Microfluidics has received extensive attention due to its ability to rapidly prepare a large number of microdroplets with controlled sizes and defined morphologies.In addition to having large surface areas and control...Microfluidics has received extensive attention due to its ability to rapidly prepare a large number of microdroplets with controlled sizes and defined morphologies.In addition to having large surface areas and controllable confinement environments,these prepared microdroplets can be used as analytical detection devices to screen and optimize various kinetic parameters.This review summarizes recent advances in the microfluidic control of droplet-based catalytic reactions and discusses the role of these droplets in both homogeneous and heterogeneous catalyzes and in the catalysis of macromolecular biological enzymes in water-in-oil and oil-in-oil environments.Additionally,the existing problems and future development directions of droplets in catalysis are highlighted to promote the development of catalytic reactions in droplet media and provide guidance for the high-throughput screening of catalysts and the directed evolution of biological enzymes.展开更多
Objective:To improve the efficiency of drug delivery,a mannose vinyl stearate mannose ligand(Man ligand)with active liver-targeting properties was synthesized.Methods:Non-aqueous enzymatic synthesis was used to modify...Objective:To improve the efficiency of drug delivery,a mannose vinyl stearate mannose ligand(Man ligand)with active liver-targeting properties was synthesized.Methods:Non-aqueous enzymatic synthesis was used to modify the structure of mannose.Glycyrrhetinic acid-tanshinone lipid nanoparticles(GT-LN)and liver-targeted glycyrrhetinic acid-tanshinone mannose-modified lipid nanoparticles(GT-MLN)were prepared.The physicochemical properties and release profiles of both formulations were evaluated,and their pharmacokinetic behavior and tissue distribution were investigated.Results:The average particle sizes of GT-LN and GT-MLN were 190.20±1.35 and 204.83±3.86 nm,respectively,with corresponding surface Zeta potentials of-28.0±1.68 and-30.24±2.10 mV.The drug release profile of GT-LN conformed to the Higuchi equation,whereas that of GT-MLN followed both the first-order kinetic and RitgerePeppas equations.Both formulations significantly enhanced the gastrointestinal stability of the drug.In vivo studies in mice demonstrated that hepatic GA and TSN concentrations in both groups were significantly higher than those in the original drug suspension group(P=.01).Notably,the concentrations in the GT-MLN group were significantly higher compared to the GTLN group(P=.01).Conclusion:Man ligand was formed via the linkage of vinyl stearate with the hydroxyl group at C-6 in mannose.The Manligand endowed these lipid nanoparticles with obvious active liver-targeting properties.Our results provide an efficient and stable route of drug delivery to the liver with improved drug availability.展开更多
Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-C...Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-Cy FE catalyzes the crosslinking between an aromatic residue(Ω1) and a non-aromatic residue(X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the Ri PP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these Ri PPs are likely class-specific and only present in gamma-proteobacteria. The 3-Cy FE Pau B from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of Pau B in vitro,showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.展开更多
Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis o...Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone(3S,4S)-4a and whisky lactone(3R,4S)-4b from α,β-unsaturatedγ-ketoesters. A new ene reductase(ER) from Swingsia samuiensi(Ss ER) and a carbonyl reductase(Ss CR)were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating(3S,4S)-4a(99% ee and87% de) and(3R,4S)-4b(99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.展开更多
Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmac...Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.展开更多
The therapeutic efficacy of cuproptosis,ferroptosis,and apoptosis is hindered by inadequate intracellular copper and iron levels,hypoxia,and elevated glutathione(GSH)expression in tumor cells.Thermoelectric technology...The therapeutic efficacy of cuproptosis,ferroptosis,and apoptosis is hindered by inadequate intracellular copper and iron levels,hypoxia,and elevated glutathione(GSH)expression in tumor cells.Thermoelectric technology is an emerging frontier in medical therapy that aims to achieve efficient thermal and electrical transport characteristics within a narrow thermal range for biological systems.Here,we systematically constructed biodegradable Cu_(2)MnS_(3-x)-PEG/glucose oxidase(MCPG)with sulfur vacancies(S_(V))using photothermoelectric catalysis(PTEC),photothermal-enhanced enzyme catalysis,and starvation therapy.This triggers GSH consumption and disrupts intracellular redox homeostasis,leading to immunogenic cell death.Under 1064 nm laser irradiation,MCPG enriched with S_(V),owing to doping,generates a local temperature gradient that activates PTEC and produces toxic reactive oxygen species(ROS).Hydroxyl radicals and oxygen are generated through peroxide and catalase-like processes.Increased oxygen levels alleviate tumor hypoxia,whereas hydrogen peroxide production from glycometabolism provides sufficient ROS for a cascade catalytic reaction,establishing a self-reinforcing positive mechanism.Density functional theory calculations demonstrated that vacancy defects effectively enhanced enzyme catalytic activity.Multimodal imaging-guided synergistic therapy not only damages tumor cells,but also elicits an antitumor immune response to inhibit tumor metastasis.This study offers novel insights into the cuproptosis/ferroptosis/apoptosis pathways of Cu-based PTEC nanozymes.展开更多
Glutathione transferases（GSTs） play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and/l-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Sacc...Glutathione transferases（GSTs） play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and/l-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Saccharo- myces cerevisiae, a novel atypical GST, is still poorly understood. In the present study, we investigated the gluta- thione（GSH） activation mechanism of Gtt2 using the density functional theory（DFT） with the hybrid functional B3LYP. The computational results show that a water molecule could assist a proton transfer between the GSH thiol and the N atom of His133. The energy barrier of proton transfer is 46.0 kJ/mol. The GSH activation mechanism and the characteristics of active site are different from those of classic cytosolic GSTs.展开更多
Here we developed a saccharic colorimetric method based on the combination of chemoselective ligation and enzyme-specific catalysis using aminooxy/ hydrazine-functionalized gold nanoparticles （AO/AuNPs or H/AuNPs）. ...Here we developed a saccharic colorimetric method based on the combination of chemoselective ligation and enzyme-specific catalysis using aminooxy/ hydrazine-functionalized gold nanoparticles （AO/AuNPs or H/AuNPs）. In the detection of galactose （Gal）, galactohexodialdose （GHDA）, the galactose oxidase （GalOx）-catalyzed product, has an aldehyde group, which allows it to chemoselectively react with an aminooxy or hydrazine group at the outer layer of AO/AuNPs or H/AuNPs by oxime/hydrazone click chemistry to form oxime or hydrozone. Consequent134 through the specific recognition of 1,4-phenylenediboronic acid （PDBA） on cis-diols, GHDA, which contains two pairs of hydroxyls in the cis form, can bind not only with AO/AuNPs or H/AuNPs, but also with PDBA to form boronate diester, thereby triggering the aggregation of AuNPs and causing the corresponding color change. As GalOx catalyzed specific substrates, the amount of Gal correlated with the production of GHDA and the extent of AuNPs aggregation, thus allowing a simple and easily operatable colorimetric method for Gal detection to be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 617 nm to that at 536 nm vary linearly with the logarithmic values of Gal concentrations within a wide range of 500 nM to 5 mM. Moreover, this colorimetric method shows anti-interference capability and high sensitivity with a detection limit of 21 nM. Thus, a universal platform for accurate and specific colorimetric analysis can be established through the integration of chemoselective ligation with enzyme specific catalysis.展开更多
Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions includ...Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.展开更多
Enzyme immobilization is a crucial step in advancing the industrial application of biocatalysts.Achieving effective enzyme encapsulation and precise tailoring of the enzyme microenvironment is essential for maximizing...Enzyme immobilization is a crucial step in advancing the industrial application of biocatalysts.Achieving effective enzyme encapsulation and precise tailoring of the enzyme microenvironment is essential for maximizing catalytic performance,yet remains a challenge.In this study,we synthesized hierarchically porous covalent organic framework(COF)aerogels,incorporating alkyl chains of varying lengths on the framework,to enable efficient enzyme immobilization,facilitate mass transfer and fine-tune the microenvironment.The introduction of alkyl chains modulated enzyme-COF interactions,inducing interfacial activation and promoting open enzyme conformation with more accessible active sites.Notably,increasing the length of alkyl chains strengthened enzyme-COF interactions,and the enzyme immobilized on the COF aerogel with the longest alkyl chains(C6)showed the highest activity and stability,achieving more than twice the conversion of the free enzyme even at low temperatures(0–20°C)and maintaining 90%of its initial conversion after thermal treatment at 80°C.Furthermore,COF aerogel-immobilized enzymes maintained high conversion in continuous-flow reactions for 8 h,demonstrating operational robustness.The generality of this approach is validated with multiple lipases,showing enhanced activity.This study highlights the potential of functionalized COF aerogels as a versatile and robust platform for developing high-performance biocatalysts.展开更多
Comprehensive Summary Imbalance in the levels of ascorbic acid(AA)can pose a risk to human health.Therefore,it's essential to establish an accurate method for the detection of AA.In this work,a novel N-doped carbo...Comprehensive Summary Imbalance in the levels of ascorbic acid(AA)can pose a risk to human health.Therefore,it's essential to establish an accurate method for the detection of AA.In this work,a novel N-doped carbon composite(MnO_(x)@NC)with dual enzyme-like activities to detect AA was prepared by calcination of Mn-MOF containing H3TTPCA ligand.展开更多
Enzyme-powered micro/nanomotors(EMNMs)represent cutting-edge research taking advantage of enzymes as biocatalysts to provide a driving force for micro/nanomotors.Up to now,EMNMs have been designed to be powered by cat...Enzyme-powered micro/nanomotors(EMNMs)represent cutting-edge research taking advantage of enzymes as biocatalysts to provide a driving force for micro/nanomotors.Up to now,EMNMs have been designed to be powered by catalase,urease,lipase,collagenase,compound enzymes,etc.They not only have good biocompatibility and biosafety but also possess the unique ability to utilize physiologically relevant fuel to achieve autonomous propulsion through in vivo catalytic reactions.This innovation has opened exciting possibilities for medical applications of EMNMs.Given the fact that the human body is naturally abundant with substrates available for enzymatic reactions,EMNMs can effectively exploit the complex microenvironment associated with diseases,enabling the diagnosis and treatment of various medical conditions.In this review,we first introduce different kinds of EMNMs applied in specific environments for the diagnosis and treatment of diseases,while highlighting their advancements for revolutionizing healthcare practices.Then,we address the challenges faced in this rapidly evolving field,and at last,the potential future development directions are discussed.As the potential of EMNMs becomes increasingly evident,continued research and exploration are essential to unlock their full capabilities and to ensure their successful integration into clinical applications.展开更多
Mesophilic and thermophilic anaerobic fermentation performance of waste activated sludge(WAS)pretreated by enzymes catalysis associated with microbial community shifts were investigated.WAS disintegration was boosted ...Mesophilic and thermophilic anaerobic fermentation performance of waste activated sludge(WAS)pretreated by enzymes catalysis associated with microbial community shifts were investigated.WAS disintegration was boosted considerably by enzymolysis with 8750 mg/L of soluble COD release within 180 min.Mesophilic anaerobic fermentation(MAF)produced nearly equal VFA accumulation with over 3200 mg COD/L compared with that of thermophilic fermentation(TAF).Bacterial community consortia showed great shifting differences in dynamics of main T⁃RFs between MAF and TAF.Moreover,MAF was conducive to form intermediate bacterial community evenness compared to TAF,which preserved a robust function of VFA production.The enzymes catalysis prompted bio⁃energy(electricity)recovery potential of WAS organics via anaerobic fermentation(MAF/TAF)with evaluating electricity conversion efficiency of 0.75-0.82 kW·h/kg VSS(3.9 times higher than control test).Finally,this study proposed some novel thinking on future WAS treatment/management towards energy recovery coupled with energy⁃sufficient wastewater treatment by co⁃locating WAS anaerobic fermentation,MFC plant with wastewater treatment plant(s).展开更多
The radical S-adenosyImethionine(SAM)enzyme NosL catalyzes the conversion of L-tryptophan((-Trp,1)to 3-methyl-2-indolic acid(MIA,2),a key in termediate in the biosynthesis of the peptide an tibiotic no siheptide.Previ...The radical S-adenosyImethionine(SAM)enzyme NosL catalyzes the conversion of L-tryptophan((-Trp,1)to 3-methyl-2-indolic acid(MIA,2),a key in termediate in the biosynthesis of the peptide an tibiotic no siheptide.Previous study showed that this remarkable recombination reaction starts from the cleavage of the Cα-COO^(-)bond to result in a·CO_(2)^(-)radical migration.In contrast to the radical SAM tyrosine lyases,NosL appears unable to cleave the Cα-Cβbond,which is intrinsically more favorable to be cleaved than the Cα-COO^(-)bond.In this study,we investigate the NosL activity with tryptamine(11)and tryptophol(12),two L-Trp analogues lacking a carboxylate moiety.We showed that NosL cleaves the C1-C2 bond of these two substrates to produce 3-methylindole(7),suggesting that the enzyme can still catalyze aβ-scission when the carboxyl group of Trp is absent.We also showed the enzyme exhibits a promiscuous activity,initiating the reaction by abstracting hydrogen atoms from two different sites to produce two sets of products.展开更多
Enzymes are natural treasure troves that hold multiple superiority.Enzymatic catalysis has become a powerful tool for asymmetric synthesis,though it is typically limited to a relatively narrow range of reaction types....Enzymes are natural treasure troves that hold multiple superiority.Enzymatic catalysis has become a powerful tool for asymmetric synthesis,though it is typically limited to a relatively narrow range of reaction types.By integrating the advantages of enzymatic catalysis with photocatalysis,photoenzymatic catalysis not only expands the catalytic capabilities of enzymes but also provides an effective strategy for the stereo-control of photochemical reactions,thereby emerging as a significant research field.Herein,we focus on new-to-nature photoenzymatic catalysis by repurposing naturally occurring enzymes with visible light.We highlight the seminal work in reshaping various classes of enzymes,emphasizing their catalytic mechanism and synthetic potentials.展开更多
The biochemical property and functional identification of three recombinant glycosyltransferases,includingβ-1,4-rhamnosyltransferase(Cps8R),β-1,4-galactosyltransferase(Cps8) and α-2,3-sialyltransferase(Cps8K)involv...The biochemical property and functional identification of three recombinant glycosyltransferases,includingβ-1,4-rhamnosyltransferase(Cps8R),β-1,4-galactosyltransferase(Cps8) and α-2,3-sialyltransferase(Cps8K)involved in the biosynthesis of the tetrasaccharide repeating unit of serotypeⅧcapsular polysaccharide(CPS)of Group B Streptococci(GBS),were systematically investigated.Subsequently,these recombinant enzymes were employed for one-pot three-enzyme efficient synthesis of the tetrasaccharide repeating unit of GBS serotypeⅧCPS using the chemically synthesized Glca-PP-(CH2)ir-OPh as the starting substrate in a satisfying yieldof 87%.展开更多
Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials,where the material properties are mainly influenced by the species of natural biomolecules,linear ...Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials,where the material properties are mainly influenced by the species of natural biomolecules,linear synthetic polymer,or small molecules,limiting their diversity.Herein,we adopted dendrimer poly(amidoamine)(PAMAM)as the inducer to synthesize organosilica-PAMAM network(OSPN)capsules for mannose isomerase(MIase)encapsulation based on a hard-templating method.The structure of OSPN capsules can be precisely regulated by adjusting the molecular weight and concentration of PAMAM,thereby demonstrating a substantial impact on the kinetic behavior of the MIase@OSPN system.The MIase@OSPN system was used for catalytic production of mannose from Dfructose.A mannose yield of 22.24% was obtained,which is higher than that of MIase in organosilica network capsules and similar to that of the free enzyme.The overall catalytic efficiency(kcat/Km)of the MIase@OSPN system for the substrate D-fructose was up to 0.556 s^(-1)·mmol^(-1)·L.Meanwhile,the MIase@OSPN system showed excellent stability and recyclability,maintaining more than 50% of the yield even after 12 cycles.展开更多
Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a...Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.展开更多
Fluoroacetate dehalogenases(FAcD),a homodimeric enzyme,catalyzes the conversion of fluoroacetic acid to glycolic acid(GoA).It has been proved that the enzyme has a half-of-the-site reactivity.Namely,its catalytic(C)su...Fluoroacetate dehalogenases(FAcD),a homodimeric enzyme,catalyzes the conversion of fluoroacetic acid to glycolic acid(GoA).It has been proved that the enzyme has a half-of-the-site reactivity.Namely,its catalytic(C)subunit converts the first substrate to a covalent intermediate;then,the non-catalytic(NC)subunit binds a second substrate and promotes the conversion of the intermediate in the C subunit into the final product.After the release of the product,the C subunit becomes the NC subunit,and the previous NC subunit becomes the C subunit.To elucidate the detailed mechanism behind this cooperative catalysis,we have conducted microsecond-scale MD simulations along the reaction pathway.The simulations indicate that the substrate in the NC subunit induces W185 and Y141 adopting an open conformation in the C subunit.The opening of W185(C)facilitates the entry of catalytic water,enhancing the catalytic activity for product formation,while the opening of Y141(C)creates an unfavorable environment for product binding,promoting its release.An interaction network analysis reveals that the substrate in the NC subunit can induce conformational changes through a conserved water chain at the interface.展开更多
The cytochrome P411 enzyme is a variant of cytochrome P450_(BM3) from Bacillus megaterium whose active site is an iron porphyrin imine([Fe(Por)(NH)]^(-))specie.This specie has been reported to successfully promote the...The cytochrome P411 enzyme is a variant of cytochrome P450_(BM3) from Bacillus megaterium whose active site is an iron porphyrin imine([Fe(Por)(NH)]^(-))specie.This specie has been reported to successfully promote the primary amination of benzylic and allylic C(sp^(3))-H bonds.We employed density functional theory to study the electronic structure of the active site of P411 enzyme and the primary amination of C-H bond reaction that it catalyzes.The calculated spin densities and orbital values indicate the existence of resonance in this specie;namely,[(por)(–OH)Fe^(Ⅳ)–N^(2-)–H]^(-)↔[(por)(–OH)Fe^(Ⅲ)–N^(·-)–H]^(-).The amination of C(sp^(3))-H bonds consists of two main reaction steps:hydrogen-atom abstraction and radical recombination,and the former is demonstrated to be the rate-determining step.Furthermore,we studied the regioselectivity of the amination of primary and secondary C(sp^(3))-H bonds.Our calculations indicated that the secondary C(sp^(3))-H bonds of the substrate would be more favored for the activation by P411 enzyme.These results provide valuable information for understanding the properties and selectivity of C-H/C-N bond-activation reactions catalyzed by the P411 enzyme or other similar enzymes.展开更多
基金supported by the National Key R&D Program of China(2021YFA1501600)National Nature Science Foundation of China(Nos.22107028 and 22103062)+3 种基金Program of Shanghai Outstanding Academic Leaders(No.21XD1421200)Science and Technology Commission of Shanghai Municipality(22JC1403900)Shanghai Pujiang Program(No.22PJ1402800)the Fundamental Research Funds for the Central Universities.
文摘Microfluidics has received extensive attention due to its ability to rapidly prepare a large number of microdroplets with controlled sizes and defined morphologies.In addition to having large surface areas and controllable confinement environments,these prepared microdroplets can be used as analytical detection devices to screen and optimize various kinetic parameters.This review summarizes recent advances in the microfluidic control of droplet-based catalytic reactions and discusses the role of these droplets in both homogeneous and heterogeneous catalyzes and in the catalysis of macromolecular biological enzymes in water-in-oil and oil-in-oil environments.Additionally,the existing problems and future development directions of droplets in catalysis are highlighted to promote the development of catalytic reactions in droplet media and provide guidance for the high-throughput screening of catalysts and the directed evolution of biological enzymes.
基金the High-level construction discipline of the National Administration of Traditional Chinese Medicine(zyyzdxk-2023272).
文摘Objective:To improve the efficiency of drug delivery,a mannose vinyl stearate mannose ligand(Man ligand)with active liver-targeting properties was synthesized.Methods:Non-aqueous enzymatic synthesis was used to modify the structure of mannose.Glycyrrhetinic acid-tanshinone lipid nanoparticles(GT-LN)and liver-targeted glycyrrhetinic acid-tanshinone mannose-modified lipid nanoparticles(GT-MLN)were prepared.The physicochemical properties and release profiles of both formulations were evaluated,and their pharmacokinetic behavior and tissue distribution were investigated.Results:The average particle sizes of GT-LN and GT-MLN were 190.20±1.35 and 204.83±3.86 nm,respectively,with corresponding surface Zeta potentials of-28.0±1.68 and-30.24±2.10 mV.The drug release profile of GT-LN conformed to the Higuchi equation,whereas that of GT-MLN followed both the first-order kinetic and RitgerePeppas equations.Both formulations significantly enhanced the gastrointestinal stability of the drug.In vivo studies in mice demonstrated that hepatic GA and TSN concentrations in both groups were significantly higher than those in the original drug suspension group(P=.01).Notably,the concentrations in the GT-MLN group were significantly higher compared to the GTLN group(P=.01).Conclusion:Man ligand was formed via the linkage of vinyl stearate with the hydroxyl group at C-6 in mannose.The Manligand endowed these lipid nanoparticles with obvious active liver-targeting properties.Our results provide an efficient and stable route of drug delivery to the liver with improved drug availability.
基金supported by grants from the National Key Research and Development Program (Nos. 2018YFA0900402 and 2021YFA0910501)the National Natural Science Foundation of China (Nos. 21822703, 21921003, and 32070050)+2 种基金the funding of Innovative research team of high-level local universities in Shanghaia key laboratory program of the Education Commission of Shanghai Municipality (No. ZDSYS14005)West Light Foundation of the Chinese Academy of Sciences (No. xbzgzdsys-202105)。
文摘Three-residue cyclophane-forming enzymes(3-Cy FEs) are a group of radical S-adenosylmethionine(SAM)enzymes involved in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides(Ri PPs). 3-Cy FE catalyzes the crosslinking between an aromatic residue(Ω1) and a non-aromatic residue(X3) in a Ω1-X2-X3 motif to produce a cyclophane ring, a key step in the biosynthesis of the Ri PP natural product triceptide. In this study, we perform a genome-wide search for the Xye-type triceptides, showing these Ri PPs are likely class-specific and only present in gamma-proteobacteria. The 3-Cy FE Pau B from Photorhabdus australis exhibits a relaxed substrate specificity on the X3 position, but glycine in this position is not suitable for cyclophane formation. We also reconstituted the activity of Pau B in vitro,showing it produces the N-terminal cyclophane firstly, and then the C-terminal ring, whereas the middle cyclophane is produced in the last step.
基金financially sponsored by the National Key Research and Development Program of China (No.2021YFC2102804)the National Natural Science Foundation of China(No.22078096)。
文摘Polysubstituted chiral γ-butyrolactones are the core structural units of many natural products and high value-added flavors and fragrances used in the food and cosmetic industry. Current enzymatic cascade synthesis of these molecules faces the problems of low enzyme activity and phase separation in batch reaction, resulting in low productivity. Herein, we report a new continuous-flow process to synthesize the optically pure Nicotiana tabacum lactone(3S,4S)-4a and whisky lactone(3R,4S)-4b from α,β-unsaturatedγ-ketoesters. A new ene reductase(ER) from Swingsia samuiensi(Ss ER) and a carbonyl reductase(Ss CR)were engineered by directed evolution to improve their activity and thermostability. The continuous-flow preparative reactions were performed in two 3D microfluidic reactors, generating(3S,4S)-4a(99% ee and87% de) and(3R,4S)-4b(99% ee and 98% de) with space-time yields 3 and 7.4 times higher than those of the batch reactions. The significant enhancement in the productivity of enzyme cascade catalysis brought by cutting-edge continuous microfluidic technology will benefit the general multi-enzyme catalytic systems in the future.
文摘Enzyme engineering is an important part of modern biotechnology.Due to its high reaction specificity,high efficiency,mild reaction conditions,and low pollution,it is also an important method widely used in the pharmaceutical field.The application of enzymes in medicine is diverse,such as:diagnosis,prevention and treatment of diseases with enzymes,manufacture of various drugs with enzymes,etc.,mainly through manual operations,to obtain enzymes required by the pharmaceutical industry,and through various means Enzymes perform their catalytic functions.This article mainly introduces the application of enzyme engineering in the pharmaceutical field,and also prospects the development trend of enzyme engineering in the pharmaceutical field.
基金supported by the National Natural Science Foundation of China(NSFC 52002091,U22A20347,and 52102344)Heilongjiang Natural Science Foundation Project of Outstanding Youth Project(YQ2023B005)+1 种基金China Postdoctoral Science Foundation(2023T160154)the Fundamental Research Funds for the Central Universities。
文摘The therapeutic efficacy of cuproptosis,ferroptosis,and apoptosis is hindered by inadequate intracellular copper and iron levels,hypoxia,and elevated glutathione(GSH)expression in tumor cells.Thermoelectric technology is an emerging frontier in medical therapy that aims to achieve efficient thermal and electrical transport characteristics within a narrow thermal range for biological systems.Here,we systematically constructed biodegradable Cu_(2)MnS_(3-x)-PEG/glucose oxidase(MCPG)with sulfur vacancies(S_(V))using photothermoelectric catalysis(PTEC),photothermal-enhanced enzyme catalysis,and starvation therapy.This triggers GSH consumption and disrupts intracellular redox homeostasis,leading to immunogenic cell death.Under 1064 nm laser irradiation,MCPG enriched with S_(V),owing to doping,generates a local temperature gradient that activates PTEC and produces toxic reactive oxygen species(ROS).Hydroxyl radicals and oxygen are generated through peroxide and catalase-like processes.Increased oxygen levels alleviate tumor hypoxia,whereas hydrogen peroxide production from glycometabolism provides sufficient ROS for a cascade catalytic reaction,establishing a self-reinforcing positive mechanism.Density functional theory calculations demonstrated that vacancy defects effectively enhanced enzyme catalytic activity.Multimodal imaging-guided synergistic therapy not only damages tumor cells,but also elicits an antitumor immune response to inhibit tumor metastasis.This study offers novel insights into the cuproptosis/ferroptosis/apoptosis pathways of Cu-based PTEC nanozymes.
基金Supported by the National Natural Science Foundation of China(No.20903045)the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20070183046)the Specialized Fund for the Basic Research of Jilin University,China(No.200810018)
文摘Glutathione transferases（GSTs） play an important role in the detoxification of xenobiotic/endobiotic toxic compounds. The α-, π-, and/l-classes of cytosolic GSTs have been studied extensively, while Gtt2 from Saccharo- myces cerevisiae, a novel atypical GST, is still poorly understood. In the present study, we investigated the gluta- thione（GSH） activation mechanism of Gtt2 using the density functional theory（DFT） with the hybrid functional B3LYP. The computational results show that a water molecule could assist a proton transfer between the GSH thiol and the N atom of His133. The energy barrier of proton transfer is 46.0 kJ/mol. The GSH activation mechanism and the characteristics of active site are different from those of classic cytosolic GSTs.
文摘Here we developed a saccharic colorimetric method based on the combination of chemoselective ligation and enzyme-specific catalysis using aminooxy/ hydrazine-functionalized gold nanoparticles （AO/AuNPs or H/AuNPs）. In the detection of galactose （Gal）, galactohexodialdose （GHDA）, the galactose oxidase （GalOx）-catalyzed product, has an aldehyde group, which allows it to chemoselectively react with an aminooxy or hydrazine group at the outer layer of AO/AuNPs or H/AuNPs by oxime/hydrazone click chemistry to form oxime or hydrozone. Consequent134 through the specific recognition of 1,4-phenylenediboronic acid （PDBA） on cis-diols, GHDA, which contains two pairs of hydroxyls in the cis form, can bind not only with AO/AuNPs or H/AuNPs, but also with PDBA to form boronate diester, thereby triggering the aggregation of AuNPs and causing the corresponding color change. As GalOx catalyzed specific substrates, the amount of Gal correlated with the production of GHDA and the extent of AuNPs aggregation, thus allowing a simple and easily operatable colorimetric method for Gal detection to be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 617 nm to that at 536 nm vary linearly with the logarithmic values of Gal concentrations within a wide range of 500 nM to 5 mM. Moreover, this colorimetric method shows anti-interference capability and high sensitivity with a detection limit of 21 nM. Thus, a universal platform for accurate and specific colorimetric analysis can be established through the integration of chemoselective ligation with enzyme specific catalysis.
基金funded by the National Natural Science Foundation of China(21576134 and 21706126)the National Key Research and Development Program(2016YFA0204300)。
文摘Here,a dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.
基金the National Natural Science Foundation of China(grant no.22271015)TaiShan Scholars Program(no.tsqn202408322)Beijing Institute of Technology Research Fund Program,and Young Elite Scientists Sponsorship Program by BAST(no.BYESS2024365)for financial support,and Analysis and Testing Center of Beijing Institute of Technology.
文摘Enzyme immobilization is a crucial step in advancing the industrial application of biocatalysts.Achieving effective enzyme encapsulation and precise tailoring of the enzyme microenvironment is essential for maximizing catalytic performance,yet remains a challenge.In this study,we synthesized hierarchically porous covalent organic framework(COF)aerogels,incorporating alkyl chains of varying lengths on the framework,to enable efficient enzyme immobilization,facilitate mass transfer and fine-tune the microenvironment.The introduction of alkyl chains modulated enzyme-COF interactions,inducing interfacial activation and promoting open enzyme conformation with more accessible active sites.Notably,increasing the length of alkyl chains strengthened enzyme-COF interactions,and the enzyme immobilized on the COF aerogel with the longest alkyl chains(C6)showed the highest activity and stability,achieving more than twice the conversion of the free enzyme even at low temperatures(0–20°C)and maintaining 90%of its initial conversion after thermal treatment at 80°C.Furthermore,COF aerogel-immobilized enzymes maintained high conversion in continuous-flow reactions for 8 h,demonstrating operational robustness.The generality of this approach is validated with multiple lipases,showing enhanced activity.This study highlights the potential of functionalized COF aerogels as a versatile and robust platform for developing high-performance biocatalysts.
基金supported by Youth Science and Technology Star Project of Dalian City(No.2022RQ042)Postgraduate Education Reform Project of Liaoning Normal University(No.YJSJG202418)+2 种基金Undergraduate Scientific Research Training Project(No.JSZDBKSXM2023009)State Key Laboratory of Inorganic Synthesis and Preparative Chemistry,College of Chemistry,Jilin University(Grant No.2024-46)Guangxi Collaborative Innovation Centre of Structure and Property for New Energy and Materials,Science Research,Technology Development Project of Guilin(No.20210102-4).
文摘Comprehensive Summary Imbalance in the levels of ascorbic acid(AA)can pose a risk to human health.Therefore,it's essential to establish an accurate method for the detection of AA.In this work,a novel N-doped carbon composite(MnO_(x)@NC)with dual enzyme-like activities to detect AA was prepared by calcination of Mn-MOF containing H3TTPCA ligand.
基金support by the Young Taishan Scholars Program of Shandong Province(Grant No.tsqn202306272)National Key Research and Development Project of China(Grant No.2023YFFO715101)+5 种基金National Natural Science Foundation of China(Grant No.22307050)Natural Science Foundation of Shandong Province(Grant No.ZR2023QB292,ZR2024YQ068,2024HWYQ-062)the Leading Project of Science and Technology of Yantai Development Zone(Grant No.2021RC016)CFG and RLR acknowledge funding from Fundaçao para a Ciencia e Tecnologia(FCT)Grants 10.54499/2022.05711.CEECIND/CP1718/CT0012(CFG)and 10.54499/2022.05737.PTDC(CFG,RLR)as well as TERM RES Hub–Scientific Infrastructure for Tissue Engineering and Regenerative Medicine,reference PINFRA/22190/2016(Norte-01-0145-FEDER-022190).
文摘Enzyme-powered micro/nanomotors(EMNMs)represent cutting-edge research taking advantage of enzymes as biocatalysts to provide a driving force for micro/nanomotors.Up to now,EMNMs have been designed to be powered by catalase,urease,lipase,collagenase,compound enzymes,etc.They not only have good biocompatibility and biosafety but also possess the unique ability to utilize physiologically relevant fuel to achieve autonomous propulsion through in vivo catalytic reactions.This innovation has opened exciting possibilities for medical applications of EMNMs.Given the fact that the human body is naturally abundant with substrates available for enzymatic reactions,EMNMs can effectively exploit the complex microenvironment associated with diseases,enabling the diagnosis and treatment of various medical conditions.In this review,we first introduce different kinds of EMNMs applied in specific environments for the diagnosis and treatment of diseases,while highlighting their advancements for revolutionizing healthcare practices.Then,we address the challenges faced in this rapidly evolving field,and at last,the potential future development directions are discussed.As the potential of EMNMs becomes increasingly evident,continued research and exploration are essential to unlock their full capabilities and to ensure their successful integration into clinical applications.
基金Sponsored by the Scientific Research Funds of Huaqiao University(Grant No.605-50Y18055).
文摘Mesophilic and thermophilic anaerobic fermentation performance of waste activated sludge(WAS)pretreated by enzymes catalysis associated with microbial community shifts were investigated.WAS disintegration was boosted considerably by enzymolysis with 8750 mg/L of soluble COD release within 180 min.Mesophilic anaerobic fermentation(MAF)produced nearly equal VFA accumulation with over 3200 mg COD/L compared with that of thermophilic fermentation(TAF).Bacterial community consortia showed great shifting differences in dynamics of main T⁃RFs between MAF and TAF.Moreover,MAF was conducive to form intermediate bacterial community evenness compared to TAF,which preserved a robust function of VFA production.The enzymes catalysis prompted bio⁃energy(electricity)recovery potential of WAS organics via anaerobic fermentation(MAF/TAF)with evaluating electricity conversion efficiency of 0.75-0.82 kW·h/kg VSS(3.9 times higher than control test).Finally,this study proposed some novel thinking on future WAS treatment/management towards energy recovery coupled with energy⁃sufficient wastewater treatment by co⁃locating WAS anaerobic fermentation,MFC plant with wastewater treatment plant(s).
基金supported in part by grants from the National Key Research and Development Program(2018Y F A0900402 and 2016Y F A0501302)from the National Natural Science Foundation of China(21822703 and 21921003).
文摘The radical S-adenosyImethionine(SAM)enzyme NosL catalyzes the conversion of L-tryptophan((-Trp,1)to 3-methyl-2-indolic acid(MIA,2),a key in termediate in the biosynthesis of the peptide an tibiotic no siheptide.Previous study showed that this remarkable recombination reaction starts from the cleavage of the Cα-COO^(-)bond to result in a·CO_(2)^(-)radical migration.In contrast to the radical SAM tyrosine lyases,NosL appears unable to cleave the Cα-Cβbond,which is intrinsically more favorable to be cleaved than the Cα-COO^(-)bond.In this study,we investigate the NosL activity with tryptamine(11)and tryptophol(12),two L-Trp analogues lacking a carboxylate moiety.We showed that NosL cleaves the C1-C2 bond of these two substrates to produce 3-methylindole(7),suggesting that the enzyme can still catalyze aβ-scission when the carboxyl group of Trp is absent.We also showed the enzyme exhibits a promiscuous activity,initiating the reaction by abstracting hydrogen atoms from two different sites to produce two sets of products.
基金the National Natural Science Foundation of China(22277053 to X.H.223B2703 to Y.X.)+3 种基金the National Key Research and Development Program of China(2022YFA0913000 to X.H.)the Natural Science Foundation of Jiangsu Province(BK20220760 to X.H.)the Fundamental Research Funds for the Central Universities(0205/14380346 to X.H.)the Excellent Research Program of Nanjing University(ZYJH004 to X.H.).
文摘Enzymes are natural treasure troves that hold multiple superiority.Enzymatic catalysis has become a powerful tool for asymmetric synthesis,though it is typically limited to a relatively narrow range of reaction types.By integrating the advantages of enzymatic catalysis with photocatalysis,photoenzymatic catalysis not only expands the catalytic capabilities of enzymes but also provides an effective strategy for the stereo-control of photochemical reactions,thereby emerging as a significant research field.Herein,we focus on new-to-nature photoenzymatic catalysis by repurposing naturally occurring enzymes with visible light.We highlight the seminal work in reshaping various classes of enzymes,emphasizing their catalytic mechanism and synthetic potentials.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant numbers 21877074 and 21672129).
文摘The biochemical property and functional identification of three recombinant glycosyltransferases,includingβ-1,4-rhamnosyltransferase(Cps8R),β-1,4-galactosyltransferase(Cps8) and α-2,3-sialyltransferase(Cps8K)involved in the biosynthesis of the tetrasaccharide repeating unit of serotypeⅧcapsular polysaccharide(CPS)of Group B Streptococci(GBS),were systematically investigated.Subsequently,these recombinant enzymes were employed for one-pot three-enzyme efficient synthesis of the tetrasaccharide repeating unit of GBS serotypeⅧCPS using the chemically synthesized Glca-PP-(CH2)ir-OPh as the starting substrate in a satisfying yieldof 87%.
基金supported by the National Key Research and Development Program of China(Grant No.2021YFC210-2300)the National Key Research and Development Program of China(Grant No.2022YFC2105902)+2 种基金Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(Grant No.TSBICIP-KJGG-003)Open Funding Project of the State Key Laboratory of Biochemical Engineering of China(Grant No.2020KF-06)Haihe Laboratory of Sustainable Chemical Transformations.
文摘Organic matter-induced mineralization is a green and versatile method for synthesizing hybrid nanostructured materials,where the material properties are mainly influenced by the species of natural biomolecules,linear synthetic polymer,or small molecules,limiting their diversity.Herein,we adopted dendrimer poly(amidoamine)(PAMAM)as the inducer to synthesize organosilica-PAMAM network(OSPN)capsules for mannose isomerase(MIase)encapsulation based on a hard-templating method.The structure of OSPN capsules can be precisely regulated by adjusting the molecular weight and concentration of PAMAM,thereby demonstrating a substantial impact on the kinetic behavior of the MIase@OSPN system.The MIase@OSPN system was used for catalytic production of mannose from Dfructose.A mannose yield of 22.24% was obtained,which is higher than that of MIase in organosilica network capsules and similar to that of the free enzyme.The overall catalytic efficiency(kcat/Km)of the MIase@OSPN system for the substrate D-fructose was up to 0.556 s^(-1)·mmol^(-1)·L.Meanwhile,the MIase@OSPN system showed excellent stability and recyclability,maintaining more than 50% of the yield even after 12 cycles.
基金supported in part by grants from the National Key Research and Development Program of China(2022YFC2303100)the National Natural Science Foundation of China(22193070,32030002,81974495 and 21977109)the Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZLCX20212401).
文摘Cytochrome P450 enzymes (P450s) belong to a large family of oxidative hemeproteins and catalyze highly diverse oxygenation reactions that are involved in the biosynthesis of various natural products. Here, we report a multifunctional cytochrome P450 enzyme, PyrE2, which catalyzes the regioselective, successive 6-electron oxidation of an inert methyl group to produce a carboxyl product through formation of the hydroxyl and aldehyde intermediates in pyrroindomycin biosynthesis. The time-course biotransformation was characterized by the presence of the hydroxyl and aldehyde intermediates, the lag of the formation of the carboxyl product, and the subsequent loss of both intermediates, indicating that each 2-electron oxidation exhibits the distributive mechanism that requires substrate binding and product releasing. Bioinformatics analysis shows that the homologs of pyrE2 are common in the gene clusters of the spirotetronates varying in the oxidative state of the corresponding exocyclic carbon, indicating the generality and diversity of P450-catalyzed oxygenation in related biosynthetic pathways.
基金supported by the Key-Area Research and Development Program of Guangdong Province(2020B0101350001)the Shenzhen Fundamental Research Program(GXWD2020123116580700720200812124825001)+1 种基金the Shenzhen Science and Technology Program(RCBS20210706092258097)supported by the Shenzhen Bay Laboratory Supercomputing Center。
文摘Fluoroacetate dehalogenases(FAcD),a homodimeric enzyme,catalyzes the conversion of fluoroacetic acid to glycolic acid(GoA).It has been proved that the enzyme has a half-of-the-site reactivity.Namely,its catalytic(C)subunit converts the first substrate to a covalent intermediate;then,the non-catalytic(NC)subunit binds a second substrate and promotes the conversion of the intermediate in the C subunit into the final product.After the release of the product,the C subunit becomes the NC subunit,and the previous NC subunit becomes the C subunit.To elucidate the detailed mechanism behind this cooperative catalysis,we have conducted microsecond-scale MD simulations along the reaction pathway.The simulations indicate that the substrate in the NC subunit induces W185 and Y141 adopting an open conformation in the C subunit.The opening of W185(C)facilitates the entry of catalytic water,enhancing the catalytic activity for product formation,while the opening of Y141(C)creates an unfavorable environment for product binding,promoting its release.An interaction network analysis reveals that the substrate in the NC subunit can induce conformational changes through a conserved water chain at the interface.
文摘The cytochrome P411 enzyme is a variant of cytochrome P450_(BM3) from Bacillus megaterium whose active site is an iron porphyrin imine([Fe(Por)(NH)]^(-))specie.This specie has been reported to successfully promote the primary amination of benzylic and allylic C(sp^(3))-H bonds.We employed density functional theory to study the electronic structure of the active site of P411 enzyme and the primary amination of C-H bond reaction that it catalyzes.The calculated spin densities and orbital values indicate the existence of resonance in this specie;namely,[(por)(–OH)Fe^(Ⅳ)–N^(2-)–H]^(-)↔[(por)(–OH)Fe^(Ⅲ)–N^(·-)–H]^(-).The amination of C(sp^(3))-H bonds consists of two main reaction steps:hydrogen-atom abstraction and radical recombination,and the former is demonstrated to be the rate-determining step.Furthermore,we studied the regioselectivity of the amination of primary and secondary C(sp^(3))-H bonds.Our calculations indicated that the secondary C(sp^(3))-H bonds of the substrate would be more favored for the activation by P411 enzyme.These results provide valuable information for understanding the properties and selectivity of C-H/C-N bond-activation reactions catalyzed by the P411 enzyme or other similar enzymes.
