogous biocatalytic and/or biomimetic reactions that proceed under mild conditions. While the associated mechanism has been well-characterized in TxtE, much less is known about RufO. Herein we present the first structure of RufO alongside a series of computational and biochemical studies investigating its unusual reactivity. We demonstrate that free l-tyrosine is not readily accepted as a substrate despite previous reports to the contrary. Instead, we propose that RufO natively modifies l-tyrosine tethered to the peptidyl carrier protein of a nonribosomal peptide synthetase encoded by the same biosynthetic gene cluster and present both docking and molecular dynamics simulations consistent with this hypothesis. Our results expand the scope of direct enzymatic nitration reactions and provide the first evidence for such a modification of a peptide synthetase-bound substrate. Both of these insights may aid in the downstream development of biocatalytic approaches to synthesize rufomycin analogues and related drug candidates.