G Model
CATTOD-9386; No. of Pages6
ARTICLE IN PRESS
2
the stability of the lipase from T. lanuginosus and the phospholi-
pase activity of the enzyme from F. oxysporum [18]. However, some
manuscripts may be found in the literature on the uses and biocata-
lyst design of this interesting enzyme [20–27], and none regarding
the use of this interesting enzyme in flavor synthesis reactions.
The reaction system for this esterification is quite complex com-
posed by the solvent, the relatively polar acid, and the alcohol, plus
the water that is present in the medium and also is formed during
the reaction. According to the literature, the adsorption of water
or some substrates (carboxylic acid and alcohol) on the biocatalyst
particle is one of the most important reasons of biocatalyst inac-
tivation [28], as they can form multi-phases around the enzyme
instances, to recover the enzyme activity prolonging its operational
stability [28]. This washing is time consuming and produces some
sound technology as a better mixing strategy for enzyme reaction
systems has been successfully proposed in the literature [34–44].
[45,46] in mixing, shearing and mass transfer [47,48]. Also, it is
reasonable to expect some stirring inside the biocatalyst particle
that cannot be obtained via conventional stirring. In spite of these
possibilities, ultrasound technology has not been extensively used
of MCI-Lecitase (by substrates mass) and n-hexane as solvent. The
mixtures were placed in either, mechanical stirring or ultrasonic
energy at 50 ◦C, for 1 h.
2.4. Effects of alcohols on the enzyme performance
The different alcohols (ethanol, 1-propanol, 2-propanol, 1-
butanol, isobutanol, 1-pentanol and 2-pentanol) were mixed with
caprylic or myristic acid (0.1 M) at a molar ratio of 1:1 in 50 mL
Erlenmeyer flasks (working volume of 10 mL), followed by the addi-
tion of 10% of MCI-Lecitase (by substrates mass) and n-hexane as
solvent. The mixtures were placed in either, mechanical stirring or
ultrasonic energy at 50 ◦C, for 1 h.
2.5. Effects of organic solvents on the enzyme performance
n-Hexane, cyclohexane, n-heptane, and n-octane were tested as
solvents in the reactions between ethanol and caprylic or myris-
tic acid (0.1 M) at a molar ratio of 1:1 in 50 mL Erlenmeyer flasks
(working volume of 10 mL), followed by the addition of 10% of MCI-
Lecitase (by substrates mass) and the solvent. The mixtures were
placed in either, mechanical stirring or ultrasonic energy at 50 ◦C,
for 1 h.
2.6. Effect of biocatalyst concentration at the reaction rate
The concentration of MCI-Lecitase varied from 2.5 to 15% by
substrates mass. The reactions were conducted using 0.1 M of sub-
strates at a molar ratio of 1:1 in 50 mL Erlenmeyer flasks (working
volume of 10 mL), using the appropriate solvent. The mixtures were
placed in either, mechanical stirring or ultrasonic energy at 50 ◦C,
measuring the initial reaction rate.
Recently, it has been shown the immobilization of this enzyme
from Candida antarctica, lipases from Rhizomucor miehei, T. lanug-
inosus) [51,52], and the biocatalysts obtained presented some
advantages compared to the commercial ones in esterification reac-
tions [29,31,53], even using ultrasound technology [54].
2.7. Effect of temperature on the enzyme activity
In this paper, the Lecitase-UItra immobilized on styrene-
divinylbenzene beads was evaluated for first time in different
esterification reactions comparing the effects of the ultrasound
technology and the traditional mechanical stirring.
Reaction temperature varied from 40 to 60 ◦C. The reactions
were conducted using 0.1 M of substrates at a molar ratio of 1:1
in 50 mL Erlenmeyer flasks (working volume of 10 mL), using the
appropriate solvent. The mixtures were placed in either, mechan-
ical stirring or ultrasonic energy at the specific temperature,
measuring the initial reaction rate.
2. Materials and methods
2.8. Effects of substrate concentration on the enzyme activity
2.1. Materials
The concentration of acids and alcohols varied from 0.1 to 0.6 M
at a molar ratio of 1:1 in 50 mL Erlenmeyer flasks (working vol-
ume of 10 mL) followed by the addition of 10% of MCI-Lecitase (by
substrates mass) and the appropriate solvent. The mixtures were
placed in either, mechanical stirring or ultrasonic energy at 50 ◦C,
measuring the initial reaction rate.
Lecitase-Ultra was a kind gift from Novozymes (Spain). The
porous styrene-divinylbenzene MCI CHP20P GEL (Supelco) sup-
port, substrates, solvents, and other chemicals were purchased
from Sigma-Aldrich (Sigma, St. Louis, USA) and were of analyti-
cal grade. Ultrasonic bath (Unique Inc., model USC 2880A, 40 kHz,
220 W, Brazil) with temperature control was used in all experi-
ments.
2.9. Enzyme reuse
2.2. Enzyme immobilization
After the esterification reaction, the immobilized enzyme was
separated from the reaction medium by vacuum filtration using a
sintered glass funnel, and placed in a fresh reaction batch. In some
instances, it was tested the repeated use of MCI-Lecitase adding
molecular sieves to the reaction medium. The sieves were collected,
as well as the immobilized enzyme, and reused in the new batch.
The immobilization of Lecitase-Ultra on the styrene-
divinylbenzene beads was carried out according to a previously
described protocol [51,52]. The immobilized preparations pre-
sented 120 mg of protein per wet g of support. The preparation
was named MCI-Lecitase.
2.3. Effect of acid chain on the enzyme activity
2.10. Reaction analysis
Butyric, caprylic, capric, lauric, myristic, palmitic and stearic
acids, at a concentration of 0.1 M were tested in the esterification
reaction using ethanol at a molar ration of 1:1 in 50 mL Erlenmeyer
flasks (working volume of 10 mL), followed by the addition of 10%
The progress of the esterification was monitored determining
the residual acid content by titration of 0.5 mL of sample with NaOH
(0.01 M) until pH 7, using ethanol as quenching agent. The amount
of ester was calculated as being equivalent to the consumed acid.
Please cite this article in press as: J.S. Alves, et al., Use of Lecitase-Ultra immobilized on styrene-divinylbenzene beads as catalyst of