CRYSTALLIZATION AND CHARACTERIZATION OF CANDIDA RUGOSA LIPASE

Abstract

A lipase isolated from the fungus Candida rugosahas beenpurified and crystallized in a form suitable for X-ray crystallographic structure determination. Several proteins with lipolytic activity having isoelectric points from 4.2 to 5.8 were separated by ion exchange chromatography.The protein having the lowestisoelectric point was crystallized from 2-methy]-2,4-pentandiol in MES bufferin the presenceof calcium (II) salts. The crystals with cell dimensions a=64.9(1) 7 b=97.2(1) A and b=175.8(2)A, grow as large colorless plates often exceeding 0.7 mm in each of two dimensions. From the diffraction pattern the apparent crystal symmetry is C222,. Experimental density determination suggests one 60,000 M,. molecule in the asymmetric unit and approximately 50% solvent by volume. Theratio unit cell volume to the molecular weightof the contents of the unit cell, Vj, is 2.3 A3/d. Diffraction is strong to a resolution of 2 A resolution and work is underway to determine the three dimensional structure of this enzyme.