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Immobilized lipase-catalyzed esterification for synthesis of trimethylolpropane triester as a biolubricant
Renewable Energy 130 (2019) 489e494
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Immobilized lipase-catalyzed esterification for synthesis of trimethylolpropane triester as a biolubricant Heejin Kim a, Nakyung Choi b, Yangha Kim c, Hak-Ryul Kim d, Junsoo Lee e, In-Hwan Kim a, b, * a
Department of Public Health Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, 03760, Republic of Korea d School of Food Science and Biotechnology, Kyungpook National University, Daegu, 702-701, Republic of Korea e Division of Food and Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, 28644, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2017 Received in revised form 25 April 2018 Accepted 21 June 2018 Available online 22 June 2018
Synthetic oleochemical esters of polyols and fatty acids are biodegradable and possess desirable technical and ecological properties. Trimethylolpropane (TMP) triester has been widely applied as a hydraulic fluid. TMP triester was effectively synthesized by lipase-catalyzed esterification from TMP and high oleic fatty acid from palm oil using an immobilized lipase. The immobilized lipase was prepared with liquid Lipozyme TL 100 L from Thermomyces lanuginosus with Duolite A568 as a carrier. The effects of temperature, enzyme loading, vacuum level, and water activity of the enzyme on the synthesis of TMP triester were investigated. The optimum temperature, enzyme loading, and vacuum level were 60 C, 15% (based on total substrate), and 6.7 kPa, respectively. The optimum water activity range of the enzyme was 0.5e0.9. Under the optimum conditions, the maximum conversion reached up to 95% after 9 h. No significant differences in physical properties were observed between TMP triester from this study and a commercial TMP triester prepared by chemical catalyst. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Biolubricant Duolite A568 Immobilized lipase Thermomyces lanuginosus Trimethylolpropane triester
1. Introduction As pollution and environmental health have become increasingly important public issues, interest in biolubricants has grown because they are biodegradable and environmentally friendly [1]. Because of this trend, biodegradable base stocks have replaced mineral oil base stocks. Vegetable oil based lubricants are biodegradable and have low eco-toxicity compared with mineral oil based lubricants. However, they have several drawbacks, namely low thermal, oxidative, and hydrolytic stabilities and poor low temperature fluidity because of high pour points [2]. Synthetic biolubricants have been developed to overcome these limitations. Among the synthetic lubricants, synthetic esters of polyols and fatty acids (FA) are considered as environmentally friendly substitutes to mineral oil based lubricants because they have suitable properties for lubricant application. Synthetic esters of polyols and FA generally show good performance at low temperatures, and
* Corresponding author. Department of Public Health Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea. E-mail address: [email protected] (I.-H. Kim). https://doi.org/10.1016/j.renene.2018.06.092 0960-1481/© 2018 Elsevier Ltd. All rights reserved.
have high thermo-oxidative stability, high viscosity indices, good antiwear performance, and low evaporation properties [1]. Therefore, these esters are suitable for use as high performance lubricants in industry. Among the polyols, trimethylolpropane (TMP) is commonly used to synthesize TMP triester, because TMP has high performance and moderate price level [3]. TMP triester is an important lubricant and has been widely applied as a hydraulic fluid, crank case lubricant, high temperature grease, and compressor oil [4]. A number of studies have investigated the synthesis of TMP esters via esterification of TMP with free FA or FA methyl esters using a homogeneous or heterogeneous chemical catalyst [3,5e7]. Meanwhile, there have also been several reports on the synthesis of TMP esters by esterification using lipases as the biocatalyst. The lipases most frequently used for synthesis of TMP esters are Novozym 435 from Candida antarctica, Lipozyme RM IM from Rhizomucor miehei, and Candida rugosa lipase. For example, Åkermanet al. [8] achieved 96% conversion using Novozym 435 for esterification of TMP ester from oleic acid in 24 h. In studies of the synthesis of TMP triester by transesterification using C. rugosa lipase or Lipozyme RM IM [9,10], conversion of 64% was achieved
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with C. rugosa lipase after 24 h and conversion of 90% was achieved with Lipozyme RM IM after 66 h. Overall, Novozym 435 is the most effective lipase considering reaction rate and conversion in the synthesis of TMP triester. However, when the immobilized lipase prepared in this study was used for synthesis of TMP triester, the reaction rate was much faster and the conversion was much higher than with Novozym 435. The goal of this study was to synthesize TMP triester from TMP and FA using an immobilized lipase. The immobilized lipase was prepared using liquid Lipozyme TL 100 L from Thermomyces lanuginosus and Duolite A568 as a carrier. The effects of reaction temperature, enzyme loading, vacuum, and water activity of the enzyme were investigated. The physical properties of TMP triester synthesized in this study were determined and compared with that of a commercial TMP triester prepared by chemical catalyst. 2. Materials and methods 2.1. Materials High oleic fatty acid (HOFA) from palm oil was used as the substrate for synthesis of TMP triester. HOFA was donated by ILSHINWELLS (Cheongju, Republic of Korea). The FA compositions of the HOFA were oleic acid (C18:1n-9, 80%), linoleic acid (C18:2n-6, 11%), palmitic acid (C16:0, 6%), stearic acid (C18:0, 2%) and myristic acid (C14:0, 1%). TMP and commercial TMP triester prepared by chemical catalyst were donated by Ohsung Chemical Ind. Co., Ltd. (Incheon, Republic of Korea). Liquid Lipozyme TL 100 L and Lipozyme TL IM from T. lanuginosus, Novozym 435 from C. antarctica and Lipozyme RM IM from R. miehei were purchased from Novozymes (Seoul, Republic of Korea). Lipase OF from C. rugosa was purchased from Meito Sangyo Co., Ltd. (Tokyo, Japan). Lipase PS from Pseudomonas fluorescens and Lipase AYS from C. rugosa were purchased from Amano Enzymes (Troy, VA, USA). Duolite A568 was purchased from Rohm and Haas (Chauny, France). All of the other chemicals used in this study were of analytical grade, unless otherwise stated.
Conversionð%Þ ¼
activity. The salts used were LiCl (aw ¼ 0.11), MgCl2 (aw ¼ 0.33), Mg(NO3)2(aw ¼ 0.53), NaCl (aw ¼ 0.75), K2CO4(aw ¼ 0.97). The equilibration process was carried out at 25 C for over 24 h.
2.4. Lipase-catalyzed esterification Lipase-catalyzed esterification of TMP with FA were carried out in a 50-mL water-jacketed glass vessel. The scheme of the reaction was shown in Scheme 1. TMP (0.4 g, 3.1 mmol) and FA (2.6 g, 9.2 mmol) were placed in a reactor preheated to the desired temperature using a water circulator. The reaction was initiated by adding enzyme to the substrate mixture with stirring at 250 rpm under vacuum. The vacuum level was controlled with a micrometering valve (Swagelok, Solon, OH, USA) and monitored using a digital vacuum gauge (Teledyne, Thousand Oaks, CA, USA). Samples (100 mL) were withdrawn from the reaction mixture at appropriate intervals and dissolved in chloroform. Individual sample was then filtered through a 0.45 mm nylon microfilter (Pall Corporation, Port Washington, NY, USA). The samples were analyzed by gas chromatography. All experiments were conducted in triplicate.
2.5. Product analysis The conversion in the reaction mixture was determined by dissolving samples (10 mL) obtained under various reaction conditions in chloroform (1 mL). A gas chromatograph (model 3800; Varian Inc., Palo Alto, CA, USA) equipped with a DB-1ht column (15 m 0.25 mm i.d.; J&W Scientific, Folsom, CA, USA) and a flame ionization detector was used for the analysis. The column temperature was held at 150 C for 2 min, increased to 370 C at a rate of 25 C/min and then held at 370 C for 5 min. Helium at a flow rate of 1.5 mL/min was used as the carrier gas and the split ratio was 1:50. The injector and detector temperatures were set at 340 and 350 C, respectively. The conversion to TMP triester was calculated using the following equation:
TMP triester 100 FA þ TMP monoester þ TMP diester þ TMP triester
(1)
2.2. Enzyme immobilization
2.6. Determination of the physical properties
Immobilization of enzyme was performed as described in our previous study [11]. The enzyme solution was prepared by mixing liquid Lipozyme TL 100 L (120 mL) with sodium phosphate buffer (30 mL, 50 mM, pH 7.0). The enzyme solution (150 mL) was added to a flask containing Duolite A568 (15 g), which acted as a carrier. This mixture was shaken at 250 rpm and incubated at 30 C for 17 h in an orbital shaker. The carrier was then separated from the enzyme solution by filtration and immediately washed with buffer solution (150 mL) to remove unbound enzyme. The carrier with the immobilized enzyme was dried overnight at room temperature and then dried in a vacuum oven for 12 h at 40 C. The immobilized enzymes were stored at 4 C before use.
To determine the physical properties of the synthesized TMP triester, a large-scale version of the lipase-catalyzed esterification was conducted under the optimum conditions. After the reaction, the final product was separated from the enzyme by filtration. The viscosity and viscosity index were determined according to the ASTM D445 and ASTM D2270 methods, respectively. The viscosity was calculated based on the time taken for the fluid to flow through a glass capillary tube (Cannon-Fenske Routine viscometer, Cannon Instrument Co., State College, PA). The kinetic viscosity was obtained as the product of this time and the tube constant. The viscosity index was calculated taking into account the product viscosities at 40 and 100 C. The pour point and cloud point were measured using a Tanaka Mini-Pour/Cloud Point Tester (Model MPC-102 S, Tanaka Scientific Ltd., Tokyo, Japan) according to the ASTM D2500 and ASTM D6749 methods, respectively. The color was determined using a colorimeter (PFX195, The Tintometer Ltd,
2.3. Equilibration of water activity The immobilized enzyme was pre-equilibrated in individual sealed containers with saturated salt solutions of known water
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Scheme 1. Lipase-catalyzed esteirification of TMP with fatty acid using immobilized lipase.
Amesbury, UK) according to ASTM D1209. All experiments were conducted in triplicate. 3. Results and discussion 3.1. Synthesis of TMP triester via lipase-catalyzed esterification 3.1.1. Enzyme screening Six commercial lipases and the immobilized lipase prepared in this study were screened for their activity in the synthesis of TMP triester. The immobilized lipase was prepared by physical binding of liquid Lipozyme TL 100 L to Duolite A568 as a carrier. The enzyme activity was defined based on the conversion to TMP triester. In these experiments, temperature, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 10% (based on total substrate), 0.67 kPa, 3:1, and 0.3, respectively. Only two lipases, namely, Novozym 435 and our immobilized lipase, were effective for the synthesis of TMP triester (Fig. 1.). Meanwhile, the other five commercial lipases did not synthesize
TMP triester (wt%)
100 80 60 40
A B C D E F G
20 0
0
2
4
6
8
10
12
Reaction time (h) Fig. 1. Enzyme screening for synthesis of TMP triester by lipase-catalyzed esterification. The following enzymes were used: A, Lipase OF (from Meito Sangyo Co., Ltd.) from Candida rugosa; B, Lipase AYS (from Amano Enzymes) from Candida rugosa; C, Lipase PS from Pseudomonas fluorescens; D, Lipozyme RM IM from Rhizomucor miehei; E, Lipozyme TL IM from Thermomyces lanuginosus; F, Novozym 435 Candida antarctica; and G, the immobilized lipase from Thermomyces lanuginosus. In these experiments, temperature, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 10% (based on total substrate), 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
TMP triester. For the experiments with Novozym 435, the maximum conversion (ca. 84%) was achieved at 12 h. On the other hand, with our immobilized lipase, a similar conversion (ca. 83%) was achieved within only 6 h and the maximum conversion (ca. 95%) was obtained after 12 h. Therefore, our immobilized lipase is more effective for the synthesis of TMP triester than Novozym 435, which is the most effective lipase identified to date. In our previous study [11], the same immobilized lipase was also effective for the triacylglycerol (TAG) synthesis from a-linolenic acid-rich FA and glycerol by esterification. Because the structure of TMP is similar to that of glycerol, the immobilized lipase was applied for synthesis of TMP triester. Consequently, TMP triester was also synthesized efficiently via esterification of TMP with FA using the immobilized lipase as a biocatalyst. Therefore, the immobilized lipase prepared in this study was chosen as the lipase to investigate the effects of the process parameters. 3.1.2. Effect of temperature The reaction temperature is a crucial factor affecting lipasecatalyzed esterification. Generally, increasing the temperature reduces the viscosity of solution and improves the solubility of a compound. This facilitates interactions between the enzyme and substrate and thus increases the reaction rate [12]. Even though high temperatures cause an increase of the reaction rate, temperatures that are too high can deactivate enzymes and reduce the enzyme stability and half-life [13]. The optimum temperature for use of an enzyme depends on its source, the nature of immobilization or chemical modification, the solvent, and the pH of the reaction medium [14]. The effect of temperature on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 2). The temperature range tested was 40e80 C. In these experiments, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 10% (based on total substrate), 0.67 kPa, 3:1, and 0.3, respectively. As the temperature increased from 40 to 60 C, both the reaction rate and conversion increased significantly, but no significant difference was observed at temperatures between 60 and 70 C. Meanwhile, the conversion decreased drastically as the temperature increased from 70 to 80 C. These results were consistent with those from our previous study [11] on the synthesis of a-linolenic acid enriched TAG using the same immobilized lipase as in this study. These results indicated that the immobilized lipase remarkably lost its activity in esterification of FA with TMP at temperatures higher than 70 C. Even though similar maximum conversions were obtained after 12 h at temperatures between 60 and 70 C, 60 C was chosen as the optimum temperature in consideration of energy requirements and stability of the lipase.
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approached equilibrium after 6 h. Although the reaction rate with an enzyme loading of 20% was slightly faster than with a loading of 15%, the conversions obtained with these loadings were not significantly different after the reaction reached equilibrium. Meanwhile, with an enzyme loading of 5%, the conversion increased very slowly and did not reach equilibrium throughout the entire period studied. Therefore, taking into consideration the cost of the operation, an enzyme loading of 15% was chosen as the optimum loading for synthesis of TMP triester.
Fig. 2. Effect of temperature on the synthesis of TMP triester by immobilized lipasecatalyzed esterification. In these experiments, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 10% (based on total substrate), 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
3.1.3. Effect of enzyme loading It is necessary to use an adequate amount of enzyme for an effective reaction. Even though the reaction rate is proportional to the enzyme loading [15,16], to reduce the enzyme loading is also important for economic feasibility. Therefore, it is important to determine the optimum enzyme loading for an enzymatic reaction to achieve the highest level of efficiency. The effect of enzyme loading on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 3). The enzyme loading range tested was 5e20% (based on total substrate). In these experiments, temperature, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 0.67 kPa, 3:1, and 0.3, respectively. During the first 4 h, the reaction rate and conversion increased remarkably as the enzyme loading increased from 5 to 20%. For the experiments using enzyme loadings of 15 and 20%, the conversion
Fig. 3. Effect of enzyme loading on the synthesis of TMP triester by immobilized lipase-catalyzed esterification. In these experiments, temperature, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
3.1.4. Effect of vacuum The amount of water in lipase-catalyzed esterification is also a crucial factor. Water affected the conversion and reaction rate for lipase-catalyzed esterification in nonaqueous media [17]. For esterification/hydrolysis reactions, if the water produced by the reaction of TMP with FA is removed, the reaction equilibrium can be moved toward esterification. There are some methods for controlling the water content in a reaction mixture [15,18]. A vacuum can be an effective method for controlling the water content in the reaction mixture [19]. The effect of the vacuum level on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 4). In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 15% (based on total substrate), 3:1, and 0.3, respectively. The range of pressures tested was 0.7e40.0 kPa. At 40.0 kPa, the conversion was only 88% at 12 h. Meanwhile, the reaction rate and conversion increased significantly as the vacuum level increased from 40.0 to 13.3 kPa. The reaction rate and conversion increased slightly when the vacuum level was increased further to 6.7 kPa. However, the reaction rate decreased when the vacuum level was increased beyond 6.7 kPa. After 6 h, no significant differences among the conversions were observed for vacuum levels between 0.7 and 6.7 kPa. Although a high vacuum level can cause a reduction of enzyme activity because of the deficiency of essential water for the lipase activity [20], application of a suitable vacuum level is essential to inhibit hydrolysis and improve the yield. Hong et al. [21] reported that Novozym 435 required a low water content for enzymatic esterification of conjugated linoleic acid with glycerol, and the equilibrium could be shifted toward the synthesis reaction by the removal of water. However, as the vacuum level was too
Fig. 4. Effect of vacuum level on the synthesis of TMP triester by immobilized lipasecatalyzed esterification. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 15% (based on total substrate), 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
H. Kim et al. / Renewable Energy 130 (2019) 489e494
high, the reaction rate decreased. A similar tendency was also observed in this study. Consequently, vacuum levels higher than 6.7 kPa were not suitable because the excessively high vacuum level removed the essential water in the lipase and reduced the reaction rate. Meanwhile, a vacuum level of 40.0 kPa was also not suitable because water formed in the reaction was not removed effectively, which could lead to hydrolysis. Hence, a vacuum of 6.7 kPa was selected as the optimum level for synthesis of TMP triester. 3.1.5. Effect of the initial water activity There is a critical lower limit for the water content, and below this, enzymes will not be able to maintain their catalytic activities [14,22]. This critical water content is needed to maintain the threedimensional configuration of the enzyme necessary for its catalytic activity. However, excessive water can induce negative effects. Therefore, to maximize the efficiency, to identify the optimum water content in the enzyme is important for an enzymatic reaction. The effect of the water activity of the enzyme on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 5). The water activity range tested was 0.11e0.97. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and vacuum level were kept constant at 60 C, 15% (based on total substrate), 3:1, and 6.7 kPa, respectively. As the water activity increased from 0.11 to 0.33, the reaction rate and the conversion increased significantly throughout the entire reaction (Fig. 5). With a water activity of 0.11, the reaction rate was significantly slower than at the other water activities tested in this study. This was probably caused by a lack of essential water for the catalytic activity of the enzyme. Meanwhile, during the first 4 h of reaction, the reaction rate increased slightly as the water activity increased from 0.33 to 0.75. However, increasing the water activity from 0.75 to 0.97 did not have a remarkable effect on the conversion. Although slight differences in the reaction rates were observed with water activities between 0.53 and 0.97, similar maximum conversions (ca. 95%) were achieved after 9 h. These results indicate that the immobilized lipase is very effective for synthesis of TMP triester when the water activity is higher than 0.5. It has been reported that lipases from different sources have
TMP triester (wt%)
100 80 60 40
aw 0.11 aw 0.33
20 0
aw 0.53 aw 0.75 aw 0.97
0
2
4
6
8
10
12
Reaction time (h) Fig. 5. Effect of the water activity of the enzyme on the synthesis of TMP triester by immobilized lipase-catalyzed esterification. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and vacuum level were kept at 60 C, 15% (based on total substrate), 3:1, and 6.7 kPa, respectively. All experiments were conducted in triplicate.
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different optimum water activities. For the synthesis of butyl butyrate by transesterification, Chowdary et al. [23] found the optimum water activities for lipases from C. rugosa and Penicillium roqueforti were both 0.33, those for lipases from Mucor javanicus and Rhizopus oryzae were both 0.54, and that of lipase from Aspergillus niger was 0.75. For the synthesis of phospholipid containing n-3 PUFA via acidolysis, Kim et al. [24] found that the optimum water activity of immobilized phospholipase was 0.65. It is clear that different lipases in organic media have quite different water activity requirements for optimum efficacy. For the synthesis of structured lipid by acidolysis with olive oil and capric acid using Lipozyme TL IM from T. lanuginosus, Oh et al. [25], indicated that capric acid incorporation into olive oil increased when the water activity was continuously increased up to 0.80. Meanwhile, Svensson and Adlercreutz [26] studied the effect of acyl migration in Lipozyme TL IM-catalyzed esterification using TAG, and found that the reaction rate with a water activity of 0.35 was the fastest and that with a water activity of 0.84 was the slowest. These results show that the optimum water activity of lipase from T. lanuginosus varies depending on the type of substrate used or reaction system. 3.2. Physical properties of the final product After the reaction parameters were optimized, the physical properties of the TMP triester were tested. As a polyolester, the physical properties of TMP triester meet the requirements for use as a lubricant. The physical properties of the TMP triester synthesized in this study were compared with those of a commercial TMP triester prepared by chemical catalyst as the reference, and to the properties of the HOFA substrate. The physical properties investigated in this study were the viscosity, viscosity index, pour point, cloud point, and color (Table 1). Viscosity is one of the most important properties of a lubricant, because it is related to the ability of the lubricant to efficiently lubricate the contact surface. The viscosities of the TMP triester from this study and the commercial TMP triester were not significantly different. The viscosity index indicates the change in viscosity depending on the temperature change. The high viscosity index implies that the change in viscosity is small. In general, mineral oils show a viscosity index of approximately 100, but vegetable oil-based lubricants have higher viscosity index than mineral oils [27]. Both the TMP triester from this study and the commercial TMP triester had high viscosity indices of approximately 200 which implies that these TMP triesters are desirable for a wide temperature range. The cloud point and pour point are also important factors to indicate the lubricants properties. The cloud point and pour point of the TMP triester from this study were as low as those of the commercial TMP triester. Generally, a lubricant with low pour point has efficiently worked in low temperature environments. Meanwhile, the TMP triester from this study was much lighter in color than the commercial TMP triester, which was similar to the result reported by Åkerman et al. [8]. Consequently, it was demonstrated that the TMP triester from this study has identical physical properties when compared to the commercial TMP triester. 4. Conclusions The immobilized lipase from T. lanuginosus with Duolite A568 as a carrier is a promising biocatalyst for synthesis of TMP triester. The conversion to TMP triester decreased markedly as temperature increased from 70 to 80 C. TMP triester was synthesized effectively when the water activity of the immobilized lipase was higher than 0.53. The final product, composed of 95 wt% TMP triester, 3.5 wt%
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Table 1 Physical propertiesa of TMP triester synthesized in this study, a commercial TMP triester prepared by chemical catalyst, and high oleic fatty acid (HOFA). Product
TMP triester Ib TMP triester IIc HOFA a b c
Viscosity (mm2/s) 40 C
100 C
46.2 46.6 18.7
9.5 9.5 4.8
Viscosity index, (VI)
Pour point ( C)
Cloud point ( C)
Color
195 194 189
48 49 7
23 25 9
40 260 97
Values represent the average of triplicate determinations from different experiments. TMP triester was synthesized by immobilized lipase-catalyzed esterification under the optimum conditions. Commercial TMP triester prepared by chemical catalyst.
TMP diester, and 1.5 wt% FA, was obtained after 9 h under the optimum conditions. These results are even better than those from earlier studies on the synthesis of TMP triester using other commercial lipases. The TMP triester synthesized in this study has physical properties that are similar to a commercial TMP triester prepared by a chemical method. Acknowledgement This research was supported by the Yang Young Foundation. References [1] P. Nagendramma, S. Kaul, Development of ecofriendly/biodegradable lubricants: an overview, Renew. Sustain. Energy Rev. 16 (1) (2012) 764e774. [2] B. Wilson, Lubricants and functional fluids from renewable sources, Ind. Lubric. Tribol. 50 (1) (1998) 6e15. [3] H.A. Hamid, R. Yunus, U. Rashid, T.S. Choong, S. Ali, A.M. Syam, Synthesis of high oleic palm oil-based trimethylolpropane esters in a vacuum operated pulsed loop reactor, Fuel 166 (2016) 560e566. €m, M.A. Mollaahmad, S. Karlsson, R. Hatti-Kaul, [4] C.O. Åkerman, A.E. Hagstro Biolubricant synthesis using immobilised lipase: process optimisation of trimethylolpropane oleate production, Process Biochem. 46 (12) (2011) 2225e2231. [5] T.-S. Chang, H. Masood, R. Yunus, U. Rashid, T.S. Choong, D.R.A. Biak, Activity of calcium methoxide catalyst for synthesis of high oleic palm oil based trimethylolpropane triesters as lubricant base stock, Ind. Eng. Chem. Res. 51 (15) (2012) 5438e5442. [6] F. Zaccheria, M. Mariani, R. Psaro, P. Bondioli, N. Ravasio, Environmentally friendly lubricants through a zero waste process, Appl. Catal. B Environ. 181 (2016) 581e586. [7] E. Wang, X. Ma, S. Tang, R. Yan, Y. Wang, W.W. Riley, et al., Synthesis and oxidative stability of trimethylolpropane fatty acid triester as a biolubricant base oil from waste cooking oil, Biomass Bioenergy 66 (2014) 371e378. €msa €, R. Hatti-Kaul, Clean synthesis of [8] C.O. Åkerman, Y. Gaber, N.A. Ghani, M. La biolubricants for low temperature applications using heterogeneous catalysts, J. Mol. Catal. B Enzym. 72 (3e4) (2011) 263e269. [9] E. Uosukainen, Y.-Y. Linko, M. L€ ams€ a, T. Tervakangas, P. Linko, Transesterification of trimethylolpropane and rapeseed oil methyl ester to environmentally acceptable lubricants, J. Am. Oil Chem. Soc. 75 (11) (1998) 1557e1563. €msa €, P. Linko, Production of trimethylol[10] Y.-Y. Linko, T. Tervakangas, M. La propane esters of rapeseed oil fatty acids by immobilized lipase, Biotechnol. Tech. 11 (12) (1997) 889e892. [11] H. Kim, N. Choi, S.-W. Oh, Y. Kim, B.H. Kim, I.-H. Kim, Synthesis of a-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase, Food Chem. 237 (2017) 654e658.
[12] H.S. Garcia, K.J. Keough, J.A. Arcos, C.G. Hill, Continuous interesterification of butteroil and conjugated linoleic acid in a tubular reactor packed with an immobilized lipase, Biotechnol. Tech. 13 (6) (1999) 369e373. [13] J.Y. Baik, N.H. Kim, S.-W. Oh, I.-H. Kim, Preparation of highly purified stearidonic acid from Echium oil via an enzymatic method combined with preparative high performance liquid chromatography, J. Oleo Sci. 64 (7) (2015) 729e736. [14] J.S. Dordick, Enzymatic catalysis in monophasic organic solvents, Enzym. Microb. Technol. 11 (4) (1989) 194e211. [15] F. Blasi, S. Maurelli, L. Cossignani, G. D’Arco, M.S. Simonetti, P. Damiani, Study of some experimental parameters in the synthesis of triacylglycerols with CLA isomers and structural analysis, J. Am. Oil Chem. Soc. 86 (6) (2009) 531e537. [16] I. Karabulut, G. Durmaz, A.A. Hayaloglu, Fatty acid selectivity of lipases during acidolysis reaction between oleic acid and monoacid triacylglycerols, J. Agric. Food Chem. 57 (21) (2009) 10466e10470. [17] J.J. Han, T. Yamane, Enhancement of both reaction yield and rate of synthesis of structured triacylglycerol containing eicosapentaenoic acid under vacuum with water activity control, Lipids 34 (9) (1999) 989e995. [18] R. Rosu, M. Yasui, Y. Iwasaki, T. Yamane, Enzymatic synthesis of symmetrical 1, 3-diacylglycerols by direct esterification of glycerol in solvent-free system, J. Am. Oil Chem. Soc. 76 (1999) 839e843. [19] Y. Watanabe, Y. Yamauchi-Sato, T. Nagao, T. Yamamoto, K. Ogita, Y. Shimada, Production of monoacylglycerol of conjugated linoleic acid by esterification followed by dehydration at low temperature using Penicillium camembertii lipase, J. Mol. Catal. B Enzym. 27 (4e6) (2004) 249e254. [20] D.S. No, T. Zhao, J. Lee, J.-S. Lee, I.-H. Kim, Synthesis of phytosteryl ester containing pinolenic acid in a solvent-free system using immobilized Candida rugosa lipase, J. Agric. Food Chem. 61 (37) (2013) 8934e8940. [21] S.I. Hong, Y. Kim, S.W. Yoon, S.Y. Cho, I.H. Kim, Synthesis of CLA-enriched TAG by Candida Antarctica lipase under vacuum, Eur. J. Lipid Sci. Technol. 114 (9) (2012) 1044e1051. [22] I.-H. Kim, H.S. Garcia, C.G. Hill, Phospholipase A1-catalyzed synthesis of phospholipids enriched in n-3 polyunsaturated fatty acid residues, Enzym. Microb. Technol. 40 (5) (2007) 1130e1135. [23] G. Chowdary, S. Prapulla, The influence of water activity on the lipase catalyzed synthesis of butyl butyrate by transesterification, Process Biochem. 38 (3) (2002) 393e397. [24] I.-H. Kim, H.S. Garcia, C.G. Hill, Synthesis of structured phosphatidylcholine containing n-3 PUFA residues via acidolysis mediated by immobilized phospholipase A1, J. Am. Oil Chem. Soc. 87 (11) (2010) 1293e1299. [25] J.-E. Oh, K.-W. Lee, H.-K. Park, J.-Y. Kim, K.-I. Kwon, J.-W. Kim, et al., Lipasecatalyzed acidolysis of olive oil with capric acid: effect of water activity on incorporation and acyl migration, J. Agric. Food Chem. 57 (19) (2009) 9280e9283. [26] J. Svensson, P. Adlercreutz, Effect of acyl migration in Lipozyme TL IMcatalyzed interesterification using a triacylglycerol model system, Eur. J. Lipid Sci. Technol. 113 (10) (2011) 1258e1265. [27] H.M. Mabarak, E.N. Mohamed, H.H. Masjuki, M.A. Kalam, K.A.H. Al Mahmud, M. Habibullah, A.M. Ashraful, The prospects of biolubricants as alternatives in automotive applications, Renew. Sustain. Energy Rev. 33 (2014) 34e43.
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Immobilized lipase-catalyzed esterification for synthesis of trimethylolpropane triester as a biolubricant Heejin Kim a, Nakyung Choi b, Yangha Kim c, Hak-Ryul Kim d, Junsoo Lee e, In-Hwan Kim a, b, * a
Department of Public Health Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea Department of Integrated Biomedical and Life Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, 03760, Republic of Korea d School of Food Science and Biotechnology, Kyungpook National University, Daegu, 702-701, Republic of Korea e Division of Food and Animal Sciences, Chungbuk National University, Cheongju, Chungbuk, 28644, Republic of Korea b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 December 2017 Received in revised form 25 April 2018 Accepted 21 June 2018 Available online 22 June 2018
Synthetic oleochemical esters of polyols and fatty acids are biodegradable and possess desirable technical and ecological properties. Trimethylolpropane (TMP) triester has been widely applied as a hydraulic fluid. TMP triester was effectively synthesized by lipase-catalyzed esterification from TMP and high oleic fatty acid from palm oil using an immobilized lipase. The immobilized lipase was prepared with liquid Lipozyme TL 100 L from Thermomyces lanuginosus with Duolite A568 as a carrier. The effects of temperature, enzyme loading, vacuum level, and water activity of the enzyme on the synthesis of TMP triester were investigated. The optimum temperature, enzyme loading, and vacuum level were 60 C, 15% (based on total substrate), and 6.7 kPa, respectively. The optimum water activity range of the enzyme was 0.5e0.9. Under the optimum conditions, the maximum conversion reached up to 95% after 9 h. No significant differences in physical properties were observed between TMP triester from this study and a commercial TMP triester prepared by chemical catalyst. © 2018 Elsevier Ltd. All rights reserved.
Keywords: Biolubricant Duolite A568 Immobilized lipase Thermomyces lanuginosus Trimethylolpropane triester
1. Introduction As pollution and environmental health have become increasingly important public issues, interest in biolubricants has grown because they are biodegradable and environmentally friendly [1]. Because of this trend, biodegradable base stocks have replaced mineral oil base stocks. Vegetable oil based lubricants are biodegradable and have low eco-toxicity compared with mineral oil based lubricants. However, they have several drawbacks, namely low thermal, oxidative, and hydrolytic stabilities and poor low temperature fluidity because of high pour points [2]. Synthetic biolubricants have been developed to overcome these limitations. Among the synthetic lubricants, synthetic esters of polyols and fatty acids (FA) are considered as environmentally friendly substitutes to mineral oil based lubricants because they have suitable properties for lubricant application. Synthetic esters of polyols and FA generally show good performance at low temperatures, and
* Corresponding author. Department of Public Health Sciences, Graduate School, Korea University, 145 Anam-Ro, Sungbuk-Gu, Seoul, 02841, Republic of Korea. E-mail address: [email protected] (I.-H. Kim). https://doi.org/10.1016/j.renene.2018.06.092 0960-1481/© 2018 Elsevier Ltd. All rights reserved.
have high thermo-oxidative stability, high viscosity indices, good antiwear performance, and low evaporation properties [1]. Therefore, these esters are suitable for use as high performance lubricants in industry. Among the polyols, trimethylolpropane (TMP) is commonly used to synthesize TMP triester, because TMP has high performance and moderate price level [3]. TMP triester is an important lubricant and has been widely applied as a hydraulic fluid, crank case lubricant, high temperature grease, and compressor oil [4]. A number of studies have investigated the synthesis of TMP esters via esterification of TMP with free FA or FA methyl esters using a homogeneous or heterogeneous chemical catalyst [3,5e7]. Meanwhile, there have also been several reports on the synthesis of TMP esters by esterification using lipases as the biocatalyst. The lipases most frequently used for synthesis of TMP esters are Novozym 435 from Candida antarctica, Lipozyme RM IM from Rhizomucor miehei, and Candida rugosa lipase. For example, Åkermanet al. [8] achieved 96% conversion using Novozym 435 for esterification of TMP ester from oleic acid in 24 h. In studies of the synthesis of TMP triester by transesterification using C. rugosa lipase or Lipozyme RM IM [9,10], conversion of 64% was achieved
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with C. rugosa lipase after 24 h and conversion of 90% was achieved with Lipozyme RM IM after 66 h. Overall, Novozym 435 is the most effective lipase considering reaction rate and conversion in the synthesis of TMP triester. However, when the immobilized lipase prepared in this study was used for synthesis of TMP triester, the reaction rate was much faster and the conversion was much higher than with Novozym 435. The goal of this study was to synthesize TMP triester from TMP and FA using an immobilized lipase. The immobilized lipase was prepared using liquid Lipozyme TL 100 L from Thermomyces lanuginosus and Duolite A568 as a carrier. The effects of reaction temperature, enzyme loading, vacuum, and water activity of the enzyme were investigated. The physical properties of TMP triester synthesized in this study were determined and compared with that of a commercial TMP triester prepared by chemical catalyst. 2. Materials and methods 2.1. Materials High oleic fatty acid (HOFA) from palm oil was used as the substrate for synthesis of TMP triester. HOFA was donated by ILSHINWELLS (Cheongju, Republic of Korea). The FA compositions of the HOFA were oleic acid (C18:1n-9, 80%), linoleic acid (C18:2n-6, 11%), palmitic acid (C16:0, 6%), stearic acid (C18:0, 2%) and myristic acid (C14:0, 1%). TMP and commercial TMP triester prepared by chemical catalyst were donated by Ohsung Chemical Ind. Co., Ltd. (Incheon, Republic of Korea). Liquid Lipozyme TL 100 L and Lipozyme TL IM from T. lanuginosus, Novozym 435 from C. antarctica and Lipozyme RM IM from R. miehei were purchased from Novozymes (Seoul, Republic of Korea). Lipase OF from C. rugosa was purchased from Meito Sangyo Co., Ltd. (Tokyo, Japan). Lipase PS from Pseudomonas fluorescens and Lipase AYS from C. rugosa were purchased from Amano Enzymes (Troy, VA, USA). Duolite A568 was purchased from Rohm and Haas (Chauny, France). All of the other chemicals used in this study were of analytical grade, unless otherwise stated.
Conversionð%Þ ¼
activity. The salts used were LiCl (aw ¼ 0.11), MgCl2 (aw ¼ 0.33), Mg(NO3)2(aw ¼ 0.53), NaCl (aw ¼ 0.75), K2CO4(aw ¼ 0.97). The equilibration process was carried out at 25 C for over 24 h.
2.4. Lipase-catalyzed esterification Lipase-catalyzed esterification of TMP with FA were carried out in a 50-mL water-jacketed glass vessel. The scheme of the reaction was shown in Scheme 1. TMP (0.4 g, 3.1 mmol) and FA (2.6 g, 9.2 mmol) were placed in a reactor preheated to the desired temperature using a water circulator. The reaction was initiated by adding enzyme to the substrate mixture with stirring at 250 rpm under vacuum. The vacuum level was controlled with a micrometering valve (Swagelok, Solon, OH, USA) and monitored using a digital vacuum gauge (Teledyne, Thousand Oaks, CA, USA). Samples (100 mL) were withdrawn from the reaction mixture at appropriate intervals and dissolved in chloroform. Individual sample was then filtered through a 0.45 mm nylon microfilter (Pall Corporation, Port Washington, NY, USA). The samples were analyzed by gas chromatography. All experiments were conducted in triplicate.
2.5. Product analysis The conversion in the reaction mixture was determined by dissolving samples (10 mL) obtained under various reaction conditions in chloroform (1 mL). A gas chromatograph (model 3800; Varian Inc., Palo Alto, CA, USA) equipped with a DB-1ht column (15 m 0.25 mm i.d.; J&W Scientific, Folsom, CA, USA) and a flame ionization detector was used for the analysis. The column temperature was held at 150 C for 2 min, increased to 370 C at a rate of 25 C/min and then held at 370 C for 5 min. Helium at a flow rate of 1.5 mL/min was used as the carrier gas and the split ratio was 1:50. The injector and detector temperatures were set at 340 and 350 C, respectively. The conversion to TMP triester was calculated using the following equation:
TMP triester 100 FA þ TMP monoester þ TMP diester þ TMP triester
(1)
2.2. Enzyme immobilization
2.6. Determination of the physical properties
Immobilization of enzyme was performed as described in our previous study [11]. The enzyme solution was prepared by mixing liquid Lipozyme TL 100 L (120 mL) with sodium phosphate buffer (30 mL, 50 mM, pH 7.0). The enzyme solution (150 mL) was added to a flask containing Duolite A568 (15 g), which acted as a carrier. This mixture was shaken at 250 rpm and incubated at 30 C for 17 h in an orbital shaker. The carrier was then separated from the enzyme solution by filtration and immediately washed with buffer solution (150 mL) to remove unbound enzyme. The carrier with the immobilized enzyme was dried overnight at room temperature and then dried in a vacuum oven for 12 h at 40 C. The immobilized enzymes were stored at 4 C before use.
To determine the physical properties of the synthesized TMP triester, a large-scale version of the lipase-catalyzed esterification was conducted under the optimum conditions. After the reaction, the final product was separated from the enzyme by filtration. The viscosity and viscosity index were determined according to the ASTM D445 and ASTM D2270 methods, respectively. The viscosity was calculated based on the time taken for the fluid to flow through a glass capillary tube (Cannon-Fenske Routine viscometer, Cannon Instrument Co., State College, PA). The kinetic viscosity was obtained as the product of this time and the tube constant. The viscosity index was calculated taking into account the product viscosities at 40 and 100 C. The pour point and cloud point were measured using a Tanaka Mini-Pour/Cloud Point Tester (Model MPC-102 S, Tanaka Scientific Ltd., Tokyo, Japan) according to the ASTM D2500 and ASTM D6749 methods, respectively. The color was determined using a colorimeter (PFX195, The Tintometer Ltd,
2.3. Equilibration of water activity The immobilized enzyme was pre-equilibrated in individual sealed containers with saturated salt solutions of known water
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491
Scheme 1. Lipase-catalyzed esteirification of TMP with fatty acid using immobilized lipase.
Amesbury, UK) according to ASTM D1209. All experiments were conducted in triplicate. 3. Results and discussion 3.1. Synthesis of TMP triester via lipase-catalyzed esterification 3.1.1. Enzyme screening Six commercial lipases and the immobilized lipase prepared in this study were screened for their activity in the synthesis of TMP triester. The immobilized lipase was prepared by physical binding of liquid Lipozyme TL 100 L to Duolite A568 as a carrier. The enzyme activity was defined based on the conversion to TMP triester. In these experiments, temperature, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 10% (based on total substrate), 0.67 kPa, 3:1, and 0.3, respectively. Only two lipases, namely, Novozym 435 and our immobilized lipase, were effective for the synthesis of TMP triester (Fig. 1.). Meanwhile, the other five commercial lipases did not synthesize
TMP triester (wt%)
100 80 60 40
A B C D E F G
20 0
0
2
4
6
8
10
12
Reaction time (h) Fig. 1. Enzyme screening for synthesis of TMP triester by lipase-catalyzed esterification. The following enzymes were used: A, Lipase OF (from Meito Sangyo Co., Ltd.) from Candida rugosa; B, Lipase AYS (from Amano Enzymes) from Candida rugosa; C, Lipase PS from Pseudomonas fluorescens; D, Lipozyme RM IM from Rhizomucor miehei; E, Lipozyme TL IM from Thermomyces lanuginosus; F, Novozym 435 Candida antarctica; and G, the immobilized lipase from Thermomyces lanuginosus. In these experiments, temperature, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 10% (based on total substrate), 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
TMP triester. For the experiments with Novozym 435, the maximum conversion (ca. 84%) was achieved at 12 h. On the other hand, with our immobilized lipase, a similar conversion (ca. 83%) was achieved within only 6 h and the maximum conversion (ca. 95%) was obtained after 12 h. Therefore, our immobilized lipase is more effective for the synthesis of TMP triester than Novozym 435, which is the most effective lipase identified to date. In our previous study [11], the same immobilized lipase was also effective for the triacylglycerol (TAG) synthesis from a-linolenic acid-rich FA and glycerol by esterification. Because the structure of TMP is similar to that of glycerol, the immobilized lipase was applied for synthesis of TMP triester. Consequently, TMP triester was also synthesized efficiently via esterification of TMP with FA using the immobilized lipase as a biocatalyst. Therefore, the immobilized lipase prepared in this study was chosen as the lipase to investigate the effects of the process parameters. 3.1.2. Effect of temperature The reaction temperature is a crucial factor affecting lipasecatalyzed esterification. Generally, increasing the temperature reduces the viscosity of solution and improves the solubility of a compound. This facilitates interactions between the enzyme and substrate and thus increases the reaction rate [12]. Even though high temperatures cause an increase of the reaction rate, temperatures that are too high can deactivate enzymes and reduce the enzyme stability and half-life [13]. The optimum temperature for use of an enzyme depends on its source, the nature of immobilization or chemical modification, the solvent, and the pH of the reaction medium [14]. The effect of temperature on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 2). The temperature range tested was 40e80 C. In these experiments, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 10% (based on total substrate), 0.67 kPa, 3:1, and 0.3, respectively. As the temperature increased from 40 to 60 C, both the reaction rate and conversion increased significantly, but no significant difference was observed at temperatures between 60 and 70 C. Meanwhile, the conversion decreased drastically as the temperature increased from 70 to 80 C. These results were consistent with those from our previous study [11] on the synthesis of a-linolenic acid enriched TAG using the same immobilized lipase as in this study. These results indicated that the immobilized lipase remarkably lost its activity in esterification of FA with TMP at temperatures higher than 70 C. Even though similar maximum conversions were obtained after 12 h at temperatures between 60 and 70 C, 60 C was chosen as the optimum temperature in consideration of energy requirements and stability of the lipase.
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approached equilibrium after 6 h. Although the reaction rate with an enzyme loading of 20% was slightly faster than with a loading of 15%, the conversions obtained with these loadings were not significantly different after the reaction reached equilibrium. Meanwhile, with an enzyme loading of 5%, the conversion increased very slowly and did not reach equilibrium throughout the entire period studied. Therefore, taking into consideration the cost of the operation, an enzyme loading of 15% was chosen as the optimum loading for synthesis of TMP triester.
Fig. 2. Effect of temperature on the synthesis of TMP triester by immobilized lipasecatalyzed esterification. In these experiments, enzyme loading, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 10% (based on total substrate), 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
3.1.3. Effect of enzyme loading It is necessary to use an adequate amount of enzyme for an effective reaction. Even though the reaction rate is proportional to the enzyme loading [15,16], to reduce the enzyme loading is also important for economic feasibility. Therefore, it is important to determine the optimum enzyme loading for an enzymatic reaction to achieve the highest level of efficiency. The effect of enzyme loading on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 3). The enzyme loading range tested was 5e20% (based on total substrate). In these experiments, temperature, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 0.67 kPa, 3:1, and 0.3, respectively. During the first 4 h, the reaction rate and conversion increased remarkably as the enzyme loading increased from 5 to 20%. For the experiments using enzyme loadings of 15 and 20%, the conversion
Fig. 3. Effect of enzyme loading on the synthesis of TMP triester by immobilized lipase-catalyzed esterification. In these experiments, temperature, vacuum level, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 0.7 kPa, 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
3.1.4. Effect of vacuum The amount of water in lipase-catalyzed esterification is also a crucial factor. Water affected the conversion and reaction rate for lipase-catalyzed esterification in nonaqueous media [17]. For esterification/hydrolysis reactions, if the water produced by the reaction of TMP with FA is removed, the reaction equilibrium can be moved toward esterification. There are some methods for controlling the water content in a reaction mixture [15,18]. A vacuum can be an effective method for controlling the water content in the reaction mixture [19]. The effect of the vacuum level on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 4). In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and water activity of the enzyme were kept constant at 60 C, 15% (based on total substrate), 3:1, and 0.3, respectively. The range of pressures tested was 0.7e40.0 kPa. At 40.0 kPa, the conversion was only 88% at 12 h. Meanwhile, the reaction rate and conversion increased significantly as the vacuum level increased from 40.0 to 13.3 kPa. The reaction rate and conversion increased slightly when the vacuum level was increased further to 6.7 kPa. However, the reaction rate decreased when the vacuum level was increased beyond 6.7 kPa. After 6 h, no significant differences among the conversions were observed for vacuum levels between 0.7 and 6.7 kPa. Although a high vacuum level can cause a reduction of enzyme activity because of the deficiency of essential water for the lipase activity [20], application of a suitable vacuum level is essential to inhibit hydrolysis and improve the yield. Hong et al. [21] reported that Novozym 435 required a low water content for enzymatic esterification of conjugated linoleic acid with glycerol, and the equilibrium could be shifted toward the synthesis reaction by the removal of water. However, as the vacuum level was too
Fig. 4. Effect of vacuum level on the synthesis of TMP triester by immobilized lipasecatalyzed esterification. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and water activity of the enzyme were kept at 60 C, 15% (based on total substrate), 3:1, and 0.3, respectively. All experiments were conducted in triplicate.
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high, the reaction rate decreased. A similar tendency was also observed in this study. Consequently, vacuum levels higher than 6.7 kPa were not suitable because the excessively high vacuum level removed the essential water in the lipase and reduced the reaction rate. Meanwhile, a vacuum level of 40.0 kPa was also not suitable because water formed in the reaction was not removed effectively, which could lead to hydrolysis. Hence, a vacuum of 6.7 kPa was selected as the optimum level for synthesis of TMP triester. 3.1.5. Effect of the initial water activity There is a critical lower limit for the water content, and below this, enzymes will not be able to maintain their catalytic activities [14,22]. This critical water content is needed to maintain the threedimensional configuration of the enzyme necessary for its catalytic activity. However, excessive water can induce negative effects. Therefore, to maximize the efficiency, to identify the optimum water content in the enzyme is important for an enzymatic reaction. The effect of the water activity of the enzyme on the synthesis of TMP triester via lipase-catalyzed esterification was investigated (Fig. 5). The water activity range tested was 0.11e0.97. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and vacuum level were kept constant at 60 C, 15% (based on total substrate), 3:1, and 6.7 kPa, respectively. As the water activity increased from 0.11 to 0.33, the reaction rate and the conversion increased significantly throughout the entire reaction (Fig. 5). With a water activity of 0.11, the reaction rate was significantly slower than at the other water activities tested in this study. This was probably caused by a lack of essential water for the catalytic activity of the enzyme. Meanwhile, during the first 4 h of reaction, the reaction rate increased slightly as the water activity increased from 0.33 to 0.75. However, increasing the water activity from 0.75 to 0.97 did not have a remarkable effect on the conversion. Although slight differences in the reaction rates were observed with water activities between 0.53 and 0.97, similar maximum conversions (ca. 95%) were achieved after 9 h. These results indicate that the immobilized lipase is very effective for synthesis of TMP triester when the water activity is higher than 0.5. It has been reported that lipases from different sources have
TMP triester (wt%)
100 80 60 40
aw 0.11 aw 0.33
20 0
aw 0.53 aw 0.75 aw 0.97
0
2
4
6
8
10
12
Reaction time (h) Fig. 5. Effect of the water activity of the enzyme on the synthesis of TMP triester by immobilized lipase-catalyzed esterification. In these experiments, temperature, enzyme loading, molar ratio of FA to TMP, and vacuum level were kept at 60 C, 15% (based on total substrate), 3:1, and 6.7 kPa, respectively. All experiments were conducted in triplicate.
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different optimum water activities. For the synthesis of butyl butyrate by transesterification, Chowdary et al. [23] found the optimum water activities for lipases from C. rugosa and Penicillium roqueforti were both 0.33, those for lipases from Mucor javanicus and Rhizopus oryzae were both 0.54, and that of lipase from Aspergillus niger was 0.75. For the synthesis of phospholipid containing n-3 PUFA via acidolysis, Kim et al. [24] found that the optimum water activity of immobilized phospholipase was 0.65. It is clear that different lipases in organic media have quite different water activity requirements for optimum efficacy. For the synthesis of structured lipid by acidolysis with olive oil and capric acid using Lipozyme TL IM from T. lanuginosus, Oh et al. [25], indicated that capric acid incorporation into olive oil increased when the water activity was continuously increased up to 0.80. Meanwhile, Svensson and Adlercreutz [26] studied the effect of acyl migration in Lipozyme TL IM-catalyzed esterification using TAG, and found that the reaction rate with a water activity of 0.35 was the fastest and that with a water activity of 0.84 was the slowest. These results show that the optimum water activity of lipase from T. lanuginosus varies depending on the type of substrate used or reaction system. 3.2. Physical properties of the final product After the reaction parameters were optimized, the physical properties of the TMP triester were tested. As a polyolester, the physical properties of TMP triester meet the requirements for use as a lubricant. The physical properties of the TMP triester synthesized in this study were compared with those of a commercial TMP triester prepared by chemical catalyst as the reference, and to the properties of the HOFA substrate. The physical properties investigated in this study were the viscosity, viscosity index, pour point, cloud point, and color (Table 1). Viscosity is one of the most important properties of a lubricant, because it is related to the ability of the lubricant to efficiently lubricate the contact surface. The viscosities of the TMP triester from this study and the commercial TMP triester were not significantly different. The viscosity index indicates the change in viscosity depending on the temperature change. The high viscosity index implies that the change in viscosity is small. In general, mineral oils show a viscosity index of approximately 100, but vegetable oil-based lubricants have higher viscosity index than mineral oils [27]. Both the TMP triester from this study and the commercial TMP triester had high viscosity indices of approximately 200 which implies that these TMP triesters are desirable for a wide temperature range. The cloud point and pour point are also important factors to indicate the lubricants properties. The cloud point and pour point of the TMP triester from this study were as low as those of the commercial TMP triester. Generally, a lubricant with low pour point has efficiently worked in low temperature environments. Meanwhile, the TMP triester from this study was much lighter in color than the commercial TMP triester, which was similar to the result reported by Åkerman et al. [8]. Consequently, it was demonstrated that the TMP triester from this study has identical physical properties when compared to the commercial TMP triester. 4. Conclusions The immobilized lipase from T. lanuginosus with Duolite A568 as a carrier is a promising biocatalyst for synthesis of TMP triester. The conversion to TMP triester decreased markedly as temperature increased from 70 to 80 C. TMP triester was synthesized effectively when the water activity of the immobilized lipase was higher than 0.53. The final product, composed of 95 wt% TMP triester, 3.5 wt%
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Table 1 Physical propertiesa of TMP triester synthesized in this study, a commercial TMP triester prepared by chemical catalyst, and high oleic fatty acid (HOFA). Product
TMP triester Ib TMP triester IIc HOFA a b c
Viscosity (mm2/s) 40 C
100 C
46.2 46.6 18.7
9.5 9.5 4.8
Viscosity index, (VI)
Pour point ( C)
Cloud point ( C)
Color
195 194 189
48 49 7
23 25 9
40 260 97
Values represent the average of triplicate determinations from different experiments. TMP triester was synthesized by immobilized lipase-catalyzed esterification under the optimum conditions. Commercial TMP triester prepared by chemical catalyst.
TMP diester, and 1.5 wt% FA, was obtained after 9 h under the optimum conditions. These results are even better than those from earlier studies on the synthesis of TMP triester using other commercial lipases. The TMP triester synthesized in this study has physical properties that are similar to a commercial TMP triester prepared by a chemical method. Acknowledgement This research was supported by the Yang Young Foundation. References [1] P. Nagendramma, S. Kaul, Development of ecofriendly/biodegradable lubricants: an overview, Renew. Sustain. Energy Rev. 16 (1) (2012) 764e774. [2] B. Wilson, Lubricants and functional fluids from renewable sources, Ind. Lubric. Tribol. 50 (1) (1998) 6e15. [3] H.A. Hamid, R. Yunus, U. Rashid, T.S. Choong, S. Ali, A.M. Syam, Synthesis of high oleic palm oil-based trimethylolpropane esters in a vacuum operated pulsed loop reactor, Fuel 166 (2016) 560e566. €m, M.A. Mollaahmad, S. Karlsson, R. Hatti-Kaul, [4] C.O. Åkerman, A.E. Hagstro Biolubricant synthesis using immobilised lipase: process optimisation of trimethylolpropane oleate production, Process Biochem. 46 (12) (2011) 2225e2231. [5] T.-S. Chang, H. Masood, R. Yunus, U. Rashid, T.S. Choong, D.R.A. Biak, Activity of calcium methoxide catalyst for synthesis of high oleic palm oil based trimethylolpropane triesters as lubricant base stock, Ind. Eng. Chem. Res. 51 (15) (2012) 5438e5442. [6] F. Zaccheria, M. Mariani, R. Psaro, P. Bondioli, N. Ravasio, Environmentally friendly lubricants through a zero waste process, Appl. Catal. B Environ. 181 (2016) 581e586. [7] E. Wang, X. Ma, S. Tang, R. Yan, Y. Wang, W.W. Riley, et al., Synthesis and oxidative stability of trimethylolpropane fatty acid triester as a biolubricant base oil from waste cooking oil, Biomass Bioenergy 66 (2014) 371e378. €msa €, R. Hatti-Kaul, Clean synthesis of [8] C.O. Åkerman, Y. Gaber, N.A. Ghani, M. La biolubricants for low temperature applications using heterogeneous catalysts, J. Mol. Catal. B Enzym. 72 (3e4) (2011) 263e269. [9] E. Uosukainen, Y.-Y. Linko, M. L€ ams€ a, T. Tervakangas, P. Linko, Transesterification of trimethylolpropane and rapeseed oil methyl ester to environmentally acceptable lubricants, J. Am. Oil Chem. Soc. 75 (11) (1998) 1557e1563. €msa €, P. Linko, Production of trimethylol[10] Y.-Y. Linko, T. Tervakangas, M. La propane esters of rapeseed oil fatty acids by immobilized lipase, Biotechnol. Tech. 11 (12) (1997) 889e892. [11] H. Kim, N. Choi, S.-W. Oh, Y. Kim, B.H. Kim, I.-H. Kim, Synthesis of a-linolenic acid-rich triacylglycerol using a newly prepared immobilized lipase, Food Chem. 237 (2017) 654e658.
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