A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1.
An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was purified by Ni-chelation affinity chromatography. It showed a single band with a molecular mass of 18kDa in SDS-PAGE. The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C. The enzyme activity was Ca(2+)-independent. Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.
Wang
Baijing
B
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China. yfeng@mail.jlu.edu.cn
Lu
Dongmei
D
Gao
Renjun
R
Yang
Zhen
Z
Cao
Shugui
S
Feng
Yan
Y
D041522:Aeropyrum / Q000201:enzymology*; D000595:Amino Acid Sequence; D004492:Edetic Acid; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D011994:Recombinant Proteins; D004492:Edetic Acid; D004950:Esterases; D010741:Phospholipases A; D054467:Phospholipases A2