[Site-directed mutagenesis and effects on the enzymatic properties of subtilisin E].
Site-directed mutagenesis was used to investigate the effects of S221C/P225A, N118S/S221C/P225A, D60N/N118S/S221C/P225A and Q103R/N118S/S221C/P225A mutations on the properties of Subtilisin E. It was found that S221C/P225A mutant is 73,000-fold decreased in amidase activity than subtilisin E and 3-fold increased than subtiligase in the ratio of esterase/amidase; N118S/S221C/P225A mutant has 3.6-fold and 15-fold decreased in amidase and esterase activity respectively and as a result, it has a 4-fold lower in the ratio of amidase/esterase than S221C/P225A mutant; Although it has no effect on the esterase activity, D60N/N118S/S221C/P225A mutant enhanced its ratio of amidase/esterase by 15 fold, 3.3-fold and 10.3 fold compared to N118S/S221C/P225A mutant, S221C/P225A mutant and subtiligase respectively; Q103R/N118S/S221C/P225A mutant, however, has a 5-fold enhanced in the amidase activity and 55-fold and 1000-fold decrease in the esterase activity and the ratio of esterase/amidase compared to N118S/S221C/P225A.
Yang
Y H
YH
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences. yonghuayang@hotmail.com
Jiang
L
L
Yang
S L
SL
Wu
Y J
YJ
Zhu
L Q
LQ
D000581:Amidohydrolases / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*
D004950:Esterases; D013381:Subtilisins; D000581:Amidohydrolases; C019665:amidase