[Expression, purification and enzymatic characterization of Thermus thermophilus HB8 aspartate aminotransferase in Escherichia coli].
To obtain thermostable aspartate aminotransferase, the gene aspC from an extremely thermophilic bacterium, Thermus thermophilus HB8 was cloned, and its product was overexpressed in Escherichia coli BL21 (DE3) and Rosetta (DE3). The expression in Rosetta (DP3) was more efficient. The optimum reactive pH was 7, and the recombinant enzyme activity changed little when incubated in the buffer of pH8 - 10 on 37 degrees C for 1 h. The optimum reactive temprature was 75 degrees C, and the recombinant enzyme was more stable on the temperature of 25 - 55 degrees C. The half life of recombinant enzyme on 65 degrees C was 3.5 h, on 75 degrees C was 2.5 h. KmKG was 7.559 mmol/L, VmaxKG was 0.086 mmol/(L x min), KmAsp was 2.031 mmol/L, VmaxAsp was 0.024 mmol/(L x min). Ca2+, Fe3+, Mn2+ inhibited enzyme activity softly.
Zhou
Hua
H
College of Life Science and Pharmacy, Nanjing University of Technology, Nanjing 210009, China.
Hong
Yuan
Y
Yan
Ming
M
Xu
Lin
L
D001219:Aspartate Aminotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D008670:Metals; D011994:Recombinant Proteins; D001219:Aspartate Aminotransferases