An advanced method for the determination of carboxyl methyl esterase activity using gas chromatography-chemical ionization-mass spectrometry.
We developed a quantitative method for the determination of methyl esterase activity, analyzing substrate specificity against three major signal molecules, jasmonic acid methyl ester (MeJA), salicylic acid methyl ester (MeSA), and indole-3-acetic acid methyl ester (MeIAA). We used a silylation reagent for chemical derivatization and used gas chromatography (GC)-mass spectroscopy in analyses, for high precision. To test this method, an Arabidopsis esterase gene, AtME8, was expressed in Escherichia coli, and then the kinetic parameters of the recombinant enzyme were determined for three substrates. Finally, this method was also applied to the direct quantification of phytohormones in petals from lilies and roses.
Koo
Yeon Jong
YJ
Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742, Republic of Korea.
Yoon
Eunsil
E
Song
Jong Tae
JT
Seo
Hak Soo
HS
Kim
Jeong-Han
JH
Lee
Yin-Won
YW
Lee
Jong Seob
JS
Cheong
Jong-Joo
JJ
Choi
Yang Do
YD
D017360:Arabidopsis / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000032:analysis*; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D010937:Plant Growth Regulators / Q000032:analysis; D010944:Plants / Q000737:chemistry; D012015:Reference Standards; D014297:Trimethylsilyl Compounds
D007202:Indicators and Reagents; D010937:Plant Growth Regulators; D014297:Trimethylsilyl Compounds; C047270:N,N-bis(trimethylsilyl)-2,2,2-trifluoroacetamide; C039293:trimethylchlorosilane; D002265:Carboxylic Ester Hydrolases