Enhanced catalytic performance of penicillin G acylase by covalent immobilization onto functionally-modified magnetic Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles.

With the emergence of penicillin resistance, the development of novel antibiotics has become an urgent necessity. Semi-synthetic penicillin has emerged as a promising alternative to traditional penicillin. The demand for the crucial intermediate, 6-aminopicillanic acid (6-APA), is on the rise. Enzyme catalysis is the primary method employed for its production. However, due to certain limitations, the strategy of enzyme immobilization has also gained prominence. The magnetic Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles were successfully prepared by a rapid-combustion method. Sodium silicate was used to modify the surface of the Ni0.4Cu0.5Zn0.1Fe2O4 nanoparticles to obtain silica-coated nanoparticles (Ni0.4Cu0.5Zn0.1Fe2O4-SiO2). Subsequently, in order to better crosslink PGA, the nanoparticles were modified again with glutaraldehyde to obtain glutaraldehyde crosslinked Ni0.4Cu0.5Zn0.1Fe2O4-SiO2-GA nanoparticles which could immobilize the PGA. The structure of the PGA protein was analyzed by the PyMol program and the immobilization strategy was determined. The conditions of PGA immobilization were investigated, including immobilization time and PGA concentration. Finally, the enzymological properties of the immobilized and free PGA were compared. The optimum catalytic pH of immobilized and free PGA was 8.0, and the optimum catalytic temperature of immobilized PGA was 50°C, 5°C higher than that of free PGA. Immobilized PGA in a certain pH and temperature range showed better catalytic stability. Vmax and Km of immobilized PGA were 0.3727 µmol·min-1 and 0.0436 mol·L-1, and the corresponding free PGA were 0.7325 µmol·min-1 and 0.0227 mol·L-1. After five cycles, the immobilized enzyme activity was still higher than 25%.

Improved delivery system for celastrol-loaded magnetic Fe3O4/α-Fe2O3 heterogeneous nanorods: HIF-1α-related apoptotic effects on SMMC-7721 cell.

Fe3O4/α-Fe2O3 heterogeneous nanorods were prepared by a rapid combustion method with α-FeOOH nanorods as precursors. Fe3O4/α-Fe2O3 heterogeneous nanorods with a saturation magnetization of 33.2 emu·g-1 were obtained using 30 mL of absolute ethanol at a calcination temperature of 300 °C. Their average length was around 140 nm, and average diameter was about 20 nm. To improve the dispersion characteristics of the Fe3O4/α-Fe2O3 heterogeneous nanorods in aqueous solution, citric acid and PEG were applied to modify the nanorod surface via the Mitsunobu reaction. The results showed that the hydrodynamic size range of Fe3O4/α-Fe2O3/CA-PEG-celastrol was 250-500 nm, the surface potential was -15 mV, and the saturation magnetization was approximately 23 emu·g-1. The drug loading capacity of Fe3O4/α-Fe2O3/CA-PEG was larger than the non-PEG modified version. Fe3O4/α-Fe2O3/CA-PEG-celastrol had slow-release characteristics and was sensitive to changes in pH. Application of a magnetic field significantly promoted the inhibition of SMMC-7721 human liver cancer cell growth after treatment with Fe3O4/α-Fe2O3/CA-PEG-celastrol. Celastrol and Fe3O4/α-Fe2O3/CA-PEG-celastrol increased the production of reactive oxygen species in SMMC-7721 cells and promoted apoptosis and apoptosis-related proteins (p53, Bax, Bcl-2) were also changed. In addition, the expression of hypoxia-inducible factor 1α (HIF-1α) was enhanced. We may conclude that celastrol-loaded magnetic Fe3O4/α-Fe2O3 heterogeneous nanorods may be applied in the chemotherapy of human cancer with good biocompatibility and delivery.

Covalent Immobilization of Cellulase onto Amino and Graphene Oxide Functionalized Magnetic Fe2O3/Fe3O4@SiO2 Nanocomposites.

Magnetic Fe2O3/Fe3O4@SiO2 nanocomposites were prepared via the citric-alcohol solution combustion process. The obtained nanocomposites were characterized with SEM, XRD, VSM, TEM, EDS, HRTEM, and FTIR techniques. The results revealed that the magnetic Fe2O3/Fe3O4@SiO2 nanocomposites were successfully obtained with the average grain size of 87 nm and the saturation magnetization of 36 emu/g. After the surface of magnetic Fe2O3/Fe3O4@SiO2 nanocomposites was functionalized by amino group, the amino-functionalized Fe2O3/Fe3O4@SiO2-NH2 nanocomposites were loaded onto graphene oxide based on Mitsunobu reaction. Subsequently, the cellulase was immobilized onto Fe2O3/Fe3O4@SiO2-NH-GO nanocomposites by a glutaraldehyde-mediated Schiff base reaction. The immobilization conditions were optimized by adjusting the pH, temperature, and cellulase dose. The results revealed that optimized immobilization conditions were determined to be temperature of 50 °C, pH of 5, and cellulase solution of 0.1 mL. 97.3% cellulase were successfully immobilized under the optimal conditions. The catalytic performances of the immobilized cellulase were also evaluated. The maximum activity was achieved at pH 4, and 50 °C with cellulase solution of 0.4 mL.

A Novel Chondroitin AC Lyase With Broad Substrate Specificity From Pedobacter rhizosphaerae: Cloning, Expression, and Characterization.

Chondroitin AC lyase (ChSaseAC) is one of the essential polysaccharides lyases in low molecular chondroitin sulfate production. In this work, a novel PrChSaseAC from Pedobacter rhizosphaerae was successfully cloned, expressed in Escherichia coli. After optimizing the induction, the recombinant PrChSaseAC could be expressed efficiently at 0.1 mM IPTG, 25°C, and 12 h induction. Then, it was purified with Ni-NTA affinity chromatography. The characterization of the purified PrChSaseAC showed that it had high specific activity and good storage stability, which would favor the production of low molecular weight chondroitin sulfate. It also displayed activity toward chondroitin sulfate C and hyaluronic acid. PrChSaseAC had the highest activity at pH 7.5, 37°C, 10 mM Ca2+, and 5 mg/ml of chondroitin sulfate A. Molecular docking of substrate and enzyme showed the interactions between the enzyme and substrate; it revealed that the enzyme showed high activity to CS-A and hyaluronic acid, but lower activity to CS-C attributed to the structure of the binding pocket. The high stability and specific activity of the enzyme will benefit the industrial production or clinical treatment.

Immobilization and Evaluation of Penicillin G Acylase on Hydroxy and Aldehyde Functionalized Magnetic α-Fe2O3/Fe3O4 Heterostructure Nanosheets.

Magnetic α-Fe2O3/Fe3O4 heterostructure nanosheets were fabricated via hydrothermal calcination. The activity of penicillin G acylase (PGA), which was covalently immobilized onto silica-decorated heterostructure nanosheets, achieved the highest activity of 387.03 IU/g after 18 h of incubation with 0.1 ml of PGA. In contrast, the activity of free PGA reached the highest level when the temperature was 45°C with a pH of 8.0. However, the activity of free PGA changed more dramatically than immobilized PGA as the relative conditions changed. Moreover, the Michaelis-Menten constant (Km) and reusability of immobilized PGA were also explored. The results showed that free PGA Km and maximum rate (Vmax) were 0.0274 M and 1.167 µl/min, respectively. Km and Vmax values of immobilized PGA were 0.1082 M and 1.294 µl/min, respectively. After 12 cycles of repetitive use, immobilized PGA remained approximately 66% of its initial activity, indicating that the PGA immobilized onto the heterostructure nanosheets showed better stability and reusability than free PGA.

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization.

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its' important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.

Immobilization and Performance of Penicillin G Acylase on Magnetic Ni0.7Co0.3Fe2O4@SiO2-CHO Nanocomposites.

Magnetic Ni0.7Co0.3Fe2O4 nanoparticles that were prepared via the rapid combustion process were functionalized and modified to obtain magnetic Ni0.7Co0.3Fe2O4@SiO2-CHO nanocomposites, on which penicillin G acylase (PGA) was covalently immobilized. Selections of immobilization concentration and time of fixation were explored. Catalytic performance of immobilized PGA was characterized. The free PGA had greatest activity at pH 8.0 and 45oC while immobilized PGA's a ctivities peaked a t pH 7.5 and 45°C. Immobilized PGA had better thermal stability than free PGA at the range of 30-50°C for different time intervals. The activity of free PGA would be 0 and that of immobilized PGA still retained some activities at 60°C after 2 h. Vmax and Km of immobilized PGA were 1.55 mol/min and 0.15 mol/l, respectively. Free PGA's Vmax and Km separately were 0.74 mol/min and 0.028 mol/l. Immobilized PGA displayed more than 50% activity after 10 successive cycles. We concluded that immobilized PGA with magnetic Ni0.7Co0.3Fe2O4@SiO2-CHO nanocomposites could become a novel example for the immobilization of other amidohydrolases.

Preparation of Magnetic NixCo(1-x)Fe2O4 Nanoparticles by the Rapid Combustion Approach and Their Adsorption of Reactive Red 2BF.

A rapid combustion approach for the preparation of magnetic NixCo(1-x)Fe2O4 nanoparticles was introduced. The experimental results showed that the content of Ni in NixCo(1-x)Fe2O4 nanoparticles, the volume of absolute alcohol and the calcination temperature were three key factors to grain size, crystallinity and properties of magnetic NixCo(1-x)Fe2O4 nanoparticles, and the influence rules of which were revealed. The magnetic Ni0.4Co0.6Fe2O4 nanoparticles that calcined at 400 °C for 2 h with the heating rate of 3 °C/min and absolute alcohol of 40 mL were employed to remove Reactive Red 2BF (RR-2BF) from aqueous solution, and the adsorption kinetic and the adsorption isotherm of RR-2BF onto them were investigated at room temperature. The kinetic data were in good agreement with the pseudo-second-order kinetic model with the initial RR-2BF concentrations of 100-500 mg/L; the adsorption equilibrium data were analyzed with Langmuir, Freundlich and Temkin models, and the adsorption isotherm was most effectively described by the Temkin model based on the correlation coefficient (0.9985); the magnetic Ni0.4Co0.6Fe2O4 nanoparticles showed better adsorption of RR-2BF under acidic condition.

Nitrogen doped small molecular structures of nano-graphene for high-performance anodes suitable for lithium ion storage.

N-doped nano-graphene derivatives were prepared by a bottom-up organic synthesis method. Through d-spacing modification and dynamic self-assembly of the structures of these molecules, ideal lithium ion-transfer aggregation formed between each monolayer. Rapid ion/electron transfer and maintenance of the structural integrity during repeated ion insertion/extraction occurred due to the lack of a covalent interaction force among the assembled structures. The doping level, i.e., number of N atoms, had a significant influence on the molecular self-assembled structures through hierarchical self-assembly. As the N concentration increased, the d-space between the nanosheets increased from 3.4 to 4.3. The capacity of the nano-graphene increased greatly from N-doping nano-graphene (NG-N4) to 1800 mA h g-1, indicating that the capacity is related to the structure. Moreover, the N-doping site of nano-graphene was defined and the relationship between the performance and structure was determined.

A Facile Solution Combustion and Calcination Process for the Preparation of Magnetic NiFe2O4/SiO2 Nanocomposites.

A facile solution combustion and calcination process for the preparation of magnetic NiFe2O4/SiO2 nanocomposites was introduced, the as-prepared NiFe2O4/SiO2 nanocomposites were characterized by XRD, VSM, BET, FTIR, SEM, and TEM techniques. The results showed that the single ferrite phase of NiFe2O4 could be formed at 400 °C; the calcination temperature, the content of silica in nanocomposites, and the volume of absolute alcohol were three key factors to the properties of the magnetic NiFe2O4/SiO2 nanocomposites: with the volume of absolute alcohol increasing from 10 mL to 100 mL, the average grain size of NiFe2O4/SiO2 nanocomposites calcined at 400 °C for 2 h with the heating rate of 3 °C/min decreased from about 16.6 nm to 8.4 nm, while the saturation magnetization decreased from about 6.1 Am2/kg to 1.5 Am2/kg; with the calcination temperature increasing from 400 °C to 800 °C, the average grain size of NiFe2O4/SiO2 nanocomposites calcined for 2 h with the heating rate of 3 °C/min and absolute alcohol of 30 mL increased from about 11.0 nm to 14.1 nm, and the saturation magnetization increased from about 3.4 Am2/kg to 24.0 Am2/kg; at the same time, with the content of silica in nanocomposites increasing from 0 to 10 wt.%, the average grain size of NiFe2O4/SiO2 nanocomposites calcined at 400 °C for 2 h with the heating rate of 3 °C/min decreased from about 18.4 nm to 11.0 nm, while the saturation magnetization decreased from about 18.2 Am2/kg to 3.4 Am2/kg.

Values of (99m)Tc-methoxyisobutylisonitrile imaging after first-time large-dose (131)I therapy in treating differentiated thyroid cancer.

OBJECTIVE: The aim of this study is to investigate the use of (99m)Tc-methoxyisobutylisonitrile (MIBI) imaging for evaluating the treatment response of differentiated thyroid cancer (DTC) after the first administration of a high dose of (131)I. METHODS: Patients with DTC who received (131)I therapy underwent (99m)Tc-MIBI imaging after successive increases in the therapeutic dose of (131)I, and the serum levels of thyroglobulin (Tg) were measured. RESULTS: A total of 191 patients were enrolled in the final analysis, including 65 metastases and/or thyroid remnant-positive patients (22 patients with metastases and 43 patients with thyroid remnants). The sensitivity of (99m)Tc-MIBI imaging for detecting positive cases and thyroid remnants was 56.9% and 39.5%, respectively, which was significantly lower than that of (131)I imaging (92.3% and 100%, respectively, P<0.01 for both). The sensitivity of (99m)Tc-MIBI imaging for detecting metastases was 90.9%, which was slightly higher than that of (131)I imaging (77.3%, P>0.05). The Tg levels in the positive group were significantly higher than that in the negative group (P<0.01). In addition, the Tg levels in the (99m)Tc-MIBI(+)/(131)I(-) group were significantly higher than that in the (131)I(+)/(99m)Tc-MIBI group (P<0.05). CONCLUSION: After the first (131)I therapy, although (99m)Tc-MIBI imaging was able to detect the existence of metastatic lesions in patients with DTC better, its assessment for the removal efficiency of thyroid remnants was unsatisfactory. The results of (99m)Tc-MIBI imaging showed good correlations with the Tg level.

[Diagnostic Value of (18)F-FDG-PET/CT for Multiple Myeloma].

OBJECTIVE: To study the diagnostic value of (18)F-FDG-PET/CT in multiple myeloma (MM). METHODS: A total of 66 patients, who were highly suspected of MM in our hospital from July 2012 to December 2014, were chosen as study objects. All patients were diagnosed or excluded by pathological examination. All patients were detected by (18)F-FDG-PET/CT, and its diagnostic value was analyzed. The number of focuses were counted. RESULTS: Out of 66 patients 59 patients (89.39%) were diagnosed with multiple myeloma. The sensitivity of PET was 98.31%, the specificity of PET was 85.71%, the Youden index was 0.8402; the sensitivity of CT was 96.61%, the specificity of CT was 85.71%, the Youden index was 0.8232; the sensitivity of PET/CT was 100.00%, the specificity of PET/CT was 83.33%, the Youden index was 0.8333. In 59 MM patients, 635 focuses were detected, out of them 572 focuses (90.08%) were detected by CT, 593 focuses (93.39%) were detected by PET, 530 focuses (83.46%) were coincided on PET/CT. CONCLUSION: (18)F-FDG-PET/CT has diagnostic value for multiple myeloma, and it can be used in locating and counting focuses, as well as in evaluating the treatment efficacy and guiding the clinical work.