Carica papaya Latex Lipase: sn-3 Stereoselectivity or Short-Chain Selectivity? Model Chiral Triglycerides Are Removing the Ambiguity P. Villeneuve, M. Pina, D. Montet and J. Graille* CIRAD-CP, Laboratoire de Lipotechnie, F34032 Montpellier Cedex, France ABSTRACT: Short-chain fatty acids are usually located at po- sition sn-3 in natural triglycerides, particulary in dairy fats. As a result, it is extremely difficult to differentiate between sn-3 stere- ospecificity and short-chain typoselectivity in many lipases and acyltransferases that perform in this way. This ambiguity can be removed through successive use of a chiral triglyceride with a short fatty acid in position sn-1 and of its racemic in controlled hydrolysis reactions. After checking that the proposed method effectively confirmed the type of activity of control biocatalysts (Candida cylindracea nonspecific lipase and Mucor miehei 1-3 regiospecific lipase), we confirmed that Carica papaya latex has a strict sn-3 stereospecificity. JAOCS 72, 7.53-755 (1995). KEY WORDS: Carica papaya latex, chiral triglyceride, enzy- matic hydrolysis, racemic triglyceride, stereospecificity, typose- lectivity. Lipases (E.C 3.1.1.3) play a predominant role in the digestion mechanism of humans and animals (1). Therefore, there is a considerable amount of literature on the hydrolysis of acyl- glycerols by these enzymes (2-4). A distinction can be made between three kinds of lipases: nonspecific; stereoselective, preferentially hydrolyzing one of the triglyceride positions; and typoselective lipases, which preferentially hydrolyze given fatty acids, irrespective of their positions. There is probably no enzyme that is specific to a single fatty acid; usually, a marked preference for a category of fatty acids, such as short-chain fatty acids from C 4 to Cl0, is ob- served. In dairy fats (5) and in the great majority of natural fats and oils, short-chain fatty acids are distributed in posi- tion sn-3. Studying sn-3 specificity is therefore intricate be- cause it becomes difficult to make a distinction between short-chain fatty acid typoselectivity and sn-3 stereoselectiv- ity. We recently proposed a procedure (6) for synthesis of chi- ral triglycerides with a short-chain fatty acid in position sn-1 and their corresponding racemics. Such molecules remove ambiguity. This article concerns the hydrolytic performance of Carica papaya latex, an acyl- transferase obtained from a plant extract known for its prefer- *To whomcorrespondence should be addressedat CIDAD-CP, Laboratoire de Lipotechnie, BP5035,F34032Montpellier Cedex,France. ence for short chains in dairy fat bioconversion operations. But, is it really short-chain fatty acid typoselective or sn-3 stereospecific? The approach taken in this work provides the answer. EXPERIMENTAL PROCEDURES Triglycerides. The synthesis and stereochemical anaIysis of triglycerides were described recently (6). Enzymes. The Cariea papaya latex was a purified extract (Sigma, Saint Priest, France), and the granular material was ground up before use. The lipozyme IM (Novo Nordisk, Copenhagen, Denmark) was the lipase from Mucor miehei, fixed on an ion exchange resin. The Candida cylindracea li- pase (Sigma) was a preparation that contained lactose. Hydrolysis. Seventy mg of triglycerides was added to 2.3 mL of 0.2M Tris HC1 buffer (pH 8); then 10 mg of enzyme preparation was added and shaken at 40°C for a time, t, in an ultraturax (IKA-Werk, Paris, France) at 10,000 rpm to obtain a hydrolysis rate of about 10% (t = 2 rain for Carica papaya, t = 30 s for Candida cylindracea and for Lipozyme IM). The hydrolysis was stopped by the addition of 1.5 mL of saline acid solution (0.1M H2SO 4, 2.5M NaC1). The fat was extracted three times with 3 mL of ether/heptane solution (75:25, vol/vol). The lipolysis products were then separated by thin-layer chromatography, and the sample was applied with an automatic applicator (Camag Linomat III; Camag, Ltd., Muttenz, Switzerland) to a preparative plate (Kieselgel 60, 0.25 Mesh; Merck, Darmstadt, Germany). Development was carried out in a hexane/ether/acetic acid mixture (50:50:1, vol/vol/vol). The diglycerides and free fatty acids bands were located by transparency and transformed into isopropyl esters. Measurement of the degree of lipolysis. The degree of lipolysis was obtained by KOH titration of the free acids in the reaction medium from a 1-mL sample taken from the 9 mL of ether/heptane extract (75:25, vol/vol). Derivation into isopropyl esters (7). The total amount of diglycerides and free fatty acids recovered were dissolved in a hexane/isopropanol mixture (3:2, vol/vol). Then, 400 ~tL of 6M H2SO 4 and l mL isopropanol were added. The tube was hermetically sealed, shaken, and placed in the oven for 1 h at 100°C. The tube was then cooled in an ice bath, and 5 mL dis- Copyright © 1995 by AOCS Press 753 IAOCS, Vol. 72, no. 6 (1995)