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Enzymatic Epoxidation of High Oleic Soybean Oil Ximing Zhang, Julia Burchell, and Nathan S. Mosier ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00884 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018
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ENZYMATIC EPOXIDATION OF HIGH OLEIC SOYBEAN OIL
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Ximing Zhang1,2, Julia Burchell1, Nathan S. Mosier1*
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1. Laboratory of Renewable Resources Engineering, Department of Agricultural and
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Biological Engineering, Purdue University, 225 S. University St., West Lafayette, IN 47907,
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USA
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2. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou
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310058, P.R. China
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* Corresponding author. Tel.: +1-765-494-7022. Fax: +1-765-494-7023.
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E-mail:
[email protected].
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KEYWORDS: enzymatic epoxidation, high oleic soybean oil, free fatty acids
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Abstract
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We report the production of epoxidized high oleic soybean oil (EHOSBO) using immobilized
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lipase as a sustainable replacement for the current acid-catalyzed process. Enzymatic
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epoxidation of conventional oil at 35°C both with and without toluene resulted in yields of
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nearly 100%. While the addition of free fatty acids (FFA) to the reaction did not prevent
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hydrolysis of triglycerides, the yields were significantly higher when oleic acid (OA) was
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added at 8.0% of the oil mass. Relative reaction rates of unsaturated fatty acids for oleic and
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linoleic acid are similar and half that of linolenic acid, although full epoxidation of linolenic
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acid was not observed. Oil from high oleic genetically modified soybeans resulted in
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epoxidation yields of 95% at 35°C without FFA added. These results suggest that toluene and
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FFA are not necessary to achieve high yields with enzymatic epoxidation at 35°C, especially
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for high OA soybean oil.
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Introduction
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Epoxidized soybean oil (ESBO) has seen increasing interest for its use in a variety of
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products such as paints, coatings, lubricants, films or food packaging and is currently
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produced at greater than 200,000 tonnes per year globally by the Prilezhaev epoxidation,
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which utilizes strong acids1–4. Due to the harsh conditions of the reaction there is low
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selectivity and ring opening of the epoxide occurs, leading to low yields and undesirable
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by-products5,6. Recent development of a lipase-catalyzed epoxidation presents a promising,
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environmentally-friendly alternative to the Prilezhaev reaction. This approach utilizes
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Novozymes 435, made by immobilization of Candida antarctica Lipase B (CALB) on acrylic
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resin, which operates at milder conditions which minimizes epoxy ring opening7,8.
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Although enzyme-catalyzed epoxidation eliminates the need for strong acids, such as
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sulfuric and acetic acid, the methods reported in the literature use toluene, a toxic
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hydrocarbon, to maintain the activity of the enzyme9,10. Toluene alleviates direct contact of
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the hydrogen peroxide with the enzyme, which is the main source of loss in enzyme activity,
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especially at high temperatures and has been shown to increase yields11–13. Although
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toluene helps to control the reaction temperature by diluting the reactants and removing
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heat through evaporative cooling, the use of toluene at commercial-scale requires careful
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handling and disposal due to its toxicity9. Additionally, a process without toluene is more
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volume efficient, thus lowering capital costs, while also simplifying separation of end
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products, thus lowering capital and operational costs14,15.
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Warwel and Klass, and Okada et al. reported that the addition of free fatty acids (FFA)
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to the reaction serves to provide carboxylic acids for forming peroxy acids that are needed
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to epoxidize the unsaturated C=C bonds, thus eliminating the need for acetic acid in the
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process16,17. Although the lipase is able to hydrolyze fatty acids from triglycerides, addition of
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FFA is still desirable as it has been reported to suppress hydrolysis of fatty acids and
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eliminate the production of di-, and monoglycerides, which are difficult to remove from the
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end product and lower end product quality16. Ideally, it would be better to eliminate the
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need for adding pure FFA as acetic acid is far less expensive.
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While conventional soybean oil is a major substrate for epoxidation, high oleic soybean
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oil (HOSBO) has shown potential for application in industry with increased stability in
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lubricant and PVC settings18–20. The ideal number of epoxide groups for commercial
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application is reported at three per triglyceride, which is the average number of double
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bonds per triglyceride in high oleic soybean oil, thus making it a suitable feedstock for these
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applications18,21.
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The objective of this work was to study the effect of toluene and the addition of FFA on
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the enzyme-catalyzed epoxidation of soybean oil, as well as high oleic soybean oil. The
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effects of toluene, FFA, and operational temperature on epoxide yields and rates were
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determined. The effects of these variables on the enzyme activity over time were also
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evaluated, toward an overall goal to decrease processing costs at commercial scale.
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Materials and Methods
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Materials
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Consumer-grade vegetable oil containing 100% soybean oil and with an iodine value of
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137 g I2/100 g of oil was purchased from a local grocery store (Great Value). Novozymes 435
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(Candida antarctica lipase B immobilized on acrylic resin, particle size around 300μm,
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enzyme activity≥5,000 U/g) was purchased from Sigma-Aldrich. Anhydrous sodium sulfate,
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potassium hydroxide, cyclo-hexane and methanol were also purchased from Sigma-Aldrich
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(Milwaukee, WI). Hydrogen peroxide (30% w/w) and glacial acetic acid were purchased from
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Fisher Scientific (Hanover Park, IL). Plenish® high oleic soybean oil was donated by DuPont
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Pioneer with an iodine value of 85 g I2/100 g. Table 1 shows the difference in fatty acid
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composition between Plenish® oleic soybean oil and commercial soybean oil.
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Table 1. Typical fatty acid composition from soybean oil and Plenish® high oleic soybean oil
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(table remade from DuPont Pioneer. https://www.plenish.com/food/oil-profile/)
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Plenish® High Oleic
C16:0
C18:0
C18:1
C18:2
C18:3
Palmitic
Stearic
Oleic
Linoleic acid
Linolenic
acid
acid
acid
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7
2
11
4
22
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8
acid
Soybean Oil Commercial Soybean Oil 79
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Enzymatic epoxidation
Epoxidations were carried out in a 250 mL three necked, round bottom flask placed in a
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water bath on top of a magnetic stirring hotplate. Soybean oil (50.0 g) was combined with
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2.0 g of Novozymes 435 (4.0 wt.% of oil) in the flask and stirred at 400 rpm with a
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polytetrafluoroethylene-coated magnetic stir bar. Hydrogen peroxide at 2:1 molar ratio to
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C=C bonds (52.0 g) was added step-wise to the flask from a separatory funnel within the first
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20 minutes of reaction. Epoxidation reactions were carried out for 24 hours at the indicated
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temperature. Samples consisting of 3-4mL were taken at a predetermined time interval and
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then centrifuged at 3,000 rpm and 20°C for 5 minutes. Three layers were formed after
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centrifugation, and the top layer containing the oil was transferred to a separate centrifuge
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tube. Sodium sulfate (10.0 wt% of oil) was added to adsorb any remaining moisture. The
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mixture was centrifuged again at 3,000 rpm and 20°C for 5 minutes. The supernatant was
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collected and stored at 4°C for further analysis. All epoxidations were conducted in triplicate
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and statistical analyses performed using Minitab.
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Fatty acid methyl ester (FAME) preparation
Epoxidized soybean oil was converted into methyl ester form based on Badings and
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Jong’s method and then analyzed by gas chromatography22. Soybean oil (40 mg) was
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combined with 2 mL cyclohexane and 0.2 mL of 2M potassium hydroxide in methanol in a
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centrifuge tube. The tubes were vortexed for two minutes and then 1.5mL of the
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supernatant was collected and transferred to a GC vial for further analysis.
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Gas chromatography
Gas chromatography system consisted of Agilent automatic liquid sampler (7693A) and
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GC (7820A) equipped with an Agilent HP-INNOWax column and flame ionization detector
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(FID). Samples (0.5 μL) were injected with a 20:1 split ratio to the detector. Column
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temperature was set at 180°C as the starting temperature, held for 1 minute and increased
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at rate of 10°C/min to 250°C, and then held for 8 minutes. The GC spectra peaks elution time
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were calibrated by ESBO and soybean FAME standards. The product composition of the
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reaction were analyzed based on corresponding GC peak areas.
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Characterization of soybean oil
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The iodine value of the oil was determined according to the procedure outlined from
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the American Oil Chemists’ Society (AOCS) in method Tg 1-6423. Acid number was
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determined through the procedure described by Hagström et al.10. Briefly summarized,
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approximately 0.1 g of epoxidized sample was added to a 50 mL flask and dissolved in 10 mL
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ethyl acetate. Phenolphthalein, 1% v/v in ethanol (4.0 mL) was added. The solution was
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titrated with 0.03 M potassium hydroxide in ethanol until a change in color was observed.
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The following equation was using to calculate the acid number:
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=
× ×
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where
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the molecular weight of KOH (g/mol), and +#,-. is the mass of the sample (g)2.
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! "#$
is the concentration of KOH (M), % !
(1)
"#$
is the volume of KOH, &' ()* is
Oxirane oxygen analysis was carried out according to AOCS method Cd 9-5724. The
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percent conversion was determined based on the oxirane oxygen measurements and the
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following equation:
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))
/ 0123 43 54 24 46 1 7/89: = ;)) < > × 100 =
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where OOex is the content of oxirane oxygen experimentally determined, and OOth is the
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theoretical maximum oxirane oxygen content. The theoretical oxirane oxygen content is
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determined by the following formula: C ⁄EF
(2)
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D 99 A = BHIIJ7C L × M × 100 ⁄EF :F
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where IV0 is the initial iodine value of soybean oil; and Ai (126.9) and Ao (16.0) are the atomic
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weights of iodine and oxygen, respectively.
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Results and Discussion
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D
K
(3)
Since epoxidation reaction is catalyzed by peroxy acid, free oleic acid (OA) was added to the soybean oil as a source of carboxylic acids for peroxidation. To determine the optimal
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operating conditions for enzyme reusability, the reaction temperature for the enzyme was
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examined. Novozymes 435 was reported to be stable from 30 to 60°C8. Because epoxidation
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reaction is exothermic, temperatures slightly below this range (23°C, 35°C, and 50°C) were
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studied for reaction times up to 24 hours. Since hydrogen peroxide begins to decompose at
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50°C, temperatures higher than that were not considered. Oleic acid (8.0 wt% of the total
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oil) was used in the first experiment and hydrogen peroxide (30% w/w) was added dropwise
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at a rate of 5 mL/min. Yields were estimated based on measurements of oxirane oxygen and
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iodine values. Figure 1 shows that 50°C has a rapid initial conversion rate which then levels
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off after approximately 7 hours for a final yield of 81% of theoretical. One explanation for
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this could be that the hydrogen peroxide decomposed before full epoxidation could occur.
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However, this is unlikely as the molar ratio of hydrogen peroxide to epoxidation sites was
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2:1. Alternatively, the enzyme may have lost its activity in the presence of higher
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temperatures and hydrogen peroxide, thus resulting in reduced yields25.
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Figure 1. Enzyme-catalyzed epoxidation of conventional soybean oil over time at 23°, 35°,
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and 50°C. Conditions: 8.0 wt% (related to oil) oleic acid, 2:1 H2O2 to C=C bond molar ratio,
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stir speed = 400 rpm, 4.0 wt% (related to oil) enzyme loading. a,b – denotes significance
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levels with p
