Novel cholesterol esterase secreted by Streptomyces persists during aqueous long-term storage.
We isolated a moderate thermophilic actinomycete, Streptomyces sp. X9, from soil and purified cholesterol esterase (CHE) from the culture medium to homogeneity. The molecular masses of the purified CHE estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration chromatography were 23.6 and 163 kDa, respectively, indicating that the enzyme assumes an oligomeric form. Heavy metals such as Hg2+ and Ag+ similarly inhibited the activity of the CHE in the same manner as those of other bacterial CHEs. The activity of Streptomyces sp. X9 CHE was susceptible to dithiothreitol, beta-mercaptoethanol and p-chloromercuribenzoate, but resistant to phenylmethylsulfonyl fluoride, unlike those of other bacterial CHEs. The purified CHE could utilize both cholesteryl and p-nitrophenyl (pNP) esters of fatty acids as substrates. Steady-state kinetics revealed respective Km values for cholesteryl myristate and pNP-myristate of 0.34 and 1.1 mM, indicating that the cholesteryl residue is important for catalysis. We also found that the Km for the pNP esters are dependent on the chain length of the substrate fatty acid residues. These results indicate that the novel CHE specifically hydrolyzes substrates by recognizing both cholesteryl and fatty acid moieties. The enzyme was stable during long-term aqueous storage at room temperature, indicating its potential application as a diagnostic reagent.
Xiang
Hongyu
H
Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
Takaya
Naoki
N
Hoshino
Takayuki
T
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D021381:Protein Transport; D002787:Sterol Esterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003902:Detergents; D004791:Enzyme Inhibitors; D002787:Sterol Esterase