Enzymatic Cascade Reaction Research Articles

Gas-shearing synthesis of core–shell multicompartmental microparticles as cell-like system for enzymatic cascade reaction

Core-shell multicompartmental microparticles have gained increasing attention as their architecture resembles the multicompartmental composition of eukaryotic cells. However, challenges remain in the fabrication of biocompatible multicompartmental microparticles at a sufficiently high scale. Herein, all-aqueous ‘mangosteen-like’ core–shell multicompartmental microparticles were created by an oil-free gas-shearing fabrication approach. Both the shell thickness and core size of thus obtained mangosteen-like microparticles can be easily finetuned. Mangosteen-like particles have well segregated (up to eight) compartments which are attractive to precisely control enzymatic cascade reactions. To illustrate the application potential, insulin releasing mangosteen-like microparticles (‘artificial pancreatic beta cells’) were developed.

Immunoglobulin E Detection Method Based on Cascade Enzymatic Reaction Utilizing Portable Personal Glucose Meter.

We herein describe a cascade enzymatic reaction (CER)-based IgE detection method utilizing a personal glucose meter (PGM), which relies on alkaline phosphatase (ALP) activity that regulates the amount of adenosine triphosphate (ATP). The amount of sandwich assay complex is determined according to the presence or absence of the target IgE. Additionally, the ALP in the sandwich assay catalyzes the dephosphorylation of ATP, a substrate of CER, which results in the changes in glucose level. By employing this principle, IgE was reliably detected at a concentration as low as ca. 29.6 ng/mL with high specificity toward various proteins. Importantly, the limit of detection (LOD) of this portable PGM-based approach was comparable to currently commercialized ELISA kit without expensive and bulky analysis equipment as well as complexed washing step. Finally, the diagnostic capability of this method was also successfully verified by reliably detecting IgE present in a real human serum sample with an excellent recovery ratio within 100 ± 6%.

Cobalt-doped MoS2 nanocomposite with NADH oxidase mimetic activity and its application in colorimetric biosensing of NADH

NADH oxidase (NOX) is an oxidoreductase that catalyzes the oxidation of NADH to NAD+ with the participation of oxygen. NOX can artificially regulate metabolic processes as an important regulatory enzyme for NAD+ regeneration. In this study, cobalt-doped molybdenum sulfide (Co-MoS2) nanocomposite was prepared by hydrothermal synthesis, which exhibited NOX mimics. The catalytic mechanism of NOX mimics was studied. The NOX-like activity of Co-MoS2 is optimal at pH 4.5 and in the temperature range of 20–45℃. Further, its catalytic activity maintained almost the same after 5 cycles of repetitive addition of NADH, suggesting that Co-MoS2 nanozyme possessed significant catalytic persistence. The produced H2O2 catalyzed by Co-MoS2 can be detected by horseradish peroxidase (HRP) and 3,3′,5,5′-tetramethylbenzidine at pH4.5. Thus, interestingly, a colorimetric NADH sensor was proposed on the basis of the enzymatic cascade reaction of NADH oxidase-like conjugated with HRP. This as-established method had wide detection range (0.5–800 μM) with the detection limit of 0.25 μM NADH, capable of detecting NADH in real sample.

Magnetic Switchable Pickering Interfacial Biocatalysis: One-Pot Cascade Synthesis of Phytosterol Esters from High-Acid Value Oil

The traditional reutilization of high-acid value oil resources causes pollution, suffers from high energy consumption, and brings relatively low economic value. In this study, high-acid value oil was converted into high-value product phytosterol esters (PEs) using a “one-pot” enzymatic cascade reaction strategy including the hydrolysis of high-acid value oils into free fatty acids (FFAs) and followed by its esterification of phytosterols. Lipase AYS was immobilized on magnetic hollow mesoporous carbon nanospheres to obtain AYS@MHMCS, forming a sustainable and solvent-free Pickering interfacial biocatalysis platform. Each droplet in the emulsion system served as a microreactor with the reactants in the oil phase; the byproduct glycerol was spontaneously excreted to the aqueous phase which would “push” the hydrolysis reaction forward. The products of hydrolysis (fatty acids) remained in the oil phase, continuing the next esterification reaction with phytosterols, which ensured the efficient and continuous operation of the tandem reaction. Benefiting from the interfacial activation effect of lipase, the catalytic efficiency of the emulsion system was 30 times that of the single-phase system. The yield of PEs was up to 90%, while the highest in the single-phase was only 30%. Additionally, the magnetic-responsive Pickering emulsion could recover AYS@MHMCS intactly in 15 min, and there would be no significant loss of catalytic activity even after continuous reuse for 15 cycles. This method not only provides a robust biocatalysis technique with a sustainable interface but also paves the way to high-value utilization of high-acid value oil resources.

A portable and quantitative detection of microRNA-21 based on cascade enzymatic reactions with dual signal outputs

MicroRNAs (miRNAs) are physiological status-related molecules which can be used as biomarkers for diseases, such as cancers. The point-of-care testing (POCT) of miRNAs has great application potential in early diagnosis and process monitoring of diseases. In this paper, a fast and dual signal outputs detection for microRNA-21 (miRNA-21) was established by using both personal glucose meter (PGM) and fluorescence spectrometer. In such an assay protocol, a dual-functional hairpin structure was rationally designed to recognize miRNA-21 and serve as the carrier of the reporter adenosine monophosphate (AMP). The hairpin structure can be specifically degraded by exonuclease T (Exo T) after hybridization with the target miRNA-21, releasing a large amount of AMP as the reporter. Then a smart signal conversion machinery composed of four enzymes and the corresponding substrates was employed to produce dual output signals through enzymatic cascade reactions. The machinery includes two parts: an adenosine triphosphate (ATP) generation system and a glucose consumption/NADPH production system. The produced AMP in the former step triggers the production of ATP, and subsequently the consumption of glucose and the production of NADPH. The changes of both glucose and NADPH are proportional to the concentration of miRNA-21, and can be determined by PGM and fluorescence spectrometer, respectively. Besides, the build-in substrate-recycling mechanism achieves signal amplification of the cascade enzymatic reactions. Under the optimal experimental conditions, the PGM signal is linearly correlated with the concentration of miRNA-21 in the range from 5 to 150 nM, with the limit of detection (LOD) of 3.65 nM. The LOD of fluorescence detection mode is even lowered to 0.03 nM. The miRNA-21-spiked serum samples, as well as the actual serum samples from cancer patients, have been successfully detected by this detection strategy. Thus the established assay provides a POCT solution for cancer diagnosis and prognosis.

Inhibitory Effect of Lentivirus-Mediated Gag-Caspase-8 on the Growth of HER-2 Overexpressing Primary Human Breast Cancer Cells.

Background: Apoptosis plays an essential role in the development and treatment of tumors, and caspase-8 (CASP8) plays an important role in the enzyme cascade reaction that leads to apoptosis. Human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer is highly aggressive and has a high recurrence rate and poor prognosis. This study investigated whether lentivirus-mediated Gag-CASP8 can effectively deliver activated CASP8 into primary human breast cancer cells overexpressing HER-2 to induce apoptosis and explore the underlying mechanism. Materials and Methods: HER-2 overexpressing primary human breast cancer cells were infected with lentivirus-like particles carrying Gag-CASP8. Results: After a 48h infection of primary human breast cancer cells with HER-2 by lentivirus-mediated Gag-CASP8, significant differences were observed in the survival rate, migration ability, S-phase number of cells, apoptosis rate, and intracellular activated CASP8 and caspase-3 levels in tumor cells compared with those in the control group (p < 0.05). Conclusions: Lentivirus-mediated Gag-CASP8 can deliver activated CASP8 into HER-2 overexpressing primary human breast cancer cells and induce apoptosis by activating caspase-3, a downstream apoptotic executive molecule. By blocking the S-phase to inhibit cell proliferation and migration, lentivirus-mediated Gag-CASP8 provides a reference for tumor gene therapy.

Functional Expression of Two Unusual Acidic Peroxygenases from Candolleomyces aberdarensis in Yeasts by Adopting Evolved Secretion Mutations.

Fungal unspecific peroxygenases (UPOs) are emergent biocatalysts that perform highly selective C-H oxyfunctionalizations of organic compounds, yet their heterologous production at high levels is required for their practical use in synthetic chemistry. Here, we achieved functional expression of two new unusual acidic peroxygenases from Candolleomyces (Psathyrella) aberdarensis (PabUPO) in yeasts and their production at a large scale in a bioreactor. Our strategy was based on adopting secretion mutations from an Agrocybe aegerita UPO mutant, the PaDa-I variant, designed by directed evolution for functional expression in yeast, which belongs to the same phylogenetic family as PabUPOs, long-type UPOs, and shares 65% sequence identity. After replacing the native signal peptides with the evolved leader sequence from PaDa-I, we constructed and screened site-directed recombination mutant libraries, yielding two recombinant PabUPOs with expression levels of 5.4 and 14.1 mg/liter in Saccharomyces cerevisiae. These variants were subsequently transferred to Pichia pastoris for overproduction in a fed-batch bioreactor, boosting expression levels up to 290 mg/liter, with the highest volumetric activity achieved to date for a recombinant peroxygenase (60,000 U/liter, with veratryl alcohol as the substrate). With a broad pH activity profile, ranging from pH 2.0 to 9.0, these highly secreted, active, and stable peroxygenases are promising tools for future engineering endeavors as well as for their direct application in different industrial and environmental settings. IMPORTANCE In this work, we incorporated several secretion mutations from an evolved fungal peroxygenase to enhance the production of active and stable forms of two unusual acidic peroxygenases. The tandem-yeast expression system based on S. cerevisiae for directed evolution and P. pastoris for overproduction on an ∼300-mg/liter scale is a versatile tool to generate UPO variants. By employing this approach, we foresee that acidic UPO variants will be more readily engineered in the near future and adapted to practical enzyme cascade reactions that can be performed over a broad pH range to oxyfunctionalize a variety of organic compounds.

A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1.

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

High-performance cascade nanoreactor based on halloysite nanotubes-integrated enzyme-nanozyme microsystem

Various enzymatic reactions or enzymatic cascade reactions occur efficiently in biological microsystems due to space constraints or orderly transfer of intermediate products. Inspired by this, the horseradish peroxidase (HRP)-like nanozyme (Fe-aminoclay) was in situ synthesized on the surface of alkali-activated halloysite nanotubes and the natural enzyme (glucose oxidase, GOx) was immobilized on it to construct a high-efficiency GOx-FeAC@AHNTs cascade nanoreactor. In which, FeAC@AHNTs can not only be used as a carrier for immobilized enzymes, but also help its catalytic activity to cooperate with glucose oxidase in a cascade reaction. The microcompartments and substrate channel effect of this enzyme-nanozyme microsystem exhibit a superior catalytic performance than that of natural enzyme system, and exhibits excellent long-term stability and recyclability. Subsequently, the GOx-FeAC@AHNTs cascade nanoreactor was employed as a glucose colorimetric platform, which displayed a low detection limit (0.47 µmol/L) in glucose detection. This enzyme-nanoenzyme nanoreactor provides a simple and effective example for constructing a multi-enzyme system with limited space, and lays the foundation for subsequent research in the fields of biological analysis and catalysis.

Conjugated Polymer-Ferrocence Nanoparticle as an NIR-II Light Powered Nanoamplifier to Enhance Chemodynamic Therapy.

Chemodynamic therapy (CDT) is a promising therapeutic modality with transition metal ions and endogenous H2O2 as reagents, but its efficiency is impaired by low endogenous H2O2 levels and nonregeneration of metal ions. Most intracellular H2O2 supplement strategies use oxidases and are intensively dependent on oxygen participation. The hypoxia microenvironments of solid tumors weaken their performance. Here, we develop a near-infrared II light powered nanoamplifier to improve the local oxygen level and to enhance CDT. The nanoamplifier CPNP-Fc/Pt consists of ferrocene (Fc)- and cisplatin prodrug (Pt(IV))-modified conjugated polymer nanoparticles (CPNPs). CPNP has a donor-acceptor structure and demonstrates a good photothermal effect under 1064 nm light irradiation, which accelerates blood flow and efficiently elevates the local oxygen content. In response to intracellular glutathione, Pt(II) is released from CPNP-Fc/Pt and triggers enzymatic cascade reactions with nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and superoxide dismutase to convert oxygen into H2O2. The enhanced oxygen level results in efficient intracellular H2O2 supply. Fc is reacted with H2O2 and converted to Fc+ via the Fenton reaction, with the generation of hydroxyl radicals for CDT. Unlike free metal ions, the Fe(III) in Fc+ is reduced to Fe(II) by intracellular NAD(P)H, which achieves the regeneration of Fc. The sufficient intracellular H2O2 supply and efficient Fc regeneration effectively enhance the Fenton reaction and demonstrate good in vivo CDT results with tumor growth suppression. This design offers a promising strategy to enhance CDT efficiency in the hypoxia microenvironment of solid tumors.

Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

AbstractWithin the cellular microenvironment of organisms, the surface‐ and volume‐ enclosed multiple enzymes perform highly complex series of chemical reactions for very selective and efficient synthesis of biomolecules. Several efforts have been carried out to mimic these timely and spatially defined natural enzyme cascade reactions onto the artificial multienzyme nano‐assemblies via immobilization of two or more enzymes on nanosupport, while maintaining the catalytic activity. These biocatalytic systems undergo cascade reactions for the efficient production of chemicals and materials of interest. Their increasing interest is based on their numerous potential applications in the area of environmental, biomedical and applied chemistry. A variety of nanosupports and multienzyme immobilization techniques have been optimized and reported. Nanoreactors usually show molecular crowding and anomalous diffusion of substances that modify the mass action kinetic laws. Thus, the kinetics is different than those shown by free diluted enzymes. Consequently, the catalytic activity considerations in cascade reactions on nanoassemblies are of great importance. Thus, in this work, important parameters of enzyme kinetics in cascade reaction and the molecular tools for the design and optimization of bi‐ and multi‐enzymatic nanobioreactors reactions are discussed.

Multifunctional lanthanide metal-organic framework based ratiometric fluorescence visual detection platform for alkaline phosphatase activity.

A turn-on/off ratiometric fluorescence detection platform based on multifunctional lanthanide metal-organic framework (Ln-MOF) and an enzymatic cascade reaction is proposed for alkaline phosphatase (ALP) activity assay. L-phosphotyrosine is hydrolyzed to levodopa (L-dopa) by two steps of enzymatic reaction. L-dopa further reacts with naphthoresorcinol to produce carboxyazamonardine with strong emission at 490 nm. In this process, multifunctional Ln-MOF (Cu@Eu-BTC, BTC is the 1,3,5-benzenetricarboxylic acid) acts not only as a nanozyme to catalyze the fluorogenic reaction between L-dopa and naphthoresorcinol but also as a fluorescence internal standard. The emission of Cu@Eu-BTC at 620 nm is quenched by phosphate anions, and the dual-response ratiometric fluorescence (F490/F620) can be achieved. A good linear relationship was obtained between Δ(F490/F620) and ALP activity in the range 0.3-24 U L-1 with the detection limit of 0.02 U L-1. In addition,a portable assay tube was designed for visual and point-of-care testing of ALP activity by color variation (ratiometric chromaticity). Both the ratiometric fluorescence detection and the visual detection methods were successfully applied to monitor ALP activity in human serum samples with recovery between 95.5%-109.0% and 94.0%-110.1%, and relative standard deviation less than 8.1% and 9.5%, respectively. As far as we know, this is the first report of ALP activity assay assisted by multifunctional Ln-MOF.Graphical abstract.

Biocatalytic micromixer coated with enzyme-MOF thin film for CO2 conversion to formic acid

In this study, a novel micromixer with a 3D helical, threaded channel was fabricated via 3D printing. The micromixer can enhance the mass transfer of reactants and product in an enzymatic cascade reaction converting CO2 to formic acid. Two enzymes, including carbonic anhydrase (CA) and formate dehydrogenase (FDH), were biomineralised in a zeolitic imidazolate framework-8 composite thin film on the micromixer channel that has been modified with polydopamine/polyethyleneimine. The biocatalytic performance of the micromixer was evaluated by testing at various liquid flow rates, and an optimum liquid flow rate at 1 mL/min (Rel = 8, Del = 3) was observed as the two-phase flow pattern in the micromixer channel transitioned from slug flow to bubbly flow. A comparison of the micromixer performance with and without threaded channels revealed ~ 170% enhancement in formic acid yield, indicating improved mixing with the presence of threads. In addition, the formic acid production rate for the micromixer with threaded channel was three folds higher than a conventional bubble column, demonstrating the superior performance of the proposed micromixer. The ease of assembling multiple micromixer units in series also enabled the immobilisation of different enzymes in separate units to carry out sequential reactions in a modular system. As a proof of concept, the solution product collected from long term biocatalysis was also tested in a direct formic acid fuel cell, which showed a promising prospect of integrating these two systems for a closed-loop energy generation system.

Copper sulfide nanoclusters with multi-enzyme-like activities and its application in acid phosphatase sensing based on enzymatic cascade reaction

Nanozymes are artificial enzymes, which can substitute natural enzymes for wide range of catalysis-based applications. However, it is challenging to explore novel mimic enzymes or multi-enzyme mimics. Herein we report the facile preparation of uniform CuS nanoclusters (NCs), which possessed outstanding tetra-enzyme mimetic catalytic activities, including peroxidase (POD)-mimics, catalase (CAT)-mimics, ascorbic acid oxidase (AAO)-mimics and superoxide dismutase (SOD)-mimics. The catalytic mechanism of POD-like was coming from the oxygen vacancies of CuS. Furthermore, the steady-state kinetics of POD-, CAT- and AAO mimics of CuS NCs were systematically explored. On basis of the enzymatic cascade reaction that acid phosphatase (ACP) involved in a weak acidic environment, in the presence of O-phenylenediamine, quinoxaline fluorescent substance will be generated. Thus, a fluorescent biosensor platform was proposed for detection of ACP with the linear range of 0.05–25 U L−1 and limit of detection of 0.01 U L−1. The as-proposed method was applicable to real serum sample detection accurately and reproducibly. Considering the simple preparation, good stability, excellent multi-enzyme activities and controllable experimental operation, CuS NCs would provide a basis for expanding to other biocatalytic and biomedical applications.

Single-cell profiling of D-2-hydroxyglutarate using surface-immobilized resazurin analogs

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.

Enzymatic Cascade Reactions Mediated by Highly Efficient Biomimetic Quasi Metal-Organic Frameworks.

The integration of chemo- and enzymatic catalysis for effective multistep cascades has presented critical challenges for decades. In this work, the biomimetic quasi NH2-MIL-101 (qNM) with highly efficient peroxidase-like activity was synthesized via a palmitic acid-induced strategy followed by pyrolysis. The effects of the amount of palmitic acid and calcination temperature on the synthesis of qNM were optimized. It was found that qNM was an excellent catalyst for oxidations of various peroxidase substrates, and a possible mechanism was proposed, i.e., the presence of FeII species in qNM was responsible for its excellent activity, which facilitated the transition between FeII and FeIII species to produce more hydroxyl radicals by H2O2 decomposition. The qNM served as the potential matrix for enzyme immobilization through a cross-linking method, and kinetic studies revealed that the catalytic efficiency (kcat/Km) for the immobilized GOx (23.7 mM-1 s-1) is comparable to that of free GOx (26.9 mM-1 s-1). The immobilized GOx also showed improved stability against high temperatures and organic solvents compared to free GOx, and analysis of the secondary structure of GOx indicated that the improved stability resulted from enzyme rigidity by the intense covalent linkage with qNM. Furthermore, qNM contributed its biomimetic activity to cooperate with a single enzyme (GOx) or two enzymes (β-Gal and GOx) for the enzymatic cascade reactions. Compared with the mixture of each component in the solution, the combination of the single-enzyme system (GOx) or the two-enzyme system (β-Gal and GOx) in qNM achieved 2.67-fold and 1.83-fold enhancements in the activity of catalytic cascades, respectively. This study provides new insights into the construction of effective and synergistic cascade reactions by integrating biomimetic MOF with natural enzyme, which holds potential for applications in biotechnology and ecofriendly and biomimetic catalysis.

Application of an enzymatic cascade reaction for the synthesis of the emeraldine salt form of polyaniline

The synthesis of the emeraldine salt form of polyaniline (PANI-ES) from aniline with Aspergillus sp. glucose oxidase (GOD), d-glucose, dissolved O2, and horseradish peroxidase isoenzyme C (HRPC) in the presence of large unilamellar vesicles of AOT (sodium bis-(2-ethylhexyl)sulfosuccinate) as templates at pH = 4.3 and T ~ 25 °C was investigated in a systematic way. In this cascade reaction mixture, the oxidation of aniline is catalyzed by HRPC with H2O2 that is formed in situ as byproduct of the GOD-catalyzed oxidation of d-glucose with O2. Under the elaborated experimental conditions which we considered ideal, the formation of PANI-ES products is evident, as judged by UV/Vis/NIR and EPR measurements. Comparison was made with a reference reaction, which was run under similar conditions with added H2O2 instead of GOD and d-glucose. Although the reference reaction was found to be superior, with the cascade reaction, PANI-ES products can still be obtained with high aniline conversion (> 90%) within 24 h as stable dark green PANI-ES/AOT vesicle dispersion. Our results show that the in situ formation of H2O2 does not prevent the inactivation of HRPC known to occur in the reference reaction. Moreover, the GOD used in the cascade reaction is inactivated as well by polymerization intermediates.

Self-propelled nanomotor reconstructs tumor microenvironment through synergistic hypoxia alleviation and glycolysis inhibition for promoted anti-metastasis

Solid tumors always exhibit local hypoxia, resulting in the high metastasis and inertness to chemotherapy. Reconstruction of hypoxic tumor microenvironment (TME) is considered a potential therapy compared to directly killing tumor cells. However, the insufficient oxygen delivery to deep tumor and the confronting “Warburg effect” compromise the efficacy of hypoxia alleviation. Herein, we construct a cascade enzyme-powered nanomotor (NM-si), which can simultaneously provide sufficient oxygen in deep tumor and inhibit the aerobic glycolysis to potentiate anti-metastasis in chemotherapy. Catalase (Cat) and glucose oxidase (GOx) are co-adsorbed on our previously reported CAuNCs@HA to form self-propelled nanomotor (NM), with hexokinase-2 (HK-2) siRNA further condensed (NM-si). The persistent production of oxygen bubbles from the cascade enzymatic reaction propels NM-si to move forward autonomously and in a controllable direction along H2O2 gradient towards deep tumor, with hypoxia successfully alleviated in the meantime. The autonomous movement also facilitates NM-si with lysosome escaping for efficient HK-2 knockdown to inhibit glycolysis. In vivo results demonstrated a promising anti-metastasis effect of commercially available albumin-bound paclitaxel (PTX@HSA) after pre-treated with NM-si for TME reconstruction. This cascade enzyme-powered nanomotor provides a potential prospect in reversing the hypoxic TME and metabolic pathway for reinforced anti-metastasis of chemotherapy.

Importance the Type 1 (CR1) and Type 3 (CR3) Complement Receptors in the Infectious Disease

The complement system is one of the host’s primary defence mechanisms against pathogens. Its activation involves proteolytic cascades of enzymatic reactions that result in products with effector functions and recognition of molecules on the surface of microorganisms [1,2].

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

Significant advances in enzyme discovery, protein and reaction engineering have transformed biocatalysis into a viable technology for the industrial scale manufacturing of chemicals. Multi-enzyme catalysis has emerged as a new frontier for the synthesis of complex chemicals. However, the in vitro operation of multiple enzymes simultaneously in one vessel poses challenges that require new strategies for increasing the operational performance of enzymatic cascade reactions. Chief among those strategies is enzyme co-immobilization. This review will explore how advances in synthetic biology and protein engineering have led to bioinspired co-localization strategies for the scaffolding and compartmentalization of enzymes. Emphasis will be placed on genetically encoded co-localization mechanisms as platforms for future autonomously self-organizing biocatalytic systems. Such genetically programmable systems could be produced by cell factories or emerging cell-free systems. Challenges and opportunities towards self-assembling, multifunctional biocatalytic materials will be discussed.