Purification and characterization of a novel caffeine oxidase from Alcaligenes species

J Biotechnol. 2006 Sep 18;125(3):319-27. doi: 10.1016/j.jbiotec.2006.03.018. Epub 2006 May 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcaligenes / chemistry
  • Alcaligenes / enzymology*
  • Bacterial Proteins / isolation & purification
  • Caffeine / pharmacology
  • Enzyme Activation
  • Enzyme Stability
  • Heavy Ions
  • Hydrogen-Ion Concentration
  • Oxygenases / chemistry*
  • Oxygenases / isolation & purification*
  • Oxygenases / metabolism
  • Temperature


  • Bacterial Proteins
  • Caffeine
  • Oxygenases
  • caffeine oxidase