ARTICLE
High Yields of Ascorbyl Palmitate
by Thermostable Lipase-Mediated Esterification
S. Bradoo, R.K. Saxena, and R. Gupta*
Department of Microbiology, University of Delhi South Campus, New Delhi-110 021, India
ABSTRACT: High yields of ascorbyl palmitate (6-O-palmitoyl obtained 68% yield in 8 h using a high substrate molar ratio.
L
-ascorbic acid) were obtained by lipase-mediated esterifica-
Sakashita et al. (1) used immobilized lipase to catalyze the
synthesis of ascorbyl stearate at 40°C in 4 h, whereas Uragaki
(6) obtained ascorbyl stearate synthesis at 20–70°C with a dif-
ferent lipase.
In the present work, attempts were made to obtain high
yields of ascorbyl palmitate in a short time using Bacillus
stearothermophilus SB1 lipase. This paper describes the ef-
fect of various parameters on the synthesis of ascorbyl palmi-
tate. Repeated use of immobilized enzyme in batch esterifica-
tion and in a solvent-free system is also discussed.
tion using Bacillus stearothermophilus SB 1 lipase. The final
yield was greatly influenced by the initial water content of the
system, quantity of enzyme, and molar ratio of palmitic acid to
L
-ascorbic acid. Reaction rates increased directly with tempera-
ture from 40 to 100°C. Maximum conversion (97%) was
achieved after 30 min at 100°C (solvent-free), 1 h at 80°C (sol-
vent-free), and 2 h at 60°C (solvent/hexane). The synthesis was
scaled up to 1-L volume with 95% conversion using 50 mmoles
of ascorbic acid and 250 mmoles of palmitic acid in hexane.
Similar yields of ester were obtained in five repetitive cycles
using 5 g enzyme immobilized on Accurel. The present B.
stearothermophilus SB 1 lipase was a more efficient catalyst
for the synthesis of ascorbyl palmitate than other commercial
lipases.
EXPERIMENTAL PROCEDURES
Chemicals. L-Ascorbic acid and palmitic acid were purchased
Paper no. J9147 in JAOCS 76, 1291–1295 (November 1999).
from SRL (Mumbai, India), molecular sieves from SD’S
(Mumbai, India), and all solvents were procured from Quali-
gens (Mumbai, India). All the chemicals used were of analyt-
ical grade. However, acetonitrile and water were of high-per-
formance liquid chromatography (HPLC) grade.
KEY WORDS: Ascorbyl palmitate, Bacillus sp., biocatalysis, en-
zyme, esterification, immobilization, lipase, 6-O-palmitoyl L-
ascorbic acid, thermostable enzyme.
Enzymes. Powdered lipases from porcine pancreas, Rhi-
zomucor miehei, Pseudomonas cepacia, and C. rugosa were
obtained from Sigma (St. Louis, MO). Lipase from B.
stearothermophilus SB-1 was produced in our laboratory.
Bacillus stearothermophilus SB-1 produced 13,000 U/L
lipase with specific activity of 25 U/mg protein in minimal
medium containing 2% corn oil (7). The production was car-
ried out at 50°C and pH 3.0 in shake flasks at 250 rpm (New
Brunswick Scientific shaker, Edison, NJ). After 36 h, the cul-
ture was centrifuged at 18,600 × g for 20 min, and the super-
natant was subjected to 85% ammonium sulfate precipitation.
The pellet was dissolved in 0.01 M sodium phosphate buffer
(pH 7.0) and subjected to dialysis against distilled water at
4°C for 24 h. After partial purification, 80% enzyme yield
with fourfold increase in specific activity was obtained. The
dialyzed enzyme was subjected to immobilization/lyophiliza-
tion and used for esterification.
Present-day demand for ecofriendly technologies has changed
the scenario in favor of natural additives in place of synthetic
compounds, especially in food and personal-care products. In
this regard, fatty acid esters of ascorbic acid occupy an impor-
tant place as potential antioxidants and as surfactants in high-
fat-content food and personal-care products. They also have
an application as browning inhibitors in fruits (1). Generally,
these esters are chemically synthesized by catalyzing esterifi-
cation of ascorbic acid with concentrated sulfuric acid (2,3).
However, the chemical synthesis is an energy-intensive
process and results in the formation of a mixture of products
with a preponderance of O-6 substitution. It also involves
cumbersome downstream processing of the final product. En-
zymatic synthesis has been suggested as an alternative method
as it is regioselective and also involves mild reaction condi-
tions (1,4). Further, use of immobilized enzyme simplifies the
downstream processing and makes the process economical.
There are very few reports on the lipase-catalyzed synthe-
sis of ascorbyl palmitate. Humeau et al. (5) studied the syn-
thesis of ascorbyl palmitate by Candida antarctica lipase and
Lipase assay. Lipase was assayed by the standard proce-
dure of titrimetry (8) using 1.5% (vol/vol) olive oil as sub-
strate at 50°C. One unit of activity is defined as the amount
that liberates 1 µmol fatty acid from olive oil per min under
the standard assay conditions.
Enzyme immobilization. Bacillus stearothermophilus SB-1
lipase was immobilized on Accurel EP 100 porous polypropy-
*To whom correspondence should be addressed.
E-mail: micro@dusc.ernet.in
Copyright © 1999 by AOCS Press
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JAOCS, Vol. 76, no. 11 (1999)