Fujii_2003_Biochim.Biophys.Acta_1631_197

Reference

Title : Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester - Fujii_2003_Biochim.Biophys.Acta_1631_197
Author(s) : Fujii R , Utsunomiya Y , Hiratake J , Sogabe A , Sakata K
Ref : Biochimica & Biophysica Acta , 1631 :197 , 2003
Abstract :

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.

PubMedSearch : Fujii_2003_Biochim.Biophys.Acta_1631_197
PubMedID: 12633686

Related information

Inhibitor Methyl-DEUP
Substrate Methyl-DEUP    Methyl-DEUP

Citations formats

Fujii R, Utsunomiya Y, Hiratake J, Sogabe A, Sakata K (2003)
Highly sensitive active-site titration of lipase in microscale culture media using fluorescent organophosphorus ester
Biochimica & Biophysica Acta 1631 :197

Fujii R, Utsunomiya Y, Hiratake J, Sogabe A, Sakata K (2003)
Biochimica & Biophysica Acta 1631 :197