Limonin D-ring lactone hydrolase, the enzyme catalysing the reversible lactonization/hydrolysis of D-ring in limonin, has been purified from Citrus seeds and immobilized on Q-Sepharose to produce homogeneous limonoate A-ring lactone solutions. The immobilized limonin D-ring lactone hydrolase showed a good operational stability and was stable after sixty-seventy operations and storing at 4 degrees C for six months.