An improved method for the enrichment of phenylalanine ammonia lyase (E.C.4.3.1.5-PAL) activity of Rhodotorula yeast is described. Whole cells of various Rhodotorula yeast strains showed low levels of PAL activity on account of membrane permeability barriers for the substrate, L-phenylalanine. Different methods of enriching PAL activity including ultra-sonication, detergent and enzyme solubilization were investigated in this study in an effort to overcome these permeability barriers. Ultra-sonication of the yeast cells was found to be the most effective of the methods and yielded over 10-fold higher enzyme activity as compared to untreated whole cells. Solubilization of the yeast cells involving a synergistic treatment with Triton-X-100 and glucuronidase gave about nine-fold enrichment while cells treated individually with Triton-X-100, glucuronidase and cetyl trimethyl ammonium bromide gave 6.8, 5.9-and 5.8-fold higher activity, respectively. Homogenization; treatment with chloroform, isopropanol. and toluene; freezing and thawing were ineffective in enhancing the enzyme activity. Of the six Rhodotorula yeast strains employed in this study, Rhodotorula glutinis, NCYC 61 exhibited maximum PAL activity. This simple, rapid and improved method of PAL activity enrichment could serve as a rich enzyme source, especially in the biosynthesis of L-phenylalanine and L-phenylalanine methyl ester. (C) 2004 Elsevier Inc. All rights reserved.