Search

Bacterial infections during wound healing impede the healing process and trigger local or systemic inflammatory reactions. Consequently, there is an urgent need to develop a new material with antimicrobial and antioxidant properties to promote infected wound healing. A synergistically antimicrobial and antioxidant hyaluronic acid hydrogel (HMn) is prepared by employing MnO 2 nanosheets into 4ARM-PEG5000-SH crosslinked methacrylated hyaluronic acid (HAMA) network. The coordination between sulfhydryl groups of 4ARM-PEG5000-SH and MnO 2 nanosheets ensures entrapment of the nanosheets within the hydrogel, while the interaction between 4ARM-PEG5000-SH and HAMA results in facile gelation through thiol-ene click reaction. MnO 2 nanosheets exhibit strong photothermal properties and reactive oxygen species (ROS) scavenging abilities, while hyaluronic acid promotes wound healing. When subjected to near-infrared (NIR) irradiation, the HMn achieves a bactericidal rate of 95.24 % for Staphylococcus aureus and nearly 100 % for Escherichia coli. In animal experiments, treatment with the HMn under NIR irradiation results in the best wound healing outcomes. Both in vitro and vivo biocompatible assays demonstrate that the HMn has rarely cell cytotoxicity and tissue damage. The HMn is easy to prepare and has good biocompatibility as well as efficient antibacterial and antioxidant properties, providing a novel method for the treatment of infected wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acyl-coA oxidase (ACO) is an important flavoenzyme responsible for the first step of peroxisomal fatty acid β-oxidation. In this study, the roles of Tyr232 and Tyr401 in flavin adenine dinucleotide (FAD) binding and enzyme catalysis of ACO were explored using site-directed mutagenesis. For mutant proteins, different levels of activity loss were observed. Wavelength scanning of Y232 and Y401 mutant proteins indicated that there is no FAD binding in Y401S and Y401G mutant ACO. Structure analysis indicated that the phenolic hydroxyl and benzene ring of the side chain could stabilize FAD binding through hydrogen bonds network and hydrophobic pocket formation. These results indicated that these two tyrosine residues play a critical role in the FAD binding of ACO.

Heterologous hosts are important platforms for engineering natural product biosynthesis. Escherichia coli is such a host widely used for expression of various biosynthetic enzymes. While numerous studies have been focused on optimizing the expression conditions for desired functional proteins, this work describes how supplement of exogenous nutrients into the fermentation broth influences the formation of natural products in E. coli. A type III polyketide synthase gene stts from Streptomyces toxytricini NRRL 15443 was heterogeneously expressed in E. coli BL21(DE3). This enzyme uses five units of malonyl-CoA to generate a polyketide 1,3,6,8-tetrahydroxynaphthalene, which can be spontaneously oxidized into a red compound flaviolin. In this work, we manipulated the fermentation broth of E. coli BL21(DE3)/pET28a-stts by supplying different nutrients including glucose and sodium pyruvate at different concentrations, from which six flaviolin derivatives 1-6 were produced. While addition of glucose yielded the production of 1-4, supplement of sodium pyruvate into the induced broth of E. coli BL21(DE3)/pET28a-stts resulted in the synthesis of 5 and 6, suggesting that different nutrients may enable E. coli to generate different metabolites. These products were purified and structurally characterized based on the spectral data, among which 2-6 are novel compounds. These molecules were formed through addition of different moieties such as acetone and indole to the flaviolin scaffold. The concentrations of glucose and sodium pyruvate and incubation time affect the product profiles. This work demonstrates that supplement of nutrients can link certain intracellular metabolites to the engineered biosynthetic pathway to yield new products. It provides a new approach to biosynthesizing novel molecules in the commonly used heterologous host E. coli.

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6'-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.

It is shown that the nonlinear optical phenomenon known as second-harmonic generation can be used for label-free, time-resolved study of the transport of molecules through living cell membranes. The adsorption and transport of a 300-Da molecular-mass hydrophobic ion at the Escherichia coli membrane is observed. Remarkably, at low ion concentrations, the second-harmonic generation technique clearly exposes a multistep molecular transport process: Transport of the molecular ion across the outer and cytoplasmic membranes of the Gram-negative bacteria is recorded, in sequence, in time. Fitting of the data to a multiprocess kinematic model reveals that the transport of this hydrophobic ion through the outer membrane is much faster than through the cytoplasmic membrane, likely reflecting the effectiveness of ion transport porins. The observations illustrate an experimental means for studying the interactions of small molecules with cell membranes., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from A. ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe(2)S(2)] cluster of the protein.

His-tags are widely used for the purification of recombinant proteins. High-cost carriers functionalized with nickel ions are commonly required for the selective immobilization of His-tagged enzymes. In this study, His-tags of varying lengths were fused to the N-terminus of D-amino acid oxidase (DAO) from Trigonopsis variabilis. The attachment of a His6 tag significantly improved the solubility of the recombinant DAO expressed in Escherichia coli. By modulating the tag lengths, a better balance between cell growth and protein solubility was achieved, resulting in a higher volume activity (His3). Furthermore, the fusion of longer tags (His6 and His9) facilitated the rapid immobilization of DAOs onto a commercial epoxy carrier without metal bearing, resulting in more selective immobilization. In conclusion, the modulation of His-tag length was preliminarily demonstrated as a simple and cost-effective approach to achieve efficient expression, as well as fast and selective immobilization of DAO. [ABSTRACT FROM AUTHOR]

Abstract: A putative type III polyketide synthase (PKS) gene, csyA, was cloned from Aspergillus oryzae. It was ligated into pET28a and expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. CsyA was identified as a pyrone synthase which has displayed broad substrate specificity toward fatty acyl-CoA starter units to yield triketide and tetraketide pyrones. It synthesizes triketide pyrones by adding two units of malonyl-CoA to the short chain starter units (C4–C8 CoAs), while yields both triketide and tetraketide pyrones from longer fatty acyl starters (C10–C18 CoAs). A series of fatty acyl N-acetylcysteamine thioesters (SNACs) (C5–C9) including odd-numbered fatty acyl starters were synthesized, all of which can be accepted by CsyA as the starter units to synthesize pyrones, indicating that this enzyme has broad substrate specificity toward fatty acyl starters and can be used to synthesize some unnatural pyrones. The optimal temperature and pH for CsyA are 55°C and 7.5, respectively. Under the optimal enzymatic conditions, CsyA exhibits high activity toward C6 and C7 starter units, and the k cat/K m values for hexanoyl- and heptanoyl-SNAC are 291.21 and 1615.58s−1 M−1, respectively. In addition to its in vitro activity, CsyA can also take fatty acid biosynthetic intermediates to synthesize a series of fatty acyl-primed pyrones in E. coli cells. [Copyright &y& Elsevier]

Abstract: IscA was proposed to be involved in the iron–sulfur cluster assembly in Acidithiobacillus ferrooxidans encoded by the iscSUA operon, but the role of IscA in the iron–sulfur cluster assembly still remains controversial. In this study, the IscA from A. ferrooxidans ATCC 23270 was successfully expressed in Escherichia coli, and purified by affinity chromatography to homogeneity. To our surprise, the purified IscA was observed to be an iron–sulfur protein according to MALDI-TOF-MS and spectra results, which was capable of recruiting intracellular iron and sulfur and hosted a stable [Fe4S4] cluster. Site-directed mutagenesis for the protein revealed that Cys35, Cys99 and Cys101 were in ligating with the [Fe4S4] cluster. The [Fe4S4] cluster could be assembled in apoIscA with Fe2+ and sulfide in vitro. The IscA from A. ferrooxidans may function as a scaffold protein for the pre-assembly of Fe–S cluster and then transfer it to target proteins in A. ferrooxidans. [Copyright &y& Elsevier]

Abstract: Rusticyanin is a small blue copper protein isolated from Acidithiobacillus ferrooxidans with extreme acid stability and redox potential. The protein is thought to be a principal component in the iron respiratory electron transport chain in this microorganism, but its exact role in electron transfer remains controversial. The gene of rusticyanin was cloned then overexpressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. It was reported that Cys138, His85 and His143 were important residues for copper binding, but the significance of Cys138 was not verified so far. We constructed the mutant expression plasmids of these three residues using site-directed mutagenesis. Mutant proteins were expressed in E. coli and purified with a nickel metal affinity column. The EPR and atomic absorption spectroscopy results confirmed that Cys138 was crucial for copper binding. Removal of the sulfhydryl group of Cys138 resulted in copper loss. Mutations of His85 and His143 showed little effect on copper binding. [Copyright &y& Elsevier]

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the β-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat peroxisomal acyl-CoA oxidase I into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5′ end of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal-affinity column in 90% yield to apparent homogeneity. The specific activity of the purified His-tagged rat peroxisomal acyl-CoA oxidase I was 1.5μmol/min/mg. It has been proposed that Glu421 is a catalytic residue responsible for deprotonation of α-proton of acyl-CoA substrate. We constructed four mutant expression plasmids of the enzyme, pACO(E421D), pACO(E421A), pACO(E421Q), and pACO(E421G) using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal-affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Glu421 is a catalytic residue of rat peroxisomal acyl-CoA oxidase I. Our overexpression in E. coli and one-step purification of the highly active N-terminal His-tagged rat peroxisomal acyl-CoA oxidase I greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by peroxisomal acyl-CoA oxidase I. [Copyright &y& Elsevier]

Mitochondrial 3-ketoacyl-CoA thiolase is a key enzyme for the β-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported. We cloned a cDNA of rat mitochondrial 3-ketoacyl-CoA thiolase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5′ end of the gene. The cloned cDNA was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 92% yield to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase was 25 U/mg. It has been proposed that His352 is a catalytic residue responsible for activation of coenzyme A by deprotonation of a sulfhydryl group. We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that His352 is a catalytic residue of rat mitochondrial 3-ketoacyl-CoA thiolase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial 3-ketoacyl-CoA thiolase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by 3-ketoacyl-CoA thiolase. [Copyright &y& Elsevier]

Congo red (CR) is a hazardous pigment, posing increasing dangerous to the environment and human health. However, the in-situ detection of CR in living cells has not been reported, as far as we know. Here, negatively-charged green-emitting Ca, N, S-doped carbon dots (Mis-mPD-CDs) were fabricated from plant and m-phenylenediamine (mPD) by facile one-step hydrothermal carbonization. Mis-mPD-CDs were capable of rapidly detecting CR on the basis of their fluorescence quenching by CR due to the inner filter effect. This CR detection based on Mis-mPD-CDs displayed a linear range of 0.2–1.2 μM and a low limit of detection (58 nM), and was not interfered by metal ions, important biological molecules, and other dyes, showing high sensitivity and selectivity. More interestingly, Mis-mPD-CDs can rapidly enter and label animal cells (A549, 4T1, and HUVEC), fungi (S. cerevisiae , C. albicans , and T. reesei), and bacteria (E. coli and S. aureus) for long term with high stability and appealing biocompatibility. Based on these compelling characteristics, we applied Mis-mPD-CDs for sensing and imaging CR in living cells (A549, C. albicans , E. coli, and S. aureus) and zebra fish. On the other hand, the quantitative detection of CR by Mis-mPD-CDs was realized in real samples like fish tissues and industrial wastewater. This is the first report on applying CDs for rapid CR detection in living cells and in vivo. Mis-mPD-CDs provides a novel efficient platform for probing intracellular CR, expanding the applications of CDs as biosensors for toxic dyes. [Display omitted] • Mis-mPD-CDs were hydrothermally synthesized from plant and mPD. • Mis-mPD-CDs can rapidly detect Congo red (CR) with high sensitivity and selectivity. • Mis-mPD-CDs can rapidly image universal cells for long time with great stability and safety. • Mis-mPD-CDs were applied for sensing CR in living cells and zebra fish. • Mis-mPD-CDs were used for the quantitative assay of CR in fish tissues and wastewater. [ABSTRACT FROM AUTHOR]