Wax Ester-Synthesizing Activity of Lipases Takahiro Tsujitaa,*, Maho Sumiyoshib, and Hiromichi Okudab a

Central Research Laboratory and bDepartment of Medical Biochemistry, School of Medicine, Ehime University, Ehime 791-0295, Japan

ABSTECT: The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of longchain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, wax ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium. Paper no. L8076 in Lipids 34, 1159–1166 (November 1999).

Wax esters (long-chain fatty alcohols esterified to long-chain fatty acids) are widely distributed in living organisms. They are especially abundant in the cuticles of leaves and insects, in marine algae, and in certain bacteria. In mammals, wax esters are synthesized in most tissues, especially the liver (1). Sebaceous glands secrete a lipid mixture containing wax esters onto the skin surface (2). The microsomal membranes of meibomian glands catalyze wax ester synthesis (3,4). Rat brain and murine ascites cells also synthesize wax esters (5,6). Cofactors such as ATP, Mg2+, and CoA reportedly stimulate wax ester formation (7,8). However, the wax ester-synthesizing enzymes have not been purified and characterized. In specific organic solvents lipases have been reported to synthesize certain esters when alcohol (acyl acceptor) is present at high concentration. For example, pancreatic lipase catalyzes the acyl transfer reaction in a 99% organic medium (9), *To whom correspondence should be addressed at Central Research Laboratory, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime, Japan 791-0295, E-mail: [email protected] Abbreviation: BSA, bovine serum albumin. Copyright © 1999 by AOCS Press

and microbial lipase synthesizes acylglycerols from fatty acids and glycerol in a glycerol medium (10). These lipases are used industrially for ester and peptide synthesis in organic media. Riley et al. (11) found that pancreatic lipase catalyzes the synthesis of alcohol ester from fatty acid and ethanol in aqueous medium. Other lipases and carboxylesterase synthesize ethyl esters (12,13). However, high concentrations of ethanol (about 1 to 1.5 M) were used for the assay of ethyl ester synthesis; the ratio of ethanol (acyl acceptor)/free fatty acid (acyl donor) was over 2500. In this study, we focused on the acyl transfer reaction catalyzed by lipases at the surface of the substrate emulsion, and demonstrated that these enzymes catalyze the formation of wax esters from free fatty acids or triacylglycerols and an equimolar amount of long-chain alcohol in an aqueous medium. Our results suggest that lipases generally catalyze wax ester formation on the surface of substrate emulsions at a low concentration of alcohol. Therefore, we postulate that wax esters can be formed in the various organs by various lipases, independently of the presence of ATP, Mg2+, and CoA. MATERIALS AND METHODS Materials. The following enzyme substrates and reagents were used: [1-14C]trioleoylglycerol (3.95 Gbq/mol) and [114 C]oleic acid (2.1 Gbq/mmol) were from Dupont NEN (Boston, MA). Trioleoylglycerol, bile salts, and colipase were from Sigma (St. Louis, MO). Oleic acid, dioleoylglycerol, monooleoylglycerol, palmityl oleate, lauryl oleate, and fatty alcohols were from Funakoshi (Tokyo, Japan). Bovine serum albumin (BSA) was from Wako Pure Chemical Industries (Osaka, Japan) and was extracted by the method of Chen to remove free fatty acid (14). Enzyme sources. Rats (Crj: Wistar) were cared for under guidelines for animal experimentation of the Laboratory Animal Center at Ehime University School of Medicine. Pancreatic lipase from rat pancreas (3200 U/mg protein, at pH 6.8) (15) and carboxylester lipase from porcine pancreas (159 U/mg protein, at pH 6.8) (16) were purified as described previously. Crystalline lipase from Pseudomonas fluorescens was from Amano Pharmaceutical Co. (Nagoya, Japan), and was purified further as described previously (4200 U/mg protein, at pH 6.8) (17). A lingual lipase fraction was prepared from rat tongues. The entire lingual serous glandular region

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was homogenized in cold 25 mM potassium phosphate buffer, pH 6.3, containing 0.9% NaCl. The homogenate was centrifuged at 100,000 × g for 60 min, and the supernatant, which was used as the enzyme solution, was stored at −80°C (0.28 U/mg protein, at pH 5.0) (18). Crude microbial lipases from Mucor lipolyticus (480 U/mg protein, at pH 8.4), Candida cylindracea (526 U/mg protein, at pH 6.8), and Rhizopus sp. (99 U/mg protein, at pH 6.8) were obtained from Amano Pharmaceutical Co. One U (unit) will produce 1.0 µmol of oleic acid from trioleoylglycerol emulsified with gum arabic per minute at 37°C. Enzyme assays. All of the enzyme assays were done in aqueous medium. Wax ester synthesis from oleic acid was determined using [14C]oleic acid. A suspension of 30 µmol [14C]oleic acid (5,000,000 dpm) and 30 µmol fatty alcohol in 2 mL 5% (wt/vol) gum arabic solution was placed in an ice bath and sonicated for 5 min with a Tomy model UD-200 (Tomy, Tokyo, Japan) equipped with a microtip at setting 2–3. The assay mixture consisted of 0.2 mL of 200 mM potassium phosphate buffer (pH 6.8) containing 0.75 µmol [14C]oleic acid (125,000 dpm), 0.75 µmol fatty alcohol, and 2.5 mg gum arabic. Colipase (0.75 µg/mL) and cholic acid (6.25 mM) for pancreatic lipase, and cholic acid (6.25 mM) for carboxylester lipase were added in the assay mixture. The incubation was carried out for 1 h at 37°C, and the reaction was stopped by adding 1.5 mL chloroform/methanol (1:2, vol/vol) containing standard lipids (10 nmol hexadecanol, palmityl oleate, and oleic acid). The mixture was shaken for 15 s, then 0.5 mL chloroform and 0.75 mL H2O were added, and the mixture was shaken again for 5 s, followed by centrifugation at 1,000 × g for 10 min. The lower phase was dried with a stream of nitrogen, solubilized in 50 µL chloroform, and separated by thin-layer chromatography (Whatman silica gel K-5). The plates were developed with hexane/diethyl ether/acetic acid (85:15:1, by vol). The radiolabeled lipid spots (substrateproduct distribution) were directly determined using BioImaging Analyzer, BAS 1000 (Fuji Film, Tokyo, Japan). The radioactivity of lipids was also determined using a liquid scintillation counter. Lipids were located with iodine vapor, and spots of wax ester were cut out for radioactivity measurement. Wax ester hydrolysis was determined using palmityl [14C]oleate or lauryl [14C]oleate as substrate. A suspension of 30 µmol [14C]wax ester (5,000,000 dpm) in 2 mL 5% (wt/vol) gum arabic solution was sonicated for 5 min as described above. The assay mixture consisted of 0.2 mL of 200 mM potassium phosphate buffer (pH 6.8) containing 0.75 µmol [14C]wax ester (125,000 dpm) and 2.5 mg gum arabic. Colipase (0.75 µg/mL) and cholic acid (6.25 mM) for pancreatic lipase, and cholic acid (6.25 mM) for carboxylester lipase were added in the assay mixture. The incubation was carried out for 1 h at 37°C. The lipids were extracted as described above and separated by thin-layer chromatography (Whatman silica gel K-5) with hexane/diethyl ether/acetic acid (85:15:1, by vol). The radiolabeled lipid spots were directly determined using the BAS 1000. The radioactivity of lipids was also determined using a liquid scintillation counter as described above. Lipids, Vol. 34, no. 11 (1999)

Wax ester synthesis from trioleoylglycerol was determined using [14C] trioleoylglycerol. A suspension of 30 µmol [14C]trioleoylglycerol (5,000,000 dpm) and 30 µmol fatty alcohol in 2 mL 5% (wt/vol) gum arabic solution was sonicated for 5 min as described above. The assay mixture consisted of 0.2 mL of 200 mM potassium phosphate buffer (pH 6.8) containing 0.75 µmol [14C]trioleoylglycerol (125,000 dpm), 0.75 µmol fatty alcohol, 2.5 mg gum arabic, and 5 mg BSA. Colipase (0.75 µg/mL) and cholic acid (6.25 mM) for pancreatic lipase, and cholic acid (6.25 mM) for carboxylester lipase were added in the assay mixture. The incubation was carried out for 1 h at 37°C. The lipids were extracted as described above and separated by thin-layer chromatography (Whatman silica gel K-5). The plates were developed with hexane/diethyl ether/acetic acid first at 60:40:2, by vol, and then at 95:5:1, by vol. The separated radioactive lipid spots were determined using the BAS 1000. Preparation of [14C] wax esters. Palmityl [14C]oleate and lauryl [14C]oleate were synthesized by pancreatic lipase-catalyzed esterification of [14C]oleic acid and isolated by preparative thin-layer chromatography. A suspension of 10 µmol [14C]oleic acid (640 kBq) and 20 µmol fatty alcohol in 1 mL 5% (wt/vol) gum arabic solution was sonicated for 5 min. The oleic acid emulsion was incubated with 500 µg of pancreatic lipase at pH 6.5. After incubation for 1 h at 37°C, lipids were extracted as described above and separated by thin-layer chromatography (Whatman silica gel K-5). The plates were developed with hexane/diethyl ether/acetic acid (85:15:1, by vol), and then the lipid spots were scraped off and extracted with chloroform/methanol (2:1, vol/vol). RESULTS Effect of enzyme concentration. When emulsified oleic acid and hexadecanol (molar ratio 1:1) were incubated with pancreatic lipase, wax ester (palmityl oleate) was synthesized in a dose- and time-dependent manner (Fig. 1). Similar results were obtained using carboxylester lipase and P. fluorescens lipase (data not shown). By using high concentrations of lipases, the ratios of palmityl oleate/free oleic acid reached constant values, and the apparent equilibrium ratios were 0.90:0.10 for pancreatic lipase and 0.94:0.06 for carboxylester lipase and P. fluorescens lipase (Fig. 2). Similar results were obtained using oleic acid and dodecanol emulsion as substrates (data not shown). Wax ester-hydrolyzing activities of these lipases were determined using palmityl [14C]oleate as a substrate. Pancreatic lipase was able to hydrolyze palmityl oleate in a concentration-dependent manner, and the ratio of palmityl oleate/free oleic acid also reached a constant value (0.89:0.11) (Fig. 3). This ratio was essentially the same as the ratio obtained for wax ester synthesis (0.90:0.10) and was not changed during long-term incubation (at 84.5 µg/mL lipase for 48 h incubation). Similar results were observed using lauryl [14C]oleate as a substrate (data not shown). Carboxylester lipase and P. fluorescens lipase were also able to hydrolyze palmityl

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FIG. 1. Effect of incubation time on the synthesis of palmityl oleate by pancreatic lipase. The synthesis of palmityl oleate was determined using [14C]oleic acid (3.75 mM) and hexadecanol (3.75 mM) as substrates. After incubation at 37°C, lipids were extracted and the molar fraction of palmityl oleate was determined.

oleate in a concentration-dependent fashion, and the apparent equilibrium ratios of palmityl oleate/free oleic acid were 0.93:0.07, essentially the same as the ratio (0.94:0.06) for the

FIG. 2. Effect of lipase concentration on the synthesis of palmityl oleate. The synthesis was determined using [14C]oleic acid (3.75 mM) and hexadecanol (3.75 mM) as substrates. After incubation at pH 6.8 and 37°C for 1 h, lipids were extracted and the molar fraction of palmityl oleate was determined. ● , Pancreatic lipase from rats; ●, carboxylester lipase from porcine pancreas; ▲, Pseudomonas fluorescens lipase.

FIG. 3. Effect of pancreatic lipase concentration on the synthesis/hydrolysis of palmityl oleate with or without bovine serum albumin (BSA). Palmityl [14C]oleate (3.75 mM) hydrolysis and palmityl oleate synthesis from [14C]oleic acid (3.75 mM) and hexadecanol (3.75 mM) were determined with or without BSA (25 mg/mL). After incubation at 37°C for 1 h, lipids were extracted and the molar fraction of palmityl oleate was determined. ● , Synthesis without BSA; ■ , synthesis with BSA; ●, hydrolysis without BSA; ■, hydrolysis with BSA.

synthesis (data not shown). In these condition, these lipases completely degraded ethyl oleate: the apparent equilibrium ratios of ethyl oleate/free oleic acid were about 0.005:0.995 (data not shown). Upon addition of 2.5% BSA, the apparent equilibrium ratio changed, the ratio of palmityl oleate/free oleic acid being 0.47:0.53 for hydrolysis and 0.51:0.49 for synthesis (Fig. 3). Effect of alcohol chain length and pH. Figure 4 shows the effect of saturated alcohol fatty acyl chain length on wax ester synthesis. The patterns of three enzymes were superimposable. The apparent equilibrium ratio of wax ester/free oleic acid was around 0.9:0.1 when fatty alcohols with acyl chains longer than decanol were used as substrates. The ratio decreased when fatty alcohols with acyl chains shorter than octanol were used, and no wax ester-synthesizing activity was detected when ethanol was used as a substrate. Figure 5 shows the pH activity curves for palmityl oleate synthesis by three lipases. The three pH activity curves were superimposable. Below pH 7.0, the apparent equilibrium ratios of palmityl oleate/free oleic acid were about 0.9:0.1. The ratio of palmityl oleate/free oleic acid decreased sharply at alkaline pH. Above pH 8.0, wax ester-synthesizing activity decreased and wax ester-hydrolyzing activity increased. When the activity of hydrolysis/synthesis was measured at pH 9.0 for 48 h using a high concentration of pancreatic lipase (84.5 µg/mL), the ratio Lipids, Vol. 34, no. 11 (1999)

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FIG. 4. Effect of fatty alcohol acyl chain length on synthesis of wax ester, as determined using [14C]oleic acid (3.75 mM) and saturated alcohols of various chain lengths (3.75 mM) as substrates. After incubation at pH 6.8 and 37°C for 1 h, lipids were extracted and the molar fraction of wax ester was determined. ● , Pancreatic lipase (84.5 µg/mL); ●, carboxylester lipase (100 µg/mL); ▲, Pseudomonas fluorescens lipase (100 µg/mL).

FIG. 5. Effect of pH on the synthesis of palmityl oleate, as determined using [14C]oleic acid (3.75 mM) and hexadecanol (3.75 mM) as substrates. After incubation at 37°C for 1 h, lipids were extracted and the molar fraction of palmityl oleate was determined. Acetate buffer (pH 5.0 and 5.5), phosphate buffer (pH 6.0–8.0), and Tris buffer (pH 8.0–9.0) were used. The ionic strength of reaction mixture was 0.1. Symbols and lipase concentration are as in Figure 4.

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of palmityl oleate/free oleic acid was 0.22:0.78 for synthesis and 0.46:0.54 for hydrolysis (data not shown). Wax ester synthesis by other lipases. Other lipases obtained from rat tongues, M. lipolyticus, C. cylindracea, and Rhizopus species also synthesized wax ester dose-dependently from oleic acid and hexadecanol (data not shown). However, the enzyme concentrations at which equilibrium was reached were not the same: the apparent equilibrium ratio (0.89:0.11 for palmityl oleate/oleic acid) was reached by 0.6 U/mL of lingual lipase, 3 U/mL C. cylindracea lipase and 5 U/mL Rhizopus sp. lipase, but the equilibrium ratio was not reached by 5 U/mL of M. lipolyticus lipase (0.51:0.49 for palmityl oleate/oleic acid); the equilibrium ratio (0.92:0.08 for palmityl oleate/oleic acid) was reached by 20 U/mL of M. lipolyticus lipase. No wax ester-synthesizing activity from oleic acid and hexadecanol was detected when carboxylesterases from rat adipose and kidney were used as enzymes (data not shown). Wax ester synthesis from triacylglycerol. Figure 6 shows the effect of lipase concentrations on palmityl oleate formation from trioleylglycerol with 2.5% BSA. When [14C]trioleylglycerol/hexadecanol (molar ratio, 1:1) emulsion was incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, the formation of oleic acid and palmityl oleate increased with increasing lipase concentration. At a high concentration of pancreatic lipase (84.5 µg/mL), the molar ratio of wax ester, oleic acid, and monooleoylglycerol was about 1:1.2:1. At a high concentration of P. fluorescens lipase (over 20 µg/mL), the molar ratio of wax ester to oleic acid was about 1:2. Rat lingual lipase and microbial lipases also synthesized wax ester from trioleoylglycerol (Table 1). The effect of saturated fatty alcohol acyl chain length on wax ester formation from trioleoylglycerol by pancreatic lipase, carboxylester lipase, and P. fluorescens lipase is shown in Figure 7. Trioleoylglycerol was almost completely degraded by high concentrations of lipases. The patterns of oleic acid formation were roughly the inverse of wax ester formation: free oleic acid formation decreased with an increase in the acyl chain length of fatty alcohol, whereas wax ester formation increased. Wax ester formation was not detected when fatty alcohols with acyl chains shorter than hexanol were used. Figure 8 shows the effect of pH on palmityl oleate synthesis from trioleoylglycerol by three lipases. Trioleylglycerol degradation and oleic acid formation increased with rising pH up to 9.0. However, palmityl oleate synthesis decreased sharply at alkaline pH. Similar results were observed using other lipases: palmityl oleate synthesis decreased at pH 9.0 compared to pH 6.8 (Table 1). DISCUSSION The typical substrates for lipase(s) are long-chain triacylglycerols, which are separated from the aqueous medium by the surface phase. Thus, lipase must be adsorbed to the lipid surface, and the quality of the surface is an important factor for lipase activity. At the lipid surface, the adsorbed lipase can

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TABLE 1 Palmityl Oleate Synthesis from Trioleoylglycerol by Various Lipasesa PO Lipase

pH

Lingual

5.0 6.8 6.8 9.0 6.8 9.0 6.8 9.0

Mucor lipolyticus Candida cylindracea Rhizopus sp.

TO

DO

MO

OA

(µmol/reaction mixture) 0.370 0.263 0.718 0.156 0.767 0.367 0.445 0.083

0.346 0.221 0.162 0.002 0.026 0.429 0.049 0.138

0.266 0.335 0.107 0.015 0.005 0.088 0.211 0.171

0.054 0.150 0.026 0.456 0.000 0.066 0.577 0.585

0.236 0.512 0.812 1.611 1.402 0.361 0.665 0.832

a Wax ester synthesis was determined using [14C]trioleoylglycerol (3.75 mM) and hexadecanol (3.75 mM) as substrates with bovine serum albumin (25 mg/mL). After incubation at 37°C for 1 h, lipids were extracted and the molar concentrations of palmityl oleate (PO), trioleoylglycerol (TO), dioleoylglycerol (DO), monooleoylglycerol (MO), and oleic acid (OA) were determined. Lingual lipase concentration was 0.45 U/mL, and M. lipolyticus, C. cylindracea, and Rhizopus sp. lipase concentrations were 10 U/mL.

FIG. 6. Effect of lipase concentration on synthesis of palmityl oleate from trioleylglycerol, as determined using [14C]trioleoylglycerol (3.75 mM) and hexadecanol (3.75 mM) as substrates with BSA (25 mg/mL). After incubation at pH 6.8 and 37°C for 1 h, lipids were extracted and their molar concentrations determined. ●, Palmityl oleate; ■ , trioleylglycerol; ● , oleic acid; ■, dioleoylglycerol; ▲ , monooleoylglycerol. For abbreviation see Figure 3.

catalyze various reactions such as hydrolysis, esterification and transesterification. Therefore, substances that affect the surface would be expected to influence the lipase reaction

rate. Fatty alcohols affect lipase activity in this way. Ferreira and Patton (19) suggested that the inhibition of lipase activity by fatty alcohols was due to inhibition of interfacial activation of the lipase by dilution of the high surface concentration of substrate. Mattson et al. (20) reported that the inhibitory effect of long-chain fatty alcohol is due to its adsorption on the substrate, thus blocking the interaction with enzyme. Both of these groups suggested that the slight inhibition of lipolysis was due to the esterification of fatty acid by fatty alcohols. However, they assayed lipase activities at alkaline pH (pH 8.0 and 9.0). If the lipase activities had been assayed at neutral pH, the contribution of esterification to the inhibition of lipolysis (fatty acid release) might have been greater, because the equilibrium favored ester formation at neutral pH. Pancreatic lipase (21,22), carboxylester lipase (23,24), and P. fluorescens lipase (25) contain the common active site sequence (Gly-Xaa-Ser-Xaa-Gly) and Asp-His-Ser triad, which are characteristics of serine proteinase. Its catalytic mechanism resembles that of serine proteinases, proceeding via an acyl-enzyme intermediate. In this study, we demonstrated that lipases catalyze the synthesis of wax ester from fatty acid/triacylglycerol and long-chain fatty acyl alcohols by the mechanism shown in Scheme 1. Incubation of lipases with free fatty acid or triacylglycerol results in the formation of an acyl-enzyme intermediate, which is deacylated by nucleophilic attack of water or alcohol. When water is the fatty acid acceptor, hydrolysis occurs, but when alcohol is the acceptor, wax ester synthesis (alcoholysis) occurs. Long-chain fatty acyl alcohols such as hexadecanol are more efficient acyl acceptors than water. One explanation for this phenomenon is that alcohol is much more hydrophobic than water. Therefore, the long-chain alcohols may transfer to the lipid–water interface more easily than to water. At neutral pH, only 10% of the wax esters were hydrolyzed by a high concentration of lipase during long-term incubation (48 h). Conversely, about 90% of oleic acid was esterified by lipase at high concentration. Therefore, the ap-

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FIG. 7. Effect of fatty alcohol acyl chain length on the synthesis of wax ester from trioleoylglycerol, as determined using [14C]trioleoylglycerol (3.75 mM) and saturated alcohols with various chain lengths (3.75 mM) as substrates with BSA (25 mg/mL). After incubation at pH 6.8 and 37°C for 1 h, lipids were extracted and their molar concentrations determined. ●, Wax ester; ■ , trioleylglycerol; ●, oleic acid; ■, dioleoylglycerol; ▲, monooleoylglycerol. For abbreviation see Figure 3.

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FIG. 8. Effect of pH on palmityl oleate synthesis from trioleylglycerol. Wax ester synthesis was determined using [14C]trioleoylglycerol (3.75 mM) and hexadecanol (3.75 mM) as substrates with BSA (25 mg/mL). After incubation at 37°C for 1 h, lipids were extracted and their molar concentrations determined. Acetate buffer (pH 5.0 and 5.5), phosphate buffer (pH 6.0–8.0) and Tris buffer (pH 8.0–9.0) were used. The ionic strength of reaction mixture was 0.1. Symbols are as in Figure 6. For abbreviation see Figure 3.

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SCHEME 1

parent equilibrium ratio of ester/oleic acid reached 0.9:0.1 (Fig. 3). However, the equilibrium ratio was affected by addition of BSA: the equilibrium ratio was changed to around 0.5/0.5 in the presence of BSA (25 mg/mL) (Fig. 3). But when triacylglycerol was used as the acyl donor, the equilibrium ratio was not affected in the presence of BSA: the molar ratio of wax ester/oleic acid/monooleoylglycerol was about 1:1.2:1 at a high concentration of pancreatic lipase, and wax ester/oleic acid was 1:2 at a high concentration of P. fluorescens lipase (Fig. 6). With a fatty acid as substrate (upper portion, Scheme 1), the enzyme competes for acyl-enzyme intermediate formation with BSA, which traps free fatty acid. With triacylglycerol as a substrate (lower portion, Scheme 1), BSA cannot affect the process of acyl-enzyme intermediate formation and nucleophilic displacement. Enzymatically catalyzed wax ester synthesis may proceed any of in three ways (26): (i) Free fatty acids are directly incorporated into wax esters. Wax ester is synthesized from palmitic acid and hexadecanol in the liver of both rat and dogfish, and this reaction is not influenced by ATP, CoA, or Mg2+ (27). (ii) Fatty acyl-CoA is used as the acyl donor for the esterification of alcohols. Wax ester synthesis in murine preputial gland tumor is catalyzed by an acyltransferase which uses fatty acyl-CoA as the acyl donor (6). AcylCoA:fatty alcohol acyltransferase in microsomal preparations from the meibomian gland catalyzes the synthesis of wax ester from acyl-CoA and long-chain fatty acyl alcohols (28). (iii) Acyl moieties are transferred from phospholipids to alcohols. Wax ester is synthesized by retinal pigment epithelial membranes via acyl transfer of a palmityl group from the sn-1 position of an endogenous lecithin to alcohols (29). The present findings suggest that lipases catalyze the synthesis of wax esters in two ways: direct esterification of free fatty acid and acyltransfer from triacylglycerol to alcohol. In conclusion, various lipases can catalyze the synthesize wax ester from free fatty acid or triacylglycerol, independent of the presence of ATP, Mg2+, and CoA. At neutral pH and by using long-chain fatty acyl alcohols, the equilibrium ratio of wax ester and free fatty acid favored ester formation: the ratio was about 0.9:0.1. Therefore, various lipases might catalyze the synthesis of wax esters in the various organs.

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[Received December 2, 1998, and in revised form April 20, 1999; final revision accepted September 20, 1999]