Novel process for enzymatic production of sterol esters in microaqueous solution with cholesterol esterase from Trichoderma sp. AS59 Atsushi Maeda a, Norihumi Hashitani a, Takayuki Mizuno a, Masanori Bunya a a Faculty of Engineering, Tokushima Bunri University, 1314-1 Shido, Sanuki 769-2193, Japan (maeda@fe.bunri-u.ac.jp) ABSTRACT Phytosterols, their structure are very similar to that of cholesterol, have promise as a functional food material. However, they have not been actively used as material for low solubility in both oil and water. The research objective is to increase the solubility by modifying the sterol using microbial enzymes and to broaden the reach of phytosterols as biomass material and a food additive in our laboratory. We recently reported the characterization of novel cholesterol esterase from Trichoderma sp. AS59 isolated from soil. Therefore, in the present study, we examined the ability of the enzyme to synthesize steryl esters from sterol and free fatty acids of varying chain lengths. As a result, all the medium- and long-chain free fatty acids used are successfully employed in cholesterol ester synthesis and the yield of cholesteryl palmitate reached over 6% in large scale such as 3 ml of solvent. Of additional interest is that the fatty acid specificities in the synthesis and hydrolysis of cholesteryl esters were entirely different from each other. Furthermore, we attempted to synthesize stigmasteryl palmitate as a food additive and found that the enzyme catalyzed the synthesis of the ester. That indicates the potential utility of the enzyme in the food industry. Keywords: Cholesteryl ester; Plant sterol ester; Cholesterol esterase; Trichoderma sp. Stigmasterol INTRODUCTION Sterol is very important material as components of biological membranes. The animal has a sterol in the form of cholesterol, which is not only the above as a constituent of biological membranes, but also the precursors for vitamin D3, steroid hormones and bile acids required for lipid absorption. Thus, cholesterol is an important matter for us humans, but excessive cholesterol deposited in the arteries will also act as triggers for serious medical problems such as atherosclerosis and myocardial infarction [1]. The excessive cholesterol intake has recently become a serious problem with the change in diet. The structures of phytosterols that are naturally occurring in plants are very similar to that of cholesterol, and they are rich in vegetable oil such as soybean, canola and corn oil. But even if we consume plant sterols, we can hardly be absorbed into the body because of the difference of structure between cholesterol and phytosterols. Furthermore, they are effective in inhibiting the absorption of cholesterol in the small intestine. An inverse correlation between plant sterol ingestion and cholesterol absorption was revealed earliest by Sugano et al [2-4]. Thus, phytosterols have promise as a functional food material. However, they have not been actively used as material for low solubility in both oil and water. Therefore, in our laboratory, the research objective is to increase the solubility by modifying the sterol using microbial enzymes and to broaden the reach of phytosterols as biomass material and a food additive. We found a novel cholesterol esterase to synthesize steryl ester from the culture filtrate of a fungal strain Trichoderma sp. AS59 isolated from soil [5]. The enzyme exhibited not only wide-ranging hydrolytic activities on cholesterol esters of short-, medium- and long-chain fatty acids. In this study, we examined the ability of the enzyme to synthesize steryl esters from sterol and free fatty acids of varying chain lengths. If the ability of the synthesis of steryl ester is similar to that of the hydrolysis, it is possible to synthesis the useful sterol esters as a functional food material. This leads to further widen the application of plant sterol to food, and it is still challenging work. MATERIALS & METHODS Enzyme and chemicals Cholesterol esterase from Trichoderma sp. AS59 was purified from a 3-day liquid culture according to the method already reported [5]. The purified enzyme was dialyzed against distilled water, and lyophilized
before use. One unit of activity was defined as the amount of the enzyme producing 1 µmol of free cholesterol per min using cholesterol linoleate as substrate. Cholesterol (purity > 99%) and oleic acid (99%) were purchased from Wako Pure Chemical Industry (Osaka, Japan). Stigmasterol (95%) was purchased from Tama Biochemical (Tokyo, Japan). Saturated fatty acids of different chain lengths (C2 - C18; > 97%), and their cholesterol esters (> 95%) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Enzyme reactions between sterol and fatty acids The substrate solution was performed using following process. After adding the excess amount of cholesterol to 5 ml of hexane, the hexane solution was shaken and stood at 27 o C for overnight. We prepared the substrate solution by adding.95 ml of oleic acid (at final concentration of 1 mmol/l) to the 3 ml of the supernatant. The enzyme solution was prepared by adding.1 ml of distilled water to 1 U of lyophilized cholesterol esterase. Then, the esterification was examined by mixing 3 ml of the substrate solution with.1 ml of the enzyme solution in 5 ml glass vial. A mixture was shaken at 12 rpm in a 27 o C water bath for 12 h. The effect of chain length of free fatty acids (FFAs) on sterol esterification was also examined in the same method. However, the final concentration of each FFA was prepared at 1 mmol/l in the substrate solution. The effect of water content on the esterification of cholesterol with palmitic acid was examined in a similar method according to the following procedures. We processed water-saturated hexane by mixing 2 ml of hexane and 5 ml of.2 mol/l phosphate buffer (ph 7.) and standing at 27 o C for overnight. Then, we prepared the substrate solution of palmitic acid using by this water-saturated hexane. Finally, we combined the substrate solution, 1 U of lyophilized cholesterol esterase, and, 2, 4, 6, 8, or 1 µl of.2 mol/l phosphate buffer (ph 7.). The whole mixture was shaken at 12 rpm in a 27 o C water bath for 12 h. The products and the remaining reactants were determined by gas chromatography. Gas chromatography Quantitative analyses of sterols, FFAs, and their esters in the reaction mixture were conducted with a Shimadzu GC17 gas chromatograph (Kyoto, Japan) connected to a DB-1ht capillary column (.25 mm x 5 m; J&W Scientific, Folson, CA). Argon at a flow rate of 8 ml/min was used as carrier gas, and the splitting ratio was 1/4. In the course of analysis, the column temperature was raised from 12 to 345 o C at 15 o C/min, and then maintained at 345 o C for 2 min. Both the injector and the flame ionization detector temperatures were set at 37 o C. A half microliter of each sample was injected with an automated injector. Peaks in the chromatograms were assigned by comparing their retention times with those of commercially available standards or that of the stigmasteryl palmitate prepared enzymatically in this study. RESULTS & DISCUSSION Before esterification of cholesterol with FFAs of different chain lengths, several factors affecting the reaction including the enzyme amount and the concentration of cholesterol were investigated using cholesterol and oleic acid as substrates. We measured the saturated concentration of cholesterol in hexane by GC, and it was about 25 mmol/l. When we tested the esterification of cholesterol with oleic acid on the condition that the concentration of cholesterol 2. Concentration of ester [mmol/l] 1.5 1..5. 5 1 15 Time [h] Figure 1. Time course of esterification of cholesterol with oleic acid
ranges from 5 to 25 mmol/l, no esterification had occurred at less than 1 mmol/l of cholesterol, and the yield of cholesteryl oleate has reached its maximum at 25 mmol/l. The formation of cholesteryl oleate monitored at 12 h increased with increasing amount of the enzyme, but it did not increase significantly when 1 U or more of the enzyme was used (data not shown). Time course of the esterification of cholesterol with oleic acid was shown in Figure 1. We confirmed that the enzymatic synthesis of cholesteryl esters had occurred because no peak of product except for cholesteryl oleate was found, and the decrease in cholesterol as substrate was equal to the increase in cholesteryl oleate as product. But, extending the reaction time even further didn t improve the yield. 4 3 2 1 3 4 6 8 1 12 14 16 18 Chain length of saturated fatty acid Figure 2. Effect of chain length of FFAs on cholesterol esterification. A substrate mixture containing 1 mmol/l of each saturated fatty acid was combined with 1 U of the enzyme dissolved in a.2 mol/l phosphate buffer (ph 7.) and shaken at 12 rpm in a 27 o C water bath for 12 h. Figure 2 shows the effect of chain length of fatty acid on cholesterol esterification. This indicates that all the medium- and long-chain FFAs used were successfully employed in ester synthesis, whereas the short-chain FFAs were unavailable for this reaction. In the previous study [5], the short chain fatty acid cholesteryl esters (especially cholesteryl butyrate) were available for hydrolysis. It is clear that the fatty acid specificity in ester synthesis is entirely different from that in hydrolysis. Similar findings have been observed with pancreatic cholesterol esterase and many lipases from different sources [6-9], with a few exceptions including Candida (C.) rugosa lipase [1,11]. 8 6 4 2 2 4 6 8 1 12 Water content [µl] Figure 3. Effect of water content on esterification of cholesterol with palmitic acid. A standard substrate mixture containing 1 mmol/l of palmitic acid was combined with 1 U of the enzyme dissolved in -1 µl of.2m phosphate buffer (ph 7.) after 12 h of incubation at 27 o C. This ester synthesis is the reverse reaction of hydrolysis. Therefore, in water-rich condition, enzymatic hydrolysis has an advantage over the ester synthesis by the chemical equilibrium. Furthermore, the trend to ester synthesis is not strong because the solubility of cholesterol in hexane is low. And so, we investigated
the feasibility of cholesteryl palmitate synthesis on very low water content. Figure 3 shows the effect of water content on esterification with 1 U of the enzyme measured after 12 h of incubation at 27 o C. When the volume of water-saturated hexane as organic solvent was 3 ml, the yield of cholesteryl palmitate was constant low for more than 5 µl of water content, and has risen sharply for 2 µl of water content. But, no synthesis had occurred for less than 5 µl of water content. We consider that this is because the equilibrium position of this reaction turned from the hydrolysis to the ester synthesis on very low water content. However, ester synthesis had not occurred for less than 5 µl of water because water is necessary for the enzymatic reaction. Under the same conditions, we examined the esterification of plant sterol with FFA. Figure 4 shows the time course of the esterification of stigmasterol with palmitic acid. The yield of stigmasteryl palmitate was reached to 7%. 8 6 4 2 2 4 6 8 1 12 14 Time [h] Figure 4. Time course of esterification of stigmasterol with palmitic acid. A substrate mixture containing 1 mmol/l of palmitic acid and 7. mmol/l of stigmasterol was combined with 1 U of the enzyme dissolved in 2 µl of.2m phosphate buffer (ph 7.) at 27 o C. However, extending the reaction time even further didn t also improve the yield. Because the saturated concentration of stigmasterol in hexane measured by GC was about 5.6 mmol/l, the chemical equilibrium was established at the time of synthesis of some stigmasteryl palmitate. In these conditions, the enzyme turned into paste and clung to the inside of the vial. It is easy to separate the enzyme and the solvent, then the reaction rate may be recovered and the mass of steryl ester may increase by replacing the substrate solution. CONCLUSION All the medium- and long-chain FFAs used are successfully employed in cholesterol ester synthesis and the yield of cholesteryl palmitate was over 6% in large scale such as 3 ml of solvent. We were also able to synthesize stigmasterol palmitate as the plant sterol ester. These results could pave the way large-scale synthesis of sterol ester and indicate the potential utility of the enzyme in the food industry. REFERENCES [1] O Keefe JHJr., Cordian L., Harris WH., Moe RLM., & Vogel R. 24. Optimal low-density lipoprotein is 5 to 7 mg /dl: lower is better and physiologically normal. J Am Coll Cardiol, 43, 2142-2146. [2] Sugano M., Kamo F., Ikeda I., & Morioka H. 1976. Lipid-lowering activity of phytostanols in rats. Atherosclerosis, 24, 31-39. [3] Sugano M., Morioka H., & Ikeda I. 1977. A comparison of hypercholesterolemic activity of beta-sitosterol and betasitostanol in rats. J Nutr, 17, 211-219. [4] Ikeda I., & Sugano M. 1978. Comparison of absorption and metabolism of beta-sitosterol and beta-sitostanol in rats. Atherosclerosis, 3, 227-237.
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