粪产碱杆菌青霉素G酰化酶的分离纯化及酶学性质

Purification and Characterization of Alcaligenes faecalis Penicillin G Acylase

采用DEAE-Sepharose阴离子交换层析和Butyl-Sepharose CL 4B疏水层析，从重组大肠杆菌DH5α菌体中分离纯化得到粪产碱杆菌青霉素G酰化酶（AfPGA）。经纯化，AfPGA纯度较粗酶提高50．6倍、比活达到212．7U／mg，经HPLC测定纯度为92．8％，收率为78％。理化性质分析表明：AfPGA含有2个亚基（α亚基和β亚基，其Mw分别为2．90×10^4和5．92×10^4；AfPGA等电点约为7．9～8．0，最适pH约为10．0，并在pH〉7．0时表现出了稳定的催化活力。

Alcaligenes faecalis penicillin G acylase（AfPGA） was isolated from recombinant Escherichia coli DH5α, and purified by a combination of DEAE-Sepharose anion-exchange chromatography and Butyl-Sepharose CL 4B hydrophobic chromatography. The purity of the AfPGA obtained was 92. 8% by HPLC assay. The specific activity attained 212. 7 U/mg protein with a purification fold of 50. 6 comparing to the crude enzyme. The total recovery yield was 78%. The investigation of physiochemical properties of the pure enzyme showed that AfPGA had two subunits（α subunit and β subunit）, whose molecular weights were 2.90×10^4 and 5.92×10^4 , respectively. The isoelectric point of the enzyme was between pH 7.9 and 8.0. Furthermore, the enzyme exhibits highest activity and good stability at pH 10.0 and above 7.0,respectively.