Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli.
The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.
Wen
Yong
Y
Department of Drug Biosynthesis, School of Pharmacy, Fudan University, Shanghai 200032, China.
Shi
Xunlong
X
Yuan
Zhongyi
Z
Zhou
Pei
P
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D044086:Kluyvera / Q000201:enzymology*; D010399:Penicillin Amidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020161:Physical Chromosome Mapping; D010957:Plasmids; D011454:Prostaglandins A / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature
D011454:Prostaglandins A; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010399:Penicillin Amidase