Efficient Substrate Research Articles

Optimization and scaling up of extracellular polysaccharide production by submerged culture of Ganoderma lucidum on starch-containing medium using response surface methodology and laboratory bioreactors of various designs.

Basidiomycetes, known for their production of bioactive compounds, traditionally use simple sugars for fermentation. However, their ability to degrade complex plant polysaccharides through enzyme secretion presents potential for the use of renewable raw materials. This study focused on the optimization of exopolysaccharide (EPS) production and efficient substrate consumption by Ganoderma lucidum using response surface methodology (RSM). Using an optimized medium containing 15 g⋅L-1 wheat starch, 0.375 g⋅L-1 NH4Cl, and 0.75 g⋅L-1 CaCl2 (C/N ratio of 40), a significant increase in EPS concentration from 121.1±10.2 mg⋅L-1 to 229.0±20.3 mg⋅L-1 and starch degradation degree (SDD) from 9.1% to 57.6% was achieved after 9 d of submerged cultivation. Scale-up experiments were conducted in both column and stirred tank bioreactors, employing submerged and immobilized cultivation modes. Submerged cultivation in the column bioreactor yielded the highest process desirability of 0.56, achieving EPS concentration of 192.5±5.4 mg⋅L-1 and 60.2% starch degradation degree within 7 d. These results highlight the potential of the used column bioreactor for efficient and rapid EPS production. Notably, bioreactor experiments revealed local maxima in EPS content at specific time points, suggesting that cell wall degradation, potentially induced by shear stress, may contribute to the release of polysaccharides into the culture broth.

A self-immolative Kdn-glycoside substrate enables high-throughput screening for inhibitors of Kdnases.

Aspergillus fumigatus, a filamentous fungus, is an opportunistic pathogen and the major causative agent of the often-fatal disease, invasive aspergillosis (IA). Current treatments for IA are limited due to their high toxicity and/or the emergence of drug resistance; therefore, a need exists for the development of new therapeutics to treat IA. The Kdnase produced by A. fumigatus plays a vital role in maintaining cell wall integrity. As there are no known Kdnases in humans, developing inhibitors of Kdnase from this fungal pathogen is a promising therapeutic approach. The rapid testing of enzymatic activity in a high-throughput screen of large chemical libraries can be an efficient way to find new small molecule lead compounds. Herein we show that a Kdn glycoside with a self-immolative cleavable aglycon is a practical and efficient substrate for a high throughput assay to identify Kdnase inhibitors. We optimized the activity assay and screened over 27,000 compounds from two bioactive chemical libraries as potential inhibitors, and we compared the hit compounds' potency towards Aspergillus terreus and Trichophyton rubrum Kdnases, two other fungal Kdnases. We validated a number of hits and these small molecules are potential leads for the development of novel therapeutics to treat invasive aspergillosis.

Synthetic Lesions with a Fluorescein Carbamoyl Group As Analogs of Bulky Lesions Removable by Nucleotide Excision Repair: A Comparative Study on Properties.

Mammalian nucleotide excision repair (NER), known for its broad substrate specificity, is responsible for removal of bulky lesions from DNA. Over 30 proteins are involved in NER, which includes two distinct pathways: global genome NER and transcription-coupled repair. The complexity of these processes, the use of extended DNA substrates, and the presence of bulky DNA lesions induced by chemotherapy have driven researchers to seek more effective methods by which to assess NER activity, as well as to develop model DNAs that serve as efficient substrates for studying lesion removal. In this work, we conducted a comparative analysis of model DNAs containing bulky lesions. One of these lesions, N-[6-{5(6)-fluoresceinylcarbamoyl}hexanoyl]-3-amino-1,2-propanediol (nFluL), is known to be efficiently recognized and excised by NER. The second lesion, N-[6-{5(6)-fluoresceinylcarbamoyl}]-3-amino-1,2-propanediol (nFluS), has not previously been tested as a substrate for NER. To evaluate the efficiency of lesion excision, a 3'-terminal labeling method was employed to analyze the excision products. The results showed that nFluS is removed approximately twice as efficiently as nFluL. Comparative analyses of the effects of nFluL and nFluS on the geometry and thermal stability of DNA duplexes - combined with spectrophotometric and spectrofluorimetric titrations of these DNAs with complementary strands - were performed next. They revealed that the absence of an extended flexible linker in nFluS alters the interaction of the bulky fluorescein moiety with neighboring nitrogenous bases in double-stranded DNA. This absence is associated with the enhanced efficiency of excision of nFluS, making it a more effective synthetic analog for studying bulky-lesion removal in model DNA substrates.

Ultrasound-Assisted Remote Benzylic C(sp3)-H Alkylation of N-Fluoroamides with Enol Silanes.

We have developed an ultrasound-assisted remote benzylic C(sp3)-H alkylation of N-fluorobenzamides with enol silanes; a series of β-aryl substituted propiophenones was generated in good to high yields. This reaction represents a formal C(sp3)-C(sp3) coupling and features simplicity, high efficiency, and wide substrate scope in a multiple-step sequential process, which involves N-F homolysis, 1,5-hydrogen atom transfer, benzylic radical addition, oxidation by copper(II) salt, and removal of the trimethylsilyl group. Also, DFT theoretical calculations and Marcus theory were employed to consolidate the proposed reaction mechanism.

Pyridine‐Boryl Radical‐Catalyzed [3 + 2] Cycloadditions of Alkyne‐Tethered Cyclopropyl Ketones

AbstractBicyclo[3.3.0]octanes represent crucial structural motifs found in various bioactive natural compounds. In this study, we present the first metal‐free formal [3 + 2] cycloadditions of alkyne‐tethered cyclopropyl ketones via pyridine‐boryl radical catalysis, affording bicyclo[3.3.0]octanes with high efficiency and broad substrate scope. This reaction features the generation of ketyl radical by pyridine‐boryl radical and a consecutive 5‐exo‐dig and 5‐exo‐trig radical cascade process.

Aerobic thermophilic digestion of fecal matter: Condensate recovery, low solids production, and water reuse

A bench-scale thermophilic sequencing batch reactor (SBR) was designed, fabricated, and tested for the recovery of clean water and the reuse of condensate. The SBR reactor was non-Fouling and membrane-free, Aerobic, low-Solids producing, and Thermophilic (FAST). Accelerated treatment of domestic wastewater organic matter enabled 99.9 % removal of total suspended solid (TSS) from influent loads and 99.8 % removal of chemical oxygen demand (COD) loads. The FAST system also ensured efficient pathogen inactivation, substrate utilization, and biomass growth that aligned with experimental results. Future research will focus on scale-up of technology and assessment of performance in diverse environments.

V-Shaped Heterostructure Nanocavities Array with CM and EM Coupled Enhancement for Ultra-Sensitive SERS Substrate.

The field of semiconductor surface-enhanced Raman scattering (SERS) substrates has experienced significant advancements, leading to a wide range of applications in several fields. However, the quest for new ultra-sensitive semiconductor SERS materials is still of utmost importance. In this regard, an efficient and novel substrate, F4TCNQ/MoS2 heterostructure is introduced, assisted by V-shaped aluminum anodic oxide (AAO) nanocavities with different depths. Utilizing the efficient charge transfer of organic/inorganic semiconducting heterostructure and the photoconfinement capability of the nanocavity structure of the AAO nanotemplate, excellent stability, fast sensing, enhanced Raman, and photodegradation activities are achieved. Due to its unique 3D structure, the optimized F4TCNQ/MoS2/AAO with 1500 nm depth achieves ultra-high sensitivity detection of 9.0×10-16 M for conventional probe molecules. Furthermore, precise detection of water contaminants is observed for the first time with a V-shaped heterostructure due to combined organic/inorganic features that differ significantly from conventional MoS2 structures or other metal/inorganic or inorganic/inorganic semiconductors. This research presents a novel and versatile strategy for SERS and demonstrates its diverse potential performance in practical applications.

Fe2O3 Nano‐Catalyst Mediated Oxidative Decarboxylation CH Acylation/Benzoylation of 3‐Alkylidene‐2‐Oxindoles With α‐Keto Acids

ABSTRACTWe successfully employed an environmentally friendly synthesized heterogeneous Fe2O3 nano‐catalyst‐mediated one pot approach to β CH bond acylation of 3‐Alkylidene‐2‐Oxindoles via oxidative decarboxylation of α‐keto acids. This method enabled the activation of Csp2‐H bonds, resulting in the formation of Csp2‐Csp2 single bonds and the generation of highly substituted derivatives of (E)‐2‐(2‐oxoindolin‐3‐ylidene)‐1‐aryl‐3‐substituted‐propane‐1,3‐diones and (E)‐3‐oxo‐2‐(2‐oxoindolin‐3‐ylidene)‐3‐substituted‐propanoates. The synthesized non‐toxic Fe2O3 nano‐material was meticulously characterized through FT‐IR, powdered XRD, HRTEM, EDX, BET, and ICP‐MS analyses. This heterogeneous catalyst can be conveniently recovered with a magnetic field and reused in subsequent reactions. This method offers excellent functional‐group tolerance, efficient substrate yield in less time, simple workup, and a recyclable catalyst.

Convenient Au@Ag Double‐Layer Nanoarray Fabricated by Rapid Thermal Annealing and Chemical Replacement Method for Surface‐Enhanced Raman Spectroscopy Sensing

Surface‐enhanced Raman spectroscopy (SERS) has a wide range of applications in molecular recognition, environmental pollutant detection, and other fields. However, the intensity and number of “hot spots” in 1D and 2D nanostructures are limited due to the scale‐dependent localized plasmonic effect of nanostructures, making it difficult to increase the detection limit. Herein, a kind of 3D substrate called Au@Ag double‐layer nanoarray (Au@Ag DLA) is prepared using rapid thermal annealing and chemical replacement methods. The energy‐dispersive spectrometer spectra confirm the successful growth of AgNPs on the gold nanoarray (Au SLA) by showing no presence of the copper element, indicating complete replacement of the Cu film deposited on Au SLA by Ag atoms. The detection limit of malachite green in Au@Ag DLA is 10−8 mol L−1, four and three orders of magnitude higher than that of Au SLA and AgNPs, respectively. This stronger SERS effect of Au@Ag DLA arises from the larger number of intense hot spots generated not only on the horizontal surface but also in the vertical direction. This finding provides a method for developing efficient and stable 3D SERS substrate, which can be utilized for the trace detection of water pollutants and pesticide residue.

Bifunctional MoO3-x/CuS Heterojunction Nanozyme-Driven "Turn-On" SERS Signal for the Sensitive Detection of Cerebral Infarction Biomarker S100B.

The use of nanozymes has become a promising auxiliary approach to "turn on" surface-enhanced Raman scattering (SERS) signals for the label-free detection of disease markers. Nevertheless, there are still major challenges to develop bifunctional nanomaterials with both excellent enzyme-like activity and high SERS performance. To this end, a novel Z-scheme MoO3-x/CuS heterojunction was first constructed as a powerful "two-in-one" substrate, which can not only catalyze leucomalachite green (LMG) to SERS-active malachite green (MG) but also serve as an efficient substrate to amplify the SERS signal of catalysate. Due to the strong interfacial coupling effect between the MoO3-x and CuS nanomaterial, which promoted the separation and transport of carriers in the heterojunction, the MoO3-x/CuS heterojunction showed higher peroxidase-like activity compared to individual components and the previously reported heterojunction nanozymes. Inspired by these results, a sandwich-type SERS immunoassay for the detection of the cerebral infarction biomarker S100 calcium-binding protein (S100B) was proposed based on the output signal of MG at 1620 cm-1. Furthermore, introducing the antifouling material chitosan on the surface of the MoO3-x/CuS heterojunction can effectively resist nonspecific protein adsorption and significantly improve the detection accuracy of the immunoassay. Therefore, the SERS immunoassay based on the MoO3-x/CuS heterojunction realized highly sensitive and selective detection of S100B in the concentration range of 0.001 to 100 ng/mL, with a low limit of detection of 0.47 pg/mL. The developed method has been successfully used for the accurate detection of S100B in clinical serum. The results showed that the level of S100B in the serum of cerebral infarction patients can be distinguished from those of healthy individuals and intracranial tumor patient controls. In addition, the acquired values of S100B in the serum of cerebral infarction patients based this strategy were well consistent with the results of electrochemiluminescence (ECL) detection with a relative error of less than ±7.3. It is expected that this work may open up a paradigm for improving detection sensitivity and accuracy for the early diagnosis and treatment monitoring of cerebral infarction in the clinic.

Ball-in-hole 3D nanoarchitectonics based on nanoporous Au film: Ultrasensitive, highly reliable, and reusable plasmonic platform

Hotspots have a locally enhanced electromagnetic field, effectively created by nanogaps in plasmonic metal nanostructures. The formation of uniform nanogaps, smaller than a few nanometers, allows for the detection of analytes at low concentrations and ensures reliable measurements across entire surface-enhanced Raman scattering (SERS) substrates. Despite the demand for SERS substrates in recent optical sensing applications, challenges remain in developing new techniques to maximize the number and optimize the size of nanogaps on large-area substrates. To address this issue, this study introduces a new ball-in-hole technique based on metal nanoarchitecture utilizing 3D nanoporous gold film (NPGF) and 0D gold nanoparticles (AuNPs). Using the ball-in-hole technique, 23 nm AuNPs are placed within the ∼ 28 nm mesopores of the metal film, creating uniform nanogaps of just a few nanometers that ensure consistent SERS signals across a large substrate area due to effective hotspots. The 23nmNP@NPGF substrate exhibited an enhancement factor (EF) of 1014 for 4-aminophenol (4-ATP), enabling detection at the attomole level. Moreover, the substrate retained its performance after five washing and reuse cycles, demonstrating excellent recyclability Finally, the substrate successfully detected femtomolar concentrations of key neurotransmitters like dopamine and glutamate, without requiring Raman reporters. This study presents a promising strategy for constructing metal nanoarchitectures with numerous hotspots, offering new possibilities for developing more efficient SERS substrates.

Facile preparation of three-dimensional copper foam incorporated with Au/CuO nanorods as a durable, reusable and efficient SERS substrate

An exceptionally durable and recyclable three-dimensional copper foam (CF) modified CuO nanorods with Au nanostructures on its surface is noticeable for the application in surface-enhanced Raman scattering (SERS). The utilization of CF as a 3D frame substrate allowed for the advancement of its beneficial characteristics, such as an increased surface-active area resulting from its high porosity and superior chemical/physical stability. By thermally oxidizing CF at a temperature of 500 °C, CuO nanorods were fabricated directly onto CF to exploit its advantageous properties, such as its high surface-active area and photodegradation effect. Following that, Au nanostructures were deposited onto the surfaces of CuO nanorods via photoreduction. The purpose of incorporating Au nanostructures was to optimize the SERS phenomenon, since Au exhibits high SERS efficiency and excellent surface stability. The Au/CuO@CF SERS substrate demonstrated the capability to measure MB at a concentration of 0.1 nM. Meanwhile, the recyclability of the Au/CuO@CF was evaluated by subjecting it to UV irradiation three times while utilizing MB samples. Subsequently, the Au/CuO@CF substrate's physical durability was evaluated via sandpaper abrasion test. In addition to confirming the long-term usability, the Au/CuO@CF demonstrated its resilience by maintaining good measurement performance even after being subjected to ambient air for up to 25 days.

An Effective and Controllable Platform with Ag-Stepped Nanocone Arrays Serving as Stable SERS Substrate

Facile, controllable and efficient SERS substrate preparation has always been the focus project in the SERS field, especially the solid-state nanoarray substrate. Herein, a simple and convenient method for preparing a series of homogeneous Ag-stepped nanocone arrays (Ag-SNCAs) using polymer nanocompression printing is presented. By analyzing the 1162[Formula: see text]cm[Formula: see text] characteristic peak of crystal violet (CV), the relative standard deviation (RSD) of the Raman intensity was only 4.71%. The detection limit of malachite green (MG) reached 0.001[Formula: see text]ppm, and it also has good SERS detection results for drug molecules such as melamine, methadone, morphine, methamphetamine and thiabendazole. This work provides a large area uniform and controllable preparation method of sharp cone nanostructure array, which has important application in plasmonic photocatalysis and deep light field regulations.

Stereo- and Regioselective Installation of Vinyl Sulfonyl Fluoride onto Indoles without Transition-Metal Catalyst.

Herein, we developed a practical method for synthesizing a class of novel and highly valuable indolyl vinyl sulfonyl fluorides. This protocol has carved out a path for constructing a broad range of vinyl sulfonyl fluorinated indoles with exclusive stereo- and regioselectivity through the Friedel-Crafts/elimination reaction without any transition-metal catalyst. This transformation features mild conditions, high efficiency, excellent selectivity, and rich substrate compatibility, highlighting its significant value in medicinal chemistry and many related disciplines.

Understanding the Functional Dynamics of the TokK Enzyme in Carbapenem Biosynthesis via MD Simulations and QM/MM Calculations.

Due to the recent surge in antibiotic resistance, developing novel antibiotics is the demand of the time, and thus, a precise understanding of the catalytic mechanisms of enzymes involved in antibiotic biosynthesis becomes crucial. Here, we present a comprehensive investigation into the catalytic mechanism of TokK, a freshly characterized B12-dependent RSMT enzyme that plays an important role in carbapenem biosynthesis. Using MD simulations, we show how the plasticity of the active site facilitates substrate recognition while the quantum mechanics/molecular mechanics calculations provide a detailed mechanistic understanding of the methyl transfer process, elucidating stereochemical preferences. Notably, we demonstrate the indispensable role of Trp215 in orchestrating the proper orientation of the 5'-dA• radical for efficient substrate methylation, which strongly correlates with the previous findings where the mutation of Trp215 has severely affected the enzyme activity.

Insights into Transient Dimerisation of Carnitine/Acylcarnitine Carrier (SLC25A20) from Sarkosyl/PAGE, Cross-Linking Reagents, and Comparative Modelling Analysis.

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping-pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC's functional dynamics and its structural parallels with other MC family members.

Hybrid lipid-AuNP clusters as highly efficient SERS substrates for biomedical applications

Although Surface Enhanced Raman Scattering (SERS) is widely applied for ultrasensitive diagnostics and imaging, its potential is largely limited by the difficult preparation of SERS tags, typically metallic nanoparticles (NPs) functionalized with Raman-active molecules (RRs), whose production often involves complex synthetic approaches, low colloidal stability and poor reproducibility. Here, we introduce LipoGold Tags, a simple platform where gold NPs (AuNPs) clusters form via self-assembly on lipid vesicle. RRs embedded in the lipid bilayer experience enhanced electromagnetic field, significantly increasing their Raman signals. We modulate RRs and lipid vesicle concentrations to achieve optimal SERS enhancement and we provide robust structural characterization. We further demonstrate the versatility of LipoGold Tags by functionalizing them with biomolecular probes, including antibodies. As proof of concept, we successfully detect intracellular GM1 alterations, distinguishing healthy donors from patients with infantile GM1 gangliosidosis, showcasing LipoGold Tags as advancement in SERS probes production.

Chiral Binaphthol-Catalyzed Enantioselective Conjugate Addition of Alkenyl and Arylboronic Acids to α,β-Unsaturated Cyclic N-Sulfonyl Ketimines.

The chiral binaphthol-catalyzed enantioselective conjugate addition of alkenylboronic acids and heteroarylboronic acids to cyclic N-sulfonyl ketimines is reported, providing the 1,4-addition products in high yields and moderate to excellent enantioselectivities (up to >99% ee). This mild, scalable catalytic system exhibits high efficiency and broad substrate scopes. Additionally, arylboronic acids were viable nucleophiles under more forcing conditions.

High-efficiency bioconversion of phytosterol to bisnoralcohol by metabolically engineered Mycobacterium neoaurum in a micro-emulsion system.

21-Hydroxy-20-methylpregn-4-en-3-one (4-HBC, bisnoralcohol) is a crucial intermediate for the synthesis of steroidal drugs. Significant challenges including by-products formation and poor substrate solubility were still confronted in its main synthetic route by microbial conversion from phytosterol. Construction of a direct bioconversion pathway to 4-HBC and an efficient substrate emulsion system is therefore urgently required. In this study, three novel isoenzymes of 3-ketosteroid-Δ1-dehydrogenase (KstD) and 3-ketosteroid 9α-hydroxylase (KsH) in Mycobacterium neoaurum were excavated and identified as KstD4, KstD5, and KsHA3. A strain capable of fully directing the synthesis of 4-HBC was metabolically engineered via serial genetic deletion combined with enhanced expression of cholesterol oxidase (ChOx2) and enoyl-CoA hydratase (EchA19). Moreover, a micro-emulsion system combined with soybean oil and hydroxypropyl-β-cyclodextrin improved substrate solubility and bioavailability. In batch fermentation, molar yield of 96.7% with 39.5 gL-1 4-HBC was obtained from 50 gL-1 phytosterol. Our findings demonstrate the potential for industrial-scale biosynthesis of 4-HBC.

N-carboxyacyl and N-α-aminoacyl derivatives of aminoaldehydes as shared substrates of plant aldehyde dehydrogenases 10 and 7

Aldehyde dehydrogenases (ALDHs) represent a superfamily of enzymes, which oxidize aldehydes to the corresponding acids. Certain families, namely ALDH9 and ALDH10, are best active with ω-aminoaldehydes arising from the metabolism of polyamines such as 3-aminopropionaldehyde and 4-aminobutyraldehyde. Plant ALDH10s show broad specificity and accept many different aldehydes (aliphatic, aromatic and heterocyclic) as substrates. This work involved the above-mentioned aminoaldehydes acylated with dicarboxylic acids, phenylalanine, and tyrosine. The resulting products were then examined with native ALDH10 from pea and recombinant ALDH7s from pea and maize. This investigation aimed to find a common efficient substrate for the two plant ALDH families. One of the best natural substrates of ALDH7s is aminoadipic semialdehyde carrying a carboxylic group opposite the aldehyde group. The substrate properties of the new compounds were demonstrated by mass spectrometry of the reaction mixtures, spectrophotometric assays and molecular docking. The N-carboxyacyl derivatives were good substrates of pea ALDH10 but were only weakly oxidized by the two plant ALDH7s. The N-phenylalanyl and N-tyrosyl derivatives of 3-aminopropionaldehyde were good substrates of pea and maize ALDH7. Particularly the former compound was converted very efficiently (based on the kcat/Km ratio), but it was only weakly oxidized by pea ALDH10. Although no compound exhibited the same level of substrate properties for both ALDH families, we show that these enzymes may possess more common substrates than expected.