Paper
RSC Advances
(Mumbai, India) and soyadeodistillate from local vendor
(Dehradun, India). Immobilized biocatalyst Fermase CalB was
obtained from FERMENTA BIOTECH INDIA. Fermase is a
recombinant lipase expressed in Pichia stripitis. All other
chemicals and reagents were of analytical grade and used as
received.
Fourier transform infrared spectra were recorded on Perkin-
Elmer spectrum RX-1 FTIR spectrophotometer using potassium
bromide window for the disappearance of double bond C–C
stretching (str). Solution state NMR spectra (in CDCl3) were
recorded on 11.7T Bruker Avance III 500.13 MHz spectrometer
Fig. 4 Results of catalytic recycling experiments.
1
using a 5 mm BBFO probe. The conventional H NMR experi-
ment is carried out using 5% w/v sample solution in CDCl3
(99.8%, Aldrich) with 64 number of scans, a p/2 pulse length of
13.4 ms, 10 s recycle delay, 64K time domain data. The FID is
exponentially multiplied and Fourier transformed with 0.3 Hz
apodization and referenced to TMS at 0 ppm.
addition, the appearance of a multiplet in the range of 2.5–
3.5 ppm, because of epoxide protons, conrmed the formation
of respective epoxidized products. The 1H NMR spectra of
linoleic acid and its corresponding epoxide are presented in
(Fig. S2†). H NMR spectra of soyadeodistillate, ester of soya-
deodistillate [(9Z,12Z)-2-ethylhexyl octadeca-9,12-dienoate] and
their corresponding epoxides are presented in (Fig. S3A–D†).
1
Experimental procedure for enzymatic epoxidation
The epoxidation experiments were carried out in a 25 ml round
bottomed ask equipped with a magnetic stirrer and reux
condenser. The reaction mixture containing fatty compound
linoleic acid conc. (3 mmol) oxidant hydrogen peroxide
(3.5 mmol) and Fermase CalB catalyst 10% of substrate wt. The
Enzyme reusability
To check the recyclability of the immobilized enzyme, epoxidation
of linoleic acid was considered as a model reaction. The recycla-
bility of the enzyme was checked under optimized reaction
conditions for subsequent four cycles (Fig. 4). At the end of each
cycle, the immobilized enzyme was recovered and washed with
distilled water. The substrate with same concentration as previous
reaction was added to start a new batch of the reaction. As shown
in Fig. 4, the recovered enzyme catalyst exhibited marginal
decrease in catalytic activity with each successive run. More than
80% of initial activity of enzyme was retained aer 4 cycles. The
loss of activity aer each cycle might be due to the mechanical
instability of support material and agitation of reaction mixture.
ꢀ
resulting mixture was stirred at 35 C at a constant rotation of
300 rpm for 12 h and the progress of reaction was monitored by
1H NMR spectroscopic analysis. All the experiments were
carried out in duplicate. Aer completion of the reaction, the
immobilized enzyme catalyst was separated via ltration and
washed with ethyl acetate to remove product. The combined
organic layer was thoroughly washed with warm water and then
dried over anhydrous MgSO4, concentrated under reduced
pressure to give pure epoxidized product.
Controlled experiments were performed and treated in the
same way. Aer completion of reaction catalyst was recovered
and washed for further use.
Conclusion
Synthesis of soyadeodistillate esters
The enzymatic process can be seen as a promising alternative to
the classical epoxidation19 reaction. Waste plant oil i.e., soya-
deodistillates and its esters were tested for epoxidation in this
study under very mild conditions and result showed their
successful conversion to corresponding epoxides. In present
study the epoxidation process is established without using
additional acid as oxygen carrier and the developed method-
ology also worked effectively for the epoxidation of branched
fatty acid esters to give excellent product yields. This is a very
important nding which makes the developed method more
attractive and superior over the existing one. These results are
crucial for the chemical industry and represent the prospect of
developing green and sustainable methods.
Esterication reaction was carried out with 1 : 1 molar ratio of
acid and alcohol in the presence of 1.5% PTSA (p-toluene
sulfonic acid) as catalyst and toluene as azeotropic solvent in a
three neck 1 L round bottomed ask with dean stark apparatus,
a condenser and a thermometer pocket. The reaction mixture
was heated at 110 ꢀC and the reactions were carried out till the
theoretical quantities of water were collected. The reaction
mixture was cooled, toluene was recovered under vacuum and
product was passed through activated basic alumina to remove
traces of residual acid and product with a 97.4% yield and a
purity >98% was achieved. The product was analyzed using IR
and 1H NMR analysis.
Saponication value and free fatty acid content (acid value) of
substrates
Experimental
Materials and methods
Saponication and acid values of substrates were determined
Aqueous hydrogen peroxide (30 wt%) was purchased from according to ASTM-D-974/11 and ASTM-D-94/07 respectively.
Merck (Mumbai, India), linoleic acid from Lobachemie Acid value titrations were performed as described using a
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 53708–53712 | 53711