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

Enhancing catalytic activity of multi-enzyme in vitro through substrate channeling effect is promis-ing yet challenging.Herein,conjugated microporous polymers(CMPs)-scaffolded integrated en-zyme cascade systems(I-ECSs)are constructed through co-entrapping glucose oxidase(GOx)and horseradish peroxidase(HRP),in which hydrogen peroxide(H_(2)O_(2)) is the intermediate product.The interplay of low-resistance mass transfer pathway and appropriate pore wall-H_(2)O_(2) interactions facilitates the directed transfer of H_(2)O_(2),resulting in 2.4-fold and 5.0-fold elevation in catalytic activ-ity compared to free ECSs and separated ECSs,respectively.The substrate channeling effect could be regulated by altering the mass ratio of GOx to HRP.Besides,I-ECSs demonstrate excellent stabili-ties in harsh environments and multiple recycling.

Engineering trienzyme cascade-triggered fluorescent immunosensor platform by sequentially integrating alkaline phosphatase, tyrosinase and horseradish peroxidase

Mulit-enzyme cascades are a major type of chemical transformations and play a crucial role in biological signal transduction and metabolism. Herein, a trienzyme cascade-triggered fluorescent immunosensor platform was constructed by sequentially integrating alkaline phosphatase(ALP), tyrosinase(TYR)and horseradish peroxidase(HRP). The proposed platform was based on HRP-induced a rapid in situ fluorogenic reaction between dopamine(DA) and 1,5-dihydroxynaphthalene(DHA) to produce a strong yellow azamonardine fluorescent compound(AFC). The obtained AFC was clearly characterized by highresolution mass spectrum,1H NMR,^(13)C NMR and theoretical calculations. The integration of the twoenzyme system(TYR and HRP) or three-enzyme system(ALP, TYR and HRP) led to a maximum of 400.0-fold and 250.0-fold fluorescence enhancements, respectively. Using cardiac troponin I(c Tn I) as the model antigen, a trienzyme cascade-triggered fluorescent immunosensor platform was developed for quantitative detecting c Tn I in a wide linear range from 2 ng/m L to 150 ng/m L with a detection limit of 0.67ng/m L. In addition, the proposed platform was successfully applied in detection of c Tn I in serum of clinical patients. Overall, the developed fluorescent immunosensor performs powerful implications for researching enzyme cascade systems in the field of biomedicine.

In Vivo Activation of Pro-Protein Therapeutics via Chemically Engineered Enzyme Cascade Reaction

Selective and temporal control over protein activity is of great importance for the advancement of the protein of interest into precise molecular medicine.Simply installing synthetic ligands to proteins for activity regulation,however,is often obscured by either nonspecificity or insufficient efficiency.This study reports a chemical approach in which enzymatic cascade reactions were designed for selective activation of pro-protein both in vitro and in vivo.Specifically,the system consisted of aromatic boronic-acid-modified nanoparticles,reactive oxygen species(ROS)-responsive pro-protein(RNase A-NBC),a small molecule drug,β-Lapachone(β-Lap),and strategically screened synthetic lipids,required for the assembly of the nanocomplexes.Once target-delivered into tumor cells,the reduction ofβ-Lap produces massive H2O2 in response to NAD(P)H quinone oxidoreductase 1(NQO1),a tumor-specific enzyme,which triggers further induction by selective chemical modification of ROS-responsive cytosolic protein ribonuclease A(RNase A)-NBC,thus,switching from“inert”pro-protein to active therapeutics,that ultimately prohibit tumor cell growth.Moreover,the designed enzymatic cascade activation of the pro-protein was effective in vivo,displaying superior therapeutic efficacy to either the pro-protein alone or theβ-Lap via tumor-targeted delivery and the consequent suppression of the tumor growth.As both RNase A andβ-Lap have been evaluated clinically as antitumor therapeutics,our chemical multi-step cascade methodology is,therefore,a promising strategy for selective modulation of pro-protein chemistry in the living system for fundamental investigations,favorable toward potential anticancer applications.

Framework nucleic acid-based confined enzyme cascade for efficient synergistic cancer therapy in vivo

Artificial enzyme cascade systems with confinement effect are highly important in synthetic biology and biomedicine.Herein,a framework nucleic acid-based confined enzyme cascade(FNA-CEC)for synergistic cancer therapy in vivo was developed.The FNA-CEC consisted of glucose oxidase and horseradish peroxidase precisely assembled on an addressable DNA tetrahedron scaffold within few nanometers.Glucose oxidase(GOx)can trigger efficient glucose depletion for tumor starvation therapy,and increase the local concentration of H_(2)O_(2) in situ for enhanced downstream horseradish peroxidase(HRP)-activated prodrug therapy.Due to the spatial-confinement on DNA tetrahedron scaffold,the efficiency of intermediate metabolites transportation between the enzyme cascades was improved.Moreover,FNA-CEC was applied for efficient synergistic cancer therapy in vitro and in vivo.As a simple and efficient approach,the FNA-CEC is expected to expand the toolbox of technologies in synthetic biology and biomedicine.

Efficient synthesis of tyrosol from L-tyrosine via heterologous Ehrlich pathway in Escherichia coli

For the efficient conversion of L-tyrosine(L-Tyr)to tyrosol,which is an aromatic compound widely used in the pharmaceutical and chemical industries,a novel four-enzyme cascade pathway based on the Ehrlich pathway of Saccharomyces cerevisiae was designed and reconstructed in Escherichia coli.Then,the expression levels of the relevant enzymes were coordinated using a modular approach and gene duplication after the identification of the pyruvate decarboxylase from Candida tropicalis(CtPDC)as the rate-limiting enzymatic step.In situ product removal(ISPR)strategy with XAD4 resins was explored to avoid product inhibition and further improve tyrosol yield.As a result,the titer and conversion rate of tyrosol obtained were 35.7 g·L^(-1) and 93.6%,respectively,in a 3-L bioreactor.Results presented here provide a potential enzymatic process for industrial production of tyrosol from cheap amino acids.

Biocatalytic bis-C-alkylation of phenolics using one-pot cascades with promiscuous C-glycosyltransferase and prenyltransferase

C-glycosylation and C-prenylation are two important C-C-bond forming reactions for preparation,diversification and structural modification of natural/unnatural products with pharmacological activities.Here,we described unprecedented enzymatic cascades to C-glycosylate/prenylate different acyl resorcinol derivatives in stepwise,one-pot reactions by combining two promiscuous enzymes,MiCGT,a C-glycosyltransferase,and AtaPT,a prenyltransferase.Five novel bis-C-alkylated products were obtained and structurally identified by MS and NMR spectroscopic data as well as comparison with the literature.This study provided a potential synthetic strategy for synthesizing structurally novel and diverse compounds bearing both C-glycosyl and C-prenyl moieties by a two-step,enzymatic bis-C-alkylation.

Dynamic assembly and biocatalysis-selected gelation endow self-compartmentalized multienzyme superactivity

Cellular metabolism in multiple organelles utilizes compartmentalized multienzyme efficient catalysis to realize substance metabolism, energy conversion and immune defenses. The convenient and biomimetic design of artificial multienzymes has become an emerging research topic. Herein, we employ a facile enzyme-initiated radical polymerization to self-anchor multienzyme in cell-like hydrogels with mesoscale compartments. The dynamic assembly of glucose oxidase/cytochrome c(GOx/Cyt c) with methacrylate-modified hyaluronic acid can form nanoaggregates, where only the bound enzyme pairs with the adjacent position can catalyze the polymerization to compartmentalize multienzymes within hydrogel. Consequently, the cascade enzymes within hydrogel display 33.9 times higher activity compared to free enzymes, as well as excellent thermostability and multiple recyclability. The mechanism study indicates that the compartmental effect of the hydrogel and the anchoring effect of Cyt c synergistically enhance GOx/Cyt c activity. According to the density functional theory(DFT) calculation, Cyt c activity increment originates from its ligand changes of Fe(Ⅲ) porphyrin, which has a smaller energy barrier of the catalytic reaction.This study provides a promising strategy for autonomous colocalization of multienzyme in biocompatible hydrogels which can be potentially applied in cascade enzyme induced catalysis applications.