Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii.

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg-1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL-1 and 647.93 U·mg-1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K-1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Electron localization in niobium doped CaMnO3 due to the energy difference of electronic states of Mn and Nb.

The electron localization in Nb-doped CaMnO3 is analyzed in terms of the space and energy distribution of electronic states employing first-principles calculations. The energy difference of Mn 3d states and Nb 4d states makes NbO6 octahedra impede electrical conduction, so the random distribution of Nb in lattices leads to the localization of electrons near the bottom of the conduction bands. Therefore, although more carriers are introduced when Nb-doping content increases, both the electrical conductivity and absolute thermopower decrease in Nb heavy doped CaMnO3. The calculated transport properties agree well with the experimental data, supporting the analysis of localization.

Degradation of naproxen by combination of Fenton reagent and ultrasound irradiation: optimization using response surface methodology.

A central composite factorial design methodology was employed to optimize the degradation of naproxen (NPX) by the combination of Fenton reagent and ultrasound (US) irradiation. In this study, the variables considered for the process optimization were the hydrogen peroxide, ferrous ion and NPX initial concentrations, while ultrasonic power amplitude was adjusted at 90% and initial pH was 3. An appropriate quadratic model was developed in order to plot the response surface and contour curves. Optimum dosage of Fenton reagent for NPX removal was found to be hydrogen peroxide concentration = 9.98 mmol L⁻¹, ferrous ion concentration = 4.83 mg L⁻¹ while NPX concentration was equal to 20 mg L⁻¹. A degradation efficiency of 100% was achieved within 10 min under US.

A Highly Active Chondroitin Sulfate Lyase ABC for Enzymatic Depolymerization of Chondroitin Sulfate.

Enzymatic preparation of low-molecular-weight chondroitin sulfate (LMWCS) has received increasing attention. In this work, a chondroitin sulfate lyase ABC (Chon-ABC) was successfully cloned, expressed, and characterized. The Km and Vmax of the Chon-ABC were 0.54 mM and 541.3 U mg-1, respectively. The maximal activity was assayed as 500.4 U mg-1 at 37 °C in pH 8.0 phosphate buffer saline. The half-lives of the Chon-ABC were 133 d and 127 min at 4 °C and 37 °C, respectively. Enzymatic preparation of LMWCS was performed at room temperature for 30 min. The changes between the substrate and product were analyzed with mass spectrometry (MS), high-performance liquid chromatography (HPLC), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR). Overall, the Chon-ABC from Bacteroides thetaiotaomicron is competitive in large-scale enzymatic preparation of LMWCS for its high activity, stability, and substrate specificity.

Using galactitol dehydrogenase coupled with water-forming NADH oxidase for efficient enzymatic synthesis of L-tagatose.

L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.

Expression, biochemical characterization, and mutation of a water forming NADH: FMN oxidoreductase from Lactobacillus rhamnosus.

Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg-1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.

[Solid phase extraction and room temperature phosphorimetry coupling technique and its application].

The principle and application in the environmental analysis of Solid Phase Extraction (SPE) combined with Room Temperature Phosphorescence (RTP) analysis were reviewed. The principle, device, separation modes, operation procedures and application of SPE were commented. The merits and shortages in the operation procedure and quantitative analysis of classic RTP were also summarized. The potential application in the trace sample analysis and the advantage of coupling technique of SPE and RTP were indicated. The sensitivity and selectivity of the coupling technique could be greatly increased because of the strong enrichment ability of the SPE materials and the high selectivity of RTP. So far the technique has been successfully used in the analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PClDBFs) in the environmental samples according to their phosphorescence emission after preconcentration on the Whatman 1PS filter paper and C18 or C8 SPE membranes.

Competition between fullerene aggregation and poly(3-hexylthiophene) crystallization upon annealing of bulk heterojunction solar cells.

Concomitant development of [6,6]-phenyl-C(61)-butyric acid methyl ester (PCBM) aggregation and poly(3-hexylthiophene) (P3HT) crystallization in bulk heterojunction (BHJ) thin-film (ca. 85 nm) solar cells has been revealed using simultaneous grazing-incidence small-/wide-angle X-ray scattering (GISAXS/GIWAXS). With enhanced time and spatial resolutions (5 s/frame; minimum q ≈ 0.004 Å(-1)), synchrotron GISAXS has captured in detail the fast growth in size of PCBM aggregates from 7 to 18 nm within 100 s of annealing at 150 °C. Simultaneously observed is the enhanced crystallization of P3HT into lamellae oriented mainly perpendicular but also parallel to the substrate. An Avrami analysis of the observed structural evolution indicates that the faster PCBM aggregation follows a diffusion-controlled growth process (confined by P3HT segmental motion), whereas the slower development of crystalline P3HT nanograins is characterized by constant nucleation rate (determined by the degree of supercooling and PCBM demixing). These two competing kinetics result in local phase separation with space-filling PCBM and P3HT nanodomains less than 20 nm in size when annealing temperature is kept below 180 °C. Accompanying the morphological development is the synchronized increase in electron and hole mobilities of the BHJ thin-film solar cells, revealing the sensitivity of the carrier transport of the device on the structural features of PCBM and P3HT nanodomains. Optimized structural parameters, including the aggregate size and mean spacing of the PCBM aggregates, are quantitatively correlated to the device performance; a comprehensive network structure of the optimized BHJ thin film is presented.

Antibody persistence at 18-20 months of age and safety and immunogenicity of a booster dose of a combined DTaP-IPV//PRP∼T vaccine compared to separate vaccines (DTaP, PRP∼T and IPV) following primary vaccination of healthy infants in the People's Republic of China.

This study assessed the antibody persistence, and the immunogenicity and safety of a booster dose of a DTaP-IPV//PRP∼T (Pentaxim®, Sanofi Pasteur's AcXim family) combined vaccine and of standalone vaccines one year after primary vaccination in the People's Republic of China. Participants (N=719) previously primed with DTaP-IPV//PRP∼T at 2, 3, 4 months (Group A, N=255), 3, 4, 5 months (Group B, N=233), or DTaP (Wuhan Institute of Biological Products), PRP-T (Act-Hib®) and IPV (Imovax® Polio) at 3, 4, 5 months (Group C, N=231) received boosters of the same vaccines at 18-20 months of age. Seroprotection (SP) and seroconversion (SC) were determined before and 1 month after the booster. Safety was monitored from parental reports. In all groups 87.6-100% of participants had pre-booster protective anti-PRP, -diphtheria, -tetanus and -poliovirus antibody titers; post-booster, all SP rates were 100% and SC was ≥ 80.4% for anti-pertussis titers ≥ 4-fold increase. Reactogenicity was low for each group. These data support the use of the DTaP-IPV//PRP∼T vaccine in the People's Republic of China compared to separate DTaP, IPV, and PRP∼T administration in terms of both safety and immunogenicity.

[Immunogenicity and safety of DTaP-IPV//PRP-T combined vaccine in infants in China].

OBJECTIVE: The aim of this study was to demonstrate the immunogenicity and safety of diphtheria, tetanus, pertussis (acellular, component), poliomyelitis (inactivated) vaccine (adsorbed) and Haemophilus influenzae type b conjugate vaccine (DTaP-IPV//PRP-T) combined vaccine compared with commercially available DTaP (diphtheria, tetanus and pertussis), Haemophilus influenzae type b (Hib), tetanus conjugate and IPV monovalent vaccine. METHODS: Subjects were randomly divided into three groups, Group A and Group B were DTaP-IPV//PRP-T combined vaccine (PENTAXIM(TM)) vaccinated at 2, 3, 4 months of age or 3, 4, 5 months of age respectively; Group C was commercially available DTaP. Hib tetanus conjugate (Act-HIB(TM)) and IPV (IMOVAX PolioTM(TM)) vaccines vaccinated at 3, 4, 5 months of age. All groups received booster dose at 18 to 20 months of age, with antibody titers tested. Non-inferiority analysis was demonstrated in terms of seroprotection/seroconversion rates between Group A, Group B respectively and Group C. Safety information was collected after each vaccination to assess the safety of investigational vaccines. RESULTS: The non-inferiority of DTaP-IPV//PRP-T combined vaccine vaccinated at 2, 3, 4 or 3, 4, 5 months of age versus DTaP, Hib tetanus conjugate and IPV vaccine was demonstrated for all vaccine antigens in both primary and booster phases in terms of seroprotection/seroconversion rates. DTaP-IPV//PRP-T combined vaccine was well tolerated. The rate of solicited/unsolicited severe adverse reactions was very low and similar to the control vaccines. CONCLUSION: DTaP-IPV//PRP-T combined vaccine was highly immunogenic with good safety profile in Chinese infants, which was comparable to the commercially available control vaccines.

Immunogenicity and safety of a pentavalent acellular pertussis combined vaccine including diphtheria, tetanus, inactivated poliovirus and conjugated Haemophilus Influenzae type b polysaccharide for primary vaccination at 2, 3, 4 or 3, 4, 5 months of age in infants in China.

The aim was to demonstrate the immunogenicity and safety of a DTaP-IPV//PRP-T combined vaccine (Pentaxim(®)) compared to individual vaccines in infants in the People's Republic of China. Infants (N=792) were randomly assigned to receive DTaP-IPV//PRP-T at 2, 3 and 4 months of age (Group A) or 3, 4 and 5 months of age (Group B), or DTaP (Wuhan Institute of Biological Products), PRP-T (Act-Hib(®)) and IPV (Imovax(®) Polio) at 3, 4 and 5 months of age (Group C). Antibody titers were measured pre- and 1 month after the third vaccination; non-inferiority analyses were performed for seroprotection/seroconversion (SP/SC) rates. Safety was assessed 1 month after the primary series. SP/SC rates for the DTaP-IPV//PRP-T vaccine were high and non-inferior to the controls. Reactogenicity was low for each group and no hypotonic hyporesponsive episode or seizure was reported. In conclusion, the DTaP-IPV//PRP-T vaccine was highly immunogenic, non-inferior to the commercially available control vaccines and had a good safety profile for both primary administration schedules.