[Studies of the active site, thermostability and thermophilicity of the thermostable alkaline phosphatase by site-directed mutagenesis].
Through PCR-mediated mutagenesis, three mutants E68S, S70A and E68SS70A around active site S(69) were obtained. Their enzymatic characteristics was determined. It was found that the specific activity of E68S ascended 8 times while its optional reactive temperature climbed 20 degrees C and its Tm descended 3 degrees C; the specific activity of S70A ascended 1 time while its optional reactive temperature climbed 5 degrees C and its Tm descended 2 degrees C; the specific activity of E68SS70A descended 50% while its optional reactive temperature climbed 5 degrees C and its Tm descended 19 degrees C. These result implied that the amino acids, beside the active site, were contributed not only to enzymatic activity but also to its thermostability and thermophilicity. The work provided the direction for mutation to improve enzymatic specific activity and studying the mechanism of thermostability and thermophilicity.
Ji
C N
CN
School of Life Science, Institute of Genetics, State Key Laboratory of Genetics, Fudan University, Shanghai 200433, China.
Zhang
B
B
Jiang
T
T
Sheng
X Y
XY
Mao
Y M
YM
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D000469:Alkaline Phosphatase