Agric. Biol Chem., 52 (5), 1203-1208, 1988 1203

Ester Synthesis by the Lipase from Pseudomonasfragi 22.39 B1" Toshiyuki Nishio, Takahide Chdcano and Minoru Kamimura Research and DevelopmentLaboratories, Sapporo Breweries Ltd., 10 Okatohme, Yaizu, Shizuoka 425, Japan Received November 18, 1987

Ester synthesis by the purified lipase from Pseudomonasfragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)- Monooleinand 1,3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was muchaffected.

The synthesis of esters by the reversal re- fined as the amount which liberated 1 fimol of free fatty action of lipase hydrolysis have been long acid per minute at 37°C and pH 9.0. recognized,1} and several reports have been Reaction mixture. Reaction mixtures for ester synthesis published on the substrate specificity of some from oleic acid and various alcohols consisted of lO mmol microbial lipases for this reaction.2~5) of oleic acid, lOmmol of alcohol, and enzyme solution In our laboratory, the lipase from (1,500 units). For ester synthesis from oleyl alcohol and Pseudomonas (Ps.) fragi 22.39 B was purified various carboxylic acids, reaction mixtures consisted of lOmmol of oleyl alcohol, lOmmol of acid, and enzyme to homogeneity, and was confirmed to be a solution (1,500 units). Reaction mixtures for glyceride thermostable alkaline lipase.6) In our previous synthesis from glycerol and oleic acid consisted of paper, substrate specificity of the enzymefor 1.0~6.0g (about 10~60mmol) of glycerol, 2.0g (about ester hydrolysis was reported.7) 7mmol) of oleic acid, and enzyme solution (300units). This paper deals with the substrate speci- Each reaction mixture was put in a 50-ml Erlenmeyer flask ficity on ester synthesis and modeof action on with a screw cap and incubated at 37°C with constant glyceride synthesis by the lipase from Ps. fragi agitation (300 rpm) by a magnetic stirrer. Estimation of degree of synthesis. Reaction was stopped by addition of 40ml of acetone-ethanol mixture (1 : 1, 22.39B.v/v), and free fatty acid was titrated with 1 n NaOH. The MATERIALS AND METHODS amount of synthesis (%) was calculated from the amount of acid consumedin the reaction mixture. Preparation oflipase. The lipase used in this study was prepared as described in our previous paper,6) and con- Identification of reaction products. The reaction products firmed to be homogeneous on polyacrylamide gel elec- were extracted fromeach reaction mixture with diethyl trophoresis. This purified lipase (lyophilized powder) was ether and identified by thin-layer chromatography (TLC) dissolved in distilled water, and it was used as the enzyme and infrared spectroscopy. Twokinds of TLCplates were solution. used in the experiment. For esters from oleic acid and alcohols or those from oleyl alcohol and carboxylic acids, Measurementof lipase activity. Lipase activity was a silica gel plate (type 60, Merck Co., Ltd.) was used and it estimated by the olive oil emulsion method as describ- was developed in petroleum ether-diethyl ether-acetic ed previously.8) One unit of the enzyme activity was de- acid (70 : 30 : 1, v/v). For glycerides from glycerol and oleic

Studies on Pseudomonasfragi 22.39 B Lipase. Part IV. For Part III, see ref. 7. 1204 T. Nishio, T. Chikano and M. Kamimura

acid, a boric acid-impregnated silica gel plate was pre- injected in triplicate and the areas of the peaks were pared and it was developed in chloroform-acetone- averaged to produce calibration curves for each of the methanol (95 :4.5 :0.5, v/v). The spots of each plate were substances. visualyzed by spraying 1U% phosphomolybdic acid in The result of separation of oleic acid and glycerides of ethanol, and heating.9) oleic acid bythe HPLCis shown in Fig. 1. Figure 2 shows the relationship between the weight of these substances Quantitative estimation of oleic acid and glycerides of and the integrator counts. The calibration curve was oleic acid. Quantitative analysis of oleic acid and glycer- applicable for estimating the amounts of both oleic acid ides of oleic acid extracted with diethyl ether was done at and each glyceride. 25°C by a high performance liquid chromatography (HPLC) with Asahipak GS-310 column (7.5 x 500mm, Chemicals. Oleyl alcohol and oleic acid were purchased Asahi Kasei Co., Ltd.) using acetone as a solvent at the from Sigma Chemical Co. Other acids and alcohols used flow rate of l.Oml/min. Detection was done with a differ- as substrates were obtained from Tokyo Kasei Kogyo Co., ential refractometer, Shodex RI SE-51 (Showa Denko Co., Ltd. Triolein, diolein, monoolein, and other esters used as Ltd.), coupled to a data processor, SIC Chromatocoder 1 1 standards were from Funakoshi Yakuhin Co., Ltd. (System Instrument Co., Ltd.). The attenuation range and chart speed of the data processor were 128 and 10 mm/min. A 10-^1 sample of each standard solution was RESULTS Synthesis of various kinds of monoesters 1) Ester synthesis from various acids and alcohols. The Ps. fragi 22.39 B lipase could catalyze the synthesis of ester from carboxylic acid and alcohol. Figure 3 shows thin-layer chromatograms of the products synthesized from oleic acid and various alcohols by the lipase when 0.2ml of the enzyme solution was added. The synthesized substances were con- firmed to be esters from each substrate by infrared spectroscopy. Table I shows the yields of these esters after an 8-hr incubation. The primary alcohols such as 1-butanol, benzyl Fig. 1. HPLCChromatogram of a Mixture of Oleic alcohol, and geraniol were esterified by the Acid and Glycerides of Oleic Acid. The experiment is descrived in detail in the text.

Fig. 3. Thin-Layer Chromatogram Showing Ester Synthesis from Oleic Acid and Various Alcohols. Mixtures consisting of lOmmolof oleic acid, lOmmolof alcohol, and 0.2ml of enzyme solution (1,500units) were stirred at 37°C for 2hr. Synthesized products were de- Fig. 2. Standard Calibration Curve of Oleic Acid and veloped in petroleum ether-diethyl ether-acetic acid Glycerides of Oleic Acid. (70:30: 1, v/v) on a silica gel plate. A, 1-butanol; B, 1- The experiment is described in detail in the text. #. oleic dodecanol; C, benzyl alcohol; D, geraniol; E, 2-butanol; F, acid; O, monoolein; å¡, diolein; A, triolein. cyclohexanol; G, tertiary butanol; H, terpineol. Ester Synthesis by Ps. fragi 22.39 B Lipase 1205

Table I. Synthesis of Various Kinds of Table II. Synthesis of Oleyl Esters of Esters of Oleic Acid Various Acids Mixtures consisting of lOmmol of oleic acid, lOmmol Mixtures consisting of lOmmol of oleyl alcohol, of alcohol, and 0.2ml of enzyme solution (1,500units) lOmmol of acid, and 0.2ml of enzyme solution (1,500 were stirred at 37°C for 8 hr. The degree of synthesis was units) were stirred at 37°C for 8hr. The degree of syn- calculated from the amount of oleic acid consumed in the thesis was calculated from the amount of acid consumed synthetic reaction. in the synthetic reaction.

Alcohol Degree of synthesis (%) Acid Degree of synthesis (%) (Primary alcohols) (Normal fatty acids) 1 -Butanol 89 Butyric acid 1 1 Benzyl alcohol 83 Laurie acid 90 Geraniol 90 Linoleic acid 91 (Secondary alcohols) (Aromatic acid) 2-Butanol 1 8 Benzoic acid 0 Cyclohexanol 47 (Others) (Tertiary alcohols) Isovaleric acid 0 Tertiary butanol 0 2-Hexyldecanoic acid 0 Terpineol 0

Fig. 4. Thin-Layer Chromatogram Showing Ester Synthesis from Oleyl Alcohol and Various Acids. Fig. 5. Relative Rate of Ester Synthesis from Oleyl Mixtures consisting of lOmmolof oleyl alcohol, lOmmol Alcohol and Various Fatty Acids with Saturated Straight of acid, and 0.2ml of enzyme solution (1,500units) were stirred at 37°C for 2hr. Synthesized products were de- Carbon Chain. veloped in petroleum ether-diethyl ether-acetic acid Mixtures consisting of lOmmol of oleyl alcohol, lOmmol of fatty acid, and 0.2ml of enzyme solution (1,500units) (70: 30: 1, v/v) on a silica gel plate. A, ^-butyric acid; B, were stirred at 37°C for 2hr. Relative activity was ex- caprylic acid; C, lauric acid; D, palmitic acid; E, linoleic acid: F, isovaleric acid; G, 2-hexyldecanoic acid; H, ben- pressed by comparison with the yield of oleyl palmitate zoic acid. . synthesis (82%), which was taken as 100%. various carboxylic acids are shown in Fig. lipase in higher yield than secondary alcohols 4. Table II shows the yields of these esters after such as 2-butanol and cyclohexanol. Within an 8-hr incubation when 0.2ml of the enzyme 8hr of incubation, the reaction with the pri- solution was added. Though the lipase could mary alcohol had reached an equilibrium, use the ftty acids with straight carbon chains as while that with secondary alcohols had not. a substrate, it did not react with carboxylic The tertiary alcohols such as tertiary butanol acids with branched carbon chains such as and terpineol were not esterified at all by the isovaleric acid and 2-hexyldecanoic acid or enzyme. Thin-layer chromatograms of the those with benzene rings such as benzoic acid. products synthesized from oleyl alcohol and After 8 hr of incubation, only the reaction with 1206 T. Nishio, T. Chikano and M. Kamimura

butyric acid did not reach an equilibrium acid and oleyl alcohol were also used. Relative among the three fatty acids with straight car- rates of ester synthesis from oleyl alcohol and bon chain. various fatty acids (C2-C16) are shown in Fig. 2) Effects of carbon chain length offatty 5. Figure 6 shows the relative rate of ester acids or primary alcohols on ester synthesis. synthesis from oleic acid and various primary Various fatty acids and primary alcohols with alcohols (C2-C16). The reaction was done for saturated straight carbon chains were used as 2 hr, adding 0.2 ml of the enzyme solution. The substrates for ester synthesis. In addition, oleic synthesis was enhanced by increasing the car- bon number of the fatty acid, but this tendency was not observed in the case of alcohols. 3) Effects of water content on monoester synthesis. The effects of water content in the reaction mixture on monoester synthesis were investigated using oleic acid and 1-butanol as substrates. To the reaction mixtures, 0.2, 1.4, and 3.4ml of water containing 1,500 units of the lipase were added, respectively (water con- tents of each mixture were about 5.3, 28, and 49%, w/w). The results are shown in Fig. 7. The synthesis of butyl oleate was not much Fig. 6. Relative Rate of Ester Synthesis from Oleic affected by the amount of water in the mixture. Acid and Various Primary Alcohols with Saturated Straight Carbon Chain. Glyceride synthesis by the lipase Mixtures consisting of lOmmol of oleic acid, lOmmol of 1) Synthesis ofglyceridesfrom glycerol and alcohol, and 0.2ml of enzyme solution (1,500units) were stirred at 37°C for 2 hr. Relative activity was expressed by oleic acid. Synthesis of glycerides by the lipase comparison with the yield of butyl oleate synthesis (61%), was investigated using a reaction mixture com- which was taken as 100%. posed of 2.0g of glycerol, 2.0g of oleic acid, and 0. 1 ml ofenzyme solution. The synthesized glycerides were extracted with diethyl ether

Fig. 8. Thin-layer Chromatogram Showing Glyceride Fig. 7. Effects of Water Content on Monoester Synthesis. Synthesis from Glycerol and Oleic Acid. Amixture consisting of2.0 g ofglycerol, 2.0 g ofoleic acid, Mixtures consisting of lOmmol of oleic acid, lOmmol of and 0.1 ml of enzyme solution (300units) was stirred at 1-butanol, and 0.2~3.4ml of enzyme solution (1,500 37°C. Products synthesized at indicated reaction times units) were stirred at 37°C. Degree of synthesis was were developed in chloroform-acetone-methanol (95 : calculated from the amount of oleic acid consumed in 4.5:0.5, v/v) on a boric acid-impregnated silica gel the synthetic reaction. O, 0.2ml enzyme solution; D, plate. OA, oleic acid; MO, monoolein; DO, diolein; TO, 1.4ml enzyme solution; A, 3.4ml enzyme solution. triolein. Ester Synthesis by Ps. fragi 22.39 B Lipase 1207

Fig. 9. Composition of Glycerides Synthesized from Fig. 10. Effects of Water Content on Glyceride Glycerol and Oleic Acid. Synthesis. A mixture consisting of2.0 g ofglycerol, 2.0 g ofoleic acid, Mixtures consisting of 2.0 g of glycerol, 2.0g of oleic acid, and 0.1 ml of enzyme solution (300units) was stirred at and 0.1 ~0.5 ml ofenzyme solution (300 units) were stirred 37°C. Amounts of glycerides and oleic acid were analyzed at 37°C. Degree of synthesis was calculated from the by HPLC. #, oleic acid; O, monoolein; å¡, diolein A, amount of oleic acid consumed in the synthetic reaction. triolein. O, 0.1 ml enzyme solution; å¡, 0.3ml enzyme solution; A, 0.5 ml enzyme solution. Table III. Effects of Glycerol Content on Glyceride Synthesis Almost equimolar mono- and diolein were pro- Mixtures consisting of2.0g ofoleic acid, 1.0~6.0g of duced as oleic acid decreased. Formation of glycerol, and 0.1 ml of enzyme solution (300units) were stirred at 37°C for 24hr. The degree of synthesis was triolein was much less than that of mono- and diolein. calculated from the amount of oleic acid consumed in the synthetic reaction. Synthesized glycerides were extracted 2) Effects of glycerol content on glyceride from the reaction mixture with diethyl ether and mea- sured by HPLC; the relative content (mol%) was synthesis. The effects of glycerol content in the compared. reaction mixture on the glyceride synthesis were investigated. The results after 24 hr of in- _r Relative content cubation are shown in Table III. The amount Glycerol ^ °f (mol%) (g) Sy"^S1S of glyceride increased with increasing glycerol Triolein Diolein Monoolein content. The proportions of formation of tri- and diolein in the mixture decreased with 1.0 75 13.6 47.2 39.2 increasing glycerol content, while that of 2.0 85 12.6 43.0 44.4 4.0 88 10.1 42.8 47.1 monoolein. increased. But such variation of 6.0 93 9.4 42.6 48.0 composition of the products due to glycerol content was not great. 3) Effects of water content on glyceride and identified by the TLC, and their amounts synthesis. To the reaction mixtures for the were measured by HPLC.Figure 8 shows the glyceride synthesis, 0.1, 0.3, and 0.5ml of thin-layer chromatogram of the glyceride syn- water containing 300 units of the lipase were thesis by the lipase. Two clear spots of the added respectively (water contents of each products were initially detected as 1(3)- mixture were about 2.4, 7.0, and 11%, w/w). monoolein and 1,3-diolein. Spots of 2-mono- The results of the glyceride synthesis are shown olein, l,2(2,3)-diolein, and triolein appeared in Fig. 10. The yields of the synthesis were whenthe reaction proceeded to some extent. greatly affected by the amount of water in the The composition of the products and oleic mixture. The composition of the synthesized acid at each reaction time are shown in Fig. 9. glycerides in each mixture after 24-hr incu- 1208 T. Nishio. T. Chikano and M. Kamimura bation was almost the same. extent. They might have been produced by the spontaneous isomerization of l(3)-monoolein and 1,3-diolein. These results indicated that DISCUSSION the lipase of Ps. fragi 22.39 B has a 1,3- The Ps. fragi 22.39 B lipase could catalyze position specificity on glyceride synthesis. It the ester synthesis from fatty acids with wasreported that the enzymeof Geo. candidum straight carbon chains and oleyl alcohol, but was nonspecific as to the position of ester benzoic acid and fatty acids with branching bonds in glyceride synthesis.2) From the above carbon chains such as isovaleric acid and 2- results, it was concluded that the lipase of Ps. hexyldecanoic acid were not used as substrates fragi 22.39 B was different from the enzyme of of ester synthesis. Theactivity wasenhanced Geo. candidum with respect to the positional by increasing the carbon number of the fatty specificity in glyceride synthesis. acid. On the contrary, alcohols such as benzyl Previously, we reported that the lipase of Ps. alcohol, cyclohexanol, and geraniol were used fragi 22.39 B hydrolyzed monoesters such as by the lipase as substrates ofester synthesis as butyl oleate besides triglycerides, and it has a well as alcohols with straight carbon chains, 1,3-position specificity in the hydrolysis of and the activity was not affected by the length triolein. The enzyme could synthesize both of the carbon chain of the primary alcohol. butyl oleate and glyceride of oleic acid, and it These results show that the enzymehas strict showed a 1,3-position specificity in glyceride substrate specificity for carboxylic acids. synthesis. These facts indicate that the ester Okumura, Iwai, and Tsujisaka investigated synthesis is effected by the reversal of ester the substrate specificity of Upases from four hydrolysis, and all esters synthesized by the kinds of microorganisms, Aspergillus {Asp.) lipase could be hydrolyzed by the same enzyme. {Pen.)niger, Geotrichumcyclopium, {Geo.)and Rhizopuscandidum, {Rh.)Penicilliumdele- Acknowledgments. The authors thank Ms. Masayo mar.3) All four Upases synthesized the esters Ohashi for her technical assistance. of various primary alcohols, but they could not esterify the tertiary alcohols at all. Among REFERENCES these four Upases, only the enzyme of Geo. 1) M. Iwai, Y. Tsujisaka and J. Fukumoto, /. Gen. candidumcould use various secondary alcohols Appl. MicrobioL, 10, 13 (1964). as substrates, though the activity was lower 2) Y. Tsujisaka, S. Okumura and M. Iwai, Biochim. than that for primary alcohols. With respect to Biophys. Ada, 489, 415 (1977). the alcohol specificity, the same tendency as 3) S. Okumura, M. Iwai and Y. Tsujisaka, Biochim. the lipase of Geo. candidum was observed with Biophys. Ada, 575, 156 (1979). 4) M. Iwai, S. Okumura and Y. Tsujisaka, Agric. Biol. the enzyme of Ps.fragi 22.39 B. The enzyme of Chem., 44, 2731 (1980). the foregoing four microorganisms and Ps. 5) S. Okumura, M. Iwai and Y. Tsujisaka, Agric. Biol. fragi 22.39 B were similar with respect to the Chem., 48, 2805 (1984). specificity for carboxylic acid. 6) T. Nishio, T. Chikano and M. Kamimura, Agric. In glyceride synthesis from glycerol and Biol Chem., 51, 181 (1987). oleic acid, mono- and diolein were mainly 7) T. Nishio, T. Chikano and M. Kamimura, Agric. produced. 1(3)-Monoolein and 1,3-diolein Biol. Chem., 51, 2525 (1987). 8) N. Watanabe, Y. Ota, Y. Minoda and K. Yamada, were initially detected on TLCplates. Asmall Agric. Biol. Chem., 41, 1353 (1977). spots of 2-monoolein and l,2(2,3)-diolein were 9) D. Kritchevsky and M. C. Krik, Arch. Biochem. appeared whenthe reaction proceeded to some Biophys., 35, 346 (1952).