Antioxidant canolol production from a renewable feedstock via an engineered decarboxylase

Article abstract of DOI:10.1039/c3gc40748a

Canolol (4-vinylsyringol, VS), a potent antioxidant and an alkylperoxyl radical scavenger originally discovered in crude canola oil (rapeseed), is produced by decarboxylation of sinapic acid (SA) during canola seed roasting. Chemical syntheses of VS from SA require thermal or microwave induced decarboxylation in the presence of a base. A laboratory-evolved enzyme, designated SA decarboxylase (SAD), was developed in this study. In a biphasic bioreactor system, SAD was shown to produce VS from SA extracts prepared from canola meal with an overall yield of 3.0 mg VS per g of canola meal. In addition, we investigated the application of VS in polymerization to produce polyvinylsyringol (PVS) as a potential biodegradable polymer. The characteristics of PVS determined by thermogravimetric analysis, differential scanning calorimetry and nanoindentation tests are described.

Full text of DOI:10.1039/c3gc40748a

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Volume 15 | Number 12 | December 2013 | Pages 3279–3490
ISSN 1463-9262
PAPER
Peter C. K. Lau et al.
Antioxidant canolol production from a renewable feedstock via an engineered
decarboxylase
1463-9262(2013)15:12;1-#

Green Chemistry
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PAPER
Antioxidant canolol production from a renewable
feedstock via an engineered decarboxylase†
Cite this: Green Chem., 2013, 15, 3312
a
a
a
a,b,c
Krista L. Morley, Stephan Grosse, Hannes Leisch and Peter C. K. Lau*
Canolol (4-vinylsyringol, VS), a potent antioxidant and an alkylperoxyl radical scavenger originally disco-
vered in crude canola oil (rapeseed), is produced by decarboxylation of sinapic acid (SA) during canola
seed roasting. Chemical syntheses of VS from SA require thermal or microwave induced decarboxylation
in the presence of a base. A laboratory-evolved enzyme, designated SA decarboxylase (SAD), was deve-
loped in this study. In a biphasic bioreactor system, SAD was shown to produce VS from SA extracts pre-
pared from canola meal with an overall yield of 3.0 mg VS per g of canola meal. In addition, we
investigated the application of VS in polymerization to produce polyvinylsyringol (PVS) as a potential bio-
degradable polymer. The characteristics of PVS determined by thermogravimetric analysis, differential
scanning calorimetry and nanoindentation tests are described.
Received 19th April 2013,
Accepted 30th September 2013
DOI: 10.1039/c3gc40748a
www.rsc.org/greenchem
5
biopolymer with desirable properties of biodegradability and
strength.
Introduction
4
Phenolic compounds found in nature as hydroxycinnamic acid
In addition to decarboxylating FA, PAD decarboxylates
derivatives, glycosides, flavonoids, lignans, etc. represent an p-coumaric acid and caffeic acid but is inactive towards sinapic
important fraction of the biomass feedstock that is of socio- acid (SA, 3,5-dimethoxy-4-hydroxycinnamic acid), also called
economic importance. They constitute a major class of the sinapinic acid. SA is simply a FA molecule with an extra
plant secondary metabolites and function as antioxidants methoxy group at the 5-position of the phenyl ring. Studies
6
,7
towards cancer and cardiovascular diseases, for example. have shown that SA is the main phenolic acid in canola.
Lignins and suberins are examples of phenolic containing
polymers where phenolic acids play a structural role.
In canola meal, SA esters (sinapine and glucopyransol sina-
1
−1
pate) make up 90–95% of the total meal phenolics (10–12 mg g )
−1 8
Among the hydroxycinnamic acids found in the plant whereas the amount of free SA is less than 10% (0.3–0.4 mg g )
world, ferulic acid (FA, 4-hydroxy-3-methoxy cinnamic acid) is (Fig. 1). SA, sinapine, and glucopyranosyl sinapate all have
9
,10
the most abundant, and corn bran with 3.1% (w/w) is one of significant antioxidative properties.
the most promising sources of this antioxidant. Bioconver-
2
,3
Wakamatsu et al. first reported that roasting of canola seed
sions of FA provide many value-added products such as vinyl led to the formation of 4-vinylsyringol (VS), a decarboxylation
2
11
guaiacol (VG) and vanillin; both are flavoring agents. Recently, product of SA that they gave the name canolol. The optimum
−
1
a proof-of-concept of polyvinylguaiacol (PVG) production by a roasting temperature for VS formation (720 µg g ) was sub-
chemo-enzymatic approach, from corn bran extracted FA, was sequently shown to be 160 °C. VS exhibits a higher antioxi-
demonstrated for the first time. A cloned phenolic acid decar- dant activity than well-known antioxidants, including
boxylase (PAD), originally derived from Bacillus pumilus strain α-tocopherol, vitamin C, β-carotene, rutin and quercetin. In
UI-670, was the requisite biocatalyst that decarboxylated FA to addition, VS exhibits antimutagenic, anti-inflammatory, and
VG prior to polymerization. PVG is a potentially useful anticarcinogenic properties.
1
2
4
1
1
1
3
1
4
Chemical methods for the decarboxylation of SA to VS
require thermal or microwave induced decarboxylation in the
presence of a base. These methods include heating SA in a
1
5
a
quinoline/copper salt at 200 °C, microwave irradiation in
the presence of the base 1,8-diazabicyclo[5.4.0]undec-7-ene
Aquatic and Crop Resource Development, National Research Council Canada,
100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada.
6
1
6
E-mail: peter.lau@cnrc-nrc.gc.ca; Fax: +(514) 496-6265; Tel: +(514) 496-6325
(DBU), microwave irradiation in the ionic liquid [hmim]Br
using sodium hydrogen carbonate (NaHCO ) as a mild base,
and thermal treatment in dimethylformamide (DMF) at
30 °C using sodium acetate as a catalyst. In addition, the
b
1
7
McGill University, Departments of Chemistry and Microbiology & Immunology,
3
McGill University, Montreal, Quebec H3A 2B4, Canada
c
FQRNT Centre in Green Chemistry and Catalysis, Montreal, Quebec, Canada
1
8
1
†
Electronic supplementary information (ESI)
0.1039/c3gc40748a
available. See DOI:
1
synthesis of VS from syringaldehyde has been carried out
3
312 | Green Chem., 2013, 15, 3312–3317
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30 m × 0.25 mm × 0.25 µm HP-5MS capillary column (Missis-
sauga, ON, Canada).
Preparative scale decarboxylation of SA (5 g L− ) in buffer
0 g of cells from 1 L of Escherichia coli JM109[pKFAD-Ile85Ala]
1
2
cells expressing sinapic acid decarboxylase (SAD) were resus-
pended in 40 mL of 10 mM sodium phosphate buffer (pH 7.0)
−1
and lysed by sonication. 23.6 mL of the crude lysate (22.4 U mL )
was added to a stirring solution of 0.25 g of SA (final
concentration: 5.0 g L− , 22 mM) in 26.4 mL of 100 mM
sodium phosphate buffer (pH 7.0). After stirring at 30 °C and
1
250 rpm for 16 h, a 95% reaction conversion was determined
by HPLC. The pH of the reaction was adjusted to 3 with 1 M
HCl, and then extracted three times with ethyl acetate (EtOAc).
The organic layers were combined, washed with 100 mM
sodium phosphate buffer (pH 8) and brine, and then dried
over anhydrous sodium sulfate. Concentration afforded 0.15 g
1
of a yellow oil (crude, 75% isolated yield). H NMR (500 MHz,
CDCl
3
) 3.874 (6H, s), 5.110–5.131 (1H, d), 5.560–5.595 (2H, d),
6
1
.560–6.621 (3H, m) (ESI Fig. S1A†). GC/MS m/z (rel. intensity)
80 (100), 165 (37), 137 (23), 122 (12), 91 (11), 77 (16) (Fig. S2†).
Preparative scale decarboxylation of SA (25 g L− ) in biphasic
1
medium and VS polymerization
Fig. 1 Sinapic acid and sinapic acid esters found in canola meal.
1
g of SA was added to 16.4 mL of 100 mM sodium phosphate
buffer (pH 7.0). The pH of the solution was adjusted to pH 7.0
by dropwise addition of 4 M NaOH. The solution was overlaid
1
9
via Wittig olefination in hexane/tetrahydrofuran (THF) or via with 20 mL of toluene and 3.6 mL of the crude SAD lysate
simultaneous condensation–double decarboxylation of syring- (22.4 U mL− ) was added. The reaction was stirred at 30 °C and
1
2
0
aldehyde and malonic acid under microwave irradiation.
250 rpm for 16 h and a 99% reaction conversion was deter-
In this paper, we describe the first enzymatic process for mined by HPLC. The two layers were separated and the
generating VS from SA using an engineered decarboxylase, a aqueous layer was extracted twice with 20 mL of EtOAc. The
new enzyme called sinapic acid decarboxylase (SAD), and organic layers were combined, dried over anhydrous sodium
establish its kinetic parameters. Process conditions were sulfate, and concentrated to afford 0.67 g of a yellow oil (crude,
1
demonstrated on SA extracts from canola meal. In addition, 83% isolated yield). H NMR (500 MHz, CDCl
) 3.874 (6H, s),
3
we investigated the application of VS in polymerization to 5.110–5.131 (1H, d), 5.560–5.595 (2H, d), 6.560–6.621 (3H, m).
produce PVS as a potential biodegradable alternative to poly- GC/MS m/z (rel. intensity) 180 (100), 165 (37), 137 (23),
vinylstyrene. The characteristics of PVS determined by TGA, 122 (12), 91 (11), 77 (16). VS (500 mg) was subjected to cationic
DSC and nanoindentation tests are also described.
polymerization using dichloromethane (3.5 mL, 4.5 g) and
boron trifluoride·(Et O) (10 mg, 0.07 mmol) at −40 °C to yield
95 mg of an off-white solid. M = 10 764, M = 14 706; T =
2
2
7
1
n
w
g
06–116 °C.
Experimental
General
Size exclusion chromatography
SA (3,5-dimethoxy-4-hydroxycinnamic acid) was purchased Size exclusion chromatography (SEC) was performed using a
from Alfa Aesar (Wardhill, MA, USA). All other chemicals were multi-detection system from Viscotek (Houston, TX, USA) con-
purchased from Sigma-Aldrich (Mississauga, ON, Canada). sisting of a Model 302 Triple Detector Platform, including a
Canola meal was a generous gift from ADM Bunge Canada refractive index detector, a four capillary viscometer and a two
(Oakville, ON, Canada). Oligonucleotide primers were syn- angle laser light scattering detector, and a GPCmax integrated
thesized by IDT (San Jose, CA, USA). DNA sequencing of pump, autosampler, and degasser. Molar masses of the
mutants was performed by Plate-forme d’Analyses Génomi- samples were determined by universal calibration using the
1
ques of Laval University (Québec City, Qc, Canada). H and refractive index detector and the viscosity detector. The univer-
1
3
C NMR analyses were carried out in CDCl using a Bruker sal calibration curve was based on polystyrene standards
3
(500 MHz) spectrometer. GC/MS analysis was performed [Polymer Laboratories (Amherst, MA, USA) and Aldrich (Oak-
using an Agilent Technologies 7890A gas chromatograph ville, ON, Canada)] using the molar masses determined by the
coupled to a 5975C quadrupole mass spectrometer and a manufacturer and the intrinsic viscosities measured by the
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apparatus. The OmniSEC software from Viscotek was used for
Results and discussion
Protein engineering of PAD to alter substrate specificity
data collection and calibration. Separation in tetrahydrofuran
1
(
THF) was performed by injecting 100 μL of 1.5–2 mg mL−
solutions of standards or samples into thermostatically con- The X-ray crystal structure of the 161-amino acid residue PAD,
21
trolled SupeRes columns [35 °C; PAS-102, PAS-102.5, PAS-103- functionally active as a dimer, was solved recently. To alter
L, each 300 mm × 8 mm; PolyAnalytik (London, ON, Canada)]. the substrate specificity of wild-type PAD, five residues in the
−
1
The flow rate was 1 mL min
.
active site were targeted for saturation mutagenesis: Tyr19,
Val38, Val70, Ile85, and Phe87 (Fig. 2). Experimental details
are provided in the ESI† (protein engineering of PAD). The
Solid-state properties characterization
The thermal properties of PVS were characterized by thermo- mutants were screened for SAD activity, using a 96-well plate
gravimetric analysis (TGA) and differential scanning calorimetry spectrophotometric assay to monitor the decrease in SA absor-
(DSC). TGA measurements were performed with a Metler Toledo bance at 310 nm. Whereas no hits were obtained from the
(
Mississauga, ON, Canada) TGA SF model analyzer under a nitro- Tyr19, Val38, Val70, Phe87 libraries, four mutants with SAD
−1
gen atmosphere at a heating rate of 20 °C min in the tempera- activity were identified from the Ile85 library: Ile85Ala,
ture range of 30 °C–800 °C. The glass transition temperature, T Ile85Gly, Ile85Ser, and Ile85Thr. Crude lysates of the four
g
,
of the samples was measured using a TA Q200 differential scan- mutants were compared in 1 mL scale decarboxylation reac-
ning calorimeter (Brossard, Quebec, Canada). The DSC runs tions containing 4.5 mg mL− of SA. Ile85Ala (referred to
1
−
1
were conducted over the temperature range −70 °C–230 °C, with a herein as SAD) showed the highest activity (1.4 U mg
;
three part sequence that included heating, cooling and reheating. Table S1†) and was selected for process development, in
The underlying heating rate and cooling rate was 20 °C min−
The second heat curve was used for the T determination. mination (ESI†).
m
Precise T values were obtained by measuring peaks on the Table 1 shows that the affinity (K ) of the wildtype PAD and
1
.
addition to protein purification and kinetic parameter deter-
g
g
derivative graph of the enthalpy vs. temperature curve.
SAD towards FA and that of SAD towards SA are very close, in
the range of 2.7–3.2 mM. However, although the evolved SAD
gained the ability to act on SA, its catalytic competence
Nanoindentation test
Polymer powders were compressed at 150 °C. The resulting towards FA was compromised by about 7-fold compared to the
2
2
samples were embedded in epoxy and polished with a colloidal wildtype PAD, largely due to a reduction in kcat
.
silica suspension of 0.04 μm. The equipment used was an
indenter G200 (Agilent/MTS, Mississauga, ON, Canada) with
Preparative scale enzymatic decarboxylation of SA
the XP head and a Berkovich tip. The continuous stiffness For the preparative scale decarboxylation of SA with SAD in
measurement (CSM) method was employed, using standard aqueous buffer (100 mM sodium phosphate buffer; pH 7.0),
conditions (harmonic frequency target of 45 Hz; harmonic dis-
placement target of 2 nm; and strain rate target of 0.05 s− ).
1
Ten indents were done to a maximum depth of 2000 nm. The
reported values of the modulus (E) and hardness (H) are an
average of the values taken between 800 and 2000 nm.
Preparation of VS from canola meal
1
25 g of canola meal was defatted with hexane for 18 hours
using a soxhlet apparatus. Residual hexane was evaporated
from the defatted meal using a rotary evaporator. 1 L of 1 M
NaOH was added to 100 g of the defatted canola meal and the
mixture was incubated at 25 °C with shaking at 200 rpm for
1
8 hours. Following centrifugation (30 min, 4 °C, 3600 rpm),
the pH of the supernatant was adjusted to pH 2.0 by the
addition of 12 M HCl and extracted three times with 1 L of
hexane. The aqueous layer was then extracted three times with Fig. 2 Model of FA in the PAD active-site. Refer to ESI† for molecular
modeling.
a mixture of diethyl ether and ethyl acetate (1 : 1). The organic
layers were combined, dried over anhydrous sodium sulphate,
filtered, and concentrated to yield crude SA as a crude brown
solid (720 mg). The crude SA was suspended in 100 mM phos-
Table 1 Kinetic parameters of wild-type PAD and SAD
phate buffer (pH 7, 14.0 mL) and pH was adjusted to 7.0 by
dropwise addition of 4 M NaOH. Toluene (15 mL) was added Enzyme Substrate
k
(s
cat/K
m
max (U mg−
1
)
K
m
(mM)
k
cat (s−1
)
−1
M
−1)
V
and decarboxylation was carried out as described above using
4
WT-PAD FA
273.1
46.4
41.3
2.7 ± 0.8 182 ± 20 6.7 × 103
SAD (43 U mL− , 1.5 mL) to afford 340 mg of VS in >90% purity
1
SAD
SAD
FA
SA
3.2 ± 0.3
3 ± 1
31 ± 8
28 ± 4
9.7 × 10
9.3 × 10
1
3
as determined by HPLC (Fig. S3†) and H NMR (Fig. S1B†).
3
314 | Green Chem., 2013, 15, 3312–3317
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Table 2 Decarboxylation of SA to VS in buffer and biphasic media
Table 3 Nanoindentation tests of PVS and PVG samples
%
yield
Isolated Sample
Modulus, E (GPa)
Hardness, H (GPa)
SA (g L−
5
1
)
SAD (U mL−1)
a
Co-solvent
b
% Conv
c
PVS
5.22 ± 0.05
6.66 ± 0.05
6.75 ± 0.05
6.5 ± 0.1
0.283 ± 0.003
0.421 ± 0.003
0.463 ± 0.006
0.384 ± 0.012
0.249 ± 0.001
10.6
2.0
2.0
2.0
4.0
2
None
None
Toluene
Toluene
Toluene
95
37
99
50
97
42
30
99.5
75
30
83
45
81
n.d.
n.d.
n.d.
PVGrad
PVGcat
PVGmixed
PS 1301
25
25
50
50
25
25
25
4.15 ± 0.02
d
Ethyl acetate
Hexane
(BMIM)PF
6
2
2
of SA (7.8 mg g−1) as determined by HPLC. We predicted a
maximum concentration of 8.8 mg g− based on previously
1
a
b
SAD was used as a crude lysate. Ratio of a co-solvent to buffer was reported concentrations of sinapine, sinapoyl glucose and SA
c
d
1
: 1 (v : v). Determined by HPLC. n.d. = not determined.
8
in canola meal. Initially, we did not attempt to isolate SA at
this stage and performed an in situ enzymatic decarboxylation
on the neutralized canola meal extracts. However, the product
VS was heavily contaminated with fatty acids and was isolated
in very low purity. As a result, we first purified the canola meal
extracts by washing with hexane at pH 2 and then extracted the
1
1
0.6 U mL− of crude lysate was required to achieve 95% reac-
−
1
tion conversion at a substrate concentration of 5 g L
Table 2). The product VS could be easily isolated (75% yield)
in high purity (99%) by ethyl acetate extraction of the aqueous
medium at acidic pH, and a slightly basic buffer (pH 8) wash
of the combined organic extracts to remove unreacted SA. The
(
2
4
SA with diethyl ether–ethyl acetate. The isolated SA was used
2
5
without further purification and decarboxylation with SAD in
a biphasic reaction (as described above) yielded 340 mg of VS
in high purity (>90%), which corresponded to an overall yield
of 3.0 mg VS g− of canola meal. This represents 42% of the
available SA under the present extraction conditions.
1
H NMR spectrum of the biotransformation product (Fig. S1A
in ESI†) was consistent with previously published spectra of
1
1
1,12
VS.
In aqueous buffer, higher substrate concentrations or
lower protein concentrations resulted in incomplete reactions.
Previous experiments with wild-type PAD indicated that VG,
the decarboxylation product of FA, inhibited the enzyme even
at low concentrations (0.25 g L ). In this case, the addition
of toluene as the immiscible organic solvent for in situ product
removal, significantly improved the efficiency of the process.
We confirmed that VS was inhibiting SAD (ESI, Fig. S4†),
and examined the decarboxylation of SA with SAD in biphasic
reactions using toluene, hexane and ethyl acetate as co-
solvents. Using toluene, 2.0 U mL of SAD was required to
convert 25 g L− of SA. After 16 hours at 30 °C, the reaction
proceeded to 99% conversion and an 83% isolation yield was
obtained. Under the same conditions, incomplete enzymatic
reactions were observed using hexane and ethyl acetate. In
addition we tried the biphasic reaction in the ionic liquid
Polymerization of VS and characterization of PVS
−
1 4
4
Following our recent studies on PVG in the laboratory, we
polymerized VS under cationic conditions to explore potential
industrial applications of PVS. Treatment with boron trifluo-
ride yielded a polymer with a number average molecular weight
(
Mn) of 10 764 and a polydispersity of 1.4. Thermal analysis of
the PVS polymer revealed a glass transition temperature (Tg)
range of 106 °C–116 °C (Fig. S5†), determined by DSC, and a
thermal stability of up to 350 °C (Fig. S6†). Hatakeyama and
−
1
1
co-authors reported a T of 108 °C for a PVS polymer obtained
g
2
3
1
5
via hydrolysis of poly(4-acetoxy-3,5-dimethoxystyrene). The
molecular weight (M ) and polydispersity of the polymer were
.9 × 10 and 3.7, respectively.
Nanoindentation characterization (Fig. S7†) of our PVS
w
4
9
(
BMIM)PF6. The biotransformation went to completion.
However, due to the high boiling point of the ionic liquid we
could not evaporate the solvent and isolate the product. Fur-
thermore, we could not isolate the product from the ionic
liquid by solvent extraction due to its high solubility in
polymer, performed on polymer films compressed at 150 °C
following the CSM, afforded H and E values that were slightly
higher than the performance measured on commercial poly-
styrene, PS 1301 (NOVA Chemicals) (H = 0.249 ± 0.001 GPa et
E = 4.15 ± 0.02 GPa), that was analyzed simultaneously for com-
parison purposes (Table 3). In contrast, the H and E values of
PVS were significantly lower than the H and E values obtained
for our PVG polymers. Based on these results, it is clear that
altering the number and location of ring substituents can sig-
nificantly influence the polymer properties.
(BMIM)PF
6
. In summary, toluene was the best solvent to
achieve both high reaction conversion and isolated yield. For
−
1
−1
the decarboxylation of 50 g L SA with 2.0 U mL of SAD only
0% conversion was observed. However, by increasing the con-
4
5
−
1
centration of SAD 2-fold (4.0 U mL ) in the reaction, 97% con-
version was observed after 16 hours and an 81% isolation yield
was obtained.
Preparation of VS from canola meal
Conclusion
To explore the potential of producing VS from canola meal in
an industrial setting, we investigated the enzymatic decarboxy- The enzymatic decarboxylation of SA with SAD in aqueous
lation of canola meal extracts. Alkaline treatment of 100 g of buffer (neutral pH) at 30 °C was limited to substrate concen-
canola meal with 1 M NaOH for 18 h at 25 °C extracted 780 mg trations of ≤5 g L− due to product inhibition of the enzyme.
1
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Decarboxylation of SA was improved by using a biphasic reac-
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−
1
5
0 g L of SA was decarboxylated, affording VS in 80% isolated
yield. VS was isolated in high purity via solvent extraction and
a slightly basic buffer wash to remove unreacted starting
material. Our synthesized PVS polymer exhibited slightly
higher E and H values than a commercial polystyrene polymer.
Further studies are necessary to determine if 4-hydroxystyrene
derivatives like VS and VG would be adequate substitutes for
styrene in the polymer industry.
The enzymatic decarboxylation of SA extracts from canola
meal yielded VS in good purity (90%) and an overall isolated
yield of 3.0 mg VS per g of canola meal. As the first successful
proof-of-concept for obtaining VS from canola biomass from a
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2
6
(
bility. Nonetheless, the present yield of VS from canola meal is
K. Kida, T. Akaike and H. Maeda, J. Agric. Food Chem.,
2004, 52, 4380.
−
1
>
4-fold higher than VS produced (720 µg g at 160 °C) during
1
2
canola seed roasting by a domestic microwave. Although the 14 X. Cao, T. Tsukamoto, T. Seki, H. Tanaka, S. Morimura,
latter thermal method is advantageous with its short time
L. Cao, T. Mizoshita, H. Ban, T. Toyoda, H. Maeda and
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M. Tatematsu, Int. J. Cancer, 2008, 122, 1445.
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viable source for canolol production.
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17 A. Sharma, R. Kumar, N. Sharma, V. Kumar and
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2
7
replacement was found to expand the substrate specificity of 18 P. Terpinc, T. Polak, N. Segatin, A. Hanzlowsky, N. Poklar
PAD using a semi-rational approach. This represents the sim- Ulrih and H. Abramovic, Food Chem., 2011, 128, 62.
plest case of a non-additive mutational effect in protein engi- 19 M. J. Rein, V. Ollilainen, M. Vahermo, J. Yli-Kauhaluoma
2
8
neering as recently discussed by Reetz.
and M. Heinonen, Eur. Food Res. Technol., 2005, 220, 239.
0 A. K. Sinha, A. Sharma and B. P. Joshi, Tetrahedron, 2007,
2
2
63, 960.
1 A. Matte, S. Grosse, H. Bergeron, K. Abokitse and
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Acknowledgements
Funding by the Agricultural Bioproducts Innovation Program
of Agriculture and Agri-Food Canada is gratefully acknowl-
edged. We thank Chris Corbeil and Traian Sulea for expert
help in molecular modeling. We are grateful to Chantale Beau-
lieu for GC/MS analysis and to Louise Paquet, Florence Perrin
and Johanne Denault for polymer characterization. We wish to
thank the anonymous reviewers for various constructive com-
ments about the manuscript.
1
2
2 We acknowledge that the kcat for SAD (28 s− ) is several
orders of magnitude lower than what is desirable for indus-
3
7
−1
trial scale enzymatic application (10 to 10 s ). Further
engineering of the enzyme to have a higher turnover fre-
quency or even increase its affinity for the substrate are
possible strategies that would overcome the shortcoming of
the present proof-of-concept.
2
3 We analyzed all biotransformations after 16 hours to
compare the reaction conversion in different biphasic
solvent systems. It is possible that the reaction requires a
shorter time period. This would require further evaluation
through an optimization study. Regarding solvent use,
although toluene is not a “preferred” solvent, it is “usable”
according to the Pfizer solvent selection guide (P. J. Dunn,
D. A. Perry, et al., Green Chem., 2008, 9, 31). In 2013 alone,
Notes and references
1
2
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J. P. N. Rosazza, Z. Huang, L. Dostal, T. Volm and
B. Rousseau, J. Ind. Microbiol., 1995, 15, 457.
3
316 | Green Chem., 2013, 15, 3312–3317
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Paper
the following papers were accepted for publication in
Green Chemistry that use toluene as the reaction solvent:
S. Wolf, et al., Green Chem., 2013, 15, 315; M. B. Gawande,
et al., Green Chem., 2013, 15, 682; C. Boshui, et al., Green
Chem., 2013, 15, 738; Z. Sahli, et al., Green Chem., 2013,
( J. Am. Oil. Chem. Soc., 2010, 87, 1081) had used super-
critical fluid extraction with carbon dioxide which appears
to be a greener alternative. However, the extraction yield
was very poor compared to that of hexane. As mentioned
above, heptane is a possible replacement solvent that
needs to be evaluated for its efficiency. Heptane extracted
oil apparently required a higher temperature and longer
time to be desolventized than the hexane extraction
(E. J. Conkerton, P. J. Wan and O. A. Richard, J. Am. Oil
Chem. Soc., 1995, 72, 963).
1
5, 775; N. Garcia, et al., Green Chem., 2013, 15, 999;
M. Al-Amin, et al., Green Chem., 2013, 15, 1142; K. Dulle,
et al., Green Chem., 2013, 15, 1238; M. Bala, et al., Green
Chem., 2013, 15, 1687; A. Kadam, et al., Green Chem., 2013,
1
5, 1880; A. Chernykh, et al., Green Chem., 2013, 15, 1834.
Ethyl acetate is actually a “preferred” solvent, again accord- 25 During process development, we used solvent extraction,
ing to the Pfizer solvent selection guide. Hexane can likely
be replaced by heptane. We used dichloromethane for
the polymerization step, not the process itself. The former
use was intended to provide a proof-of-concept of the
experiment.
rather than column chromatography, to remove impurities
from our canola meal extracts and to isolate our biotrans-
formation product which would be the preferred method
on scale to avoid extra steps and costs.
26 G. P. Prpich and A. J. Daugulis, Biotechnol. Bioeng., 2007,
98, 1008.
2
4 K. Krygier, F. Sosulski and L. Hogge, J. Agric. Food Chem.,
1
982, 30, 330; We used hexane to defat the canola meal as 27 K. Shrestha, C. V. Stevens and B. de Meulenaer, J. Agric.
a pretreatment step as it is widely used and also to extract
Food Chem., 2012, 60, 7506.
residual fatty acids from the sinapic acid extracts. Li et al. 28 M. T. Reetz, Angew. Chem., Ind. Ed., 2013, 52, 2658.
This journal is © The Royal Society of Chemistry 2013
Green Chem., 2013, 15, 3312–3317 | 3317