A Novel Cold-Active and Alkali-Stable β-Glucosidase Gene Isolated from the Marine Bacterium <Emphasis Type="BoldItalic">Martelella mediterranea</Emphasis>

A β-glucosidase gene designated gluc3m was cloned through construction of a genomic library of Martelella mediterranea 2928. The gluc3m consisted of 2,496 bp and encoded a peptide of 832 amino acids that shared the greatest amino acid similarity (59%) with a β-glucosidase of family 3 glycoside hydrolase from Agrobacterium radiobacter K84. The optimum reaction temperature and pH of Gluc3M were 45 °C and 8.0, respectively. The K _m and V _max for p-nitrophenyl-β-d-glucopyranoside were 0.18 mg/ml and 196.08 μmol/min/mg enzyme, respectively. Gluc3M was found to be highly alkali stable, retaining 80% of its maximum enzymatic activity after treatment with pH 11.0 buffers for 24 h. Furthermore, the activity of Gluc3M improved remarkably in the presence of univalent metal ions, whereas it was inhibited in the presence of divalent ions. Gluc3M also exhibited significant activities toward various substrates including pNPGlu, pNPGal, salicin, and konjac powder. It is important to note that Gluc3M is a cold-active enzyme that showed over 50% of the maximum enzymatic activity at 4 °C. SWISS-MODEL revealed that the amino acids near the conserved domain SDW of Gluc3M contributed to the cold-active ability. Based on these characteristics, Gluc3M has the potential for use in additional studies and for industrial applications.     

A Moderately Thermostable Alkaline Phosphatase from Geobacillus thermodenitrificans T2: Cloning, Expression and Biochemical Characterization

A gene-encoding alkaline phosphatase (AP) from thermophilic Geobacillus thermodenitrificans T2, termed Gtd AP, was cloned and sequenced. The deduced Gtd AP protein comprises 424 amino acids and shares a low homology with other known AP (<35% identity), while it exhibits the conservation of the active site and structure element of Escherichia coli AP. The Gtd AP protein, without a predicted signal peptide of 30 amino acids, was successfully overexpressed in E. coli and purified as a hexa-His-tagged fusion protein. The pH and temperature optima for purified enzyme are 9.0 and 65 °C, respectively. The enzyme retained a high activity at 45–60 °C, while it could be quickly inactivated by a heat treatment at 80 °C for 15 min, exhibiting a half-life of 8 min at 70 °C. The K _m and V _max for pNPP were determined to be 31.5 μM and 430 μM/min at optimal conditions. A divalent cation is essential, with a combination of Mg²⁺ and Co²⁺ or Zn²⁺ preferred. The enzyme was strongly inhibited by 10 mM ethylenediaminetetraacetic acid (EDTA) and vanadate but highly resistant to urea and dithiothreitol. The properties of Gtd AP make it suitable for application in molecular cloning or amplification.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.     

Nanosized cation exchanger for the electrophoretic separation and preconcentration of catecholamines and amino acids

The first example of application of nanosized polystyrene-based cation exchanger (NSCE) with sulfo groups as a dynamic coating of capillary walls was demonstrated. The conditions of dynamic coating formation were optimized and ensured the long-term stability of the coating. Capillary-to-capillary and day-to-day repeatabilities were 4% and 3%, correspondingly. The NSCE coating stability at various pH and influence of pH on the EOF mobility were investigated. The developed NSCE-modified coated capillaries provided improved resolution (R_s = 0.9–3.2 for catecholamines and R_s = 1.7–2.8 for amino acids) and efficiencies (330–520 ×10³ t.p./m) of basic analytes, which are 1.5 times higher compared to untreated capillary. The optimized conditions were as follows: 50 mM phosphate buffer solution at pH 2.2 with 5 μM NSCE. The effect of the NSCE concentration in BGE on the electrophoretic mobilities of the analytes was investigated. The various online concentration techniques were tested in order to decrease the LODs. The simultaneous application of NSCE capillaries and field-amplified sample stacking provided the lowest LODs of catecholamines and amino acids and allowed to determine these analytes in human urine.     

A Novel Catalytic Function of Synthetic IgG‐Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine

The synthetic IgG‐binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine‐utilizing luciferase. The Z domain derivatives (ZZ‐gCys, Z‐gCys and Z‐domain) were purified and the luminescence properties were characterized by comparing with coelenterazine‐utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ‐gCys (100%) > Z‐gCys (36.8%) > Z‐domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ‐gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z‐gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG‐binding region of the Z domain.     

An Optimized Firefly Luciferase Bioluminescent Assay for the Analysis of Free Fatty Acids

A firefly luciferase (LUC)‐based bioluminescent assay for total free fatty acids (FFA) is presented. It is based on LUC's capability of converting FFA into fatty acyl‐adenylates with consumption of adenosine 5′‐triphosphate (ATP). Since ATP is a cosubstrate in LUC's bioluminescent reaction, together with firefly d ‐luciferin (d ‐LH₂) and atmospheric oxygen (O₂), any reduction in the assay's ATP content will lead to a decrease in the bioluminescent signal, which is proportional to the amount of FFA. Using FFA mixtures containing myristic (14:0), palmitic (16:0), stearic (18:0), oleic (18:1) and arachidonic acid (20:4) in ethanol, the assay was optimized through statistical experimental design methodology, namely fractional factorial (screening) and central composite (optimization) designs. The optimized method requires 2 μL of sample per tube in a final reaction volume of 50 μL. It is linear in the concentration range from 1 to 20 μm , with limits of detection (LOD) and quantitation (LOQ) of 1.3 and 4.5 μm , respectively. The method proved to be simple to perform, demands low reagent volumes, it is sensitive and robust and may be adapted to high‐throughput screening.     

Green micellar stability-indicating high-performance liquid chromatography method for determination of rupatadine fumarate in the presence of its main impurity desloratadine: Oxidative degradation kinetics study

A green micellar stability-indicating high-performance liquid chromatography method was developed for rupatadine fumarate determination in existence with its main impurity desloratadine. Separation was attained using Hypersil ODS column (150 × 4.6 mm, 5 μm), the micellar mobile phase consisted of 0.13 M sodium dodecyl sulfate, 0.1 M disodium hydrogen phosphate adjusted by phosphoric acid to pH 2.8 and 10% n-butanol. The column was maintained at 45^◦C and detection was carried out at 267 nm. A linear response was achieved over the range of 2–160 μg/ml for rupatadine and 0.4–8 μg/ml for desloratadine. The method was applied for rupatadine determination in alergoliber tablets and alergoliber syrup without the interference of methyl paraben and propyl paraben present as main excipients. Rupatadine fumarate revealed pronounced susceptibility to oxidation; further study of oxidative degradation kinetics was carried out. Rupatadine was found to follow pseudo-first-order kinetics when exposed to 10% H₂O₂ at 60 and 80°C and the activation energy was found to be 15.69 Kcal/mol. At a lower temperature (40°C), degradation kinetics regression was best fitted as a polynomial quadratic relationship, thus rupatadine oxidation at a lower temperature tends to adopt a second-order kinetics rate. Oxidative degradation product structure was revealed using infrared and found to be rupatadine N-oxide at all temperature values.     

Single-walled carbon nanotube-arrayed microelectrode chip for electrochemical analysis

The development of a single-walled carbon nanotube (SWCNT)-arrayed microelectrode chip is reported here. SWCNT-arrayed electrodes were formed directly on Pt surfaces, and were also arrayed on the chip. The electrochemical characteristics of the devices were investigated using potassium ferricyanide, K₃[Fe(CN)₆] in connection with cyclic voltammetry (CV). The electrochemical signals of electro-active amino acids; L-Tyrosine (Tyr), L-Cysteine (Cys) and L-Tryptophan (Trp) were detected using differential pulse voltammetry (DPV). The chip operated at a lower oxidation potential (vs. Ag/AgCl) compared with conventional carbon and Pt disc electrodes in 50 mM phosphate buffer solution (PBS, pH 7.4). The linear response was observed between 0.1–1 μM and 100 μM for the amino acids with correlation coefficients higher than 0.99. The electrochemical measurements of K₃[Fe(CN)₆] and amino acids revealed that the peak current intensities using SWCNT-arrayed electrodes were about 100-fold higher than those using bare Pt-arrayed microelectrodes. Additionally, the surface area dependence of the peak current responses was plotted. We concluded that our chips with SWCNT-arrayed microelectrodes provided a promising platform for electrochemical applications.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel Pyrazolo[3,4‐d]pyrimidines as Potential Telomerase Inhibitors

Series of novel pyrazolo[3,4‐d]pyrimidines as potential telomerase inhibitors were synthesized. Results of the antitumor assay indicated that compounds 4b , 5a – b , 13b , c , and 14a , b exhibited the most potent activity (IC₅₀ from 39 to 43 μM) against Ehrlich ascites carcinoma cells (EAC). Also, the newly synthesized compounds were examined for telomerase inhibition by the known a TRAP assay. The results showed that compound 13c has remarkable inhibition activity with IC₅₀ value of 30 μM. On the other hand, computational studies were performed to the titled compounds to get insight in their degree of recognition with the conserved amino acids of the telomerase enzyme active site (code: 3DU6) as promising lead in the cancer cure era.     

Purification and Properties of a Psychrotrophic <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K _m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.     

Quantum Yields and Kinetics of the Firefly Bioluminescence Reaction of Beetle Luciferases

Quantum yields of firefly bioluminescence reactions were determined for beetle luciferases from the three main families of luminous beetles emitting different bioluminescence colors. Quantum yield (QY) was significantly correlated with luminescence spectrum. The green light-emitting luciferase of the Brazilian click beetle, Pyrearinus termitilluminans, whose luminescence spectrum had the shortest peak wavelength of all the luciferases investigated, had the highest QY (0.61). Mutant analyses of active site-substituted Pyrocoelia miyako luciferases showed that, although k_cat was decreased by the mutations, the QY was not significantly affected.     

Establishing a sensitive capillary electrophoresis‐UV method for direct determination of amino acids to evaluate vinegar quality

In this study, the capillary electrophoresis method with ultraviolet detection was established to directly determine amino acids in vinegar, according to the coordination interaction between amino acids (AAs) and copper ions. The online sweeping technique was combined to improve the detection sensitivity. The quality of vinegar was evaluated with AAs as parameters by United Nations Food Agriculture Organization/Word Health Organization AAs model and principal component analysis. Optimum conditions were obtained under 50 mM CuSO₄ and adjusted pH 4.40 with 8 mM acetate, 70 s injection time, 22.5 kV separation voltage, and 254 nm detected wavelength. Method validation, indicating good linearity (R² > 0.9989), precision with an RSD less than 8.0% (n = 5), LOD (0.13–0.25 μg/mL), LOQ (0.43–0.83 μg/mL) and recovery (80.5–112.6%). Under the optimal conditions, AAs in vinegar can be directly separated which is propitious for the quality evaluation of vinegar.     

Cloning and Identification of a Novel P-II Class Snake Venom Metalloproteinase from <Emphasis Type="Italic">Gloydius halys</Emphasis>

Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation–inhibition ability.     

Uniform In2S3 octahedron-built microspheres: Bioinspired synthesis and optical properties

Uniform In₂S₃ octahedron-built microspheres were synthesized by using a mild hydrothermal treatment in the presence of L-glutamic acid at 180 °C. The microsphere with an average size of 5 μm was composed of interconnected octahedrons with diameters in the range from 100 to 150 nm. The products were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and X-ray photoelectron spectroscopy (XPS). The synthesis of various hollow and solid sphere structures and flower structures was achieved using different amino acids. This is the first report on synthesizing In₂S₃ nanostructures using different amino acids to modulate the morphology of the final products. Primary photoluminescence studies on the prepared In₂S₃ microspheres show promising results.     

Some transition metal ions complexes of tricine (Tn) and amino acids: pH-titration,synthesis and antimicrobial activity

Equilibrium studies have been carried out on complex formation of M(II) (M = Co(II), Cu(II) and Zn(II)) with tricine (Tn) and L = amino acids in aqueous solution, at 25 °C and ionic strength of I = 0.1 M (NaNO₃). The ternary complexes of amino acids are formed by simultaneous reactions. The concentration distribution of the complexes is evaluated. The solid complexes of [M(II)–Tn–Histidine (Hist)] have been synthesized and characterized by elemental analysis, infrared, magnetic and conductance measurements. The synthesized complexes have been screened for their antibacterial activities and the complexes show a significant antibacterial activity against four bacterial species: Staphylococcus aureus (Gram +ve), Streptococcus pyogenesr (Gram +ve), Serratia marcescens (Gram −ve) and Escherichia coli (Gram −ve). The activity increases by increasing the concentration of the complexes.