Aggregation behavior of Candida rugosa lipase Yih-Cherng Liou, Alejandro G. Marangoni* & Rickey Y. Yada Department of Food Science, University of Guelph, Guelph, ON, Canada N1G2W1 Candida rugosa lipase aggregated in aqueous media. The emission maximum wavelengths for both intrinsic tryptophan ¯uorescence and surface hydro- phobicity, determined using the probe 8-anilino-1-naphthalene sulfonate, were blue-shifted upon aggregation of CRL. Dynamic light scattering also showed an increase in the molecular weight of CRL as a function of increasing protein con- centration. Apparent dissociation constants derived from the three techniques were 0.48, 3.5 and 1.9 mg/ml, respectively. The speci®c activity of aggregated species (3135 U/mg) was higher than the monomer's (860 U/mg). The aggrega- tion state of lipases may in¯uence kinetic properties, speci®city and interfacial behavior. # 1999 Canadian Institute of Food Science and Technology. Pub- lished by Elsevier Science Ltd. All rights reserved Keywords: lipase, aggregation, activity. INTRODUCTION Two distinct lipase isoforms of Candida rugosa lipase (CRL) have been identi®ed (Veeraragavan and Gibbs, 1989). These two isoforms, however, both had an apparent molecular weight of 58 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophor- esis (SDS-PAGE). On the other hand, under non-dena- turing conditions, the molecular weight of lipase I was 92 kDa, whereas that of lipase II was 58 kDa. Isoelectric focusing showed that these two lipases had isoelectric points of 5.6 and 5.8, respectively. Diering results, however, have been reported by other researchers. Shaw et al. (1989) reported that three distinct forms of lipases were present in a commercial CRL preparation. The apparent molecular weights of lipases A, B, and C, as determined by nondenaturing polyacrylamide gel elec- trophoresis (NDPAGE) were 362, 200, and 143 kDa, respectively. However, results from SDS-PAGE showed that lipases A and C both had molecular weights of 62 kDa. These authors reported that lipase B was con- verted to lipase A upon addition of 1% Triton X-100. Wu et al. (1990) reported on the isolation of two CRL isoforms. Their lipase A had a molecular weight of 120 kDa and an isoelectric point of pH 4.2. Lipase B had the same apparent physical properties as lipase A in terms of electrophoretic mobility, circular dichroic pat- terns, and UV spectra, however, it diered in enantio- meric speci®city. Moreover, when lipase B was treated with sodium deoxycholate and ether-ethanol (1:1), it was converted to the A form with similar enantiomeric speci®city. Brahimi-Horn et al. (1990) reported that a commercial preparation of a CRL displayed six major bands with hydrolytic activity towards a-naphthyl acet- ate (esterase activity stain), detected on an NDPAGE gel, and two bands on an isoelectric focusing gel (pI 4.3 and 4.7). SDS-PAGE showed that the molecular weight of the proteins was 60 kDa. Rua and co-workers (1992) detected two isoforms in a commercial CRL prepara- tion (lipases A and B). Both lipases ran as a single band with similar electrophoretic mobilities under denaturing conditions (64 and 62 kDa, respectively). In addition, the amino acid composition and N-terminal sequences were similar. Under NDPAGE, lipase A showed a sin- gle band when stained for activity (a-naphthyl acetate), protein (silver), or for carbohydrate (I 125 -Con A (Con- canavalin A). On the other hand, lipase B was resolved into four bands after staining for protein or for carbo- hydrate. Using two-dimensional electrophoresis, lipase A migrated as a 60 kDa band with a pI of 5.5, while lipase B was resolved into four species having pI's of 4.80, 4.84, 4.95 and 5.04, all with a molecular weight of 60 kDa. Recently, Chang et al. (1994) compared CRL preparations from three dierent manufacturers using SDS-PAGE in the presence of urea. Lipase type VII (Sigma) contained two major forms and one minor Food Research International, Vol. 31, No. 3, pp. 243±248, 1998 # 1999 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII:S0963-9969(98)00099-4 0963-9969/98/$19.00+0.00 243 *To whom correspondence should be addressed. Fax: +1- 519-824-6631; e-mail: amarango@ uoguelph.ca