a kinetic study of starch palmitate synthesis by immobilized lipase-catalyzed esterification in...
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Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79
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Journal of Molecular Catalysis B: Enzymatic
jo ur nal home p age: www.elsev ier .com
A kinetic study of starch palmitate synthesis by imlipase- yst
Yan Wan u Xa Key Laborator Chinab State Key Lab ChineChina
a r t i c l
Article history:Received 28 OReceived in re28 December 2Accepted 4 JanAvailable onlin
Keywords:Synthesis of stLipase NovozySolvent-free syKinetic model
pose tion o
data a kinegh coCstarch
testes. Thol too 70
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74 Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79
and the inhibition effect of substrates and products was alsoinvestigated since this phenomenon is quite often in enzymaticallycatalyzed reactions.
2. Materia
2.1. Chemic
Corn staCompany, Cof analyticaChina. Novolized on mwas purchaare of analy
2.2. Starch
Accordinhydroxide/ua solvent foThen the simmediatelstarch (NS)3000 r/min parent starreached neutral starch various durwashed by HCl remainto remove w50 C for 24
As showof starch depretreatmeto VH-type abeen decreation of the cstarch withof corn star
2.3. Water
Before tpretreatme3d in a sealsolid adsorbPre-equilibmolecular sprepared w(MgNO3)6H(aw: 0.98) [
2.4. Genera
Water acysis in a sosubstrates wenclosed wties (aw < 0out in 25 macid and pconditions pretreated to dissolve
lipase. The palmitic acid acted as the solvent in the solvent freesystem when the reaction temperature was above of its meltingpoint (6364 C). The esterication was initiated by adding immo-bilized lipase (Novozym 435) into each glass vial. Glass vials were
upr0 r/mtarch
of p
lcula
ial rer mie co
etersed ree co
lcula
mall wasnol sfor 4and 1in al estee GCry cotecto/mine thvers
1
0
CP isitic
ults
effe to a
fect o
ious e pa(TLIMn a m
enzsterirted ed an wi
1).his P(CRLwhilrableto bemmog chasis ite panl and methods
als and enzyme
rch was purchased from Harbin Mei Wang Reagenthina and pretreatment by our laboratory. Palmitic acidlly grade was purchased from Shanghai Chemical Co.,zym 435 (Lipase B from Candida Antarctica immobi-
acroporous acrylic resin; specic activity: 10,000 U/g)sed from Novozymes, Denmark. All the other chemicalstically grade.
pretreatment
g to [17] the 9% aqueous solution containing sodiumrea at the desired ratio of 2:1 by weight was used asr starch. The solvent was pre-cooled to below 10 C.tarch sample in the given amount of 5% was addedy at ambient temperature of below 25 C. The native
was completely dissolved within 5 min by stirring atand the resultant solution was transparent. The trans-ch solution was neutralized with HCl (15%) until ittrality. Then, starch was precipitated out from the neu-solution by adding 50 mL of ethanol drop-wise. Afterations of dropping treatment, the precipitates weresuccessive centrifugations in 95% of ethanol until noed. Thereafter, they were washed with 100% of ethanolater. The resulting precipitates were vacuum dried at
h. in our previous studies [16,18], the average particle sizecreased to nanometer level from 4 m to 15 m afternt. The crystalline type of corn starch shift from A-typend the relative degree of crystallinity of corn starch hadsed to 10.32%. The smaller particle size and the destruc-rystal structure of starch after pretreatment endowed
higher cold-water solubility. The esterication activitych had been signicantly improved after pretreatment.
activity pre-equilibration of reaction medium
he start of the reaction, the substrates (palmitic acid,nt starch and Lipase) were pre-equilibrated for at leasted containers enclosed with saturated salt solutions orent to establish xed water activities for esterication.
ration was done at 25 C. The solid adsorbent was 3 Aieves (aw < 0.01). The saturated salt solutions used wereith LiBr (aw: 0.05), LiCl (aw: 0.11), CH3COOK (aw: 0.23),2O (aw: 0.54), NaCl (aw: 0.75), KCl (aw: 0.85), K2Cr2O7
19].
l procedure for lipase esterication
tivity or aw is an important consideration for biocatal-lvent free medium. Before the start of reaction, all theere pre-equilibrated for at least 3d in sealed containers,
ith a molecular sieve to establish xed water activi-.01). The reaction setup for esterication was carriedL closed, screw-capped glass vials containing palmiticretreated starch. To conduct the reaction under neat(without solvents), a 5:1 mol ratio of palmitic acid tostarch is needed to provide enough solution volume
solid starch and to stir the suspended immobilized
placed4024from s100 mL
2.5. Ca
Initacid pethe timparamproposwith th
2.6. Ca
A sDMSOmethareux water for 1 mmethyinto thcapillathe de5.5 mL
Oncthe con
CP = MM
whereof palm
3. Res
Thestudied
3.1. Ef
VarPorcinlipase lized o
Thecase. Ea repois denreactio(Table
Of tlipase tively, compafound erally Ion lonsynthePorcinight on a magnetic stirrer and incubated at 5575 C,in for 424 h. The removal of nonesteried palmitic acid
palmitate was accomplished by washed again withure ethanol and then dried in a hot air oven at 75 C.
tion of the Initial reaction rates
action rates, expressed as m mol consumed palmiticnute and per gram of enzyme, were determined fromurse of palmitic acid concentration. In order to get the
of the kinetic model, initial velocities were tted to theaction rate equation by non-linear regression analysismputer program Microsoft Matlab.
tion of the conversion of palmitic acid
sample 30 mg of starch palmitate dissolved in 1 mL mixed with 1 mL of sodium methoxide (0.07 M) inolution. This mixture was then heated (70 C) under0 min, while shaken, then cooled and 1 mL of deionized
mL of n-heptane were added. The mixture was shakennd left to settle. The top organic phase contained ther of palmitic acid and could be removed and injectedFID (Perkin-Elmer Autosystem XL with a CP Simdistlumn, oven set at 220 C, the injector at 250 C andr at 260 C, ow rate of N2 and air is 4.5 mL/min and, ow rate of tail-blowing is 5.0 mL/min).e methyl oleate was quantied by GC chromatograph,ion of palmitic acid (CP) was calculated as Eq. (1).
100% (1)
the conversion of palmitic acid; M0 is the initial moleacid, mol; M1 is the mole of esteried oleic acid, mol.
and discussions
ct of various parameters on the rate of reaction wererrive at a suitable kinetic model.
f different catalysts
catalysts such as Candida cylindracea lipase (CRL),ncreas lipase (PPL), Immobilized thermophilic fungal), Novozym 435 (Candida Antarctica lipase immobi-acroporous polyacrylic resin) were tested (Fig. 1).yme activity per mg enzyme was different in eachcation activity of various lipases was determined byesterication method [20]. The unit of enzyme activitys mol of palmitic acid consumed (in an estericationth pretreatment starch) per min per mg of the enzyme
orcine pancreas lipase (PPL) and Candida cylindracea), led to poor conversions of 3% and 7% in 24 h, respec-e Immobilized thermophilic fungal lipase (TLIM) offered
conversions around 23% in 24 h. Novozym SP 435 was the best catalyst with a conversion of 57% in 24 h. Gen-bilized thermophilic fungal lipase (TLIM) is very activein fatty acids. However, in the case of starch palmitate
was less effective. Candida cylindracea lipase (CRL) andcreas lipase (PPL) has been reported to be a very good
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Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79 75
0
10
20
30
40
50
60
70
80co
nver
sion
of p
alm
itic
acid
(%)
N 435 TLIM CRL PPL
Fig. 1. Effect oment starch = 1lipase at 60 C
Table 1Enzyme activi
Enzyme
Novozym 43ImmobilizedCandida cyliPorcine panc
(Reaction conalyzed by 110
catalyst forlow activity
3.2. Effect o
In esterinot only efftion. Suitabconguratiolibrium moto pay attencatalyzed estudy, the lover a widepalmitic aci
As showcation withdependencethe conversThese resulsatisfy the rlayer to per
Table 2Effect of water
1 2 3 4 5
(Reaction conalyzed by 10%
hand, aw above the optimum value allowed the enzyme completelyhydrated, but the competitive hydrolysis of the products took placeand hence limited the acylation. The optimal initial water activity(aw < 0.01) represented the most appropriate water condition forthe balance
3.3. Effect o
The effeillustrated shaking spereached a mremained arate of reac
of im
fect o
effee con6 mgeaselyst lally,
cataf 176hichore, f
fect oitic a
peran, anwn in
C, 0 5 10 15 20 25
time (h )
f different catalysts on esterication. (Reaction condition: pretreat-0 mmol, palmitic acid = 50 mmol, catalyzed by 110 mg Novozym 435
, 200 r/min for 24 h).
ty.
Activity (mol/(min/mg)
5 7.814 thermophilic fungal lipase (TLIM) 2.345ndracea lipase (CRL) 0.003reas lipase (PPL) 0.001
dition: pretreatment starch = 10 mmol, palmitic acid =50 mmol, cat-mg Novozym 435 lipase at 60 C, 200 r/min for 24 h)
the hydrolysis of aliphatic esters [21] but it had very in the current study.
f initial water activity (aw)
tation
3.4. Ef
Theand th4417an incrin catamargintion ofcase o20 h, wTheref
3.5. Efof palm
TemreactioAs showas 65cation reaction, initial water activity of the mediumects the rate of reaction but also the equilibrium posi-le initial water activity can keep the enzyme activen, but higher initial water activity will inhibit the equi-
ve to the product. Therefore, it is particularly importanttion to initial water activity control in the case of lipasesterication of starch in a solvent free system. In thisipase catalyzed esterication of starch was carried out
range of aw to see the effect of aw on the conversion ofd.n in Table 2, Novozym 435 catalyzed starch esteri-
palmitic acid in solvent free system had a clear aw. When aw value was below 0.75 in reaction media,ion of palmitic acid decreased with the increase of aw.ts suggest that a very small amount of water couldequirement of Novozym 435 for holding essential waterform its catalytic functions properly [22]. On the other
activity (aw) on the conversion of palmitic acid.
aw conversion of palmitic acid (%)
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76 Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79
0.4
0.5
-1 (m
g)-1)
30
0.6
0.7
-1 (m
g)-1)
A B
Fig. 2. Effect oand the conver60 C for 4 h; (Baw < 0.01, cata435 at 60 C, 2
3.7. Kinetic
As showrelative degafter pretreof the cryssolubility acorn starchAlthough thtallinity of has not beeis still carrietural unit o
The effeof reactiontity of pretr0 50 100 150 200 250
0.0
0.1
0.2
0.3
speed of agitation (r min-1)initi
al ra
te o
f rea
ctio
n (m
mol
h
0 0.0
0.1
0.2
0.3
0.4
0.5
initi
al ra
te o
f rea
ctio
n (m
mol
h
50
60
70 44(mg ) 77(mg ) 110(mg ) 143(mg ) 176(mg )iti
c ac
id (%
)
60
80
tic a
cid
(%)
C
D0 5 10 15 20 250
10
20
30
40
conv
ersi
on o
f pal
m
time (h )50
20
40
conv
ersi
on o
f pal
mi
0 5 10 15 20 25 3030
40
50
60
70
80 conve rsion of pa lmitic aci d initial ra te of r eacti on
quantity of pr etreat ment starch (mmol )
conv
ersi
on o
f pal
miti
c ac
id (%
)
0.3
0.4
0.5
0.6
0.7
0.8
0.9
initial rate of reaction (mm
ol h-1 m
g-1)
0 10
1020
3040
5060
7080
qua
conv
ersi
on o
f pal
miti
c ac
id (%
)
E F
f speed of agitation (A), catalyst loading (B) (C), temperature (D), quantity of pretreatmention of palmitic acid. (Reaction condition of effect of (A): pretreatment starch = 10 mmol, p) (C): pretreatment starch = 10 mmol, palmitic acid = 50 mmol, aw < 0.01, 60 C, 200 r/min
lyzed by110 mg Novozym 435 at 200 r/min for 24 h; (E) or (F): palmitic acid =50 mmol or pr00 r/min for 24 h)
s and mechanism
in our previous studies, the average particle size and theree of crystallinity of corn starch had been decreasedatment. The smaller particle size and the destructiontal structure endowed starch with higher cold-waternd dispersion stability. The esterication activity of
had been signicantly improved after pretreatment.e average particle size and the relative degree of crys-corn starch decreased, the basic composition of starchn changed. So the esterication of pretreatment starchd out at the hydroxyl groups of d-glucopyranosyl struc-f the starch polymer.ct of the quantity of both substrates on the initial rate
was investigated. It was found that when the quan-eatment starch (B) was increased, the rate of reaction
increased asequent incinitial rate.pretreatmequantity ofconversionmay be con10 mmol nocomplex. T(A) at any qreleased bebi-bi.
The Lineshows thatincreases, twith the as60 90 120 150 180 210catalyst loading (mg )
convertion of p almi tic aci dinitial rate of react ion
0.7
0.8
0.9
initial rate of re55 60 65 70tempera ture (OC)
0.3
0.4
0.5
0.6
action (mm
ol h-1 m
g-1)
0 20 30 40 50 60 70 80
convers ion of pal mitic aci d initi al rate of re actio n
ntity of palm itic ac id (m mol )
0.00.10.20.30.40.50.60.70.8
initial rate of reaction (mm
ol h-1 m
g-1)
t starch (E), quantity of palmitic acid (F) on the initial rate of reactionalmitic acid =50 mmol, aw < 0.01, catalyzed by 110 mg Novozym 435 atfor 24 h; (D): pretreatment starch = 10 mmol, palmitic acid = 50 mmol,etreatment starch =10 mmol, aw < 0.01, catalyzed by110 mg Novozym
nd reached a maximum at a critical quantity. A sub-rease in pretreatment starch quantity decreased the
For the determination of initial rate, the quantity ofnt starch was varied from 2.5 to 10 mmol at a xed
palmitic acid. Reactions were carried out up to 5% and the initial rates were determined. Therefore, itcluded that the quantity of pretreatment starch belowt reacts with the enzyme to form dead end inhibitoryhere was no evidence of inhibition by palmitic aciduantity tested. A mechanism in which the product is
tween the addition of two reactants is called Ping-Pong
weaverBurk plot, using initial rate and initial quantity, as the quantity of pretreatment starch or palmitic acidhe slope increases (Figs. 3 and 4). These results agreesumed Ping-Pong bi-bi mechanism.
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Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79 77
0.0 0. 1 0. 2 0. 3 0. 4 0. 50.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8 10mmo l 20mmo l 30mmo l
1/v
(hg/
mm
ol)
1/st arch (1/mm ol)
Fig. 3. Line weaver Burk plot 1/V vs. 1/[starch] for esterication of pretreatmentstarch with palmitic acid.
Table 3Kinetic parameters obtained for esterication of pretreatment starch with palmiticacid.
Parameter Ping-pong bi-bi
Vmax(mmol/h/mg) 1.7350 0.0032KmA (mmol/mg) 0.0156 0.0048KmB (mmol/mSSE
The rateno inhibitio[24]:
V =(
KmA[B
where v is thand KmA anfor both sub
Once it parametersof the expe
-0.02
1/v
(hg/
mm
ol)
Fig. 4. Line wement starch w
Lipas e Pal mitic acid-LipaseLipas e
Pal mitic acid H2OPretreat ment
starchPal mitic acidstarch este rs
Lipas e Pal mityl-Lipase Lipas e
Palmitic acidstarch esters starch H2O Palmitic acid
Fig. 5. The sequential reaction sequence of esterication of palmitic acid with pre-treatment starch.
In this reaction the lipase may react with palmitic acid toyield the effective lipase palmitic acid complex. Then the lipasepalmitic acid complex is transferred to an enzymeacyl intermedi-ate and water is released. This is followed by the interaction of theenzymeacyl complex with pretreatment starch to form anotherbinary complex, which then yields the ester and free lipase. But,the ester wwas accumhydrolysis ester to yiestarch. Thento yield pal
reaclot oove pg th
as gi. 6 an
predreatmhe ne frog) 2.3947 0.00780.008 0.0052
equation for this kind of mechanism, assuming there isn of both substrates and products is given by Segel as
Vmax[A][B]] + KmB[B] + KmAKmB
)(2)
e initial reaction rate, Vmax the maximum reaction rated KmB are the binding constants (Michaelis constants)
TheA p
the abshowinvalues
Figsway toof pretThus, tpalmat
(strates, palmitic acid (A) and pretreatment starch (B).was conrmed the Ping-Pong mechanism, the kinetic
of Eq. (2) were calculated by multiple regression ttingrimental values. The results are shown in Table 3.
0.00 0.02 0.04 0.06 0.08 0.101.82.02.22.42.62.83.03.23.43.63.84.04.2 4mmo l
6mmo l 8mmo l
1/pal mitic a cid (1/mm ol)
aver Burk plot 1/V vs. 1/[palmitic acid] for esterication of pretreat-ith palmitic acid.
V =Cfatty
where Cfattyinitial quan
0.0
0.0
0.1
0.1
0.2
0.2
0.3
initi
al ra
te o
f rea
ctio
n (m
mol
h-1
mg-
1 )
Fig. 6. Compasimulated rateill be hydrolyzed by lipase if the aw of reaction systemulate to arouse the hydrolytic activity of lipase. In thereaction the lipase may react with palmitic acid starchld the palmitylenzyme intermediate and released the
the palmitylenzyme intermediate reacted with H2Omitic acid and lipase.tion sequence may be given as follows (Fig. 5):f experimental rate versus simulated rate by usingarameters gives a straight-line passing through originat the experimental rate data match with the simulatedven in Figs. 6 and 7.d 7 illustrated that the tting dynamic model is a goodict the initial reaction rate of enzymatic estericationent starch with palmitic acid in solvent free system.
al kinetic equation for the enzymatic synthesis of starchm palmitic acid is the following:
1.735 Cfatty-acid Cstarch-acid Cstarch + 0.0156 Cstarch + 2.3947 Cfatty-acid
)
(3)
-acid is the initial quantity of palmitic acid, Cstarch is thetity of pretreatment starch (mmol).
0
5
0 experimen tal rat e si mulated rat e5 10 15 20 25 30
0
5
0
5
quantity of pal mitic acid (mmol )
rison effect of quantity of palmitic acid on experimental rate and.
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78 Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79
5 10 15 20 25 300.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18 experimental ra te simulated rat e
initi
al ra
te o
f rea
ctio
n (m
mol
h-1 m
g-1 )
quantity of pre treat ment sta rch (mmol )
Fig. 7. Comparison effect of quantity of pretreatment starch on experimental rateand simulated rate.
3.8. Reusability of catalyst
The catalyst was ltered, washed with heptane, dried at roomtemperature for 4 h and reused. After using six times, the conver-sion decreased marginally and it was due to the reduction in theeffective catalyst loading since there was a loss of some catalystduring ltration (Fig. 8).
1 2 3 4 5 6 771
72
73
74
75
76
77
78
79
80
81
82
conv
ersi
on o
f pal
miti
c ac
id (%
)
using times (n )
Fig. 8. Batch wise stability of lipase Novozym 435.
3.9. 1H NMR analyses
Almost all of the starch palmitate products are soluble in DMSO-d6 except for the products with a DS higher than 0.26. Theseproducts were only partially soluble in DMSO-d6. To improve thesolubility in DMSO-d6, one drop of TFA-d1 was added to the mix-tures. Fig. 9a and b show the typical 1H NMR spectra of nativeFig. 9. 1H NMR spectra of pretreatment starch (a) and starch palmitate (b).
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Y. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73 79 79
starch and starch palmitate product, respectively. The broad andoverlapped peaks in the region 3.35.6 ppm are assigned to thestarch protons [25,26]. The peaks at 0.82.2 ppm correspond tothe aliphatic hydrogen atoms of the fatty acid chain (Fig. 9b) [27].The absence of resonances in the olenic region ( 77.2 ppm) indi-cates that the products are free from un-reacted palmitic acid andthat the work-up procedure involving thorough washing of theproduct with ethanol was successful.
4. Conclusions
Synthesis of starch palmitate in solvent free system was con-ducted by employing different lipases, among which Novozym 435was found to be the most active catalyst. The effects of variousparameters on the conversion and initial rates of reaction werestudied in the presence of Novozym 435. Initial rate data andprogress curve data were used to arrive at a suitable model. Theinitial rate studies showed that the Michaelis constant for pretreat-ment starch was very low indicating lower afnity between theenzyme and the reactant. The apparent t of the kinetic data tothe assumed Ping-Pong bi-bi mechanism. The various parameterswere 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 acyla-tion of pretreatment starch with palmitic acid catalyzed byNovozym 435 follows a Ping-Pong Bi-Bi mechanism with-out pretreatment starch inhibition for quantity less than10 mmol. Tuses four regression eters wereKmB = 2.394of Novozymacid (KmB < tion rates starch quan
Acknowled
The autof China (2
Heilongjiang Provincial Education Department (2010td04) and theHeilongjiang Provincial Funds for Distinguished Young Scientists(JC201106) for support.
References
[1] W.M. Doane, Starch 44 (1992) 293295.[2] R.L. Shogren, G.F. Fanta, W.M. Doane, Starch 45 (1994) 276280.[3] V.D. Miladinov, M.A. Hanna, Ind. Crops Prod. 11 (2000) 5157.[4] A. Apostolos, J.H. Peter, Bioresour. Technol. 115 (2011) 4147.[5] S. Adachi, T. Kobayashi, J. Biosci. Bioeng. 99 (2) (2005) 8794.[6] L. Marcin, Biocatalytic esterication of common polysaccharidesstarch
modication using lipases, in: Proceedings of the 14th InternationalElectronic Conference on Synthetic Organic Chemistry, Santiago, Chile,130 November, 2010, Available online: http://www.sciforum.net &http://www.usc.es/congresos/ecsoc/ (November 3, 2010).
[7] R.T. Akhila, A. Emilia, Bioprocess Biosyst. Eng. 29 (2006) 6571.[8] H. Habib, C. Moncef, G. Youssef, S. Adel, Carbohydr. Polym. 79 (2010)
466474.[9] A.C. Kshirsagar, R.S. Singhal, Carbohydr. Polym. 69 (2007)
455461.[10] B. Atanu, R.L. Shogren, S. Gordon, J. Salch, J.L. Willett, M.B. Charles, Carbohydr.
Polym. 74 (2008) 137141.[11] A. Jorge, H. Hassina, M.B. Genevive, S. Franc ois, A. Isabelle, B. Elisabeth, Starch
Strk 51 (1999) 302307.[12] A.L. Paiva, V.M. Balcao, F.X. Malcata, Kinetics and mechanisms of reac-
tions catalyzed by immobilized lipases, Enzyme Microb. Technol. 27 (2000)187204.
[13] G.D. Yadav, A.H. Trivedi, Enzyme Microb. Technol. 32 (2003) 783789.[14] S. Hari Krishna, N.G. Karanth, Biochim. Biophys. Acta 1547 (2001)
262267.[15] Jia-Ying Xin, Yan Wang, Tie Liu, Kai Lin, Le Chang, Chun-Gu Xia, Int. J. Mol. Sci.
13 (2012) 72277234.[16] Jiaying Xin, Yan Wang, Tie Liu, Adv. J. Food Sci. Technol. 4 (5) (2012) 270276.
Zhou, L. Zhang, Polymer 32 (2000) 866870. Wan, Adv. Wehtj230apati. Rom277. Salina, Elecegn, W
Segel, ady-StElomaym. 57nistia
rke 60nistia
rke 61he equation rate proposed to describe this modelkinetic constants that were obtained by multipleanalysis of the experimental data. The tted param-: Vmax = 1.7350 mmol/h/mg, KmA = 0.0156mmol/mg,7mmol/mg. These values demonstrate a higher afnity
435 for pretreatment starch rather than to the palmiticKmA). From the obtained kinetic equation, initial reac-were successfully predicted for initial pretreatmenttity below 10 mmol.
gments
hors thank the National Natural Science Foundation0873034, 21073050), the Scientic Research Fund of
[17] J.P.[18] Yan
Xia[19] E.
221[20] Gan[21] M.D
269[22] M.R
Raj[23] P. D[24] H.
Ste[25] M.
Pol[26] L. Ju
Sta[27] L. Ju
Stag, Jia-ying Xin, Tie Liu, Kai Lin, Chao-yue Zhang, Chun-gu Xia, Chun-guMater. Res. 549 (2012) 183187.e, D. Costes, P. Adlercreutz, J. Mol. Catal. B: Enzym. 3 (1997).
D. Yadav, Piyush S. Lathi, Biochem. Eng. J. 16 (2003) 245252.ero, L. Calvo, C. Alba, A. Daneshfar, J. Biotechnol. 127 (2007)
.a, B.S. Abu, A. Arbakariya, M. Rosfarizan, A.R. Mohd Basyaruddin, n.z.tron. J. Biotechnol. 8 (3) (2005) 07173458.. Zimmermann, Biotechnol Bioeng. 74 (6) (2001) 483491.
Enzyme Kinetics. Behaviour and Analysis of Rapid Equilibrium andate Enzyme Systems, John Wiley & Sons, Inc., New York, NY, 1975.a, T. Asplund, P. Soininen, R. Laatikainen, S. Peltonen, Carbohydr.
(2004) 261267., A.K. Sugih, R. Manurung, F. Picchioni, L. Janssen, H.J. Heeres, Starch-
(2008) 667675., A.K. Sugih, R. Manurung, F. Picchioni, L. Janssen, H.J. Heeres, Starch-
(2009) 6980.
A kinetic study of starch palmitate synthesis by immobilized lipase-catalyzed esterification in solvent free system1 Introduction2 Material and methods2.1 Chemicals and enzyme2.2 Starch pretreatment2.3 Water activity pre-equilibration of reaction medium2.4 General procedure for lipase esterification2.5 Calculation of the Initial reaction rates2.6 Calculation of the conversion of palmitic acid
3 Results and discussions3.1 Effect of different catalysts3.2 Effect of initial water activity (aw)3.3 Effect of speed of agitation3.4 Effect of catalyst loading3.5 Effect of temperature on the initial rate and the conversions of palmitic acid3.6 Effect of mole ratio of substrate3.7 Kinetics and mechanism3.8 Reusability of catalyst3.9 1H NMR analyses
4 ConclusionsAcknowledgmentsReferences