Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester.
The fluorescent organophosphorus esters, diethyl 4-methylumbelliferyl phosphate (1), ethyl hexyl 4-methylumbelliferyl phosphate (2) and ethyl 4-methylumbelliferyl heptylphosphonate (3) have been synthesized and evaluated as a sensitive active-site titrant of lipase. The phosphorus esters 1, 2 and 3 inactivated the lipase from Pseudomonas aeruginosa (LPL-312) with a second-order rate constant for enzyme inactivation (k(on)) of 1.8, 32 and 5600 s(-1) M(-1), respectively. The long-chain phosphonate 3 turned out to be the most potent inactivator of the lipase to release a stoichiometric amount of highly fluorescent 4-methylumbelliferone (4MU) as a leaving group. By using the phosphate 3 as an active-site titrant, the low concentration (4.5 nM) of the active lipase was titrated successfully. The highly sensitive active-site titration with 3 enabled the direct determination of the concentration of the active lipase expressed in a microscale culture medium. Although the expression level differed significantly from one culture to another, the titrated concentration of the active lipase was proportional to the apparent activity for all the independent cultures. The molecular activity calculated for the expressed lipase was found to be the same as that of the purified lipase. The present active-site titration method is widely applicable to the biocatalytic engineering of lipases such as directed evolution, site-directed mutagenesis, chemical modification and immobilization.
Fujii
Ryota
R
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Utsunomiya
Yuji
Y
Hiratake
Jun
J
Sogabe
Atsushi
A
Sakata
Kanzo
K
D001665:Binding Sites; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D007202:Indicators and Reagents; D008994:Monoacylglycerol Lipases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017186:Titrimetry / Q000379:methods*
D003470:Culture Media; D007202:Indicators and Reagents; D009943:Organophosphorus Compounds; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone; D008994:Monoacylglycerol Lipases