Purification and characterization of a novel caffeine oxidase from Alcaligenes species.
Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.
Mohapatra
B R
BR
Water and Watershed Research Program, Department of Biology, University of Victoria, Petch Building 116, 3800 Finnerty Road, Victoria, BC, Canada, V8P5C2. bmohapat@uvic.ca <bmohapat@uvic.ca>
Harris
N
N
Nordin
R
R
Mazumder
A
A
D000421:Alcaligenes / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification; D002110:Caffeine / Q000494:pharmacology; D004789:Enzyme Activation; D004795:Enzyme Stability; D020450:Heavy Ions; D006863:Hydrogen-Ion Concentration; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D002110:Caffeine; D010105:Oxygenases; C121802:caffeine oxidase