This paper describes the enhanced separation of adenine (A), hypoxanthine (HX), 8-azaadenine (8-AA), thymine (T), cytosine (C), uracil (U) and guanine (G) by CZE dispersing carboxylic multiwalled carbon nanotubes (c-MWNTs) into the running buffer. The effect of important factors such as c-MWNT nanoparticle concentration, the acidity and concentration of running buffer, and separation voltage were investigated to acquire the optimum conditions. The seven purine and pyrimidine bases could be well separated within 16 min in a 35 cm effective length fused-silica capillary at a separation voltage of +8.0 kV in a 23 mM tetraborate buffer (pH 9.2) containing 8.0 x 10(-5) g/mL c-MWNTs. Under the optimal conditions, the linear ranges were of 2-250 microg/mL for A (R2 = 0.995), 3-200 microg/mL for U (R2 = 0.990) and G (R2 = 0.992), 3-250 microg/mL for T (R2 = 0.998), 2-200 microg/mL for C (R2 = 0.985) and 4-200 microg/mL for HX (R2 = 0.988) and 8-AA (R2 = 0.990). The detection limits were 0.9 microg/mL for A (S/N = 3), 2.4 microg/mL for U, 2.0 microg/mL for T, 1.5 microg/mL for C, 2.5 microg/mL for G and 3.0 microg/mL for HX and 8-AA. The proposed method was successfully applied for determining five purine and pyrimidine bases in yeast RNA.

The development of a novel [Ag(NH 3 ) 2 ]1 probe chemiluminescence (CL)-based imaging method for the detection of various proteins after PAGE is described. The detection is based upon the probe [Ag(NH 3 ) 2 ]1 catalyzing the CL reaction of the luminol-potassium persulfate system. The proposed method detects various proteins labeled by [Ag(NH 3 ) 2 ]1 and expands the application scope to SDS gels. It also detects proteins directly in polyacrylamide gels, without tedious transferring procedures. Furthermore, successful identification of proteins by peptide mass profiling using ionization MS was easily performed, and no pretreatments of gel prior to digestion are needed. Detection limits for standard marker proteins match CBB-R250 staining and the linear dynamic range is superior to CBB-R250 staining and silver staining. The CL imaging conditions, including luminescent reagents, silver ion concentration, the ammonia-controlled system and the washing reagents parameters have also been optimized.

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A novel extracellular enzyme with strong fibrinolytic activity, produced by , which was isolated from the soil of Zhuhai City (China) was purified and characterized. The enzyme was secreted by cultured in solid state and purified at a high efficiency using the combination of salting out, ion exchange chromatography, and size exclusion chromatography. The enzyme was estimated to have a molecular weight of approximately 27 kDa, pI of 8.9 ± 0.1, to stable at pH 5.0-12.0 and up to 50 °C; the optimum pH and temperature are 10.5 and 45 °C (2373.59 ± 54.81 U/mg), respectively. The fibrinolytic activity was enhanced by K, Na, Mg, Mn, Ca, and Ba and inhibited by Cu, Zn, and Fe. Moreover, the activity was slightly enhanced by PMSF and EDTA at low concentrations and inhibited by β-mercaptoethanol. The N-terminal amino acid sequence is AQSVPYGISQI. The enzyme has a higher enzymatic activity than most other fibrinolytic enzymes. The high thermal stability indicated that it is easy to preserve and could be activated under high-temperature conditions.

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An aqueous protocol was developed for nattokinase purification under scalable conditions using a combination of salting out, anion exchange chromatography and ultra-filtration. The enzyme was firstly washed off from the fermented soybeans. The whole proteins were precipitated by ammonium sulfate and purified by anion exchange chromatography. The purified enzyme was finally refined by size exclusion chromatography and ultra-filtration at laboratory and industry-compatible level, respectively. There was a 476.18-fold increase in enzymatic activity with a 48.3% yield at the laboratory level and 429.75-fold increase in enzymatic activity with a 42.7% yield at the industrialcompatible scale. The protocol was universally adaptive for purifying the fibrinolytic enzymes produced by bacillus tequilensis, bacillus amyloliquefaciens, and bacillus cereus, showing a purification efficiency of 329.76-fold with 42.7% yield, 221.78-fold with 32.5% yield, and 288.56fold with 38.7% yield, respectively. All procedures are scalable, aqueous, simple, cost-effective, and suitable for fibrinolytic enzymes industrial production.

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