Biosynthesis and characterization of silver nanoparticles using strawberry seed extract and evaluation of their antibacterial and antioxidant activities

Synthesis of nanomaterials is an emerging field due to their fascinating properties for applications in different field and green synthesis offers various advantages versus physical and chemical methods. Herein, green protocol has been adopted for the synthesis of silver nanoparticles (Ag NPs) using seeds extract of strawberry. The Ag NPs were characterized using advanced techniques comprising UV/Vis, XRD, FTIR, SEM, DLS and EDX. The λ_max for the Ag NPs was recorded at 405 nm. The functional groups present in the extract and involved in Ag ions reduction were determined using FTIR analysis. The SEM-EDX analysis confirmed the mono-dispersive nature of Ag NPs along with confirmation of elemental composition. The nanoparticles size distribution was recorded in 50-70 nm range. Bio-fabricated Ag NPs were appraised for antioxidant activity (DPPH with % inhibition 56.61 and ABTS with % inhibition 77.81) and antimicrobial activity, i.e., Escherichia coli, Salmonella typhimurium, Shigella sonnei, Halomonas halophile, Staphylococcus aureus and Bacillus subtilis. It is concluded that these synthesized NPs could probably be applied as potent antibacterial and antioxidant materials.     

Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

Background  

17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions.     

Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and M?ssbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.     

Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.     

Biosynthesis of aminoglycoside antibiotics: cloning,expression and characterisation of an aminotransferase involved in the pathway to 2-deoxystreptamine

The gene btrR from Bacillus circulans has been cloned and expressed and shown to produce a protein which catalyses the transamination of 2-deoxy-scyllo-inosose to give 2-deoxy-scyllo-inosamine, an intermediate in the biosynthesis of 2-deoxystreptamine.     

Expression,purification and biochemical characterization of Schizosaccharomyces pombe Mcm4, 6 and 7

Background

Hepatoma-derived growth factor (HDGF) is a protein which is highly expressed in a variety of tumours. HDGF has mitogenic, angiogenic, neurotrophic and antiapoptotic activity but the molecular mechanisms by which it exerts these activities are largely unknown nor has its biological function in tumours been elucidated. Mass spectrometry was performed to analyse the HDGFStrep-tag interactome. By Pull–down-experiments using different protein and nucleic acid constructs the interaction of HDGF and nucleolin was investigated further.

Results

A number of HDGFStrep-tag copurifying proteins were identified which interact with RNA or are involved in the cellular DNA repair machinery. The most abundant protein, however, copurifying with HDGF in this approach was nucleolin. Therefore we focus on the characterization of the interaction of HDGF and nucleolin in this study. We show that expression of a cytosolic variant of HDGF causes a redistribution of nucleolin into the cytoplasm. Furthermore, formation of HDGF/nucleolin complexes depends on bcl-2 mRNA. Overexpression of full length bcl-2 mRNA increases the number of HDGF/nucleolin complexes whereas expression of only the bcl-2 coding sequence abolishes interaction completely. Further examination reveals that the coding sequence of bcl-2 mRNA together with either the 5′ or 3′ UTR is sufficient for formation of HDGF/nucleolin complexes. When bcl-2 coding sequence within the full length cDNA is replaced by a sequence coding for secretory alkaline phosphatase complex formation is not enhanced.

Conclusion

The results provide evidence for the existence of HDGF and nucleolin containing nucleoprotein complexes which formation depends on the presence of specific mRNAs. The nature of these RNAs and other components of the complexes should be investigated in future.     

Xylose reductase and xylitol dehydrogenase activities of Candida guilliermondii as a function of different treatments of sugarcane bagasse hemicellulosic hydrolysate employing experimental design

The sugarcane bagasse hydrolysate, which is rich in xylose, can be used as culture medium for Candida guilliermondii in xylitol production. However, the hydrolysate obtained from bagasse by acid hydrolysis at 120°C for 20 min has by-products (acetic acid and furfural, among others), which are toxic to the yeast over certain concentrations. So, the hydrolysate must be pretreated before using in fermentation. The pretreatment variables considered were: adsorption time (15,37.5, and 60 min), type of acid used (H2So4 and H3Po4), hydrolysate concentration (original, twofold, and fourfold. concentrated), and active charcoal (0.5, 1.75 and 3.0%). The suitability of the pretreatment was followed by measuring the xylose reductase (XR) and xylitol dehydrogenase (XD) activity of yeast grown in each treated hydrolysate. The response surface methodology (2⁴ full factorial design with a centered face) indicated that the hydrolysate might be concentrated fourfold and the pH adjusted to 7.0 with CaO, followed by reduction to 5.5 with H₃PO₄. After that it was treated with active charcoal (3.0%) by 60 min. This pretreated hydrolysate attained the high XR/XD ratio of 4.5.     

The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster

BACKGROUND: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2x D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2x L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. RESULTS: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) (urdZ3, urdQ, urdR, urdS, urdT) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs (urdZ3, urdQ, urdR, urdS) as well as urdZ1, a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3, urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. CONCLUSIONS: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.     

Biosynthesis of the fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid and mechanism of self-resistance

Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure–activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in the HA cluster was characterized to be less sensitive to HA, and therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which was confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions, knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors.

The structural basis and self-resistance mechanism of fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid are uncovered.     

Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH₄)₂SO₄, 0.1 g/L of CaCl₂·2H₂O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K _M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10⁻² M (xylose) and 9.5×10⁻³ mM (NADPH); for XD, 1.6×10⁻¹ M (xylitol) and 9.9×10⁻² mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the V _max, some interference on the overall xylitol production by the yeast could be expected.     

Enhancement of the biochemical activity of some market antibiotics by chemical modification: Synthesis,characterization, and biochemical evaluation

The reaction of isatin with the Ampicillin gave the new compound: (6R)‐3,3‐dimethyl‐7‐oxo‐6‐(2‐(([E]‐2‐oxoindolin‐3‐ylidene)amino)‐2‐phenylacetamido)‐4‐thia‐1‐azabicyclo[3.2.0]hept ‐ane‐2‐carboxylic acid (HAI). The new complexes derived from HAI and Co(II), Ni(II), Cu(II), Eu(III), and Gd(III) were obtained in pure form. The obtained compounds were characterized by elemental analysis, FTIR, UV–Vis, Fluorescence, ¹HNMR, Mass spectra, DTA, TGA, Magnetic susceptibility, X‐ray, AAS, and the conductivity of 0.001 M in DMSO. The obtained data indicated the formation of the target complexes: [Co(HAI)(H₂O)(NO₃)]NO₃.4H₂O, [Ni(AI)(H₂O)₂]Cl.2H₂O, [Cu(AI)]Cl.H₂O, [Eu(AI)(H₂O)Cl]Cl.5H₂O and [Gd(AI)(H₂O)(NO₃)]NO₃.3H₂O. The ligation sites were predicted from the guide of the FTIR and thermal analysis meanwhile the stereochemistry was proved by the UV–Vis and magnetic moment. Co(II) and Ni(II) gave an octahedral structure while Cu(II) gave a square planar form. Molecular modeling, molecular mechanics, and DFT calculations were carried out for the synthesized compounds. The active lone pair and surface properties were obtained and discussed in the silico level. The x‐ray analysis indicates the nanoparticle behavior of the Cu‐AI complex with a monoclinic structure. The interactions of the synthesized complexes with FM‐DNA moiety were investigated through spectrometric titration (UV–vis. spectra) and by using fluorescence spectroscopy. The modes and binding affinities were evaluated and discussed using Benesi–Hildebrand method. Antimicrobial activities of the synthesized compounds have been screened using the disc diffusion method. HAI and Cu‐AI gave activity exceeded the Ampicillin. The docking work was carried using the targeting protein of Escherichia coli FabH (PDB code: 1HNJ). The obtained binding energy was compared and discussed in terms of the in vitro studies.     

Synthesis,characterization and antimicrobial activities of copper complexes derived from 4-aminoantipyrine derivatives

Cu(II) complexes have been synthesized from the Schiff base ligands derived from furfurlyidene-4-aminoantipyrine and aniline (L¹)/p-nitroaniline (L²)/p-hydroxyaniline (L³). They were characterized using analytical and spectral techniques. All the Cu(II) complexes exhibit square planar geometry. The X-band ESR spectra of the copper complexes in DMSO solution at 300 and 77 K were recorded and their salient features are reported. The in vitro biological screening effects of the investigated compounds were tested against the bacterial species, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa and fungal species, Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola and Candida albicans by serial dilution method. A comparative study of inhibition values of the Schiff base ligands and their complexes indicate that the complexes exhibit higher antimicrobial activity than the Schiff base ligands. Superoxide dismutase and reducing power activities of the copper complexes have also been studied. Depending on the molecular structure, the [CuL²(OAc)₂] complex possess promising SOD mimetic activities.     

Twins in polyhedral 26-facet Cu7S4 cages: synthesis, characterization and their enhancing photochemical activities

Unusual polyhedral 26-facet Cu(7)S(4) cages wholly exposed with amazingly unique nanotwinned structures as building blocks were successfully synthesized via a facile ethanol-assisted sacrificial Cu(2)O templates approach. Furthermore, a solvent-controlled fabrication of polyhedral copper sulfides (Cu(7)S(4) and CuS) with different stoichiometries and microstructures can be artificially achieved, which is determined by the intrinsic difference of the surface states of Cu(2)O templates. Structural and morphological evolutions were investigated by X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and field-emission scanning electron microscopy (FESEM). Stoichiometric-dependent characteristics were demonstrated in the UV-vis absorption investigations. The as-prepared Cu(7)S(4) microcages exhibit higher photocatalytic activity for enhancing degradation of methylene blue, which might be attributed to their special nanotwinned building blocks. This study is of great importance in "bottom-up" assembly of unusual ordering hollow copper sulfides superstructures, but also offers a good opportunity to understand the fundamental significance of nanotwinned structures for their potential applications.     

Nickel(II) complexes of polyhydroxybenzaldehyde N4-thiosemicarbazones: synthesis, structural characterization and antimicrobial activities

Nickel(II) complexes with 2,3-dihydroxybenzaldehyde N4-substituted thiosemicarbazone ligands (H₃L¹–H₃L⁴) have been synthesized and characterized with the aim of evaluating the effect of N4 substitution in the thiosemicarbazone moiety on their coordination behavior and biological activities. Two series of nickel(II) complexes with the general formulae [Ni(H₃L)(H₂L)]ClO₄ and [Ni₂(HL)₂] were characterized by analytical and spectral techniques. The molecular structure of one of the complexes, namely, [Ni(H₃L⁴)(H₂L⁴)]ClO₄ was established by single crystal X-ray diffraction studies. The crystal structure of this complex revealed that two H₃L⁴ ligands are coordinated to nickel(II) in different modes; one as a neutral tridentate ONS ligand and the other is as a monoanionic tridentate (ONS^?) ligand. The antimicrobial activities of the compounds were tested against 25 bacterial strains via the disc diffusion method, and their minimum inhibitory concentration (MIC) and minimum microbicidal concentration were evaluated using microdilution methods. With a few exceptions, most of the compounds exhibited low-to-moderate inhibitory activities against the tested bacterial strains. However, the complexes [Ni₂(HL³)₂] (7) and [Ni₂(HL⁴)₂] (8) indicated higher inhibitory activity against Salmonella enterica ATCC 9068 (MIC values 15.7 and <15.7 μg/ml, respectively), compared with gentamicin as the positive control (MIC 25 μg/ml). Complex (7) also inhibited Streptococcus pneumoniae more efficiently (MIC 31.2 μg/ml), compared with gentamicin (MIC > 50 μg/ml). The toxicities of the compounds were tested on brine shrimp (Artemia salina), where no meaningful toxicity level was noted for both the free ligands and the complexes. The cytotoxicities of the compounds on cell viability were determined on MCF7, PC3, A375, and H413 cancer cells in terms of IC₅₀; complexes [Ni(H₃L³)(H₂L³)]ClO₄ (3), [Ni₂(HL³)₂] (7) and [Ni₂(HL⁴)₂] (8) exhibited significant cytotoxicity on the tested cell lines.     

Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial compound FR900098

The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent antibacterial and antimalarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.     

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg_prot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg_prot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.     

Comparative and evolutionary studies of mammalian arylsulfatase and sterylsulfatase genes and proteins encoded on the X-chromosome

At least 19 sulfatase genes have been reported on the human genome, including four arylsulfatase (ARS) genes (ARSD; ARSE; ARSF; ARSH) and a sterylsulfatase (STS) gene located together on the X-chromosome. Bioinformatic analyses of mammalian genomes were undertaken using known human STS and ARS amino acid sequences to study the evolution of these genes and proteins encoded on eutherian and marsupial genomes. Several domain regions and key residues were conserved including signal peptides, active site residues, metal (Ca²⁺) and substrate binding sequences, transmembranes and N-glycosylation sites. Phylogenetic analyses describe the relationships and potential origins of these genes during mammalian evolution. Primate ARSH enzymes lacked signal peptide sequences which may influence their biological functions. CpG117 and CpG92 were detected within the 5′ region of the human STS and ARSD genes, respectively, and miR-205 within the 3′-UTR for the human STS gene, using bioinformatic methods A proposal is described for a primordial invertebrate STS-like gene serving as an ancestor for unequal cross over events generating the gene complex on the eutherian mammalian X-chromosome.     

A beta-glucosidase from Sclerotinia sclerotiorum: biochemical characterization and use in oligosaccharide synthesis

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one beta-glucosidase (beta-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that beta-glu x may be a homodimer. For p-nitrophenyl beta-d-glucopyranoside hydrolysis, apparent Km and Vmax values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55-60 degrees C and pH 5.0, respectively. beta-Glu x was strongly inhibited by Fe2+ and activated about 35% by Ca2+. beta-Glu x possesses strong transglucosylation activity in comparison with commercially available beta-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50 degrees C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and 3C nuclear magnetic resonance spectroscopy.