A kinetic study of starch palmitate synthesis by immobilized lipase-catalyzed esterification in solvent free system

Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73–79

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A kinetic study of starch palmitate synthesis by immobilized lipase-catalyzed esterification 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 fit 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 fits 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 modification starch is often required to better suit its properties to specific applications. Many reports exist in literature pertaining to the preparation of starch esters or its components with the ultimate aim of significantly modifying the physical–chemical 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 esterification of starch [6]. Normally, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and pyridine are used to dissolve the starch to make it more reactive toward esterification [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: [email protected] (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 esterification modification, 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 modification 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 esterification kinetic publications are quite rare [12]. Some of the models proposed for ester synthesis consider a simple Michaelis–Menten 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 findings [12–14]. In a previous paper [15] we have studied the influence 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 first carried out a deep study of the reaction

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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; specific 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 esterification activity of corn starch had been significantly 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 fixed water activities for esterification. 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 esterification 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 fixed water activities (aw < 0.01). The reaction setup for esterification 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 (63–64 ◦ C). The esterification was initiated by adding immobilized lipase (Novozym 435) into each glass vial. Glass vials were placed upright on a magnetic stirrer and incubated at 55–75 ◦ C, 40–240 r/min for 4–24 h. The removal of nonesterified 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 fitted 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 reflux 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 GC–FID (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, flow rate of N2 and air is 4.5 mL/min and 5.5 mL/min, flow rate of tail-blowing is 5.0 mL/min). Once the methyl oleate was quantified 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 esterified 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. Esterification activity of various lipases was determined by a reported esterification method [20]. The unit of enzyme activity is defined as ␮mol of palmitic acid consumed (in an esterification 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) 73–79

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 conflicts.

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

5

10

15

20

25

3.4. Effect of catalyst loading

time (h) Fig. 1. Effect of different catalysts on esterification. (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 44–176 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 significant 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 esterification 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 configuration, 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 esterification of starch in a solvent free system. In this study, the lipase catalyzed esterification 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 esterification 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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-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

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

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

5

0.9 0.8

F

0

120 150 180 210

O

E

30

90

catalyst loading (mg)

time (h)

80

60

-1

5

0.4

-1

0

0.5

80

40

0

0.6

D

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) )

B

conversion of palmitic acid (%)

-1

-1

initial rate of reaction (mmol h (mg) )

A

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 esterification activity of corn starch had been significantly 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 esterification 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 fixed 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 weaver–Burk 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) 73–79

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 esterification of pretreatment starch with palmitic acid. Table 3 Kinetic parameters obtained for esterification 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 confirmed the Ping-Pong mechanism, the kinetic parameters of Eq. (2) were calculated by multiple regression fitting 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 enzyme–acyl intermediate and water is released. This is followed by the interaction of the enzyme–acyl 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 palmityl–enzyme intermediate and released the starch. Then the palmityl–enzyme 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 fitting dynamic model is a good way to predict the initial reaction rate of enzymatic esterification of pretreatment starch with palmitic acid in solvent free system. Thus, the final 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 esterification 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 esterification 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.

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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)

2

3

4

5

6

7

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 filtered, 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 filtration (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.

1

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) 73–79

starch and starch palmitate product, respectively. The broad and overlapped peaks in the region ı 3.3–5.6 ppm are assigned to the starch protons [25,26]. The peaks at ı 0.8–2.2 ppm correspond to the aliphatic hydrogen atoms of the fatty acid chain (Fig. 9b) [27]. The absence of resonances in the olefinic region (ı 7–7.2 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 affinity between the enzyme and the reactant. The apparent fit 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 fitted parameters were: Vmax = 1.7350 mmol/h/mg, KmA = 0.0156mmol/mg, KmB = 2.3947mmol/mg. These values demonstrate a higher affinity 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. References [1] [2] [3] [4] [5] [6]

[7] [8] [9] [10] [11] [12]

[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]

Acknowledgments The authors thank the National Natural Science Foundation of China (20873034, 21073050), the Scientific Research Fund of

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