An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass ...An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass of 35 × 10^3 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inbibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Further more, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.展开更多
To study the thermostability of Nattokinase（subtilisin NAT,NK）,three double mutant plasmids（pET-28a-NKG61C/S98C,pET-28a-NKT22C/S87C,pET-28a-NKS24C/S87C）were constructed by site-directed mutagenesis.Target enzymes ...To study the thermostability of Nattokinase（subtilisin NAT,NK）,three double mutant plasmids（pET-28a-NKG61C/S98C,pET-28a-NKT22C/S87C,pET-28a-NKS24C/S87C）were constructed by site-directed mutagenesis.Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis.Thermostability was tested by rates of inactivation at certain temperature.The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK.The thermostability of NK performed in Ca2＋was stronger than in ethylenediaminetetraacetic acid（EDTA）.But when the temperature reached 62℃,the enzymes rapidly denatured and inactivated even in the presence of Ca2＋.Although the thermostability of mutants was not increased,this study shows a tendency of improving thermostability of NK in protein engineering.展开更多
Thermokinetics of the dismutation of superoxide anion(O_(2)^(.-))catalyzed by superoxide dismutase(SOD),in which the decomposition of hydrogen peroxide catalyzed by catalase is utilized as a source of oxygen and the a...Thermokinetics of the dismutation of superoxide anion(O_(2)^(.-))catalyzed by superoxide dismutase(SOD),in which the decomposition of hydrogen peroxide catalyzed by catalase is utilized as a source of oxygen and the autoxidation of pyrogallod as a source of the substrate(O_(2)^(.-)),has been studied by microcalorimetry.The molar reaction enthalpies of the SOD reaction and the pyrogallol autoxidation have been measured as-160.1 and-218 kJ·mol^(-1),respectively,at 298.15 K and pH 7.0.A novel method for the determination of SOD activity,the microcalorimetric assay for SOD,has been proposed.The experimental results showed that SOD had no effect on both kinetic parameters and the mechanism for the pyrogallol autoxidation.This autoxidation followed the second-order reaction kinetics in the presence of limited oxygen(the first order with respect to both pyrogallol and O2),and the second-order rate constants were hermined at 298.15 K and pH 8.0 to be 1.25 and 1.30 L·mol^(-1)·s^(-1 )in the absence and presence of SOD,respectively.A possible mechanism for the autoxidation of pyrogallol inhibited by SOD was also suggested.展开更多
基金Supported by the National Natural Science Foundation of China(39770200) the Natural Science Foundation of Hubei Province (2004ABA228)
文摘An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass of 35 × 10^3 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inbibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Further more, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.
基金Supported by the National Natural Science Foundation of China(30670464,20873092,30800190)Science and Technology Project of Wuhan(200960323115)
文摘To study the thermostability of Nattokinase（subtilisin NAT,NK）,three double mutant plasmids（pET-28a-NKG61C/S98C,pET-28a-NKT22C/S87C,pET-28a-NKS24C/S87C）were constructed by site-directed mutagenesis.Target enzymes were detected using SDS-PAGE and disulfide bond formation was detected using Western blotting analysis.Thermostability was tested by rates of inactivation at certain temperature.The results showed that disulfide bond was not formed within two cysteines and the thermostability of three double mutants was not increased compared with the wild-type NK.The thermostability of NK performed in Ca2＋was stronger than in ethylenediaminetetraacetic acid（EDTA）.But when the temperature reached 62℃,the enzymes rapidly denatured and inactivated even in the presence of Ca2＋.Although the thermostability of mutants was not increased,this study shows a tendency of improving thermostability of NK in protein engineering.
基金Project supported by the National Natural Science Foundation of China(Grant No.29273121)
文摘Thermokinetics of the dismutation of superoxide anion(O_(2)^(.-))catalyzed by superoxide dismutase(SOD),in which the decomposition of hydrogen peroxide catalyzed by catalase is utilized as a source of oxygen and the autoxidation of pyrogallod as a source of the substrate(O_(2)^(.-)),has been studied by microcalorimetry.The molar reaction enthalpies of the SOD reaction and the pyrogallol autoxidation have been measured as-160.1 and-218 kJ·mol^(-1),respectively,at 298.15 K and pH 7.0.A novel method for the determination of SOD activity,the microcalorimetric assay for SOD,has been proposed.The experimental results showed that SOD had no effect on both kinetic parameters and the mechanism for the pyrogallol autoxidation.This autoxidation followed the second-order reaction kinetics in the presence of limited oxygen(the first order with respect to both pyrogallol and O2),and the second-order rate constants were hermined at 298.15 K and pH 8.0 to be 1.25 and 1.30 L·mol^(-1)·s^(-1 )in the absence and presence of SOD,respectively.A possible mechanism for the autoxidation of pyrogallol inhibited by SOD was also suggested.
