Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate

Article abstract of DOI:10.1016/j.procbio.2013.04.019

Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil support via Schiff base formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization performance was evaluated in terms of bound

Full text of DOI:10.1016/j.procbio.2013.04.019

Process Biochemistry 48 (2013) 819–830
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric
acylation of (± )-2-amino-1-phenylethanols with vinyl acetate
∗
Deniz Yildirim , S. Seyhan Tükel
University of Cukurova, The Faculty of Sciences and Letters, Department of Chemistry, 01330 Adana, Turkey
a r t i c l e i n f o
a b s t r a c t
Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil® support via Schiff base
formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization
performance was evaluated in terms of bound protein per gram of support (%) and recovered activity
Article history:
Received 30 October 2012
Received in revised form 25 April 2013
Accepted 26 April 2013
(
%). A 4-factor and 3-level Box–Behnken design was applied for the acylation of (± )-2-(propylamino)-1-
Available online 7 May 2013
phenylethanol, a model substrate, with vinyl acetate and the asymmetric acylations of other (± )-2-amino-
1
1
-phenylethanols with different alkyl substituents onto nitrogen atom such as (± )-2-(methylamino)-
-phenylethanol, (± )-2-(ethylamino)-1-phenylethanol, (± )-2-(butylamino)-1-phenylethanol and (± )-2-
Keywords:
Enantiopure 2-amino-1-phenylethanols
Asymmetric acylation
Response surface methodology
Box–Behnken design
(hexylamino)-1-phenylethanol were performed under the optimized conditions. The optimal conditions
◦
were bulk water content of 1.8%, reaction temperature of 51.5 C, initial molar ratio of vinyl acetate to
−1
amino alcohol of 1.92, and immobilized lipase loading of 47 mg mL . (R)-enantiomers of tested amino
alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-
amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an
increase in the acylation yield and ee values of (S)-enantiomers. Enantiomeric ratio values were >200 for
all the reactions. Our results demonstrate that the immobilized lipase is a promising biocatalyst for the
preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation
of (± )-2-amino-1-phenylethanols with vinyl acetate.
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
esters, amines and amides in non-aqueous media [14–17]. How-
ever, due to the denaturation, deactivation and recycling problems
of lipases in free form, lipases have been immobilized onto var-
ious types of supports to prepare more active, cost-effective and
more enantioselective biocatalysts toward their free forms in the
preparation of aforementioned enantiopure compound groups in
non-aqueous media [15,18–20]. Furthermore, immobilization of
lipase occasionally helps to positively modulate catalytic prop-
erties of lipases such as improvement of stability and activity
in drastic reaction conditions, enhancement of enantioselectiv-
ity toward non-natural substrates [21–23]. For example, Palomo
et al. [24] reported that the enantioselectivity of Rhizopus oryzae
lipase toward (± )-glycidyl butyrate was modulated depending on
the used immobilization technique. Chaubey et al. [25] reported
that the catalytic and enantioselectivity properties of Arthrobac-
ter sp. was modulated by immobilizing onto Arthrobacter sp. with
different techniques such as hydrophobic binding, covalent bind-
ing and sol–gel entrapment. Palomo et al. [26] immobilized a
new lipase from porcine pancreas with two techniques. First, the
lipase was immobilized onto polyethyleneimine-coated agarose
support by adsorption and then the adsorbed lipase was treated
with glutaraldehyde. The results showed that the enantioselectiv-
ity of lipase treated with glutaraldehyde was enhanced 10-fold
toward (± )-glycidyl butyrate. Volpato et al. [27] reported that a
Beta-adrenergic receptor blocking agents (␤-blockers) are
important drugs consumed for the treatment of high blood pres-
sure, heart failure, hypertension and myocardial ischemic diseases
[
1–5]. Many ␤-blockers available in markets are still used as race-
mates in therapy although it is well known that chirality is a key
factor in the efficacy of ␤-blockers and distomers are possible to
cause the adverse effects [6–8]. Therefore, the syntheses of single
enantiomeric forms of ␤-blockers have found increasing demands
in pharmaceutical industry due to supplying more effective and
safer drugs [8]. Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3)
have been known as unsurpassed biocatalysts in organic syntheses
because they catalyze a variety of reactions including esterifica-
tion, interesterification, transesterification and aminolysis [9–12].
They do not require any cofactors for their activities and show
good to excellent enantioselectivity on their substrates and are
ubiquitous in nature [13]. Due to their aforementioned proper-
ties, lipases have been used in free or immobilized forms for the
preparations of pharmaceutically important enantiopure alcohols,
∗ Corresponding author. Tel.: +90 322 3386081/25; fax: +90 322 3386070.
E-mail addresses: dozyildirim@gmail.com, dyildirim@cu.edu.tr (D. Yildirim).
1
359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2013.04.019

8
20
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
lipase from Staphylococcus warneri EX17 immobilized onto gly-
oxyl, cyanogen bromide or octyl agarose beads exhibited different
enantioselectivities for the hydrolysis of chiral esters, such as
(
± )-methyl mandelate, (± )-2-O-butyryl-2-phenylacetic acid and
(
± )-2-hydroxy-4-phenyl-butyric acid ethyl ester depending on the
immobilization protocol used.
Enantiopure 2-amino-1-phenylethanol derivatives are the
group of sympathomimetic drugs. They generally interact with
␣
- and ␤-adrenergic receptors, resulting in numerous physio-
logical effects such as vascular constriction, reduction of blood
flow or decrease in mucus secretion into nasal passages [28]. The
structure–activity relationship for ligands interacting with adren-
ergic receptors showed that the essential structural features for
direct activity on the receptors were the 2-amino-1-phenylethanol
skeleton, various substituents on the phenyl ring and substituent
onto amino group [29]. Lundell et al. reported that ␣-adrenoceptor
agonist activity of (R)-noradrenaline decreased notably and its
Fig. 1. The modification steps of Florisil® support and the immobilization of Pseu-
domonas sp. lipase onto the modified Florisil® support.
subsequently rinsed with distilled water. To 1 g of acid-washed Florisil® support
2
5 mL of 3-APTES solution (4% in acetone, v/v) was added and the mixture was kept
◦
®
at 45 C until dryness [39]. The silanized-Florisil support was extensively rinsed
with distilled water and dried overnight at 120 C. The glutaraldehyde activation
◦
␤
-activity increased dramatically when (R)-noradrenaline was N-
®
of silanized-Florisil support was performed according to Alptekin et al. [40]. To
substituted and the substituent was larger than methyl group [28].
Response surface methodology (RSM) is, an effectual opti-
mization process, combination of statistical and mathematical
techniques based on the multivariate non-linear model and pro-
vides more advantageous compared to other methods due to its
simplicity and reduction of time requirement. RSM includes three
steps: (1) proposing a mathematical model to predict response;
1 g of the silanized-Florisil® support, 25 mL of glutaraldehyde solution (2.5%, w/v,
in phosphate buffer (50 mM, pH 7.0)) was added and the mixture was agitated at
room temperature for 2 h. The glutaraldehyde-activated Florisil® support was col-
lected by the filtration on a Buchnel funnel, extensively washed with distilled water
until the detection of glutaraldehyde was not observed in the filtrate. The detection
of glutaraldehyde in the filtrate was determined according to Boratynski and Zal
◦
[41]. Then, the obtained support was dried overnight at 120 C. The amounts of free
®
primary amine (−NH2) groups onto Florisil support after 3-APTES treatment and
glutaraldehyde activation were measured using ninhydrin reagent [40]. To 10 mg
of Florisil® support obtained after 3-APTES treatment and glutaraldehyde activa-
tion, 100 ␮L H2O and 200 ␮L ninhydrin reagent were added and the mixture was
kept in a boiling water bath for 30 min. Subsequently, 5 mL of a 50/50 (v/v) mix-
ture of ethanol/water was added onto the mixture and mixed well. The absorbance
of the colored complex known as Ruhemann’s purple was measured at 570 nm. A
calibration curve was plotted using ethylenediamine as standard to determine the
amounts of NH2 groups after 3-APTES treatment and glutaraldehyde activation.
The amount of unreacted NH2 groups was calculated by subtracting the amount of
NH2 groups remaining after glutaraldehyde activation from the amount of NH2
groups formed after 3-APTES treatment.
(
2) calculating the coefficients of proposed mathematical model;
and (3) testing model adequacy [30–32]. In the literature, RSM has
been employed for optimization of lipase-catalyzed synthesis of
various enzymatic reactions [33–37]. Box–Behnken designs (BBDs)
are rotatable or nearly rotatable designs and require 3 levels of
each factor. BBDs necessitate fewer experiments than full factorial
designs (FFDs) or central composite designs (CCDs) with the same
number of factors [38].
In this study, RSM was performed to optimize for the asymmet-
ric acylation of (± )-2-amino-1-phenylethanols with vinyl acetate
catalyzed by immobilized Pseudomonas sp. lipase, a facile approach
for the preparation of enantiopure 2-amino-1-phenylethanols
which may be used as potential new ␤-blockers. For this purpose,
a 4-factor and 3-level BBD was applied for the optimization study.
The independent parameters investigated were the bulk water con-
tent of reaction medium, reaction temperature, initial molar ratio
of vinyl acetate to amino alcohol and immobilized lipase loading.
The effects of alkyl chain length substituted onto amino group were
investigated onto the product yield and enantioselectivity of the
immobilized lipase. For this aim, (± )-2-amino-1-phenylethanols
with different alkyl chain lengths substituted onto amino group
were tested under the optimized conditions.
2
.2.2. Immobilization of lipase
The immobilization procedure was carried out under predetermined optimal
®
conditions. A total 2 g of glutaraldehyde-activated Florisil support was suspended
in 8 mL of lipase solution (1 mg mL−1) prepared in a citrate buffer (50 mM, pH 6.0) at
◦
5
C. Then, 10 mL of NaCNBH3 solution (0.25 M, pH 6.0) was slowly added onto it and
the mixture was gently stirred for 2 h (Fig. 1). The immobilized lipase preparations
were filtrated and rinsed with the same acetate buffer having different concen-
trations (50–200 mM) to remove the unbound proteins and dried under vacuum
to eliminate inter-particle water. The protein amounts of filtrates were measured
using the Lowry protein assay [42].
2
.2.3. Optimization of immobilization conditions
The optimal conditions for the immobilization of lipase were investigated
by changing individually the immobilization pH, immobilization temperature,
immobilization time and initial loading protein concentration. The optimal immo-
bilization pH of lipase was determined by performing the immobilization process
at different pH values ranging from 5.0 to 8.0 (50 mM acetate buffers for pH 5.0–5.5;
2
. Materials and methods
5
0 mM citrate buffer for pH 6.0; 50 mM phosphate buffers for pH 6.5–8.0). The
◦
2.1. Materials
immobilization process was carried out at the temperature values of 5, 15 and 25
C
in order to determine optimal immobilization temperature. The immobilization of
lipase was conducted for 1, 2, 3, 6, 12 and 24 h at the predetermined optimal immo-
bilization pH and temperature. The effect of initial loaded protein concentration was
Pseudomonas sp. lipase (Type XIII, lyophilized powder, ≥15 U/mg solid), (3-
aminopropyl)triethoxysilane (3-APTES), glutaraldehyde solution (Grade I, 50% in
H2O), para-nitrophenol (p-NP), para-nitrophenol palmitate (p-NPP) and (R/S)-
styrene oxide ((R/S)-SO) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
−
1
investigated in the range of 0.25–2.0 mg mL
.
®
Florisil (magnesium silicate, particle size 150–250 ␮m, pore size 6–8 nm and
2.2.4. Measurement of hydrolytic activity
2
surface area 170–300 m /g), sodium cyanoborohydride (NaCNBH3), methylamine
The hydrolytic activity of lipase was measured using p-NPP as substrate [43]. A
hydrochloride (99%), ethylamine (70%, v/v), propylamine (99%, v/v), butylamine
defined amount of immobilized lipase (10 mg) was added onto 1 mL of phosphate
buffer (50 mM, pH 7.0) and incubated for 5 min at 35 C. The reaction was started
◦
(
99%, v/v), hexylamine (99%, v/v), acetonitrile, diisopropyl ether (DIPE), diethyl ether
(
DEE), dioxane (DIOX), tetrahydrofurane (THF), tert-butyl methyl ether (TBME),
by the addition 1 mL of p-NPP solution (0.2% in absolute ethanol, w/v) and the mix-
ture was agitated at 100 rpm for 5 min. The reaction was stopped by adding 2 mL
of Na2CO3 solution (0.25 M in water) and centrifuged at 5000 rpm for 5 min. One
milliliter of supernatant was diluted to 5 mL with distilled water and the quantity of
formed p-NP was detected at 410 nm by a spectrophotometer (Shimadzu UV-1800).
acetic acid and triethyl amine (99%, v/v) were supplied from Merck (Darmstadt,
Germany). All other chemicals were of analytical grade.
2
2
.2. Methods
.2.1. Preparation of Florisil® support for lipase immobilization
2.2.5. Chemical synthesis of racemic amino alcohols
Florisil® support was silanized with 3-APTES and activated with glutaraldehyde
(± )-2-Amino-1-phenylethanols were synthesized by reacting (R/S)-SO with dif-
ferent primary amines, such as methylamine, ethylamine, propylamine, butylamine
and hexylamine at room temperature. To 20 mmol of (R/S)-SO, 25 mmol of each
®
before the lipase immobilization as follows. Ten grams of Florisil supports were
washed with 50 mL of HNO3 solution (5% in water, v/v) at 80–90 C for 60 min and
◦

D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
821
Table 1
squared effect and the interaction effect, respectively. The experimental design and
data analysis were performed by using Design Expert statistical software (Design-
Expert 8.0.7).
The coded values and levels of each independent variable.
Variables
Symbol
Variable level
Low Center
High
+1
2
.2.7. Lipase catalyzed asymmetric acylation reaction
−
1
0
The immobilized lipase catalyzed asymmetric acylation reaction was performed
in a glass vial equipped with a screw stopcock. The reactions designed by RSM were
carried out at different experimental conditions as given in Table 3. Briefly, to 800 ␮L
of TBME containing desired amount of water (0–4%), 100 ␮L of desired concentration
of vinyl acetate solution (1, 1.5 and 2 M in TBME) and 100 ␮L of ± -2-(propylamino)-
Water content of reaction medium (%)
Reaction temperature ( C)
X1
X2
X3
X4
0
2
4
60
2.0
50
◦
40
1.0
10
50
1.5
30
Molar ratio of VA/AA
Immobilized lipase loading (mg mL−1)
1
-phenylethanol solution (1 M in TBME) were added and then the acylation reaction
was initiated by adding the defined amount of immobilized lipase preparation and
allowed to continue by agitating at 100 rpm for 2 h (Fig. 2). Subsequently, 100 ␮L
of sample was taken out, dried over sodium sulfate and then analyzed by HPLC
equipped with a Shodex ORpak CDC-453 HQ chiral column (4.6 mm × 150 mm)
at 220 nm after dilution to 500 ␮L with acetonitrile/water mixture (1/4, v/v). The
calibration graphs were plotted via peak area of the each compound versus its con-
centration and the amount of the each compound was calculated from their peak
areas. The mobile phase consisted of water/acetonitrile/acetic acid/triethylamine
primary amine was separately added. The mixtures were stirred for 12 h and sub-
sequently the unreacted amines were extracted with distilled water (3× 10 mL).
The racemic amino alcohols formed were extracted with diethyl ether and the sol-
vent was evaporated under vacuum then the racemic amino alcohols were dried
on anhydrous Na2SO4. The characterizations of amino alcohols were achieved by
high-performance liquid chromatography (HPLC), Fourier transform infrared spec-
troscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR).
H NMR and C NMR spectra in DMSO-d6 were recorded on a Bruker Ultra-
shield TM NMR 300 MHz spectrometer with tetramethylsilane (TMS) as the internal
standard. Chemical shifts (ı) were expressed in parts per million (ppm), multiplicity
−
1
mixture (80/20/1.4/0.7, v/v/v/v) and its flow rate was adjusted to 0.25 mL min
.
1
13
◦
The column temperature was kept at 25 C during analysis. The enantiomeric excess
values (ee) of amino alcohols and esters were calculated from the equations given
below:
(
s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; m, multi-
plet) and coupling constants (J) were expressed as Hertz (Hz). A FTIR instrument
Perkin Elmer Spectrum RX/FTIR system) was used to determine the functional
[S − R]
[R − S]
[R + S]
alcohol
ester
ester
eealcohol =
and eeester =
(
[
S + R]alcohol
groups after the pellet was prepared in KBr. HPLC analyses were performed on a
Shodex ORpak CDC-453 HQ chiral column at 220 nm. The mobile phase used was
water/acetonitrile/acetic acid/triethylamine mixture (80/20/1.4/0.7, v/v/v/v) at a
The enantiomeric ratio values (E) of immobilized lipase were calculated from
the ee values of related ester (eeester) and amino alcohol (eealcohol) and conversion
degree (C) [44].
−1
flow rate of 0.25 mL min . The retention times of the amino alcohols and formed
products were given in Section 2.2.11.
2
.2.8. Asymmetric acylation of different N-substituted 2-amino-1-phenylethanols
The reactions were carried out under the optimized conditions estimated by
2
.2.6. Experimental design and data analysis
A 4-factor and 3-level Box–Behnken design (BBD) was applied to assess optimal
RSM. Five reactions were made and only one aliquot of 100 ␮L was taken from each
mixture for each time interval (15, 30, 60, 120 and 240 min). Aliquots were diluted
to 500 ␮L with acetonitrile/water mixture (1/4, v/v) and analyzed by the chiral HPLC
for the quantification of substrate and product concentrations, their ee values and
E values of the immobilized lipase.
To purify the reaction products, the TBME layer was loaded onto a silica gel col-
umn after 240 min reaction time, and the products were eluted with hexane–ethyl
acetate mixture (9/1, v/v). After the removal of solvent, the products were charac-
terized by chiral HPLC, FTIR and NMR techniques.
reaction conditions and to understand the relationships between reaction vari-
ables. (± )-2-(propylamino)-1-phenylethanol was selected as model substrate for
the optimization studies. Several preliminary studies have been performed before
the choosing of reaction parameters and the bulk water content of reaction medium
(
(
X1), reaction temperature (X2), initial molar ratio of vinyl acetate to amino alcohol
VA/AA) (X3) and immobilized lipase loading (X4) were determined as important
parameters. The low, zero and high levels of parameters were symbolized as −1, 0
and +1, respectively, and are given in Table 1. The experimental studies were per-
formed according to conditions demonstrated in Table 3. The results were analyzed
by RSM to fit the following quadratic model:
2
.2.9. Thermal stability
N
N
N−1
N
◦
ꢀ
ꢀ
ꢀꢀ
The thermal stability of immobilized lipase was investigated at 40, 50 and 60 C
2
Y = ˇ0 +
ˇi × Xi +
ˇii × Xi
+
ˇij × Xij
(1)
under the optimal conditions. The first-order inactivation constant (ki) and half-life
t1/2) of the lipase were calculated from the equation:
(
i=1
i=1
i=1 j=i+1
where Y is the predicted response (acylation yield), N, number of variables, Xi the
independent variable, ˇ0, ˇi, ˇii and ˇij are the intercept term, the linear effect, the
ln Vt = ln V0 − kit
Table 2
®
The results of immobilization of Pseudomonas sp. lipase onto Florisil at different immobilization conditions.
Immobilization pH
Immobilization
temperature
Immobilization
time
Initial protein concentration
Bound protein
(mg/g support)
Bound protein
(%)
Specific activity
(U/mg protein)
Recovered
activity (%)
−
1
(mg mL
)
5
5
6
6
7
7
8
.0
.5
.0
.5
.0
.5
.0
2 h
3.0 ± 0.08
3.0 ± 0.08
3.2 ± 0.10
3.2 ± 0.10
2.8 ± 0.08
2.8 ± 0.07
2.4 ± 0.07
75
75
80
80
70
70
60
1130 ± 48
1130 ± 45
1250 ± 40
1190 ± 56
1060 ± 52
1050 ± 40
880 ± 42
33.5
33.5
37.0
35.2
31.4
31.1
26.1
◦
5
5
C
1.0
◦
C
3.2 ± 0.10
3.2 ± 0.10
3.2 ± 0.10
80
80
80
1250 ± 35
1250 ± 40
1130 ± 55
37.0
37.0
33.5
◦
◦
6
.0
15
5
C
C
2 h
1.0
1.0
2
1
2
3
6
1
2
h
h
h
h
2 h
4 h
2.4 ± 0.08
3.2 ± 0.09
3.2 ± 0.09
3.2 ± 0.09
3.2 ± 0.10
3.2 ± 0.10
60
80
80
80
80
80
1010 ± 40
1260 ± 40
1260 ± 50
1260 ± 45
1200 ± 30
1130 ± 50
29.9
37.3
37.3
37.3
35.5
33.5
◦
6
.0
.0
15
15
C
C
0
0
.25
.5
1.0 ± 0.02
2.0 ± 0.05
3.2 ± 0.06
5.1 ± 0.15
7.2 ± 0.18
100
100
80
85
90
390 ± 15
670 ± 25
1290 ± 60
1030 ± 50
950 ± 40
11.5
19.8
38.2
30.5
28.1
◦
6
2 h
1.0
1
2
.5
.0

8
22
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
Fig. 2. The immobilized Pseudomonas sp. lipase catalyzed the asymmetric acylation reaction of ± -2-amino-1-phenylethanols.
where V0 and Vt are the initial activity and the activity after t time incubation, respec-
tively. t1/2 values of the free and immobilized lipase preparations were estimated
from the equation:
2.2.11.3. 2-(Propylamino)-1-phenylethanol (3a). (C11H17NO): Yellow liquid, purity
99% (HPLC).
1H NMR ı (ppm): 0.85 (t, 3H, J = 12.50 Hz, CH2CH3), 1.39 (m, 2H, CH2CH3),
−
ln(0.5)
ki
0.693
ki
t1/2 =
=
2.48 (s, 1H, NH), 2.59 (t, 2H, J = 5.81 Hz, CH2CH2CH3), 2.62 (d, 1H, J = 5.81 Hz,
NHCH2 ), 3.37 (t, 1H, J = 5.57 Hz, NHCH2 ), 3.63 (s, 1H, OH), 4.62 (dd, 1H,
where ln(0.5) is residual activity of 50%.
J = 4.55 Hz, J = 3.21 Hz, ArCHOH), 7.31–7.32 (m, 5H, HAr ).
C NMR ı (ppm): 12.2 ( CH3), 40.2 ( CH2CH3), 51.4 ( NCH2 CH2), 58.2
HCNH CH2 ), 71.5 ( HCOH), 127.2–142 (ArC).
13
2.2.10. Reusability of immobilized lipase
The operational stability of immobilized lipase was investigated in a batch
HPLC: Retention times of (S)-3a and (R)-3a are 5.33 and 8.85 min, respectively.
IR (film) cm : 3416, 3032, 2532, 1994, 1729, 1618, 1500, 1394, 1324, 1296, 1185,
1048, 1026.
−
1
type reactor. To 8 mL of TBME, 1 mL of ± -2-(hexylamino)-1-phenylethanol solu-
tion (5.0 M) and 1 mL of vinyl acetate (9.60 M) were added. The reaction was started
◦
by the addition 1 g of immobilized lipase at 50 C. After 4 h reaction time, the separa-
tion of immobilized lipase preparations from the reaction mixture was achieved by
filtration. After each experiment, the immobilized lipase preparations were washed
with 5 mL of TBME and then dried under vacuum. For the next operation, the fresh
reaction medium was loaded onto the reactor and these procedures were repeated
2
9
.2.11.4. 2-(Butylamino)-1-phenylethanol (4a). (C12H19NO): Yellow liquid, purity
9% (HPLC).
1
0 times.
1H NMR ı (ppm): 0.86 (t, 3H, J = 12.50 Hz, CH CH ), 1.31 (m, 2H, CH CH ), 1.36
3
2 2 3
(m, 2H, CH2CH2CH3), 2.49 (t, 2H, J = 6.00 Hz, CH2CH2 ), 2.60 (s, 1H, NH), 2.99
2
2
9
.2.11. Spectral studies of reaction products
.2.11.1. 2-(Methylamino)-1-phenylethanol (1a). (C9H13NO): Yellow liquid, purity
9% (HPLC).
(d, 1H, J = 6.95 Hz, NHCH2 ), 3.31 (t, 1H, J = 9.57 Hz, CHHNH CH2 ), 3.65 (s,
1H, OH), 4.63 (dd, 1H, J = 4.18 Hz, J = 3.21 Hz, ArCHOH), 7.31–7.33 (m, 5H, HAr ).
13
C NMR ı (ppm): 14.4 ( CH3), 20.4 ( CH2CH3), 39.9 ( CH2 CH2 CH2 ), 49.2
NCH2 CH2), 58.2 ( CH2NH CH2 CH2 ), 70.6 ( HCOH), 127.2–142 (ArC).
(
1H NMR ı (ppm): 2.17 (s, 1H, NH), 2.26 (d, 1H, J = 5.70 Hz, NHCH2 ), 3.44 (t,
H, J = 12.09 Hz, NHCH2 ), 3.46 (s, 3H, NCH3), 3.60 (s, 1H, OH), 4.67 (dd, 1H,
J = 2.32 Hz, J = 2.16 Hz, ArCHOH), 7.29–7.32 (m, 5H, HAr ).
HPLC: Retention times of (S)-4a and (R)-5a are 5.27 and 8.84 min, respectively.
−
1
IR (film) cm : 3422, 2975, 2958, 2738, 2491, 2365, 1621, 1475, 1433, 1397, 1171,
036.
1
1
1
3
C NMR ı (ppm): 39.8 ( NCH3), 56.5 ( NCH2 ), 70.2 ( HCOH), 127.7–138.7
ArC).
HPLC: Retention times of (S)-1a and (R)-1a are 5.27 and 9.10 min, respectively.
(
2
.2.11.5. 2-(Hexylamino)-1-phenylethanol (5a). (C14H23NO): White solid, purity 99%
(
HPLC).
−
1
IR (film) cm : 4056, 3244, 3086, 2881, 1952, 1885, 1758, 1659, 1555, 1493, 1338,
268, 1201, 1091, 1026.
1
1H NMR ı (ppm): 0.86 (t, 3H, J = 11.90 Hz, CH2CH3), 1.24 (m, 4H, J = 12.50 Hz,
CH2CH2CH2CH3), 1.28 (m, 2H, CH2CH2CH3), 1.38 (m, 2H, CH2CH2CH2 ), 2.52
2.2.11.2. 2-(Ethylamino)-1-phenylethanol (2a). (C10H15NO): Pale yellow solid,
(
t, 2H, J = 9.24 Hz, NHCH2CH2 ), 2.62 (s, 1H, NHCH2 ), 2.64 (d, 1H, J = 4.50 Hz,
CHHNH CH2 ), 3.41 (t, 1H, J = 9.60 Hz, CHHNH CH2 ), 3.65 (s, 1H, COH),
purity 99% (HPLC).
4
.62 (dd, 1H, J = 3.73 Hz, J = 2.30 Hz, ArCHOH), 7.30–7.32 (m, 5H, HAr ).
C NMR ı (ppm): 14.3 ( CH3), 22.6 ( CH2CH3), 25.4 ( CH2CH2CH3), 26.9
CH2CH2CH2CH3), 30.0 ( CH2CH2CH2 ), 49.5 ( NCH2 CH2 ), 58.2 ( CH2N),
1H NMR ı (ppm): 1.0 (t, 3H, J = 12.50 Hz, CH2CH3), 2.58 (q, 2H, J = 12.09 Hz,
CH2CH3), 2.60 (s, 1H, NH), 2.61 (d, 1H, J = 4.23 Hz, NHCH2 ), 2.63 (t, 1H,
J = 6.96 Hz, NHCH2 ), 3.65 (s, 1H, OH), 4.63 (dd, 1H, J = 2.35 Hz, J = 2.33 Hz,
13
(
7
1.8 ( COH), 126.4–142 (ArC).
ArCHOH), 7.31–7.33 (m, 5H, HAr ).
HPLC: Retention times of (S)-5a and (R)-5a are 5.58 and 8.84 min, respectively.
IR (film) cm : 3418, 2977, 2940, 2739, 2491, 2359, 1475, 1397, 1172, 1036.
1
3C NMR ı (ppm): 15.6 ( CH3), 43.6 ( NCH2 CH3), 58 ( CH2N), 70.6 ( HCOH),
−1
1
27.2–142 (ArC).
HPLC: Retention times of (S)-2a and (R)-2a are 5.3 and 9.14 min, respectively.
−
1
IR (film) cm : 4046, 3224, 3086, 2881, 1955, 1885, 1758, 1659, 1555, 1416, 1268,
201, 1065, 1026.
2.2.11.6. 2-(Methylamino)-1-phenylethyl acetate (1b). (C11H15NO2): Brown liquid,
purity 96% (HPLC).
1

D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
823
1H NMR ı (ppm): 2.36 (s, 3H,
H, J = 12.57 Hz, CHHNH CH3), 3.37 (s, 3H, NH CH3), 3.93 (d, 1H, J = 4.90 Hz,
CHHNH CH3), 5.90 (dd, 1H, J = 4.90 Hz, J = 4.90 Hz, ArCHO ), 7.35–7.37 (m, 5H,
O
C
CH3), 2.72 (s, 1H, NH CH3), 3.35 (t,
on the surface of Florisil® support should be modified and acti-
1
vated in order to bind enzymes. In this study, the surface of Florisil®
was modified with 3-APTES to provide free primary amine groups
followed by glutaraldehyde activation to provide free aldehyde
groups that can bind the enzyme molecules. The amount of free
HAr).
1
3C NMR ı (ppm): 21.4 (O
C
CH3), 39.9 ( HNCH3), 52.6 ( CH2HNCH3), 73.6
(
COH), 127.1–149.8 (ArC), 172.6 ( C O).
HPLC: Retention time of (R)-1b is 6.90 min.
NH groups introduced after 3-APTES treatment was measured as
2
IR (film) cm−1: 4068, 3400, 3033, 2601, 1970, 1715, 1597, 1490, 1371, 1264, 1025,
−1
2
50 ␮mol g support . After glutaraldehyde activation, the amount
1
005.
−
1
of NH2 group was 45 ␮mol g support . These results indicate
that 86% of total NH2 groups introduced after 3-APTES treatment
are activated with glutaraldehyde. These results also show that a
small portion of the support may behave as anion exchanger due
2.2.11.7. 2-(Ethylamino)-1-phenylethyl acetate (2b). (C10H15NO): Brown liquid,
purity 96% (HPLC).
1H NMR ı (ppm): 1.90 (t, 3H, J = 12.50 Hz, CH2CH3), 2.02 (s, 3H,
to protonation of free NH groups remaining after glutaraldehyde
O
C
CH3), 2.11
2
(
q, 2H, J = 12.09 Hz, CH2CH3), 2.58 (s, 1H, NHCH2 CH3), 3.20 (d, 1H, J = 5.57 Hz,
CHHNH CH2), 3.22 (t, 1H, J = 6.0 Hz, CHHNH CH2 ), 5.37 (dd, 1H, dd, 1H,
activation [47]. However, the immobilized lipase preparations were
washed with the different concentrations of immobilization buffer
J = 4.60 Hz, J = 4.50 Hz, ArCHO ), 7.36–7.38 (m, 5H, HAr ).
(50–200 mM) to prevent ionic adsorption of the lipase. The covalent
1
3
C NMR ı (ppm): 14.0 ( CH3), 21.3 (O
CH2NHCH2), 73.6 ( COH), 128.2–149.7 (ArC), 172.7 (
C
CH3), 44.4 ( HNCH2 CH3), 50.6
O).
immobilization of Pseudomonas sp. lipase onto glutaraldehyde-
activated Florisil® support was achieved via Schiff base formation
reaction between the free carbonyl group of glutaraldehyde and
the amino functional groups of the lipase. Due to instability of Schiff
base, they were reduced using sodium cyanoborohydride and stabi-
lized as highly stable alkylamine bonds. Sodium cyanoborohydride
was preferred as reducing agent because it was reported that it
was milder reductant than sodium borohydride and would rapidly
reduce the Schiff base. However, it would not reduce aldehyde
groups unlike sodium borohydride [48]. It was reported that the
activities of immobilized enzymes, proteins and also some labile
monoclonal antibodies preserved after the treatment of sodium
cyanoborohydride [49–51].
(
C
HPLC: Retention time of (R)-2b is 6.93 min.
IR (film) cm−1: 3401, 3034, 2604, 1969, 1719, 1624, 1490, 1375, 1269, 1006.
2.2.11.8. 2-(Propylamino)-1-phenylethyl acetate (3b). (C11H17NO): Brown liquid,
purity 97% (HPLC).
1H NMR ı (ppm): 0.80 (t, 3H, J = 12.80 Hz, CH2CH3), 1.47 (m, 2H, CH2CH3),
2
.19 (s, 3H,
O C CH3), 2.55 (t, 2H, J = 11.40 Hz, CH2CH2CH3), 2.65 (s, 1H,
NHCH2 ), 3.07 (d, 1H, J = 4.80 Hz, CHHNH CH2 ), 3.61 (t, 1H, J = 12.57 Hz,
CHHNH CH2 ), 5.90 (dd, 1H, J = 4.58 Hz, J = 4.50 Hz, ArCHO ), 7.35–7.37 (m,
H, HAr ).
5
1
3
C
NMR
CH2NHCH2 ), 56.8 ( HNCH2 CH2 ), 73.6 ( COH), 127.3–149.9 (ArC), 172.6
O).
ı (ppm): 11.5 ( CH3), 21.4 (O C CH3), 39.7 ( CH2CH3), 51.0
(
(
C
Since success of an immobilization study (high activity and
protein recovery yields) affects from various parameters such as
immobilization pH, temperature and immobilization time and ini-
tial loaded enzyme amount, the conditions of lipase immobilization
were optimized for the obtention of maximum performance from
the immobilized lipase preparation. In this study, the immobi-
lization of lipase onto glutaraldehyde-activated Florisil® support
was investigated at different pH values and the results are given
in Table 2. For the immobilized lipase, the same specific activity
was observed as 1130 ± 45 U/mg protein at pH 5.0 and 5.5 and
the maximum specific activity was determined at pH 6.0. At the
immobilization pHs above 6.0, the specific activity of immobilized
lipase decreased and observed as 880 ± 42 U/mg protein for the
immobilization pH of 8.0. These results indicate that the activity
of immobilized lipase was affected from immobilization pH. The
nearly same behavior was observed for the amount of bound lipase
and the highest amounts of bound protein were determined for the
immobilization pH 6.0 and 6.5. The pH stability of free Pseudomonas
sp. lipase was evaluated for various pH values and the results were
given in Fig. 3. The three-dimensional structure of Pseudomonas sp.
lipase nearly protected during 2 h incubation time and a 90% of rel-
ative activity of Pseudomonas sp. lipase was protected at the end of
6 h incubation time for all the investigated pHs. At the end of 24 h
incubation time, the relative activities observed were 86, 90, 88, 87
and 82%, respectively, for pH values of 5.0, 6.0, 6.5, 7.0 and 8.0.
The effect of immobilization temperature on the relative activity
of immobilized lipase and bound protein amount was studied for 5,
HPLC: Retention time of (R)-3b is 6.99 min.
IR (film) cm−1: 3063, 3033, 2970, 2939, 2881, 2597, 1960, 1721, 1619, 1440, 1371,
1
256, 1066, 1035, 1006.
2.2.11.9. 2-(Butylamino)-1-phenylethyl acetate (4b). (C12H19NO): Brown liquid,
purity 97% (HPLC).
1H NMR ı (ppm): 0.87 (t, 3H, J = 6.90 Hz, CH2CH3), 1.26 (m, 2H, CH2CH3),
1
.39 (m, 2H, CH2CH2CH3), 2.19 (s, 3H,
O C CH3), 2.51 (t, 2H, J = 6.90 Hz,
NHCH2CH2 ), 2.68 (s, 1H, NHCH2 ), 3.19 (d, 1H, J = 3.90 Hz, CHHNH CH2 ),
.55 (t, 1H, J = 12.90 Hz, CHHNH CH2 ), 5.90 (dd, 1H, J = 4.50 Hz, J = 4.60 Hz,
3
ArCHO ), 7.37–7.39, (m, 5H, HAr ).
1
3
C
NMR
ı
(ppm): 13.8
(
CH3), 21.0 (O
C
CH3), 21.4
(
CH2CH3), 39.5
COH),
(
1
CH2 CH2 CH2 ), 40.1
26.8–149.9 (ArC), 172.5 (
(
C
HNCH2 CH2), 50.4
O).
(
CH2NH ), 73.6 (
HPLC: Retention time of (R)-4b is 7.17 min.
IR (film) cm−1: 3033, 2961, 2939, 2875, 2593, 1959, 1723, 1623, 1439, 1372, 1237,
065, 1027, 1005.
1
2.2.11.10. 2-(Hexylamino)-1-phenylethyl acetate (5b). (C14H23NO): Brown liquid,
purity 97% (HPLC).
1H NMR
CH2CH2CH2CH3), 1.20 (m, 2H, CH2CH2CH3), 1.41 (m, 2H, CH2CH2CH2 ),
.90 (t, 2H, J = 12.50 Hz, NHCH2CH2 ), 2.0 (s, 1H, NHCH2 ), 2.05 (s, 3H,
CH3), 3.17 (d, 1H, J = 3.60 Hz, CHHNH CH2 ), 3.54 (t, 1H, J = 12.30 Hz,
CHHNH CH2 ), 5.90 (dd, 1H, J = 4.50 Hz, J = 4.50 Hz, ArCHO ), 7.37–7.39 (m, 5H,
ı
(ppm): 0.82 (t, 3H, J = 11.90 Hz,
CH2CH3), 1.19 (m, 4H,
1
O
C
HAr ).
1
3
C
NMR
CH2 CH2 CH3), 26.4
HNCH2 CH2 ), 53.1 ( CH2NHCH2 ), 73.6 ( COH), 126.8–149.8 (ArC), 172.5
O).
ı
(ppm): 14.2
(
CH3), 21.3 (O
CH2 CH2 CH2 ), 31.3
C
CH3), 22.4
( CH2CH3), 31.5
(
(
(
(
(
NHCH2 CH2 ), 49.9
◦
C
15 and 25 C at the immobilization pH of 6.0, immobilization time
HPLC: Retention time of (R)-5b is 7.21 min.
−1
of 2 h and initial loaded protein concentration of 1.0 mg mL . The
IR (film) cm−1: 3572, 2933, 2598, 1958, 1720, 1597, 1490, 1440, 1370, 1263, 1005.
amount of bound protein did not affect from the change of immobi-
◦
lization temperature investigated in the range of 5–25 C and found
3
. Results and discussion
as 80% of initial loaded protein. The specific activities of immobi-
lized lipase were determined as the same for the immobilization
Florisil® is mainly composed of MgO (15%) and SiO2 (85%) and
has been used as support material for the immobilizations of dif-
ferent enzymes since it exhibits an excellent mechanical character
and resists against microbial attack and organic solvents [40,45,46].
Before covalent enzyme immobilization, the inert hydroxyl groups
◦
temperature of 5 and 15 C whereas the relative activity decreased
slightly for the immobilization temperature 25 ◦C (Table 2).
The percentage of bound protein increased from 60 to 80% when
the immobilization time was changed from 1 to 2 h at the immo-
bilization pH of 6.0, immobilization temperature of 5 ◦C and initial

8
24
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
Table 3
BBD results of asymmetric acylation of 2-(propylamino)-1-phenylethanol with vinyl acetate catalyzed by the immobilized Pseudomonas sp. lipase. The experimental results
were average of three replicates. The predicted results were estimated using Eq. (3).
Run
X1/water
content (%)
X2/reaction
temperature ( C)
X3/molar ratio
of VA/AA
X4/immobilized lipase
Y/yield (%)
(experimental)
Y/yield (%)
(predicted)
◦
−1
loading (mg mL
)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2
4
4
0
0
4
4
2
2
2
2
4
2
2
0
0
2
2
0
2
2
2
2
2
2
0
2
4
2
60
50
60
40
50
50
40
50
40
40
50
50
60
50
50
50
40
60
50
50
40
50
50
50
50
60
50
50
60
1.0
1.5
1.5
1.5
1.0
1.5
1.5
2.0
2.0
1.5
2.0
1.0
1.5
1.5
1.5
2.0
1.0
2.0
1.5
1.5
1.5
1.0
1.5
1.5
1.0
1.5
1.5
2.0
1.5
30
50
30
30
30
10
30
10
30
10
50
30
50
30
10
30
30
30
50
30
50
50
30
30
10
30
30
30
10
41.1
43.1
41.5
37.7
37.5
19.3
37.7
21.0
38.6
19.1
46.8
37.7
45.2
45.2
19.1
41.2
37.4
45.0
42.8
45.2
42.7
42.7
45.3
45.1
19.1
42.6
45.4
41.5
20.9
41.0
43.2
41.6
37.7
38.1
19.1
37.7
21.6
40.2
18.2
45.8
38.1
46.1
45.2
19.1
41.2
37.1
44.1
43.2
45.2
42.3
42.7
45.2
45.2
18.5
41.6
45.2
41.2
22.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
loaded lipase concentration of 1.0 mg mL−1. After that point, the
increase in immobilization time did not affect on relative per-
centage of bound protein under the same conditions. The specific
activity of immobilized lipase was calculated as 1010 ± 40 U/mg
protein after 1 h immobilization time. For 2–6 h immobilization
times, the specific activities reached a maximum value (Table 2).
Further increase in immobilization time resulted in decrease of
relative activity of immobilized lipase. These findings may be
explained that glutaraldehyde causes distortion effect in three
dimensional structure of the lipase for the immobilization times
of 12 and 24 h due to the high reactivity of glutaraldehyde with
other nucleophilic groups of the lipase [52,53]. In view of these
results, the optimal immobilization time was chosen as 2 h for the
next investigation.
initial protein concentration was increased, the percentage of
−
1
bound protein decreased. For 1.0, 1.5 and 2.5 mg mL
protein
concentrations, the percentage of bound protein were determined
as 80, 85 and 90%, respectively. However, the immobilized lipase
−
1
activity was higher for the protein concentration of 1.0 mg mL
−
1
than those of 1.5 and 2.5 mg mL . At the protein loadings of 1.5
−
1
and 2.5 mg mL , the possibility of reaction between glutaralde-
hyde and other nucleophilic groups of the lipase increases and this
situation cause distortion of the three-dimensional structure of
the lipase, resulting in decrease of the immobilized lipase activity.
Besides, the diffusion of substrate to active site of the lipase may be
prevented at the protein loadings of 1.5 and 2.5 mg mL [52,53].
In view of these results, the optimal immobilization conditions
were selected as pH value of 6.0, immobilization temperature of
−
1
◦
All the amount of loaded protein bound on the support for
5 or 15 C, immobilization time of 2 h and initial protein con-
centration of 1.0 mg mL . Under the optimized conditions, the
−
1
−1
the initial protein concentrations of 0.25 and 0.5 mg mL . When
amount of loaded protein on the support was determined as
−
1
3
.2 mg protein g support
which corresponds to 80% of initial
loaded protein and the lipase preserved 38.2% of its hydrolytic
activity with the immobilization.
In this study, the immobilized lipase-catalyzed asymmetric acy-
lation reaction of ± -2-(propylamino)-1-phenylethanol with vinyl
acetate was optimized by using RSM. For this purpose, a 4-factor
and 3-level BBD was applied as given in Table 3. The results of
2
9 experiments were analyzed by using Design-Expert software
and the coefficients of quadratic model proposed (Eq. (2)) were
estimated as given below:
Y = +45.24 − 8.333 × 10− X + 1.92X + 1.55X + 12.07X
3
1
2
3
4
−
+
0.28X × X + 0.025X × X + 0.025X × X + 0.67X × X
1
2
1
3
1
4
2
3
2
2
0.18X × X + 0.55X × X − 3.29X − 2.32X2
2
4
3
4
1
2
2
Fig. 3. The pH stability of free Pseudomonas sp. lipase at different pH values.
−2.31X3 − 10.78X4
(2)

D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
825
Table 4
ANOVA analysis of the proposed quadratic model.
Source
Model
Sum of squares
2588.81
Degree of freedom
14
Mean square
184.92
F value
F tabulated
p-Value, Prob > F
366.28
2.48
<0.0001
Significant
4
−4
−3
X1
X2
X3
X4
X1X2
X1X3
X1X4
X2X3
X2X4
X3X4
8.333 × 10−
44.47
28.83
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8.333 × 10
1.651 × 10
88.08
57.11
3460.97
0.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
4.60
0.9682
<0.0001
<0.0001
<0.0001
0.4518
0.9449
0.9449
0.0782
0.6299
0.1439
<0.0001
<0.0001
<0.0001
<0.0001
44.47
28.83
1747.25
0.30
Significant
Significant
Significant
1747.25
0.30
2.5 × 10
2.5 × 10
1.82
−
−
3
3
−3
−3
−3
2.5 × 10
4.952 × 10
4.952 × 10
3.61
−3
2.5 × 10
1.82
0.12
1.21
70.42
34.91
34.54
754.13
0.12
1.21
70.42
34.91
34.54
754.13
0.24
2.40
139.50
69.16
68.41
2
X1
X2
X3
X4
Significant
Significant
Significant
Significant
2
2
2
1493.79
Residual
Lack of Fit
7.07
7.02
0.052
14
10
4
0.50
0.70
0.013
53.97
2.98
0.0008
Significant
Pure Error
2
Adj R2 = 0.9946
R
= 0.9973
Eq. (2) was reduced by discarding the insignificant model terms
and Eq. (3) was obtained.
0 to 2%. This increase in activity may be ascribed to the change of
conformational flexibility of lipase in order to access substrate eas-
ily. Further increase in water content of reaction medium caused a
slight decrease in the acylation yield. This may be occurrence of the
hydrolysis reaction. Meanwhile, the increase in reaction tempera-
Y = +45.24 + 1.92X + 1.55X + 12.07X − 3.29X − 2.32X22
2
2
3
4
1
2.31X32 − 10.78X42
−
(3)
◦
ture from 40 to 50 C had a slight positive effect on the acylation
where Y is the predicted response (% acylation yield), X1 is the
bulk water content of reaction medium, X2 is the reaction tem-
perature, X3 is the initial molar ratio of vinyl acetate to amino
alcohol (VA/AA) and X4 is the immobilized lipase loading. A
positive sign before a term shows a synergistic effect, while a
negative sign shows an antagonistic effect [32]. The equation
yield and the further increase in reaction temperature was no effect
on the acylation yield.
Fig. 4b demonstrates the contour graph of the influence of
reaction temperature and initial molar ratio of vinyl acetate to
± -2-(propylamino)-1-phenylethanol at bulk water content of 2%
−
1
and immobilized lipase loading of 30 mg mL . In the literature,
enol esters, particularly vinyl acetate, were reported as more effec-
tive acylating reagents for lipase catalyzed acylation reaction due
to their high affinity to serine residues in catalytic site of lipases.
Furthermore, the acylation reaction is irreversible because of keto-
enol tautomerization [50,54]. The increase of reaction temperature
was no significant effect on the yield of reaction, however, the
increase in initial molar ratio of vinyl acetate to ± -2-(propylamino)-
1-phenylethanol was significant effect on the yield. The acylation
yield increased from 41.1 (run number 1) to 45.0% (run number
18) by increasing the initial molar ratio of vinyl acetate to ± -2-
(propylamino)-1-phenylethanol from 1.0 to 2.0, respectively, when
indicates that the immobilized lipase loading (X ) with a coef-
4
ficiency of 12.07 was the greatest positive linear effect while
the bulk water content of reaction medium was the negative
linear effect on the yield of asymmetric acylation reaction. The
results of analysis of variance (ANOVA) test are given in Table 4.
F-value of proposed quadratic model and F-tabulated value are
3
66.28 and 2.48, respectively, implying that the model is signifi-
cant. The model terms with p level value lower than 0.05 (i.e. at
a 95% confidence level) are accepted as significant term. Values
between 0.05 < X < 0.1 are considered marginally with significance.
Then values higher than 0.1 are insignificant. In this case X ,
2
X , X , X , X2 , X3 and X42 are significant model terms. The
2
2
2
◦
the reaction temperature was 60 C.
3
4
1
2
coefficient of determination value (R ) was estimated as 0.9973
Enzyme loading is an important factor for application of an
immobilized enzyme in industry. Fig. 4c shows the response sur-
face graph of the effect of immobilized lipase loading and initial
molar ratio of vinyl acetate to ± -2-(propylamino)-1-phenylethanol
showing that the model explains 99.73% of total variations. The
2
adjusted coefficient of determination value (Adj R = 0.9946) is
2
in agreement with the R . As shown in Table 3, the predicted
◦
results fit very well with the experimental results within the
range investigated indicating that the proposed quadratic model
provides an adequate approximation for explaining the asym-
metric acylation reaction of ± -2-(propylamino)-1-phenylethanol
with vinyl acetate catalyzed by immobilized Pseudomonas
sp. lipase.
at bulk water content of 2% and 50 C. As shown in Fig. 4c, the acyla-
tion yield affected markedly from the immobilized lipase loading.
The acylation yield increased linearly with the increase of immo-
−
1
bilized lipase loading from 10 to 30 mg mL . The acylation yield
was observed as 19.1% when initial molar ratio of vinyl acetate
−
1
to ± -2-(propylamino)-1-phenylethanol was 1.0 and 10 mg mL of
Fig. 4a illustrates the contour graph of the influence of reaction
temperature and initial water content of reaction medium on the
asymmetric acylation yield of ± -2-(propylamino)-1-phenylethanol
when initial molar ratio of vinyl acetate to ± -2-(propylamino)-
immobilized lipase used as catalyst (run number 25). When the
−
1
immobilized lipase loading was increased to 50 mg mL and the
other reaction conditions were kept constant, the acylation yield
increased to 42.7% (run number 22). The increasing of initial molar
ratio of vinyl acetate to ± -2-(propylamino)-1-phenylethanol had
a very small positive effect on the acylation yield. The acylation
yield reached from 42.7% (run number 22) to a maximum point as
46.8% (run number 11) when the initial molar ratio of vinyl acetate
to ± -2-(propylamino)-1-phenylethanol was increased from 1.0 to
2.0.
1
3
-phenylethanol and immobilized lipase loading were 1.5 and
0 mg mL , respectively. Water plays an important role for lipase
−
1
activity in organic media and a small amount of water should be
present in the reaction medium to acquire a proper lipase confor-
mation. As shown in Fig. 4a, the acylation yield slightly increased
with increasing the bulk water content of reaction medium from

8
26
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
Fig. 5. The performance of the lipase catalyzed acylation reaction in different ether
solutions.
To assess the optimum conditions for the acylation reaction, the
desired acylation yield was chosen as “maximize” and the bulk
water content of reaction medium, reaction temperature, initial
molar ratio of vinyl acetate to amino alcohol and immobilized
lipase loading were selected as “within the range” on the desirabil-
ity section of software menu. For this way, the optimal conditions
◦
were bulk water content of 1.8%, reaction temperature of 51.5 C,
initial molar ratio of vinyl acetate to amino alcohol of 1.92, and
−
1
immobilized lipase loading of 47 mg mL . At these conditions,
the acylation yields were obtained as 47.7 ± 1.2, 48.2 ± 0.8 and
4
8.1 ± 1.1% for 3 individual studies.
The performance of the immobilized Pseudomonas sp. lipase
catalyzed acylation reaction was compared in different ether sol-
vents such as DEE (log P 0.83), DIPE (log P 1.40), DIOX (log P −0.27),
TBME (log P 0.94) and THF (log P 0.45) for ± -2-(propylamino)-1-
phenylethanol because yield of a lipase catalyzed acylation reaction
and enantioselectivity of lipase depend strictly on the nature of the
solvent used as the reaction medium that different solvents may
exhibit different behaviors to solvate the substrate and, thus, may
influence enzyme activity. We selected ether solvents for the rea-
son that the dissolution of amino alcohols used was higher in these
solvents than the other commonly used reaction media for lipase
catalyzed reaction such as hexane, heptane. The reaction was car-
ried out under the determined optimum conditions. As shown in
Fig. 5, the maximum acylation yield was obtained as 48% in TBME.
The immobilized lipase catalyzed acylation of ± -2-(propylamino)-
1
-phenylethanol with vinyl acetate performed sluggishly in DIOX
and THF and the acylation yields were determined as 8.7% for the
both solvents. The lower lipase activity in DIOX and THF may be
attributed to higher hydrophilic character of both solvents than
other used ether solvents which cause to strip the essential water
molecule around lipase molecule [55,56]. However, the ee values of
formed ester did not affect from hydrophobicity or hydrophilicity
of the solvents and were determined as >99% for all the reaction
media.
Fig. 4. (a) The contour graph for the asymmetric acylation of ± -2-(propylamino)-
-phenylethanol with vinyl acetate as a function of reaction temperature and
Thermal stabilities of the free and immobilized lipase were
1
−
1
◦
water content of reaction medium at a constant lipase loading (30 mg mL ) and
vinyl acetate/± -2-(propylamino)-1-phenylethanol molar ratio (1.5/1.0). (b) The
contour graph for the asymmetric acylation of ± -2-(propylamino)-1-phenylethanol
studied at 40, 50 and 60 C. The relative activities of free lipase
almost decreased linearly for each investigated temperature. After
7
2 h incubation time, the relative activities of free lipase were
with vinylacetate as
a function of reaction temperature and initial vinyl
◦
measured as 44, 39 and 31%, respectively, for 40, 50 and 60 C
acetate/± -2-(propylamino)-1-phenylethanol molar ratio at a constant lipase load-
−1
(Fig. 6a). k values of the free lipase were calculated as 10.5 × 10−3,
ing (30 mg mL ) and bulk water content (2%) of reaction medium. (c) The contour
graph for the asymmetric acylation of ± -2-(propylamino)-1-phenylethanol with
vinylacetate as a function of immobilized lipase loading and initial vinyl acetate/± -
i
−
3
−3 −1 ◦
, respectively, for 40, 50 and 60 C
12.3 × 10 and 15.5 × 10
h
and the corresponding t1 values were estimated as 66.0, 56.3
/2
2
-(propylamino)-1-phenylethanol molar ratio at a constant reaction temperature
◦
and 44.7 h. After 4 h incubation time, the relative activities of the
immobilized lipase were observed as 100, 95 and 92%, respec-
(
50 C) and bulk water content (2%) of reaction medium.
◦
tively, for 40, 50 and 60 C. At the end of 72 h incubation time,
the corresponding relative activities of immobilized lipase were
measured as 86, 81 and 71% (Fig. 6b). For the immobilized lipase, ki

Table 5
The yield and ee values of remaining aminoalcohols and formed esters and E values of the immobilized lipase.
Remaining aminoalcohol
Yield (%)
ee of remaining aminoalcohol (%)
Formed ester
Yield (%)
ee of formed ester (%)
E value
50
80
44
>99
>200
50
80
45
>99
>200
50
90
48
>99
>200
50
93.2
48.2
>99
>200
50
>99
50
>99
>200

8
28
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
Fig. 6. The thermal stability curves of free and immobilized lipase preparations.
values were estimated as 2.5 × 10− , 3.4 × 10 and 5.9 × 10
3
−3
−3 −1
h
,
◦
activity within 75 min incubation at 55 C, while the immobilized
◦
respectively, for 40, 50 and 60 C and the corresponding t
lipase retained 90% of its initial activity under the same conditions.
1/2
values were determined as 277.2, 203.8 and 117.4 h. These results
indicated that thermal stability of the free lipase considerably
increased by the immobilization. Mendes et al. [53] showed that
the t1/2 value of Thermomyces lanuginosus lipase immobilized
onto chitosan modified with 2,4,6-trinitrobenzene sulfonic acid
followed by activation with glutaraldehyde was around 3.90 h at
The
immobilized
lipase-catalyzed
range of N-substituted ± -2-amino-1-
resolution
studies
were examined for
a
phenylethanols under the optimized conditions. The results
showed that the selectivity of immobilized lipase for these sub-
strates obeyed the Kazlauskas rule [60]. This rule suggests that
the enantiodiscrimination of secondary alcohols by a lipase is
based on the size of the substituents [61]. As shown in Fig. 7a–e,
(R)-enantiomers of tested amino alcohols reacted faster than
their (S)-enantiomers and the acylation yields of the tested amino
alcohols increased depending on the alkyl chain length substituted
onto amino group. (R)-ester yields obtained were 44, 45, 48, 48.2
and 50%, respectively, for 1b 5b and their ee values were found to
be >99% for all the formed esters (Table 5).
E value represents the ability of the enzyme to discriminate
between enantiomers. It is generally accepted that a resolution
reaction proceed with E value above 20 is useful for industrial appli-
cations [13]. E values of immobilized lipase were determined as
>200 toward all the tested substrates, indicating that the immo-
bilized lipase was promising biocatalyst for industrial production
◦
7
0 C. Rodrigues et al. [57] reported that Candida antarctica lipase
B immobilized onto agarose and chitosan beads via glutaraldehyde
retained around 15 and 30% of their initial activities, respectively,
◦
at 50 C after 24 h incubation time whereas the initial activity
of free lipase nearly depleted at the same conditions. The corre-
sponding t1/2 values were 6.3 and 5.4 h for the aforementioned
conditions. Pahujani et al. [58] reported that an alkaline lipase
from thermo tolerant Bacillus coagulans BTS-3 immobilized onto
glutaraldehyde activated Nylon-6 protected 88% of its initial
◦
activity at 55 C after 2 h incubation. Zhu and Sun [59] studied the
thermal stability of free and immobilized Candida rugosa lipase
onto glutaraldehyde-activated nanofibrous membrane and they
reported that the free lipase retained around 20% of its initial
Fig. 7. Time courses of asymmetric acylation of tested ± -2-amino-1-phenylethanols.

D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
829
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.procbio.2013.04.019.
References
[
[
1] Agustian J, Kamaruddin AH, Bhatia S. Single enantiomeric ␤-blockers – the
existing technologies. Process Biochem 2010;45:1587–604.
2] Cullington D, Yassin A, Cleland J. Beta-blockers in the treatment of cardiovas-
cular disease. Prescriber 2008;19:31–9.
[
[
3] Frishman WH. Beta-adrenergic blockers. Circulation 2003;107:117–9.
4] Kennedy HL. Current utilisation trends for beta blockers in cardiovascular dis-
ease. Am J Med 2001;110:2–6.
[
[
[
5] Mehvar R, Brocks DR. Stereospecific pharmacokinetics and pharmacodynamics
of beta-adrenergic blockers in humans. J Pharm Sci 2001;4:185–200.
6] Barret AM. Cardio beta-adrenoceptor blockade: the guest for selectivity. J
Pharm Sci 1985;2:95–108.
7] Maciejewski M, Półtorak K, Kami n´ ska JE. Lipase resolution of new (± )-3-
aryloxy-1-halogenopropan-2-ols: versatile building blocks for ␤-adrenergic
receptor antagonists. J Mol Catal B: Enzym 2010;62:248–56.
Fig. 8. The repeated-batch operation for the immobilized lipase. The acylation yield
and ee values of formed ester are symbolized with (ꢀ) and (ꢁ), respectively.
of enantiopure 2-amino-1-phenylethanols and their corresponding
esters. The chiral HPLC analyses showed that only (R)-enantiomer
of the related racemic amino alcohol was acylated for each tested
amino alcohol by the immobilized lipase and no spontaneous con-
version and byproducts were determined. The results of IR and NMR
spectra indicated that the acylation reaction purely took place on
the hydroxyl group of 2-amino-1-phenylethanols.
[8] Patel RN. Biocatalysis for synthesis for chiral pharmaceutical intermediates. In:
Patel RN, editor. Biocatalysis in the Pharmaceutical and Biotechnology Indus-
tries. Boca Raton: CRC Press; 2006. p. 105–58.
[
9] Baig MN, Santos RCD, Zetzl C, King J, Pioch D, Bowra S. Evaluation and modelling
the utility of SCCO2 to support efficient lipase mediated esterification. Enzyme
Microb Technol 2011;49:420–6.
[
[
10] Couturier L, Taupin D, Yvergnaux F. Lipase-catalyzed chemoselective aminol-
ysis of various aminoalcohols with fatty acids.
2009;56:29–33.
J Mol Catal B: Enzym
The reusability has a crucial importance for biocatalysts and an
enhancement of operational stability utilizes more advantageous.
The reusability studies were conducted for only ± -2-(hexylamino)-
11] Edwinoliver NG, Thirunavukarasu K, Naidu RB, Gowthaman MK, Kambe TN,
Kamini NR. Scale up of a novel tri-substrate fermentation for enhanced pro-
duction of Aspergillus niger lipase for tallow hydrolysis. Bioresour Technol
2010;101:6791–6.
1
-phenylethanol since the highest acylation yield was obtained
[
[
12] Majumder AB, Gupta MN. Stabilization of Candida rugosa lipase during
transacetylation with vinyl acetate. Bioresour Technol 2010;101:2877–9.
13] Bornscheuer UT, Kazlauskas RJ. Hydrolases in organic synthesis: regio- and
stereoselective biotransformations. Weinheim: Wiley-VCH Verlag GmbH & Co.
KGaA; 2005. p. 61.
14] Andrade LH, Silva AV, Pedrozo EC. First dynamic kinetic resolution of selenium-
containing chiral amines catalyzed by palladium (Pd/BaSO4) and Candida
antartica lipase (CAL-B). Tetrahedron Lett 2009;30:4331–4.
when it was used as substrate in the experimental studies. To
show the possibility of large scale industrial application, the reac-
tion was carried out at the substrate concentration of 5 M. As
shown in Fig. 8, the initial activity of immobilized lipase was pro-
tected after 5 reuses and 98% of its initial activity was retained
at the end of 10 recycles. The ee value of formed ester was
determined as >99% after each reuse. This result indicates that
the lipase catalyzed asymmetric acylation of ± -2-(hexylamino)-
[
[15] Chaubey A, Parshad R, Gupta P, Taneja SC, Qazi GN, Rajan CR, Ponrathnam S.
Arthrobacter sp. lipase immobilization for preparation of enantiopure masked
␤-amino alcohols. Bioorg Med Chem 2009;17:29–34.
1
-phenylethanol may be attractive for large scale industrial
[
[
[
16] Pilissão C, Nascimento MG. Effects of organic solvents and ionic liquids on
the aminolysis of (RS)-methyl mandelate catalyzed by lipases. Tetrahedron:
Asymmetry 2006;17:428–33.
17] Yilmaz E, Can K, Sezgin M, Yilmaz M. Immobilization of Candida rugosa lipase on
glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester.
Bioresour Technol 2011;102:499–506.
18] Sanfilippo C, Nicolosi G. Enantiomeric resolution by lipase-catalysed esterifica-
tion of a trans-5,6-dihydro-1,10-phenanthroline possessing helical and central
chirality. Tetrahedron: Asymmetry 2007;18:1828–32.
application.
4
. Conclusion
The results of RSM studies indicated that the proposed math-
ematical model provided a statistically significant accuracy in
the prediction of the response. The results of acylation reac-
tions showed that the immobilized lipase reacted preferentially
with (R)-enantiomers of tested amino alcohols. The acylation
yields increased with the increase of alkyl chain length substi-
tuted onto nitrogen atom and the highest acylation yield was
obtained as 50% for (R)-2-(hexylamino)-1-phenylethyl acetate. E
values of immobilized lipase were calculated as >200 for all the
asymmetric acylation reaction. A retained activity of 98% was
obtained for the immobilized lipase after 10 repeated-batch oper-
ations. The results of IR and NMR spectra analyses proved that the
acylation reaction purely took place on the hydroxyl group of 2-
amino-1-phenylethanols. As a result, the asymmetric acylations
of (± )-2-amino-1-phenylethanol derivatives with vinyl acetate
catalyzed by immobilized Pseudomonas sp. lipase can be a very
promising tool for the production of (S)-2-amino-1-phenylethanol
derivatives and their corresponding (R)-esters.
[
19] Gotor-Fernández V, Brieva R, Gotor V. Lipases: useful biocatalysts for the prepa-
ration of pharmaceuticals. J Mol Catal B: Enzym 2006;40:111–20.
20] Kamal A, Malik MS, Shaik AA, Azeeza S. Lipase mediated resolution of
␥-azidoalcohols in aqueous and organic media: synthesis of (R)- and (S)-
fluoxetine and duloxetine. J Mol Catal B: Enzym 2009;58:132–7.
21] Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R.
Improvement of enzyme activity, stability and selectivity via immobilization
techniques. Enzyme Microb Technol 2007;40:1451–63.
[
[
[22] Hernandez K, Fernandez-Lafuente R. Control of protein immobilization: cou-
pling immobilization and site-directed mutagenesis to improve biocatalyst or
biosensor performance. Enzyme Microb Technol 2011;48:107–22.
[
23] Garcia-Galan C, Berenguer-Murcia Á, Fernandez-Lafuente R, Rodrigues RC.
Potential of different enzyme immobilization strategies to improve enzyme
performance. Adv Synth Catal 2011;353:2885–904.
24] Palomo JM, Segura RL, Fernandez-Lorente G, Guisan JM, Fernandez-Lafuente R.
Enzymatic resolution of (± )-glycidyl butyrate in aqueous media. Strong mod-
ulation of the properties of the lipase from Rhizopus oryzae via immobilization
techniques. Tetrahedron: Asymmetry 2004;15:1157–61.
25] Chaubey A, Parshad R, Koul S, Taneja SC, Qazi GN. Enantioselectivity modu-
lation through immobilization of Arthrobacter sp. lipase: kinetic resolution of
fluoxetine intermediate. J Mol Catal B: Enzym 2006;42:39–44.
26] Palomo JM, Segura RL, Fernandez-Lorente G, Fernandez-Lafuente R, Guisan JM.
Glutaraldehyde modification of lipases adsorbed on aminated supports: a sim-
ple way to improve their behaviour as enantioselective biocatalyst. Enzyme
Microb Technol 2007;40:704–7.
[
[
[
Acknowledgement
[
27] Volpato G, Filice M, Rodrigues RC, Heck JX, Guisan JM, Mateo C, Marco AAZ.
Modulation of a lipase from Staphylococcus warneri EX17 using immobilization
techniques. J Mol Catal B: Enzym 2009;60:125–32.
This study was financially supported by Cukurova University
with the project number of FEF 2005D15.

8
30
D. Yildirim, S.S. Tükel / Process Biochemistry 48 (2013) 819–830
[
[
28] Lundell K, Katainen E, Kiviniemi A, Kanerva LT. Enantiomers of adrenaline-type
amino alcohols by Burkholderia cepacia lipase-catalyzed asymmetric acylation.
Tetrahedron: Asymmetry 2004;15:3723–9.
29] Lee TJF, Stitzel RE. Adrenomimetic drugs. In: Craig CR, Stitzel RE, editors. Mod-
ern pharmacology with clinical applications. Philadelphia: Lippincott Williams
[45] Tukel SS, Alptekin O. Immobilization and kinetics of catalase onto magnesium
silicate. Process Biochem 2004;39:2149–50.
[46] Ozyilmaz G, Tukel SS, Alptekin O. Activity and storage stability of immo-
bilized glucose oxidase onto magnesium silicate.
2005;35:154–60.
J Mol Catal B: Enzym
&
Wilkins; 2004. p. 109–21.
[47] Barbosa O, Torres R, Ortiz C, Fernandez-Lafuente R. Versatility of glutaraldehyde
to immobilize lipases: effect of the immobilization protocol on the properties
of lipase B from Candida antarctica. Process Biochem 2012;47:1220–7.
[48] Borch RF, Bernstein MD, Durst HD. Cyanohydridoborate anion as a selective
reducing agent. J Am Chem Soc 1971;93:2897–904.
[
[
30] Box GEP, Draper NR. Empirical model building and response surfaces. New
York: John Wiley & Sons; 1987. p. 477.
31] Sarabia LA, Ortiz MC. Response surface methodology. In: Brown SD, Tauler R,
Walczak B, editors. Comprehensive chemometrics chemical and biochemical
data analysis. Amsterdam: Elsevier; 2009. p. 345–90.
[49] Hansen EH, Mikkelsen HS. Enzyme-immobilization by the glutardialdehyde
procedure. An investigation of the effects of reducing the Schiff-bases gener-
ated, as based on studying the immobilization of glucose oxidase to silanized
controlled pore glass. Anal Lett 1991;24:1419−1430.
[
32] Myers RH, Montgomery DC, Anderson-Cook CM. Response surface
methodology: process and product optimization using designed exper-
iments. Weinheim: Wiley-VCH Verlag GmbH
p. 219.
&
Co. KGaA; 2009.
[50] Park SW, Lee JW, Hong SI, Kim SW. Immobilization of glutaryl-7-
aminocephalosporanic acid acylase on silica gel and enhancement of its
stability. Appl Biochem Biotechnol 2003;104:185–98.
[
[
33] Soo EL, Salleh AB, Basri M, Rahman RNZA, Kamaruddin K. Response surface
methodological study on lipase-catalyzed synthesis of amino acid surfactants.
Process Biochem 2004;39:1511–8.
34] Sharma M, Rastogi NK, Lokesh BR. Synthesis of structured lipid with balanced
omega-3:omega-6 ratio by lipase-catalyzed acidolysis reaction: optimiza-
tion of reaction using response surface methodology. Process Biochem
[51] Hermanson GT. Bioconjugate techniques. London: Academic Press; 2008. p.
231.
[52] Mendes AA, Giordano RC, Giordano RLC, de Castro HF. Immobilization and
stabilization of microbial lipases by multipoint covalent attachment on
aldehyde-resin affinity: application of the biocatalysts in biodiesel synthesis. J
Mol Catal B: Enzym 2011;68:109–15.
[53] Mendes AA, de Castro HF, Rodrigues DS, Adriano WS, Tardioli PW, Mammarella
EJ, Giordano RC, Giordano RLC. Multipoint covalent immobilization of lipase
on chitosan hybrid hydrogels: influence of the polyelectrolyte complex type
and chemical modification on the catalytic properties of the biocatalysts. J Ind
Microbiol Biotechnol 2011;38:1055–66.
[54] Wielechowska M, Plenkiewicz J. Lipase-catalyzed separation of the enan-
tiomers of 1-substituted-3-arylthio-2-propanols. Tetrahedron: Asymmetry
2005;16:1199–205.
[55] Hazarika S, Goswami P, Dutta NN, Hazarika AK. Ethyl oleate synthesis by Porcine
pancreatic lipase in organic solvents. Chem Eng J 2002;85:61–8.
[56] Pilissão C, Carvalho PO, Nascimento MG. Enantioselective acylation of (RS)-
phenylethylamine catalysed by lipases. Process Biochem 2009;44:1352–7.
[57] Rodrigues DS, Mendes AA, Adriano WS, Gon c¸ alves LRB, Giordano RLC. Mul-
tipoint covalent immobilization of microbial lipase on chitosan and agarose
activated by different methods. J Mol Catal B: Enzym 2008;51:100–9.
[58] Pahujani S, Kanwar SS, Chauhan G, Gupta R. Glutaraldehyde activation of poly-
mer Nylon-6 for lipase immobilization: enzyme characteristics and stability.
Bioresour Technol 2008;99:2566–70.
2
009;44:1284–8.
[
[
35] Wu J, Wang J-L, Li M-H, Lin J-P, Wei D-Z. Optimization of immobilization for
selective oxidation of benzyl alcohol by Gluconobacter oxydans using response
surface methodology. Bioresour Technol 2010;101:8936–41.
36] Martins AB, Graebin NG, Lorenzoni ASG, Fernandez-Lafuente R, Ayub MAZ,
Rodrigues RC. Rapid and high yields of synthesis of butyl acetate catalyzed
by Novozym 435: reaction optimization by response surface methodology.
Process Biochem 2011;46:2311–6.
37] Yücel Y. Optimization of immobilization conditions of Thermomyces lanugi-
nosus lipase on olive pomace powder using response surface methodology.
Biocatal Agric Biotechnol 2012;1:20–4.
38] Ray S, Lalman JA, Biswas N. Using the Box–Benkhen technique to statistically
model phenol photocatalytic degradation by titanium dioxide nanoparticles.
Chem Eng J 2009;150:15–24.
[
[
[
[
39] Weetall HH. Covalent coupling methods for inorganic support materials. In:
Mosbach K, editor. Methods in enzymology. London: Academic Press; 1976. p.
1
34–48.
40] Alptekin Ö, Tükel SS, Yildirim D, Alagöz D. Covalent immobilization of catalase
onto spacer-arm attached modified Florisil: characterization and applica-
tion to batch and plug-flow type reactor systems. Enzyme Microb Technol
2
011;49:547–54.
[59] Zhu J, Sun G. Lipase immobilization on glutaraldehyde-activated nanofibrous
membranes for improved enzyme stabilities and activities. React Funct Polym
2012;72:839–45.
[60] Kazlauskas RJ, Weissfloch ANE, Rappaport AT, Cuccia LA. A rule to predict
which enantiomer of a secondary alcohol reacts faster in reactions catalyzed by
cholesterol esterase, lipase from Pseudomonas cepacia, and lipase from Candida
rugosa. J Org Chem 1991;56:2656–65.
[61] Sorgedrager MJ, Malpique R, van Rantwijk F, Sheldon RA. Lipase catal-
ysed resolution of nitro aldol adducts. Tetrahedron: Asymmetry 2004;15:
1295–9.
[
41] Boratynski J, Zal T. Colorimetric micromethods for glutaraldehyde determina-
tion by means of phenol and sulfuric acid or phenol and perchloric acid. Anal
Biochem 1990;184:259–62.
42] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the
Folin-phenol reagents. J Biol Chem 1951;193:265–75.
43] Chiou S-H, Wu W-T. Immobilization of Candida rugosa lipase on chitosan with
activation of the hydroxyl groups. Biomaterials 2004;25:197–204.
44] Faber K, Hönig H. The computer program was obtained in the ftp-server at
ftp://borg185.kfunigraz.ac.at/2001
[
[
[