Zhaowu Zeng

Stable and efficient immobilization technique of aldolase under consecutive microwave irradiation at low temperature.

To establish a stable and efficient immobilization technique under microwave irradiation, a focused microwave reaction system was used, where the temperature was set appropriately in the microwave system and cooling module to produce consecutive microwave irradiation. 2-Deoxy-D-ribose-5-phosphate aldolase (DERA) was rapidly and efficiently immobilized in mesocellular siliceous foams (MCFs) under microwave irradiation. When the output power in the microwave system was set to 30 W, after 3 min, 88.4% of the enzyme protein was coupled to the wall of the support pores and the specific activity of the immobilized enzyme was 2.24 U mg(-1), 149.2% higher than that of the free enzyme and 157.0% higher than that of the non-microwave-assisted immobilized enzyme. In catalysis, microwave-assisted immobilized DERA tolerated a wider range of both pH and temperature than other DERA preparations. The thermal and storage stabilities were also significantly improved. This focused; microwave-assisted immobilization technique has proven to be simple, stable and highly efficient. This technique could also be applied to other enzyme immobilizations.

Preparation, characterization and relative bioavailability of oral elemene o/w microemulsion

The objective was to develop an elemene oil/water (o/w) microemulsion and evaluate its characteristics and oral relative bioavailability in rats. Elemene was used as the oil phase and drug, polysorbate 80 as a surfactant along with ethanol, propylene glycol, and glycerol as the cosurfactants. The microemulsion was prepared by mixing method, or ultrasonication method in an ultrasonic bath. Its three-dimensional response surface diagram was drawn by Mathcad software. The microemulsion was characterized by visual observation, cross-polarized microscopy, size, zeta potential, acidity, viscosity, and surface tension measurement. The drug content and entrapment efficiency were determined by ultra fast liquid chromatography (UFLC) and liquid surface method. Blood was drawn from rats at different time points after oral administration of an elemene microemulsion or a commercial elemene emulsion for measurement of the drug in plasma by UFLC to establish the pharmacokinetic parameters and relative bioavailability. The elemene microemulsion as a clarified and isotropic system containing 1% elemene (w/v), 5% ethanol (v/v), 15% propylene glycol (v/v), 15% glycerol (v/v), and 5% polysorbate 80 (w/v), was characterized as (57.7 ± 2.8) nm in size, 0.485 ± 0.032 in polydispersity index, (3.2 ± 0.4) mv in zeta potential, (5.19 ± 0.08) in pH, 6 mpa·s in viscosity, (31.8 ± 0.3) mN·m−1 in surface tension, (8.273 ± 0.018) mg·mL−1 in content of β-elemene, and (99.81 ± 0.24)% in average entrapment efficiency. The area under the concentration-time curves from 0 h to 24 h (AUC0→24h) of the elemene microemulsion and commercial elemene emulsion were integrated to be 3.092 mg·h·L−1 and 1.896 mg·h·L−1 respectively, yielding a relative bioavailability of 163.1%. The present study demonstrates the elemene microemulsion as a new formulation with ease of preparation, high entrapment efficiency, excellent clarity, good stability, and improved bioavailability.

Cloning, recombinant expression and characterization of a new glucoamylase gene from Aureobasidium pullulans NRRL 12974 and its potential application in raw potato starch degradation

A new amylase gene APGA1 was cloned from Aureobasidium pullulans NRRL 12974 and expressed in Pichia pastoris . This is the first report on cloning and expression of amylolytic gene from the industrially important microorganism A. pullulans . The purified recombinant protein with MW of 66 kDa and specific activity of 298.02 Umg -1 protein was verified as a glucoamylase by its hydrolytic mode. This recombinant glucoamylase with optimal pH of 4.5, and temperature of 60°C, showed good hydrolytic activity against raw potato starch. At 60°C, 83.1% of raw potato starch slurry (150 gl -1 ) was hydrolysed into glucose by 0.1 U mg -1 starch purified recombinant glucoamylase in less than 2.5 h. This is the highest raw starch hydrolysis efficiency report about recombinant fungal glucoamylase. This useful property indicated that this glucoamylase may find important applications in the starch saccharification industry and in bioethanol production. Key words: Glucoamylase, Aureobasidium pullulans, expression, raw starch degradation.

Drug delivery systems for elemene, its main active ingredient β-elemene, and its derivatives in cancer therapy

β-elemene is a noncytotoxic Class II antitumor drug extracted from the traditional Chinese medicine Curcuma wenyujin Y. H. Chen et C. Ling. β-elemene exerts its effects by inhibiting cell proliferation, arresting the cell cycle, inducing cell apoptosis, exerting antiangiogenesis and antimetastasis effects, reversing multiple-drug resistance (MDR), and enhancing the immune system. Elemene injection and oral emulsion have been used to treat various tumors, including cancer of the lung, liver, brain, breast, ovary, gastric, prostate, and other tissues, for >20 years. The safety of both elemene injection and oral emulsion in the clinic has been discussed. Recently, the secondary development of β-elemene has attracted the attention of researchers and made great progress. On the one hand, studies have been carried out on liposome-based systems (including solid lipid nanoparticles [SLNs], nanostructured lipid carriers [NLCs], long-circulating liposomes, active targeting liposomes, and multidrug-loaded liposomes) and emulsion systems (including microemulsions, self-emulsion drug delivery systems [SEDDSs], and active targeting microemulsion) to solve the issues of poor solubility in water, low bioavailability, and severe phlebitis, as well as to improve antitumor efficacy. The pharmacokinetics of different drug delivery systems of β-elemene are also summarized. On the other hand, a number of highly active anticancer β-elemene derivatives have been obtained through modification of the structure of β-elemene. This review focuses on the two drug delivery systems and derivatives of β-elemene for cancer therapy.

Molecular cloning, characterization and functional analysis of a new isopentenyl diphosphate isomerase gene (IPI) from Curcuma wenyujin

A new isopentenyl diphosphate isomerase gene cwipi (GenBank: GU724874) was cloned from the Curcuma Wenyujin cDNA library. The full-length cDNA contained a 708 bp open reading frame (ORF) encoding a protein of 235 amino acids with calculated molecular mass of 27.36 kDa and isoelectric point of 5.14. Phylogenetic analysis demonstrated that cwipi has a high level similarity to other IPIs belonged to angiosperm group. The protein encoded by cwipi was subcellularly localized to the cytoplasm using TargetP prediction software. Semi-quantity RT-PCR results indicated that the transcriptional level of cwipi in roots was higher than that of in stems and leaves. Cwipi could accelerate the accumulation of β-carotene when it was expressed in Escherichia coli harbouring reconstructed carotenoid synthetic pathway. The cloning, characterization and functional analysis of cwipi will be useful for understanding the role of IPI in isoprenoid biosynthesis of C. Wenyujin.