PURPOSE: To obtain a hydroxylase purified to electrophoretic uniformity, by subjecting a crude cell-free extract of hydroxylase by sound treatment to a specific treatment including a chromatography stage and eluting a linked protein.

CONSTITUTION: A crude cell-free extract of hydroxylase obtained by destruction of a hydroxylase-containing cell by a controlled sound treatment is subjected to chromatography to elute a linked protein. An eluted solution is fractioned with ammonium sulfate, the fraction is concentrated, the concentrate is chromatographed to pool an eluted fraction containing about 70% of the total hydroxylase activity. Then the eluted solution is chromatographed by using a strongly anionic resin, a linked protein is eluted with 0.1 M-0.5 M KCl linear concentration gradient to give the objective hydroxylase having about 70% purity.