A Practical System for High-Throughput Screening of Mutants of <Emphasis Type="Italic">Bacillus fastidiosus</Emphasis> Uricase

For high-throughput screening (HTS) of Bacillus fastidiosus uricase mutants, a practical system was proposed. By error-prone PCR with final 1.5 mM MnCl₂, two focused libraries of mutants for A1-V158 and V150-D212 were generated separately. After induced expression of individual clones in 48-well microplates, Escherichia coli cells (BL21) were lyzed by 1.0 M Tris-HCl at pH 9.0 in 96-well microplates at 25 °C for 7.5 ~ 10.5 h; uricase reaction was continuously monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm to estimate V _m/K _m by kinetic analysis of reaction curve for comparison. V _m/K _m was resistant to initial uric acid levels with an upper limit 3-fold over that of initial rates. By receiver-operator-characteristic analysis of the recognition of the one of higher activity in uricase pair whose specific activity ratio was 1.8 or 3.3, the area-under-the-curve was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was thus developed to recognize positive mutants of 1-fold higher activity. Indeed, mutant L171I/Y182F/Y187F/A193S of higher activity but lower thermostability at pH 7.4 and mutant V144A of higher activity and consistent thermostability were discovered. Therefore, the proposed system was practical for HTS of uricase mutants.     

Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from <Emphasis Type="Italic">Musca domestica</Emphasis> Larva

Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of V_max and K_m of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.     

Decomposition and Debromination of Monobromoacetic Acid by Radio Frequency Discharge in an Aqueous Solution

In this study, efficient decomposition and debromination of monobromoacetic acid (MBAA) induced by radio frequency discharge in an aqueous solution in the concentration range from 0.1 to 8.0 mM were investigated. The decomposition and debromination intermediate byproducts were analyzed by ion chromatography. The experimental results showed that the decay of MBAA followed first-order kinetics. Increasing pH and adding organic additives to the solution enhanced MBAA removal and debromination. Acetic acid, bromate ion, oxalic acid and formic acid were determined as the major intermediate byproducts. Final products were bromide ion and carbon dioxide. Hydrated electrons are the primary species for the debromination and reactive oxygen species are the ones for the decomposition. A probable reaction pathway was proposed. The present study may provide a promising alternative for the complete mineralization of MBAA.     

Ring-contraction from benzo[b][1,4]thiazines to benzo[d]thiazolines induced by oxygen

An unexpected ring-contraction from benzo[b]pyrazino[1,2-d][1,4]thiazine-1,4-diones (6) to benzo[4,5]thiazolo[3,2-a]pyrazine-1,4-diones (7) has been developed. The preliminary mechanistic studies showed the transformation contained two independent steps: the first step is the formation of a Michael adduct upon the addition of the protic solvent, in the presence of base, to the C-2-C-3 double bond of compound 6, and the second step is a ring-contraction induced by oxygen via the migration of sulfur atom from C-2 to C-3 position. And its scope is also studied.     

Synthesis of polysubstituted 5-hydroxyhydantoins via ring-opening of isatins

A simple and efficient tandem reaction approach was developed for the synthesis of 5-hydroxyhydantoins from one-pot reaction of isatins, phthalic anhydride or succinic anhydride, and 1,3-dimethylurea (1,3-diethylurea). The products were gained through the ring-opening of isatins process. The advantages of this report are simple operation, mild reaction conditions, good yields and easily available raw materials. It was very important for us to obtain the intermediate product and that provided a solid basis for the correct interpretation of the reaction mechanism.     

Tea-leaves based nitrogen-doped porous carbons for high-performance supercapacitors electrode

Journal of Solid State Electrochemistry - Nitrogen-doped porous activated carbons have been fabricated through a simple and efficient carbonization method at 700&nbsp;°C with the waste...     

Patterning of MgO epitaxial films on LaAlO3 substrate using photosensitive sol-gel method

Journal of Sol-Gel Science and Technology - Herein, the chelated precursor solution (Mg/BzAc) was synthesized using magnesium acetate as raw material and benzoylacetone as chemical modifier. The...     

In situ synthesis of CdS/ZnS composite nanoparticles from ZIF-8 for visible light disposal of Cr(VI)

Journal of Sol-Gel Science and Technology - Zeolitic Imidazolate Framework (ZIF-8) micropolyhedra were employed as a raw material for the synthesis of fine cadmium sulfide/zinc sulfide (CdS/ZnS)...     

A Furan-based Phosphaphenanthrene-containing Derivative as a Highly Efficient Flame-retardant Agent for Epoxy Thermosets without Deteriorating Thermomechanical Performances

In order to reduce greenhouse gas emissions, developing flame retardants from bio-based resources has aroused extensive interest in recent years. In this work, we utilized furfural(biomass) and 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide(DOPO) to synthesize a biobased co-curing agent(FGD) to combine with 4,4'-diaminodiphenyl methane(DDM) for obtaining a low-phosphorus loading flame-retardant epoxy thermosets. The introduction of FGD decreased the activation energy of the curing progress, enhanced the mechanical properties of the epoxy thermosets, and did not affect the glass transition temperature of the epoxy thermosets. EP-5.0 had a lower thermal degradation rate and a doubled char yield compared with EP-0. The phosphorus content of EP-5.0 was only 0.45 wt%, while EP-5.0 reached the UL-94 V-0 rating with a high LOI value of 32%. Compared with EP-0, the PHRR of EP-2.5 and EP-5.0 decreased by 22.3% and 31.3%, respectively. The SEM results showed that the addition of FGD made the char residues more uniform and denser, which could effectively prevent combustible volatiles from escaping from the degradation area to the flame area and isolate the heat transfer so that the epoxy thermosets had an excellent flame-retardant performance.     

DNA molecular configurations in flows near adsorbing and nonadsorbing surfaces

We investigate experimentally -phage and T2-coliphage DNA molecules near both non-adsorbing glass and adsorbing 3-aminopropyltriethoxysilane (APTES)-coated glass surfaces in a simple steady shearing flow generated by a torsional flow cell. The DNA molecular deformations near the surface are found to be considerably weaker than in bulk flow at the same shear rate. This affects the DNA molecules deposition and stretching on the adsorbing surface. Surprisingly, for a simple shearing flow in the torsional shearing device, the observed stretch, for molecules both near (<10 µm) the surface and adsorbed to it, is much less than predicted by simulations.