a kinetic study of starch palmitate synthesis by immobilized lipase-catalyzed esterification in...

7
Journal of Molecular Catalysis B: Enzymatic 101 (2014) 73–79 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic jo ur nal home p age: www.elsevier.com/locate/molcatb 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 b State Key Laboratory for Oxo Synthesis & Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China 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 × C fatty-acid × C starch )/(C fatty-acid × C starch + 0.0156 × C starch + 2.3947 × C fatty-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 a w 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). 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 disad- vantages 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 com- mercial 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 1381-1177/$ see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2014.01.003

Upload: brian-dixon

Post on 30-Sep-2015

5 views

Category:

Documents


2 download

TRANSCRIPT

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

    Contents lists available at ScienceDirect

    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

  • 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

  • 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 (%)

  • 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.

  • 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.

  • 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).

  • 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