Enzymatic Cascade Research Articles

Chemo-Enzymatic Depolymerization of Functionalized Low-Molecular-Weight Polyethylene.

Polyethylene (PE) is the most commonly used plastic type in the world, contributing significantly to the plastic waste crisis. Microbial degradation of PE in natural environments is unlikely due to its inert saturated carbon-carbon backbones, which are difficult to break down by enzymes, challenging the development of a biocatalytic recycling method for PE waste. Here, we demonstrated the depolymerization of low-molecular-weight (LMW) PE using an enzyme cascade that included a catalase-peroxidase, an alcohol dehydrogenase, a Baeyer Villiger monooxygenase, and a lipase after the polymer was chemically pretreated with m-chloroperoxybenzoic acid (mCPBA) and ultrasonication. In a preparative experiment with gram-scale pretreated polymers, GC-MS and weight loss determinations confirmed ~27% polymer conversion including the formation of medium-size functionalized molecules such as ω-hydroxy acids and α,ω-carboxylic acids. Additional polymer property analyses using AFM showed that enzymatic depolymerization reduced the particle sizes of this mCPBA- and enzyme-treated LMWPE. This multi-enzyme catalytic concept with distinct chemical steps represents a unique starting point for future development of bio-based recycling methods for polyolefin waste.

Chemo‐Enzymatic Depolymerization of Functionalized Low‐Molecular‐Weight Polyethylene

Polyethylene (PE) is the most commonly used plastic type in the world, contributing significantly to the plastic waste crisis. Microbial degradation of PE in natural environments is unlikely due to its inert saturated carbon‐carbon backbones, which are difficult to break down by enzymes, challenging the development of a biocatalytic recycling method for PE waste. Here, we demonstrated the depolymerization of low‐molecular‐weight (LMW) PE using an enzyme cascade that included a catalase‐peroxidase, an alcohol dehydrogenase, a Baeyer Villiger monooxygenase, and a lipase after the polymer was chemically pretreated with m‐chloroperoxybenzoic acid (mCPBA) and ultrasonication. In a preparative experiment with gram‐scale pretreated polymers, GC‐MS and weight loss determinations confirmed ~27% polymer conversion including the formation of medium‐size functionalized molecules such as ω‐hydroxy acids and α,ω‐carboxylic acids. Additional polymer property analyses using AFM showed that enzymatic depolymerization reduced the particle sizes of this mCPBA‐ and enzyme‐treated LMWPE. This multi‐enzyme catalytic concept with distinct chemical steps represents a unique starting point for future development of bio‐based recycling methods for polyolefin waste.

Chemical tools for probing the Ub/Ubl conjugation cascades.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubl's), essential for many cellular processes, employs muti-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

Novel enzyme cascade colorimetric nanosensing platform based on 3D diatomic nanozymes synergistically enhancing peroxidase-like activity for the detection of xanthine

Abnormal xanthine (XA) levels could cause various diseases, making sensitive and reliable XA detection crucial for early diagnosis. In this study, novel enzyme cascade colorimetric nanosensing platform based on 3D PtAu/PCN/S-rGO diatomic nanozymes with peroxidase-like (POD-like) activity was designed and fabricated. The doping of S and P increased the electron transfer rate, and the synergy of 3D PCN/S-rGO carriers increased the POD-like activity of diatomic nanozymes. Additionally, an efficient enzyme cascade nanosensing platform XOD@3D PtAu/PCN/S-rGO for the colorimetric detection of XA was constructed by utilizing this nanocomposite as a carrier for immobilizing xanthine oxidase (XOD) which could effectively enhance cascade efficiency. The intermediate •OH was produced during the cascade reaction, and the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by •OH was catalyzed by the nanocomposites to form blue oxTMB, which increased the absorbance at 652 nm and thus generated a colorimetric signal for the detection of XA. The linear range of XA detection was 1.5–250 μM, and the limit of detection was 1.29 μM. This nanosensing platform exhibited significant reproducibility, specificity, and stability, making it suitable for detecting XA in real human blood samples. Therefore, this enzyme cascade colorimetric diatomic nanosensing platform has potential applications for the detection of XA and other biomarkers in actual clinical samples.

In vitro transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade.

In vitro metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such in vitro networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an in vitro transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of in vitro metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component in vitro metabolic system that converts CO2 into glycolate. As proof of concept, we constructed a novel glycolate sensor module that is based on the transcriptional repressor GlcR from Paracoccus denitrificans and established an IVT biosensing workflow that allows us to quantify glycolate from CETCH samples in the micromolar to millimolar range. We investigate the influence of 13 (shared) cofactors between the two in vitro systems to show that Mg2+, adenosine triphosphate , and other phosphorylated metabolites are critical for robust signal output. Our optimized IVT biosensor correlates well with liquid chromatography-mass spectrometry-based glycolate quantification of CETCH samples, with one or multiple components varying (linear correlation 0.94-0.98), but notably at ∼10-fold lowered cost and ∼10 times faster turnover time. Our results demonstrate the potential and challenges of IVT-based systems to quantify and prototype the activity of complex reaction cascades and in vitro metabolic networks.

Avoiding Replicates in Biocatalysis Experiments: Machine Learning for Enzyme Cascade Optimization

The optimization of enzyme cascades is a complex and resource‐demanding task due to the multitude of parameters and synergistic effects involved. Machine learning can support the identification of optimal reaction conditions, for example, in the case of Bayesian optimization (BO), by proposing new experiments based on Gaussian process regression (GPR) and expected improvement (EI). Here, we used BO to optimize the concentrations of the reaction components of an enzyme cascade. The productivity‐cost‐ratio was chosen as the optimization objective in order to achieve the highest possible productivity, which was normalized to the costs of the materials used to prevent convergence to ever‐increasing enzyme concentrations. To reduce the experimental effort, contrary to common practice in biological experiments, we did not use replicates but instead relied on the algorithm’s proposed experiments and inherent uncertainty quantification. This approach balances parameter space exploration and exploitation, which is critical for the efficient and effective identification of optimal reaction conditions. At the optimized reaction conditions identified in our study, the productivity‐cost ratio was doubled to 38.6 mmol L‐1 h‐1 €‐1 compared to a reference experiment. The parameter optimization required only 52 experiments while being robust to outlying experimental results.

Cascade Enzymes Confined in DNA Nanoanchors for Antitumor Therapy.

Cascade-enzyme reaction systems have emerged as promising tools for treating malignant tumors by efficiently converting nutrients into toxic substances. However, the challenges of poor localized retention capacity and utilization of highly active enzymes often result in extratumoral toxicity and reduced therapeutic efficacy. In this study, we introduced a cell membrane-DNA nanoanchor (DNANA) with a spatially confined cascade enzyme for in vivo tumor therapy. The DNANAs are constructed using a polyvalent cholesterol-labeled DNA triangular prism, ensuring high stability in cell membrane attachment. Glucose oxidase (GOx) and horseradish peroxidase (HRP), both modified with streptavidin, are precisely confined to biotin-labeled DNANAs. Upon intratumoral injection, DNANA enzymes efficiently colonize the tumor site through cellular membrane engineering strategies, significantly reducing off-target enzyme leakage and the associated risks of extratumoral toxicity. Furthermore, DNANA enzymes demonstrated effective cancer therapy in vitro and in vivo by depleting glucose and producing highly cytotoxic hydroxyl radicals in the vicinity of tumor cells. This membrane-engineered cascade-enzyme reaction system presents a conceptual approach to tumor treatment.

Janus Separation-Sensing Membrane Hosted with Enzyme@MOF Nanoreactor for Real-Time Blood Sensing.

Plasma separation, rich in biomarkers crucial for diagnosis, is conventionally achieved via high-speed centrifugation, a method hindered by its blood usage, lengthy processes, and complex operations, which delays detection. We introduced a novel real-time blood sensing method based on a Janus membrane and enzymes @MOFs. Asymmetric driving of the janus membrane can realize spontaneous separation of plasma and prevent hemolysis during direct separation. Glucose oxidase (GOx), uric acid oxidase (UOx) and horseradish peroxidase (HRP) were encapsulated in a hydrophilic organometallic framework (MOFs) to construct an enzyme cascade nanoreactor. Embedding enzyme in hydrophilic MOFs not only retains the natural conformation of free enzyme but also improves the brittleness of enzyme, endows MOFs with new biological functions, and expands its sensing application. Using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen and a custom app for color interpretation, we achieved real-time visualization of glucose (Glu) and uric acid (UA) at a 50 μM limit. The system accurately analyzed serum samples, matching commercial kits and showing promise for portable, personalized diagnostics.

Characterizing the nonmonotonic behavior of mutual information along biochemical reaction cascades.

Cells sense environmental signals and transmit information intracellularly through changes in the abundance of molecular components. Such molecular abundances can be measured in single cells and exhibit significant heterogeneity in clonal populations even in identical environments. Experimentally observed joint probability distributions can then be used to quantify the covariability and mutual information between molecular abundances along signaling cascades. However, because stationary state abundances along stochastic biochemical reaction cascades are not conditionally independent, their mutual information is not constrained by the data-processing inequality. Here, we report the conditions under which the mutual information between stationary state abundances increases along a cascade of biochemical reactions. This nonmonotonic behavior can be intuitively understood in terms of noise propagation and time-averaging stochastic fluctuations that are short-lived compared to an extrinsic signal. Our results reemphasize that mutual information measurements of stationary state distributions of cellular components may be of limited utility for characterizing cellular signaling processes because they do not measure information transfer.

Multifunctional Silver‐Enzyme Nanogels Assembly with Efficient Trienzyme Cascades for Synergistic Diabetic Wound Healing

AbstractDiabetic wound healing presents a persistent clinical challenge, often characterized by prolonged healing times, and can be particularly difficult to achieve in a hyperglycemic environment. In this study, a multi‐functional silver‐enzyme nanogels assembly (Ag‐nGHC) is designed by focusing on the complex diabetic wound environment. Glucose oxidase (GOX), horseradish peroxidase (HRP), and catalase (CAT) are modified within polymeric nanogel layers and assembled into a large enzyme cluster with silver ions. The close attachment of three enzymes ensures fast and continuous consumption of a high level of glucose, generation of oxygen, and hydroxyl radicals (•OH) around the wound site. Meanwhile, the silver ions within the Ag‐nGHC assembly act as an effective agent to kill bacteria. This cascade enzyme system significantly improves the microenvironment of the wound site by reducing bacterium infection and alleviating hypoxia as well as hyperglycemia. Sequentially, the improved environment facilitates the later processes including anti‐inflammatory, re‐epithelialization, and angiogenesis, evidenced by enhancing polarization toward M2 macrophages and increasing CD31 signals in this study. Overall, the Ag‐nGHC materials are proven to achieve multifunctional properties toward complicated diabetic wound healing processes (attributes such as adaptability, hypoxia‐alleviated, anti‐hyperglycemic, antimicrobial, anti‐inflammatory, and angiogenic) and showed great potential for the treatment of chronic diabetic wound.

Enzyme Cascade with Four Enzymes in One Pot for the Synthesis of L‐Phosphinothricin

AbstractThe biocatalytic oxidative deamination‐ reductive amination process for the production of L‐phosphinothricin (L‐PPT) from D,L‐phosphinothricin (D,L‐PPT) is a green and environmentally friendly approach with significant development potential. In this study, the adopted technological route involves the oxidative deamination of the D‐PPT component in D,L‐PPT to 2‐oxo‐4‐(hydroxymethylphosphinyl)butanoic acid (PPO) by utilizing D‐amino acid oxidase (DAAO), followed by the catalytic reductive amination of PPO to L‐PPT using phosphinothricin dehydrogenase (PpDH). In order to enhance the catalytic rate and reduce the inhibitory effect of the intermediate product PPO on the catalytic process, optimization of both oxidative deamination and reductive amination phases was conducted through separate strategies of constant‐pressure oxygen supply and substrate feeding, respectively. A “two‐step one‐pot” method was employed to link the optimized reactions, significantly shortening the production time. Furthermore, the “two‐step one‐pot” catalytic strategy was optimized, ultimately achieving a high 97.7% yield of L‐PPT with ee of L‐PPT>99.9% after 5.5 h of reaction under 800 mM D,L‐PPT catalytic conditions, D‐PPT and PPO could not bedetected in the reaction system, further enchanced the catalytic efficiency, production efficiency and product quality. The aforementioned results demonstrate that this proposed approach has great potential for industrial‐scale production of L‐PPT as well as significant competitive advantages in terms of economic feasibility.

Alcohol Oxidase Linked Enzyme Cascade as Anodic Biofuel Cell Catalyst

Alcohol Oxidase Linked Enzyme Cascade as Anodic Biofuel Cell Catalyst

Detecting Bacteria with Ultralow Concentrations by Enzymatic Cascade Reaction-Amplifying Strategy.

Bacteria can cause infectious diseases even at ultralow concentrations (<1 CFU/mL). It is important to rapidly identify bacterial contamination at ultralow concentrations. Herein, FITC-labeled gelatinase-sensitive nanoparticles (GNPs@FITCs) and NFM@GNP@FITCs are designed and fabricated as ultralow concentration bacteria detection platforms based on an enzymatic cascade reaction-amplifying strategy. Bacterial secretions could trigger the dissociation of GNPs@FITCs to release FITC, with gelatinase used as the model secretion. The detectable signal of ultralow concentration bacteria could be amplified effectively by the gelatinase-triggered cascade reaction. Bacterial concentration was evaluated by the change in fluorescence density. The results showed that the GNPs@FITCs and NFM@GNP@FITCs could be used for identifying bacterial contamination qualitatively, even when the bacterial contamination is lower than 1 CFU/mL. Moreover, the method has better timeliness and convenience, when compared with national standards. As solid films, NFM@GNP@FITCs have better long-term storage stability than GNPs@FITCs. The potential applications of GNPs@FITC and NFM@GNP@FITCs were proved by detecting pathogenic bacteria in food. All the results showed that the method has great potential for screening pathogenic bacterial contamination qualitatively.

Protein SUMOylation and Its Functional Role in Nuclear Receptor Control

Post-translational protein modifications (PTMs) significantly enhance the functional diversity of proteins and are therefore important for the expansion and the dynamics of the cell’s proteome. In addition to structurally simpler PTMs, substrates also undergo modification through the reversible attachment of small proteins. The best understood PTM of this nature to date is the covalent conjugation of ubiquitin and ubiquitin-like proteins (UBLs) to their substrates. The protein family of small ubiquitin-like modifier (SUMO) is one of these UBLs that has received increasing scientific attention. The pathway of SUMOylation is highly conserved in all eukaryotic cells and is crucial for their survival. It plays an essential role in many biological processes, such as the maintenance of genomic integrity, transcriptional regulation, gene expression, and the regulation of intracellular signal transduction, and thereby influences DNA damage repair, immune responses, cell cycle progression, and apoptosis. Several studies have already shown that in this context protein SUMOylation is involved in the control mechanisms of various cellular receptors. This article unites data from different studies focusing on the investigation of the strictly conserved three-step enzyme cascade of protein SUMOylation and the functional analysis of the involved proteins E1, E2, and E3 and SUMOylation target proteins. Furthermore, this review highlights the role of nuclear receptor SUMOylation and its importance for the cellular functionality and disease development arising from defects in correct protein SUMOylation.

Redesigning glycolaldehyde synthase for the synthesis of biorefinery feedstock D-xylulose from C1 compounds.

Global climate deterioration intensifies the demand for exploiting efficient CO2 utilization approaches. Converting CO2 to biorefinery feedstock affords an alternative strategy for third-generation biorefineries. However, upcycling CO2 into complex chiral carbohydrates remains a major challenge. Previous attempts at sugar synthesis from CO2 either produce mixtures with poor stereoselectivity or require ATP as a cofactor. Here, by redesigning glycolaldehyde synthase, the authors constructed a synthetic pathway for biorefinery feedstock D-xylulose from CO2 that does not require ATP as a cofactor. The artificial D-xylulose pathway only requires a three-step enzyme cascade reaction to achieve the stereoselective synthesis of D-xylulose at a concentration of 1.2gL-1. Our research opens up an alternative route toward future production of chemicals and fuels from CO2.

Taxifolin-iron nanozymes with excellent RONS scavenging ability for alleviating ethanol-induced gastric ulcer

Gastric ulcer, a chronic disease of the digestive system, presents a high incidence rate and poses significant health risks. It is closely associated with the excessive production of reactive nitrogen and oxygen species (RONS), inflammation and cell apoptosis. In this study, taxifolin (Tax)-iron nanozymes (Fe-Tax) was developed by conjugating Tax with iron ions (Fe3+), which exhibited the activities of catalase and superoxide dismutase in the gastrointestinal environment. Our results revealed that Fe-Tax nanozymes effectively scavenge RONS, mitigate oxidative damage, inflammation and cell apoptosis in vitro. Additionally, Fe-Tax could alleviate tissue inflammation and gastric mucosal damage by regulating NRF2, NF-κB, Bax/Bcl-2, and VEGF signal pathways in gastric ulcer ethanol-induced. Crucially, the Fe-Tax has been shown to have excellent biocompatibility in vitro and in vivo. Overall, this study developed Fe-Tax nanozymes with enzyme cascade reactions and provided a highly efficient strategy to prevent and alleviate gastric ulcers.

Green and Enantioselective Synthesis via Cascade Biotransformations: From Simple Racemic Substrates to High-Value Chiral Chemicals.

Asymmetric synthesis of chiral chemicals in high enantiomeric excess (ee) is pivotal to the pharmaceutical industry, but classic chemistry usually requires multi-step reactions, harsh conditions, and expensive chiral ligands, and sometimes suffers from unsatisfactory enantioselectivity. Enzymatic catalysis is a much greener and more enantioselective alternative, and cascade biotransformations with multi-step reactions can be performed in one pot to avoid costly intermediate isolation and minimise waste generation. One of the most attractive applications of enzymatic cascade transformations is to convert easily available simple racemic substrates into valuable functionalised chiral chemicals in high yields and ee. Here, we review the three general strategies to build up such cascade biotransformations, including enantioconvergent reaction, dynamic kinetic resolution, and destruction-and-reinstallation of chirality. Examples of cascade transformations using racemic substrates such as racemic epoxides, alcohols, hydroxy acids, etc. to produce the chiral amino alcohols, hydroxy acids, amines, and amino acids are given. The product concentration, ee, and yield, scalability, and substrate scope of these enzymatic cascades are critically reviewed. To further improve the efficiency and practical applicability of the cascades, enzyme engineering to enhance catalytic activities of the key enzymes using the latest microfluidics-based ultrahigh-throughput screening and artificial intelligence-guided directed evolution could be a useful approach.

Dihydro-β-ionone production by a one-pot enzymatic cascade of a short-chain dehydrogenase NaSDR and enoate reductase AaDBR1

Dihydro-β-ionone, a high-value compound with distinctive fragrance, is widely utilized in the flavor and fragrance industries. However, its low abundance in plant sources poses a significant challenge to its application through traditional extraction methods. Development of an enzyme cascade reaction with artificial design offers a promising alternative. Herein, a short-chain dehydrogenase NaSDR, was identified from Novosphingobium aromaticivorans DSM 12444, which exhibited a high activity in converting β-ionol to β-ionone. A novel biosynthesis route to produce dihydro-β-ionone from β-ionol was developed, by utilizing alcohol dehydrogenase NaSDR and enoate reductase AaDBR1. Under the optimized conditions (0.29 mg/mL NaSDR, 0.39 mg/mL AaDBR1, 1 mM NADP+ and 2.5 mM β-ionol at 40 °C for 2 h), a maximum yield (173.11 mg/L) of dihydro-β-ionone was achieved with a molar conversion rate of 35.6 %, which was 2.7-fold higher than that before optimization. Additionally, this cascade reaction achieved self-sufficient NADPH regeneration through the actions of NaSDR and AaDBR1. This study offered a fresh perspective for achieving a green and sustainable synthesis of dihydro-β-ionone and could inspire on another natural products biosynthesis.

Abstract Or109: Regulation of UFM1 modification from ER dynamics to cardiac homeostasis

Cardiovascular disease (CVD) is the leading cause of mortality and morbidity globally. Understanding the molecular mechanism governing CVD has become a major focus in developing new therapeutics. Ubiquitin (Ub) like protein post-translational modifications (PTMs) have been known to play an important role in the normal functioning of the heart. UFM1 (ubiquitin-fold modifier 1) is a novel Ub-like protein that covalently modifies protein substrates via a highly conserved E1 (UBA5)-E2 (UFC1)-E3 (UFL1) enzymatic cascade, named ufmylation. Ufmylation regulates diverse cellular processes and is implicated in various human diseases. However, its importance in the heart is unknown. For the very first time, we have reported dysregulation of ufmylation in dilated cardiomyopathy, hypertrophic human patients, and animal models. It remains unclear whether ufmylation of cardiac proteins is required for heart function and if so, how ufmylation regulates cardiac function. To determine the role of UFM1 modification in cardiomyocytes we have generated cardiac-specific UFM1-knockout (UFM1CKO) mice. The UFM1CKO mice developed dilated cardiomyopathy at resting condition, as indicated by the left ventricle (LV) wall thinning, chamber dilatation, and impaired contractility at 4 months of age, which were further exacerbated by 6 months. It appears that UFM1 modification constrains pathological cardiac remodeling by maintaining ER homeostasis and regulating ER stress response. To test the hypothesis that UFM1 modification, instead of UFM1 itself is required to maintain cardiac function, we replenished UFM1CKO mice with cardiac-specific WT or conjugation-deficient UFM1 via AAV9. By echocardiography, we revealed that, unlike WT UFM1, overexpression of the conjugation-deficient mutant failed to attenuate the cardiac remodeling and dysfunction of the UFM1CKO heart. Interestingly, the conjugation-deficient mutant further exacerbated the cardiac dysfunction of the UFM1CKO mice. Extensive fibrosis and upregulation of ER stress response protein were also noted in conjugation-deficient mutant UFM1 injected UFM1CKO heart. Overall this study indicates that modification of proteins by UFM1 is critical to cardiac function via maintaining ER homeostasis.

Conjugated microporous polymers-scaffolded enzyme cascade systems with enhanced catalytic activity

Conjugated microporous polymers-scaffolded enzyme cascade systems with enhanced catalytic activity