Anming Wang

Prospecting metagenomic enzyme subfamily genes for dna family shuffling by a novel pcr-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64–99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.

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.

A novel and efficient method for the immobilization of thermolysin using sodium chloride salting-in and consecutive microwave irradiation.

Sodium chloride salting-in and microwave irradiation were combined to drive thermolysin molecules into mesoporous support to obtain efficiently immobilized enzyme. When the concentration of sodium chloride was 3 M and microwave power was 40 W, 93.2% of the enzyme was coupled to the support by 3 min, and the maximum specific activity of the immobilized enzyme was 17,925.1 U mg(-1). This was a 4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a 1.6-fold increase versus free enzyme. Additionally, the thermal stability of the immobilized thermolysin was significantly improved. When incubated at 70°C, there was no reduction in activity by 3.5h, whereas free thermolysin lost most of its activity by 3h. Immobilization also protected the thermolysin against organic solvent denaturation. The microwave-assisted immobilization technique, combined with sodium chloride salting-in, could be applied to other sparsely soluble enzymes immobilization because of its simplicity and high efficiency.

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.