Subscriber access provided by University of Massachusetts Amherst Libraries
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sustainable Chemistry & Engineering
2
ENZYMATIC EPOXIDATION OF HIGH OLEIC SOYBEAN OIL
3
Ximing Zhang1,2, Julia Burchell1, Nathan S. Mosier1*
4
1. Laboratory of Renewable Resources Engineering, Department of Agricultural and
5
Biological Engineering, Purdue University, 225 S. University St., West Lafayette, IN 47907,
6
USA
7
2. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou
8
310058, P.R. China
9
* Corresponding author. Tel.: +1-765-494-7022. Fax: +1-765-494-7023.
1
10
E-mail:
[email protected].
11 12
KEYWORDS: enzymatic epoxidation, high oleic soybean oil, free fatty acids
13 14
Abstract
15
We report the production of epoxidized high oleic soybean oil (EHOSBO) using immobilized
16
lipase as a sustainable replacement for the current acid-catalyzed process. Enzymatic
17
epoxidation of conventional oil at 35°C both with and without toluene resulted in yields of
18
nearly 100%. While the addition of free fatty acids (FFA) to the reaction did not prevent
ACS Paragon Plus Environment
ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
19
hydrolysis of triglycerides, the yields were significantly higher when oleic acid (OA) was
20
added at 8.0% of the oil mass. Relative reaction rates of unsaturated fatty acids for oleic and
21
linoleic acid are similar and half that of linolenic acid, although full epoxidation of linolenic
22
acid was not observed. Oil from high oleic genetically modified soybeans resulted in
23
epoxidation yields of 95% at 35°C without FFA added. These results suggest that toluene and
24
FFA are not necessary to achieve high yields with enzymatic epoxidation at 35°C, especially
25
for high OA soybean oil.
26
Introduction
27
Epoxidized soybean oil (ESBO) has seen increasing interest for its use in a variety of
28
products such as paints, coatings, lubricants, films or food packaging and is currently
29
produced at greater than 200,000 tonnes per year globally by the Prilezhaev epoxidation,
30
which utilizes strong acids1–4. Due to the harsh conditions of the reaction there is low
31
selectivity and ring opening of the epoxide occurs, leading to low yields and undesirable
32
by-products5,6. Recent development of a lipase-catalyzed epoxidation presents a promising,
33
environmentally-friendly alternative to the Prilezhaev reaction. This approach utilizes
34
Novozymes 435, made by immobilization of Candida antarctica Lipase B (CALB) on acrylic
35
resin, which operates at milder conditions which minimizes epoxy ring opening7,8.
36
Although enzyme-catalyzed epoxidation eliminates the need for strong acids, such as
37
sulfuric and acetic acid, the methods reported in the literature use toluene, a toxic
38
hydrocarbon, to maintain the activity of the enzyme9,10. Toluene alleviates direct contact of
ACS Paragon Plus Environment
Page 2 of 25
Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sustainable Chemistry & Engineering
39
the hydrogen peroxide with the enzyme, which is the main source of loss in enzyme activity,
40
especially at high temperatures and has been shown to increase yields11–13. Although
41
toluene helps to control the reaction temperature by diluting the reactants and removing
42
heat through evaporative cooling, the use of toluene at commercial-scale requires careful
43
handling and disposal due to its toxicity9. Additionally, a process without toluene is more
44
volume efficient, thus lowering capital costs, while also simplifying separation of end
45
products, thus lowering capital and operational costs14,15.
46
Warwel and Klass, and Okada et al. reported that the addition of free fatty acids (FFA)
47
to the reaction serves to provide carboxylic acids for forming peroxy acids that are needed
48
to epoxidize the unsaturated C=C bonds, thus eliminating the need for acetic acid in the
49
process16,17. Although the lipase is able to hydrolyze fatty acids from triglycerides, addition of
50
FFA is still desirable as it has been reported to suppress hydrolysis of fatty acids and
51
eliminate the production of di-, and monoglycerides, which are difficult to remove from the
52
end product and lower end product quality16. Ideally, it would be better to eliminate the
53
need for adding pure FFA as acetic acid is far less expensive.
54
While conventional soybean oil is a major substrate for epoxidation, high oleic soybean
55
oil (HOSBO) has shown potential for application in industry with increased stability in
56
lubricant and PVC settings18–20. The ideal number of epoxide groups for commercial
57
application is reported at three per triglyceride, which is the average number of double
58
bonds per triglyceride in high oleic soybean oil, thus making it a suitable feedstock for these
59
applications18,21.
ACS Paragon Plus Environment
ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
60
The objective of this work was to study the effect of toluene and the addition of FFA on
61
the enzyme-catalyzed epoxidation of soybean oil, as well as high oleic soybean oil. The
62
effects of toluene, FFA, and operational temperature on epoxide yields and rates were
63
determined. The effects of these variables on the enzyme activity over time were also
64
evaluated, toward an overall goal to decrease processing costs at commercial scale.
65
Materials and Methods
66
Materials
67
Consumer-grade vegetable oil containing 100% soybean oil and with an iodine value of
68
137 g I2/100 g of oil was purchased from a local grocery store (Great Value). Novozymes 435
69
(Candida antarctica lipase B immobilized on acrylic resin, particle size around 300μm,
70
enzyme activity≥5,000 U/g) was purchased from Sigma-Aldrich. Anhydrous sodium sulfate,
71
potassium hydroxide, cyclo-hexane and methanol were also purchased from Sigma-Aldrich
72
(Milwaukee, WI). Hydrogen peroxide (30% w/w) and glacial acetic acid were purchased from
73
Fisher Scientific (Hanover Park, IL). Plenish® high oleic soybean oil was donated by DuPont
74
Pioneer with an iodine value of 85 g I2/100 g. Table 1 shows the difference in fatty acid
75
composition between Plenish® oleic soybean oil and commercial soybean oil.
76 77
Table 1. Typical fatty acid composition from soybean oil and Plenish® high oleic soybean oil
78
(table remade from DuPont Pioneer. https://www.plenish.com/food/oil-profile/)
ACS Paragon Plus Environment
Page 4 of 25
Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sustainable Chemistry & Engineering
Plenish® High Oleic
C16:0
C18:0
C18:1
C18:2
C18:3
Palmitic
Stearic
Oleic
Linoleic acid
Linolenic
acid
acid
acid
11
4
76
7
2
11
4
22
55
8
acid
Soybean Oil Commercial Soybean Oil 79
80
81
Enzymatic epoxidation
Epoxidations were carried out in a 250 mL three necked, round bottom flask placed in a
82
water bath on top of a magnetic stirring hotplate. Soybean oil (50.0 g) was combined with
83
2.0 g of Novozymes 435 (4.0 wt.% of oil) in the flask and stirred at 400 rpm with a
84
polytetrafluoroethylene-coated magnetic stir bar. Hydrogen peroxide at 2:1 molar ratio to
85
C=C bonds (52.0 g) was added step-wise to the flask from a separatory funnel within the first
86
20 minutes of reaction. Epoxidation reactions were carried out for 24 hours at the indicated
87
temperature. Samples consisting of 3-4mL were taken at a predetermined time interval and
88
then centrifuged at 3,000 rpm and 20°C for 5 minutes. Three layers were formed after
89
centrifugation, and the top layer containing the oil was transferred to a separate centrifuge
90
tube. Sodium sulfate (10.0 wt% of oil) was added to adsorb any remaining moisture. The
91
mixture was centrifuged again at 3,000 rpm and 20°C for 5 minutes. The supernatant was
92
collected and stored at 4°C for further analysis. All epoxidations were conducted in triplicate
93
and statistical analyses performed using Minitab.
ACS Paragon Plus Environment
ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
94
95
Fatty acid methyl ester (FAME) preparation
Epoxidized soybean oil was converted into methyl ester form based on Badings and
96
Jong’s method and then analyzed by gas chromatography22. Soybean oil (40 mg) was
97
combined with 2 mL cyclohexane and 0.2 mL of 2M potassium hydroxide in methanol in a
98
centrifuge tube. The tubes were vortexed for two minutes and then 1.5mL of the
99
supernatant was collected and transferred to a GC vial for further analysis.
100
101
Gas chromatography
Gas chromatography system consisted of Agilent automatic liquid sampler (7693A) and
102
GC (7820A) equipped with an Agilent HP-INNOWax column and flame ionization detector
103
(FID). Samples (0.5 μL) were injected with a 20:1 split ratio to the detector. Column
104
temperature was set at 180°C as the starting temperature, held for 1 minute and increased
105
at rate of 10°C/min to 250°C, and then held for 8 minutes. The GC spectra peaks elution time
106
were calibrated by ESBO and soybean FAME standards. The product composition of the
107
reaction were analyzed based on corresponding GC peak areas.
108
Characterization of soybean oil
109
The iodine value of the oil was determined according to the procedure outlined from
110
the American Oil Chemists’ Society (AOCS) in method Tg 1-6423. Acid number was
111
determined through the procedure described by Hagström et al.10. Briefly summarized,
112
approximately 0.1 g of epoxidized sample was added to a 50 mL flask and dissolved in 10 mL
113
ethyl acetate. Phenolphthalein, 1% v/v in ethanol (4.0 mL) was added. The solution was
ACS Paragon Plus Environment
Page 6 of 25
Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sustainable Chemistry & Engineering
114
titrated with 0.03 M potassium hydroxide in ethanol until a change in color was observed.
115
The following equation was using to calculate the acid number:
116
=
× ×
117
where
118
the molecular weight of KOH (g/mol), and +#,-. is the mass of the sample (g)2.
119
! "#$
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
120
percent conversion was determined based on the oxirane oxygen measurements and the
121
following equation:
122
))
/ 0123 43 54 24 461 7/89: = ;))< > × 100 =
123
where OOex is the content of oxirane oxygen experimentally determined, and OOth is the
124
theoretical maximum oxirane oxygen content. The theoretical oxirane oxygen content is
125
determined by the following formula: C ⁄EF
(2)
126
D 99 A = BHIIJ7C L × M × 100 ⁄EF :F
127
where IV0 is the initial iodine value of soybean oil; and Ai (126.9) and Ao (16.0) are the atomic
128
weights of iodine and oxygen, respectively.
129
Results and Discussion
130 131
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
ACS Paragon Plus Environment
ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
132
operating conditions for enzyme reusability, the reaction temperature for the enzyme was
133
examined. Novozymes 435 was reported to be stable from 30 to 60°C8. Because epoxidation
134
reaction is exothermic, temperatures slightly below this range (23°C, 35°C, and 50°C) were
135
studied for reaction times up to 24 hours. Since hydrogen peroxide begins to decompose at
136
50°C, temperatures higher than that were not considered. Oleic acid (8.0 wt% of the total
137
oil) was used in the first experiment and hydrogen peroxide (30% w/w) was added dropwise
138
at a rate of 5 mL/min. Yields were estimated based on measurements of oxirane oxygen and
139
iodine values. Figure 1 shows that 50°C has a rapid initial conversion rate which then levels
140
off after approximately 7 hours for a final yield of 81% of theoretical. One explanation for
141
this could be that the hydrogen peroxide decomposed before full epoxidation could occur.
142
However, this is unlikely as the molar ratio of hydrogen peroxide to epoxidation sites was
143
2:1. Alternatively, the enzyme may have lost its activity in the presence of higher
144
temperatures and hydrogen peroxide, thus resulting in reduced yields25.
ACS Paragon Plus Environment
Page 8 of 25
Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sustainable Chemistry & Engineering
145 146
Figure 1. Enzyme-catalyzed epoxidation of conventional soybean oil over time at 23°, 35°,
147
and 50°C. Conditions: 8.0 wt% (related to oil) oleic acid, 2:1 H2O2 to C=C bond molar ratio,
148
stir speed = 400 rpm, 4.0 wt% (related to oil) enzyme loading. a,b – denotes significance
149
levels with p