Purification of recombinant wheat cytochrome P450 monooxygenase expressed in yeast and its properties.
To investigate the properties of wheat cytochrome P450 and the characteristics of herbicide metabolism by cytochrome P450 in vitro, deeply understand the mechanisms of herbicide selectivity, recombinant wheat cytochrome P450 monooxygenase (CYP71Cv1) heterologously expressed in yeast was purified by DE-52 cellulose chromatography and fast protein liquid chromatography (FPLC) with Mono-Q column. The degree of purification was 1366-fold. The specific activity of purified cytochrome P450 reached to 512 nmol min-1 mg-1 protein with herbicide chlorsulfuron as substrate. The purified cytochrome P450 exhibited one band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular mass was 52.5 kDa. Kinetic parameter was determined in vitro. The Km values for chlorsulfuron and triasulfuron were 57 (+/-15) and 38 (+/-16) microM, respectively; and Vmax for chlorsulfuron and triasulfuron were 4.1 (+/-0.7) and 2.7 (+/-0.5) nmol min-1 mg-1protein in vitro, respectively.
Wen-Sheng
Xiang
X
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Science, Beijing 100080, China.
Xiang-Jing
Wang
W
Tian-Rui
Ren
R
Su-Qin
Ci
C
D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006540:Herbicides / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013449:Sulfonamides / Q000737:chemistry; D013997:Time Factors; D014227:Triazines / Q000737:chemistry; D014908:Triticum / Q000201:enzymology* / Q000235:genetics
D006540:Herbicides; D011994:Recombinant Proteins; D013449:Sulfonamides; D014227:Triazines; D003577:Cytochrome P-450 Enzyme System; C037137:chlorsulfuron