Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

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Journal of Molecular Catalysis B: Enzymatic

journal homepage: www.elsevier.com/locate/molcatb

A kinetic study of starch palmitate synthesis by immobilized

lipase-catalyzed esterication in solvent free system
Yan Wang a , Jiaying Xin a,b, , Jia Shi a , Wenlong Wu a , Chungu Xia b
a

Key Laboratory for Food Science & Engineering, Harbin University of Commerce, Harbin 150076, PR China
State Key Laboratory for Oxo Synthesis & Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR
China
b

a r t i c l e

i n f o

Article history:
Received 28 October 2013
Received in revised form
28 December 2013
Accepted 4 January 2014
Available online 14 January 2014
Keywords:
Synthesis of starch palmitate
Lipase Novozym 435
Solvent-free system
Kinetic model

a b s t r a c t
The objective of this work was to propose a reaction mechanism and to develop a rate equation for the
synthesis of starch palmitate by acylation of the corn starch with palmitic acid using the lipase Novozym
435 in solvent-free system. Initial rate data and progress curve data were used to arrive at a suitable model.
The initial rate studies showed that the kinetics obey the Ping-Pong bi-bi mechanism. An attempt to obtain
the best t of this kinetic model through computer simulation yielded in good approximation, the kinetic
equation was v = (1.735 Cfatty-acid Cstarch )/(Cfatty-acid Cstarch + 0.0156 Cstarch + 2.3947 Cfatty-acid ). The
mathematical expressions have been tested using several sets of data obtained from reactions carried
out under different reaction conditions. The predicted values provide very good ts of the experimental
data for the molar of starch from 2 mmol to 10 mmol, the molar of palmitic acid from 5 mmol to 70 mmol,
the reaction temperature from 50 C to 70 C, amount of lipase from 44 mg to176 mg, rotate speed from
100 r/min to 240 r/min, initial aw from <0.01 to 0.57.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Starch is an abundant renewable polysaccharide in nature that is
inexpensive, fully biodegradable and widely used in the production
of both food and industrial products [1,2]. Chemical modication
starch is often required to better suit its properties to specic
applications. Many reports exist in literature pertaining to the
preparation of starch esters or its components with the ultimate
aim of signicantly modifying the physicalchemical properties of
starches and imparting suitable mechanical characteristics so as
to render them more useful as engineering materials than native
starch [3,4].
Interest in an enzymatic route to esterify starch is fairly recent
and most works have been published after 2005 [5], with the
exception of one earlier investigation. A number of groups have
recently reported the use of organic solvents for esterication
of starch [6]. Normally, dimethyl sulfoxide (DMSO), dimethyl
formamide (DMF) and pyridine are used to dissolve the starch to
make it more reactive toward esterication [7]. Some authors [8]
have reported the preparation of a high degree of starch esters
in the presence of organic solvents using microwave heating.

Corresponding author at: Key Laboratory for Food Science & Engineering, College
of Food Engineering, Harbin University of Commerce, No. 138 Tongda Road, Daoli
District, Harbin150076, Heilongjiang, PR China. Tel.: +86 451 84838194.
E-mail address: Xinjiaying@yahoo.com.cn (J. Xin).
1381-1177/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2014.01.003

Unlike chemical esterication modication, an enzymatic one is

an environmentally friendly method which occurs under milder
conditions. The use of lipase as catalyst for ester production has
great potential. In fact, using a biocatalyst eliminates the disadvantages of the chemical process by producing very high purity
compounds with fewer or no downstream operations [9,10].
Although the introduction of an ester group into starch is an
important chemical modication task [11], little information is
available about the kinetic models and their parameters. Most
of the lipase kinetic studies are relative to hydrolysis reactions,
while the esterication kinetic publications are quite rare [12].
Some of the models proposed for ester synthesis consider a simple
MichaelisMenten mechanism, but are only valid for the simplest
enzymatic reactions. However, most approaches have proposed a
Ping-Pong Bi-Bi mechanism which seems to give the best results in
reproducing experimental ndings [1214].
In a previous paper [15] we have studied the inuence of the
acyl donor, granule shape and crystal structure of corn starch and
the type of enzyme, as well as the main operating parameters [16],
in the enzymatic production of starch ester. The best yields were
obtained when using palmitic acid as acyl donor, pretreatment
starch by sodium hydroxide/urea aqueous solution and the commercial immobilized lipase Novozym 435 as catalyst in solvent free
system.
The aim of this work was to conduct a kinetic study of the
enzyme synthesis of starch palmitate in solvent free system. With
that purpose, it was rst carried out a deep study of the reaction

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Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

and the inhibition effect of substrates and products was also

investigated since this phenomenon is quite often in enzymatically
catalyzed reactions.
2. Material and methods
2.1. Chemicals and enzyme
Corn starch was purchased from Harbin Mei Wang Reagent
Company, China and pretreatment by our laboratory. Palmitic acid
of analytically grade was purchased from Shanghai Chemical Co.,
China. Novozym 435 (Lipase B from Candida Antarctica immobilized on macroporous acrylic resin; specic activity: 10,000 U/g)
was purchased from Novozymes, Denmark. All the other chemicals
are of analytically grade.
2.2. Starch pretreatment
According to [17] the 9% aqueous solution containing sodium
hydroxide/urea at the desired ratio of 2:1 by weight was used as
a solvent for starch. The solvent was pre-cooled to below 10 C.
Then the starch sample in the given amount of 5% was added
immediately at ambient temperature of below 25 C. The native
starch (NS) was completely dissolved within 5 min by stirring at
3000 r/min and the resultant solution was transparent. The transparent starch solution was neutralized with HCl (15%) until it
reached neutrality. Then, starch was precipitated out from the neutral starch solution by adding 50 mL of ethanol drop-wise. After
various durations of dropping treatment, the precipitates were
washed by successive centrifugations in 95% of ethanol until no
HCl remained. Thereafter, they were washed with 100% of ethanol
to remove water. The resulting precipitates were vacuum dried at
50 C for 24 h.
As show in our previous studies [16,18], the average particle size
of starch decreased to nanometer level from 4 m to 15 m after
pretreatment. The crystalline type of corn starch shift from A-type
to VH -type and the relative degree of crystallinity of corn starch had
been decreased to 10.32%. The smaller particle size and the destruction of the crystal structure of starch after pretreatment endowed
starch with higher cold-water solubility. The esterication activity
of corn starch had been signicantly improved after pretreatment.
2.3. Water activity pre-equilibration of reaction medium
Before the start of the reaction, the substrates (palmitic acid,
pretreatment starch and Lipase) were pre-equilibrated for at least
3d in a sealed containers enclosed with saturated salt solutions or
solid adsorbent to establish xed water activities for esterication.
Pre-equilibration was done at 25 C. The solid adsorbent was 3 A
molecular sieves (aw < 0.01). The saturated salt solutions used were
prepared with LiBr (aw : 0.05), LiCl (aw : 0.11), CH3 COOK (aw : 0.23),
(MgNO3 )6H2 O (aw : 0.54), NaCl (aw : 0.75), KCl (aw : 0.85), K2 Cr2 O7
(aw : 0.98) [19].
2.4. General procedure for lipase esterication
Water activity or aw is an important consideration for biocatalysis in a solvent free medium. Before the start of reaction, all the
substrates were pre-equilibrated for at least 3d in sealed containers,
enclosed with a molecular sieve to establish xed water activities (aw < 0.01). The reaction setup for esterication was carried
out in 25 mL closed, screw-capped glass vials containing palmitic
acid and pretreated starch. To conduct the reaction under neat
conditions (without solvents), a 5:1 mol ratio of palmitic acid to
pretreated starch is needed to provide enough solution volume
to dissolve solid starch and to stir the suspended immobilized

lipase. The palmitic acid acted as the solvent in the solvent free
system when the reaction temperature was above of its melting
point (6364 C). The esterication was initiated by adding immobilized lipase (Novozym 435) into each glass vial. Glass vials were
placed upright on a magnetic stirrer and incubated at 5575 C,
40240 r/min for 424 h. The removal of nonesteried palmitic acid
from starch palmitate was accomplished by washed again with
100 mL of pure ethanol and then dried in a hot air oven at 75 C.
2.5. Calculation of the Initial reaction rates
Initial reaction rates, expressed as m mol consumed palmitic
acid per minute and per gram of enzyme, were determined from
the time course of palmitic acid concentration. In order to get the
parameters of the kinetic model, initial velocities were tted to the
proposed reaction rate equation by non-linear regression analysis
with the computer program Microsoft Matlab.
2.6. Calculation of the conversion of palmitic acid
A small sample 30 mg of starch palmitate dissolved in 1 mL
DMSO was mixed with 1 mL of sodium methoxide (0.07 M) in
methanol solution. This mixture was then heated (70 C) under
reux for 40 min, while shaken, then cooled and 1 mL of deionized
water and 1 mL of n-heptane were added. The mixture was shaken
for 1 min and left to settle. The top organic phase contained the
methyl ester of palmitic acid and could be removed and injected
into the GCFID (Perkin-Elmer Autosystem XL with a CP Simdist
capillary column, oven set at 220 C, the injector at 250 C and
the detector at 260 C, ow rate of N2 and air is 4.5 mL/min and
5.5 mL/min, ow rate of tail-blowing is 5.0 mL/min).
Once the methyl oleate was quantied by GC chromatograph,
the conversion of palmitic acid (CP) was calculated as Eq. (1).
CP =

M1
100%
M0

(1)

where CP is the conversion of palmitic acid; M0 is the initial mole

of palmitic acid, mol; M1 is the mole of esteried oleic acid, mol.
3. Results and discussions
The effect of various parameters on the rate of reaction were
studied to arrive at a suitable kinetic model.
3.1. Effect of different catalysts
Various catalysts such as Candida cylindracea lipase (CRL),
Porcine pancreas lipase (PPL), Immobilized thermophilic fungal
lipase (TLIM), Novozym 435 (Candida Antarctica lipase immobilized on a macroporous polyacrylic resin) were tested (Fig. 1).
The enzyme activity per mg enzyme was different in each
case. Esterication activity of various lipases was determined by
a reported esterication method [20]. The unit of enzyme activity
is dened as mol of palmitic acid consumed (in an esterication
reaction with pretreatment starch) per min per mg of the enzyme
(Table 1).
Of this Porcine pancreas lipase (PPL) and Candida cylindracea
lipase (CRL), led to poor conversions of 3% and 7% in 24 h, respectively, while Immobilized thermophilic fungal lipase (TLIM) offered
comparable conversions around 23% in 24 h. Novozym SP 435 was
found to be the best catalyst with a conversion of 57% in 24 h. Generally Immobilized thermophilic fungal lipase (TLIM) is very active
on long chain fatty acids. However, in the case of starch palmitate
synthesis it was less effective. Candida cylindracea lipase (CRL) and
Porcine pancreas lipase (PPL) has been reported to be a very good

Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

80

conversion of palmitic acid (%)

hand, aw above the optimum value allowed the enzyme completely

hydrated, but the competitive hydrolysis of the products took place
and hence limited the acylation. The optimal initial water activity
(aw < 0.01) represented the most appropriate water condition for
the balance between the above mentioned conicts.

N 435
TLIM
CRL
PPL

70

75

60
50

3.3. Effect of speed of agitation

40

The effect of shaking speed on the initial rate of the reaction is

illustrated in Fig. 2A. The initial rate followed the increase of the
shaking speed when it was less than 200 r/min, and the initial rate
reached a maximum at 200 r/min. Above this speed the initial rate
remained almost constant. This can be regarded as that the initial
rate of reaction were no longer limited by the mass transfer limitation of immobilized enzyme at shaking speeds above 200 r/min.

30
20
10
0
0

10

15

20

25

3.4. Effect of catalyst loading

time (h)
Fig. 1. Effect of different catalysts on esterication. (Reaction condition: pretreatment starch = 10 mmol, palmitic acid = 50 mmol, catalyzed by 110 mg Novozym 435
lipase at 60 C, 200 r/min for 24 h).
Table 1
Enzyme activity.
Enzyme

Activity (mol/(min/mg)

Novozym 435
Immobilized thermophilic fungal lipase (TLIM)
Candida cylindracea lipase (CRL)
Porcine pancreas lipase (PPL)

7.814
2.345
0.003
0.001

The effect of catalyst loading on the initial rate of the reaction

and the conversion of palmitic acid were studied in the range of
44176 mg. The initial rate of reaction was found to increase with
an increase in catalyst loading (Fig. 2B). However, with an increase
in catalyst loading from 110 mg to 143 mg, the conversion increased
marginally, which might be due to the increase in the concentration of catalyst above the substrate concentration (Fig. 2C). In the
case of 176 mg catalyst-loading conversion did not increase after
20 h, which could have been due to the attainment of equilibrium.
Therefore, further experiments were done at 110 mg.

(Reaction condition: pretreatment starch = 10 mmol, palmitic acid =50 mmol, catalyzed by 110 mg Novozym 435 lipase at 60 C, 200 r/min for 24 h)

3.5. Effect of temperature on the initial rate and the conversions

of palmitic acid

catalyst for the hydrolysis of aliphatic esters [21] but it had very
low activity in the current study.

Temperature has a signicant effect on the equilibrium of the

reaction, and on the activity and stability of immobilized lipase [23].
As shown in Fig. 5, for the initial rate, the most suitable temperature
was 65 C, but for the conversion of palmitic acid was 60 C. The
conversion of palmitic acid increased with increasing temperature
until 60 C and then decreased slowly. Fig. 2D illustrates that the
optimal temperature was 60 C, and that the immobilized lipase
has good thermostability.

3.2. Effect of initial water activity (aw )

In esterication reaction, initial water activity of the medium
not only effects the rate of reaction but also the equilibrium position. Suitable initial water activity can keep the enzyme active
conguration, but higher initial water activity will inhibit the equilibrium move to the product. Therefore, it is particularly important
to pay attention to initial water activity control in the case of lipase
catalyzed esterication of starch in a solvent free system. In this
study, the lipase catalyzed esterication of starch was carried out
over a wide range of aw to see the effect of aw on the conversion of
palmitic acid.
As shown in Table 2, Novozym 435 catalyzed starch esterication with palmitic acid in solvent free system had a clear aw
dependence. When aw value was below 0.75 in reaction media,
the conversion of palmitic acid decreased with the increase of aw .
These results suggest that a very small amount of water could
satisfy the requirement of Novozym 435 for holding essential water
layer to perform its catalytic functions properly [22]. On the other
Table 2
Effect of water activity (aw ) on the conversion of palmitic acid.

1
2
3
4
5

aw

conversion of palmitic acid (%)

<0.01
0.11
0.33
0.54
0.75

76.50
50.52
37.61
12.38
4.75

(Reaction condition: pretreatment starch = 10 mmol, palmitic acid =50 mmol, catalyzed by 10% Novozym 435 lipase at 60 C, 200 r/min for 24 h).

3.6. Effect of mole ratio of substrate

The quantity of palmitic acid was kept constant at 50 mmol. The
quantity of pretreatment starch was varied as 2.5 mmol, 5.0 mmol,
10 mmol, 20 mmol and 30 mmol. The conversions of palmitic acid
were 48%, 51%, 68%, 58% and 52% at 24 h, respectively. The initial rate of reaction was found to increase with increasing the
quantity of pretreatment starch until 10 mmol and then decreased
marginally (Fig. 2E), which could be attributed to the formation of
dead end complex between enzyme and excesses of pretreatment
starch. Therefore, all further reactions were carried out by using
10 mmol of pretreatment starch.
In another set of experiments, pretreatment starch was kept
constant at 10 mmol and the quantity of palmitic acid was varied as
5 mmol, 10 mmol, 20 mmol, 30 mmol, 40 mmol, 50 mmol, 60 mmol
and 70 mmol. It was found that there was an increase in the rate
of reaction with an increase in quantity of palmitic acid (Fig. 2F).
The conversions increased with increasing the quantity of palmitic
acid until 50 mmol and then decreased slowly (Fig. 2F). Therefore,
all further reactions were carried out by using 50m mol of palmitic
acid.
Thus, it was found that 10 mmol of pretreatment starch and
50 mmol of palmitic acid are the optimum quantities at 60 C and
200 r/min in solvent free system.

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Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

-1

0.5

-1

0.4
0.3
0.2
0.1
0.0
0

50
100
150
200
250
-1
speed of agitation (r min )

C
60
50

30
20
10

0.3
0.2
0.1
0.0

30

10

15

20

25

convertion of palmitic acid

initial rate of reaction

0.7

60

0.6
0.5

40

0.4
20

50

50

0.5

40

0.4

65

70

0.3

quantity of pretreatment starch (mmol)

70

0.8
0.7

60

0.6

50

0.5

40

0.4

30

0.3

20

0.2

10

0.1

0.0
10 20 30 40 50 60 70 80
quantity of palmitic acid (mmol)

-1

30

conversion of palmitic acid

initial rate of reaction

80

-1

25

-1

20

0.3

conversion of palmitic acid (%)

0.6

-1

conversion of palmitic acid (%)

0.8
0.7

60

15

60

initial rate of reaction (mmol h mg )

0.9

initial rate of reaction (mmol h mg )

70

10

55

temperature ( C)

conversion of palmitic acid

initial rate of reaction

0.9
0.8

120 150 180 210

30

90

catalyst loading (mg)

time (h)

80

60

-1

0.4

-1

0.5

80

40

0.6

44(mg)
77(mg)
110(mg)
143(mg)
176(mg)

70

0.7

initial rate of reaction (mmol h mg )

conversion of palmitic acid (%)

initial rate of reaction (mmol h (mg) )

conversion of palmitic acid (%)

-1

-1

initial rate of reaction (mmol h (mg) )

Fig. 2. Effect of speed of agitation (A), catalyst loading (B) (C), temperature (D), quantity of pretreatment starch (E), quantity of palmitic acid (F) on the initial rate of reaction
and the convertion of palmitic acid. (Reaction condition of effect of (A): pretreatment starch = 10 mmol, palmitic acid =50 mmol, aw < 0.01, catalyzed by 110 mg Novozym 435 at
60 C for 4 h; (B) (C): pretreatment starch = 10 mmol, palmitic acid = 50 mmol, aw < 0.01, 60 C, 200 r/min for 24 h; (D): pretreatment starch = 10 mmol, palmitic acid = 50 mmol,
aw < 0.01, catalyzed by110 mg Novozym 435 at 200 r/min for 24 h; (E) or (F): palmitic acid =50 mmol or pretreatment starch =10 mmol, aw < 0.01, catalyzed by110 mg Novozym
435 at 60 C, 200 r/min for 24 h)

3.7. Kinetics and mechanism

As show in our previous studies, the average particle size and the
relative degree of crystallinity of corn starch had been decreased
after pretreatment. The smaller particle size and the destruction
of the crystal structure endowed starch with higher cold-water
solubility and dispersion stability. The esterication activity of
corn starch had been signicantly improved after pretreatment.
Although the average particle size and the relative degree of crystallinity of corn starch decreased, the basic composition of starch
has not been changed. So the esterication of pretreatment starch
is still carried out at the hydroxyl groups of d-glucopyranosyl structural unit of the starch polymer.
The effect of the quantity of both substrates on the initial rate
of reaction was investigated. It was found that when the quantity of pretreatment starch (B) was increased, the rate of reaction

increased and reached a maximum at a critical quantity. A subsequent increase in pretreatment starch quantity decreased the
initial rate. For the determination of initial rate, the quantity of
pretreatment starch was varied from 2.5 to 10 mmol at a xed
quantity of palmitic acid. Reactions were carried out up to 5%
conversion and the initial rates were determined. Therefore, it
may be concluded that the quantity of pretreatment starch below
10 mmol not reacts with the enzyme to form dead end inhibitory
complex. There was no evidence of inhibition by palmitic acid
(A) at any quantity tested. A mechanism in which the product is
released between the addition of two reactants is called Ping-Pong
bi-bi.
The Line weaverBurk plot, using initial rate and initial quantity,
shows that as the quantity of pretreatment starch or palmitic acid
increases, the slope increases (Figs. 3 and 4). These results agree
with the assumed Ping-Pong bi-bi mechanism.

Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

1.8

10mmol
20mmol
30mmol

1.6

Lipase

Lipase

Palmitic acid-Lipase

Palmitic acid
starch esters

1.0
0.8

Palmitic acid

Palmityl-Lipase

0.1

0.2

0.3

0.4

0.5

1/starch (1/mmol)

Lipase

Fig. 3. Line weaver Burk plot 1/V vs. 1/[starch] for esterication of pretreatment
starch with palmitic acid.
Table 3
Kinetic parameters obtained for esterication of pretreatment starch with palmitic
acid.
Parameter

Ping-pong bi-bi

Vmax (mmol/h/mg)
KmA (mmol/mg)
KmB (mmol/mg)
SSE

1.7350 0.0032
0.0156 0.0048
2.3947 0.0078
0.008 0.0052

The rate equation for this kind of mechanism, assuming there is

no inhibition of both substrates and products is given by Segel as
[24]:
Vmax [A][B]
KmA [B] + KmB [B] + KmA KmB

(2)

where v is the initial reaction rate, Vmax the maximum reaction rate
and KmA and KmB are the binding constants (Michaelis constants)
for both substrates, palmitic acid (A) and pretreatment starch (B).
Once it was conrmed the Ping-Pong mechanism, the kinetic
parameters of Eq. (2) were calculated by multiple regression tting
of the experimental values. The results are shown in Table 3.

4.2
4.0
3.8


V=

Cfatty-acid Cstarch + 0.0156 Cstarch + 2.3947 Cfatty-acid

(3)

0.30
0.25

initial rate of reaction (mmol h mg )

3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
0.02 0.04 0.06 0.08
1/palmitic acid (1/mmol)

1.735 Cfatty-acid Cstarch

where Cfatty-acid is the initial quantity of palmitic acid, Cstarch is the

initial quantity of pretreatment starch (mmol).

4mmol
6mmol
8mmol

3.6

1.8
0.00

In this reaction the lipase may react with palmitic acid to

yield the effective lipase palmitic acid complex. Then the lipase
palmitic acid complex is transferred to an enzymeacyl intermediate and water is released. This is followed by the interaction of the
enzymeacyl complex with pretreatment starch to form another
binary complex, which then yields the ester and free lipase. But,
the ester will be hydrolyzed by lipase if the aw of reaction system
was accumulate to arouse the hydrolytic activity of lipase. In the
hydrolysis reaction the lipase may react with palmitic acid starch
ester to yield the palmitylenzyme intermediate and released the
starch. Then the palmitylenzyme intermediate reacted with H2 O
to yield palmitic acid and lipase.
The reaction sequence may be given as follows (Fig. 5):
A plot of experimental rate versus simulated rate by using
the above parameters gives a straight-line passing through origin
showing that the experimental rate data match with the simulated
values as given in Figs. 6 and 7.
Figs. 6 and 7 illustrated that the tting dynamic model is a good
way to predict the initial reaction rate of enzymatic esterication
of pretreatment starch with palmitic acid in solvent free system.
Thus, the nal kinetic equation for the enzymatic synthesis of starch
palmate from palmitic acid is the following:

-1

0.2
0.0

1/v (hg/mmol)

H2O

Fig. 5. The sequential reaction sequence of esterication of palmitic acid with pretreatment starch.

0.4

-0.02

starch

Lipase

-1

1/v (hg/mmol)

Palmitic acid
starch esters

1.2

0.6

Pretreatment
starch

Palmitic acid H2O

1.4

V=

77

0.10

Fig. 4. Line weaver Burk plot 1/V vs. 1/[palmitic acid] for esterication of pretreatment starch with palmitic acid.

experimental rate
simulated rate

0.20
0.15
0.10
0.05
0.00
5

10

15

20

25

30

quantity of palmitic acid (mmol)

Fig. 6. Comparison effect of quantity of palmitic acid on experimental rate and
simulated rate.

78

Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

experimental rate
simulated rate

-1

initial rate of reaction (mmol h mg )

0.18

82

-1

conversion of palmitic acid (%)

0.16
0.14
0.12
0.10
0.08
0.06

81
80
79
78
77
76
75
74
73
72

0.04
5

10

15

20

25

30

71
1

quantity of pretreatment starch (mmol)

using times (n)

Fig. 7. Comparison effect of quantity of pretreatment starch on experimental rate

and simulated rate.

Fig. 8. Batch wise stability of lipase Novozym 435.

3.8. Reusability of catalyst

3.9.

The catalyst was ltered, washed with heptane, dried at room

temperature for 4 h and reused. After using six times, the conversion decreased marginally and it was due to the reduction in the
effective catalyst loading since there was a loss of some catalyst
during ltration (Fig. 8).

Almost all of the starch palmitate products are soluble in DMSOd6 except for the products with a DS higher than 0.26. These
products were only partially soluble in DMSO-d6 . To improve the
solubility in DMSO-d6 , one drop of TFA-d1 was added to the mixtures. Fig. 9a and b show the typical 1 H NMR spectra of native

Fig. 9.

1H

NMR analyses

H NMR spectra of pretreatment starch (a) and starch palmitate (b).

Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 7379

starch and starch palmitate product, respectively. The broad and

overlapped peaks in the region 3.35.6 ppm are assigned to the
starch protons [25,26]. The peaks at 0.82.2 ppm correspond to
the aliphatic hydrogen atoms of the fatty acid chain (Fig. 9b) [27].
The absence of resonances in the olenic region ( 77.2 ppm) indicates that the products are free from un-reacted palmitic acid and
that the work-up procedure involving thorough washing of the
product with ethanol was successful.
4. Conclusions
Synthesis of starch palmitate in solvent free system was conducted by employing different lipases, among which Novozym 435
was found to be the most active catalyst. The effects of various
parameters on the conversion and initial rates of reaction were
studied in the presence of Novozym 435. Initial rate data and
progress curve data were used to arrive at a suitable model. The
initial rate studies showed that the Michaelis constant for pretreatment starch was very low indicating lower afnity between the
enzyme and the reactant. The apparent t of the kinetic data to
the assumed Ping-Pong bi-bi mechanism. The various parameters
were estimated. This model was used to simulate the rate data,
which were in excellent agreement with the experimental values.
The activity of Novozym 435 can be used for more than six time.
The analysis of the kinetic data showed that the acylation of pretreatment starch with palmitic acid catalyzed by
Novozym 435 follows a Ping-Pong Bi-Bi mechanism without pretreatment starch inhibition for quantity less than
10 mmol. The equation rate proposed to describe this model
uses four kinetic constants that were obtained by multiple
regression analysis of the experimental data. The tted parameters were: Vmax = 1.7350 mmol/h/mg, KmA = 0.0156mmol/mg,
KmB = 2.3947mmol/mg. These values demonstrate a higher afnity
of Novozym 435 for pretreatment starch rather than to the palmitic
acid (KmB < KmA ). From the obtained kinetic equation, initial reaction rates were successfully predicted for initial pretreatment
starch quantity below 10 mmol.

Heilongjiang Provincial Education Department (2010td04) and the

Heilongjiang Provincial Funds for Distinguished Young Scientists
(JC201106) for support.
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Acknowledgments
The authors thank the National Natural Science Foundation
of China (20873034, 21073050), the Scientic Research Fund of

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