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Ni H.,South China Normal University | Guo P.-C.,South China Normal University | Jiang W.-L.,South China Normal University | Fan X.-M.,South China Normal University | And 3 more authors.
Journal of Biotechnology | Year: 2016

Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1 mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20 mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8 IU/mg and 79.3 IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0 IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution. © 2016 Elsevier B.V. Source


Hu R.-S.,South Central Agriculture Experimental Station of China Tobacco | Hu R.-S.,South China Normal University | Wang J.,South China Normal University | Li H.,South China Normal University | And 6 more authors.
Separation and Purification Technology | Year: 2015

The tobacco (Nicotiana tabacum L.) cultivation and cigarettes manufacture industries discard huge amount of waste tobacco materials that have strong nicotine smell and contaminate the environment. A high-efficient procedure was developed for simultaneously extracting nicotine and solanesol from the waste tobacco leaf vein through the column-chromatographic extraction (CCE), followed by automatically separating and simply purifying them. Dried material powder was loaded into columns and eluted with the optimal extraction solvent of petroleum ether (PE):95% alkali ethanol (4:6). Greater than 96% extraction efficiency for both nicotine and solanesol was obtained with a 2-fold excess solvent of the material (v/w) through a cyclic CCE procedure in small-scale and scaled-up experiments. The extraction solution was separated into an ethanol-aqueous phase containing 98% nicotine and an ether phase containing 96% solanesol at pH 2.0. The ethanol-aqueous phase was vacuum-concentrated to aqueous, and 99% purity nicotine was obtained by ether fractionation of the aqueous at pH 10.0. Solanesol in the ether phase was purified to 93.1% by one time silica gel column chromatography. All processes were completed at room temperature and all solvents used were completely recovered for reuse. This work provides an extensively simplified procedure to economically utilize the tobacco wastes. © 2015 Elsevier B.V. All rights reserved. Source


Cheng S.,South China Normal University | Zhang Y.-F.,South China Normal University | Zeng Z.-Q.,South China Normal University | Lin J.,South China Normal University | And 4 more authors.
Applied Microbiology and Biotechnology | Year: 2014

Polyphenol oxidase (PPO) has multiple functions, and the lack of commercially available enzyme sources limits its widespread application in various industries. An accurate PPO assay was developed by HPLC determination of the substrate oxidation. Resources screening indicated that sweet potato (Ipomoea batatas L.) wastewater in starch production has high PPO activity. A procedure was developed for separately recovering PPO, β-amylase, sporamins, and small molecular nutrients (SMNs) from sweet potato wastewater. The wastewater was adjusted to pH 3.5 to precipitate PPO, and then adjusted to 50 % acetone to precipitate β-amylase and further to 80 % acetone to precipitate sporamins. The SMNs were obtained after acetone recovery. Purified powders of 4.3 × 105 units of PPO, 4.0 × 106 units of β-amylase, 8.70 g sporamins, and 20.2 g SMNs were obtained from the wastewater of 1 kg sweet potato. More than 50 million tons of sweet potato is used for starch production annually around the world. Through this simple procedure, huge amount of biochemical resources can be recovered from the wastewater, which greatly increases the economic value of the crop and saves the environment. © 2014, Springer-Verlag Berlin Heidelberg. Source


Zhang Y.-W.,South China Normal University | Zhang Y.-W.,Guangzhou Huichuan Medical Technology Ltd. | Fan W.-W.,South China Normal University | Li H.,South China Normal University | And 4 more authors.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2015

Abscisic acid (ABA), a universal signaling molecule, plays important roles in regulating plant growth, development and stress responses. The low contents and complex components in plants make it difficult to be accurately analyzed. A novel one-step sample preparation method for ABA in plants was developed. Fresh peanut (Arachis hypogaea) plant materials were fixed by oven-drying, microwave drying, boiling or Carnoy's fixative, and loaded onto a mini-preparing column. After washed the impurities, ABA was eluted with a small amount of solvent. ABA in plant materials was completely extracted and purified in 2mL solution and directly analyzed by HPLC, with a 99.3% recovery rate. Multiple samples can be simultaneously prepared. Analyses using this method indicated that the endogenous ABA in oven-dried peanut leaves increased 20.2-fold from 1.01 to 20.37μgg-1 dry weight within 12h and then decreased in 30% polyethylene glycol 6000 treated plants, and increased 3.34-fold from 0.85 to 2.84μgg-1 dry weight in 5 days and then decreased in soil drought treated plants. The method combined the column chromatographic extraction and solid-phase separation technologies in one step and can completely extracted plant endogenous ABA in a purified and highly concentrated form for direct HPLC analysis. © 2015 Elsevier B.V. Source

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