Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas).
Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.
Fu
Xue-Yan
XY
Department of Food Science and Technology, Ocean University of China, Qingdao, Shandong Province, 266003, People's Republic of China.
Xue
Chang-Hu
CH
Miao
Ben-Chun
BC
Liang
Jun-Ni
JN
Li
Zhao-Jie
ZJ
Cui
Feng-Xia
FX
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D049832:Decapodiformes / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013696:Temperature
D004791:Enzyme Inhibitors; D000446:Aldehyde-Lyases; C023188:trimethylamine-oxide aldolase