Co-reporter:Shu-tao Liu, Zhao-hua Chen, Jun-bin Xie, Juan Lin, Zhan-jie Chen, and Ping-fan Rao
Analytical Chemistry 2010 Volume 82(Issue 20) pp:8544
Publication Date(Web):September 28, 2010
DOI:10.1021/ac101542s
The feasibility of separating intact bacterial cells by high-performance ion exchange chromatography typical for macromolecular study was investigated. A common HPLC system coupled with a light scattering detector was employed; TOYOPEARL SuperQ-650C, a beaded anion exchanger, was used as the column media; bacterial cell samples were prepared by suspending colonies in the equilibrium buffer. Piperazine-hydrochloric acid buffer (0.02 M, pH 7.0) was chosen to be the equilibrium buffer after screening different buffer systems. The absorbed cells were eluted by a linear gradient of NaCl from 0 to 1.00 M in the equilibrium buffer within 30 min, at a flow rate of 1.0 mL/min. A distinctive chromatographic profile with high reproducibility and accuracy was obtained with all of the six kinds of bacteria tested. The eluted fractions for each sample were confirmed by microscopic analysis and physiological or biochemical identification to the bacterial cells applied. More importantly, the eluted cells retained full viability. It is apparent that living bacteria cells exhibited similar behaviors and properties with molecules in anion exchange chromatography, implying that chromatographic methods can become an effective approach in the studies of intact bacterial cells.
Co-reporter:SHAOYUN WANG;BIAO SHAO;XIUYUN YE ;PINGFRAN RAO
Journal of Food Biochemistry 2008 Volume 32( Issue 1) pp:32-45
Publication Date(Web):
DOI:10.1111/j.1745-4514.2007.00144.x
ABSTRACT
A chitinase was isolated from peanut (Arachis hypogaea L.) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography on POROS 20 HQ. There was a 133-fold increase in specific activity of purified chitinase compared with that of the crude extract. The protein exhibited a molecular mass of 34.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions, indicating that it is a monomeric protein. The isoelectric point was 5.1 by isoelectric focusing electrophoresis. Optimal pH activity was 5.4 and optimal temperature was 40–50C. The enzyme was stable below 55C, but was rapidly inactivated when incubated at temperatures above 60C. These results demonstrated that the purified protein was a kind of relatively thermostable chitinase from the peanut seeds.
PRACTICAL APPLICATIONS
This paper describes the isolation and characterization of a chitinase from Arachis hypogaea L. seeds. Chitinases are listed as the class of pathogenesis-related proteins, and they have become a popular research field because of their resistance to plant disease. The research we are presenting should help in understanding chitinase functions in plant defense mechanisms. A relatively thermostable enzyme newly obtained, which was stable below 55C, would certainly be significant for its utilization both in enzymology and in plant defense. In particular, a relatively heat-resistant enzyme from A. hypogaea L. seeds would certainly be helpful for elaborating strategies for constitutive expression of chitinase genes and their manipulation and general utilization in plant defense mechanisms through more recent techniques of genetic manipulation.
Co-reporter:P.-F. RAO;S.-T. LIU;Z. WEI;J.-C. LI;R.-M. CHEN;G.-R. CHEN;Y.-Q. ZHENG
Journal of Food Biochemistry 1996 Volume 20(Issue 4) pp:473-479
Publication Date(Web):23 FEB 2007
DOI:10.1111/j.1745-4514.1996.tb00570.x
A single-step method to isolate flavoprotein from chicken egg white is described. Egg white diluted 10 fold with 50 mM sodium phosphate buffer, pH 6.5, was applied to a DEAE (Diethylaminoethyl cellulose)-Toyopearl 650 M column. The adsorbed proteins were fractionated into two peaks by a linear salt gradient. The second peak was electrophoretically pure flavoprotein, with a total yield of about 75%.
Co-reporter:Lijing Ke, Jianwu Zhou, Wei Lu, Guanzhen Gao, Pingfan Rao
Trends in Food Science & Technology (September 2011) Volume 22(Issue 9) pp:492-497
Publication Date(Web):1 September 2011
DOI:10.1016/j.tifs.2011.06.004
Not only enjoyed as delicacy, soups of vegetables, fish or meat are also served as folk remedies in many cultures. The formation of nanostructures was observed both in the hepato-protective freshwater clam soup and the anti-diabetic bitter melon soup. It was in accompany with either the migration of clam compositions into the soup or the progression of Maillard reaction in bitter melon. The biochemical compositions, bioactivities and colloidal characteristics of those nano-particles were preliminarily investigated. It implies that the nanostructures may probably be the functioning unit, and provide a novel perspective to understanding the complicated food system.
Co-reporter:Jianru Pan, Ying Su, Xiaojun Hou, Huocong He, Shutao Liu, Junxin Wu, Pingfan Rao
Life Sciences (21 August 2012) Volume 91(Issues 3–4) pp:89-93
Publication Date(Web):21 August 2012
DOI:10.1016/j.lfs.2012.06.003
AimsRadiation-induced lung injury is one of the limiting factors for radiation therapy. SOD-TAT, a fusion protein of HIV-1 Tat protein transduction domain and hCuZn-superoxide dismutase (SOD), has been proved to be effective in preventing and treating the damage of the skin of guinea pigs by UVB radiation. In this study, we demonstrated SOD-TAT's radioprotective effects on lung injury in irradiated mice.Main methodsSOD-TAT was purified from yeast culture with ion exchange chromatography. Kunming mice were randomly divided into three groups: a control group, a group injected with wild SOD and a group injected with SOD-TAT. Pulmonary SOD activity of mice was determined 4.5 h after injection. C57BL/6 mice were randomly divided into four groups: a control group, an irradiation group, an irradiation group treated with amifostine 0.5 h before the irradiation and an irradiation group treated with SOD-TAT 4.5 h before irradiation. The monthly growth rate of every mouse's weight was calculated and the level of hydroxyproline content and antioxidant activity in lung were determined 5 months after irradiation.Key findingsSOD-TAT was transduced into the lung in vivo. SOD-TAT pretreatment could improve the growth rate of irradiated mice, significantly reduce the pulmonary hydroxyproline content, and maintain the SOD activity, glutathione peroxidase (GSH-Px) activity and total anti-oxidation capacity (T-AOC). Compared with amifostine, SOD-TAT was more effective in increasing the activities of pulmonary antioxidant enzymes.SignificanceCompared with amifostine, SOD-TAT treatment more effectively enhanced pulmonary antioxidant ability, reduced radiation-induced pulmonary fibrosis and improved the living quality of irradiated mice.
