research article microwave assisted enzymatic kinetic...

Research ArticleMicrowave Assisted Enzymatic Kinetic Resolution of(plusmn)-1-Phenyl-2-propyn-1-ol in Nonaqueous Media

Saravanan Devendran and Ganapati D Yadav

Department of Chemical Engineering Institute of Chemical Technology Matunga Mumbai 400 019 India

Correspondence should be addressed to Ganapati D Yadav gdyadavyahoocom

Received 12 April 2013 Revised 20 October 2013 Accepted 2 December 2013 Published 23 February 2014

Academic Editor Luciana Rocha Barros Goncalves

Copyright copy 2014 S Devendran and G D Yadav This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Kinetic resolution of 1-phenyl-2-propyn-1-ol an important chiral synthon was studied through trans-esterification with acylacetate to investigate synergism between microwave irradiation and enzyme catalysis Lipases from different microbial originswere employed for the kinetic resolution of (RS)-1-phenyl-2-propyn-1-ol among which Candida antarctica lipase B immobilizedon acrylic resin (Novozym 435) was found to be the best catalyst in n-hexane as solvent Vinyl acetate was the most effectiveamong different acyl esters studied The effect of various parameters was studied in a systematic manner Definite synergismbetween microwave and enzyme was observed The initial rate was improved around 128 times under microwave irradiation thanconventional heating Under optimum conditions maximum conversion (4878) and high enantiomeric excess (9325) wereobtained in 2 h From modeling studies it is concluded that the reaction follows the Ping-Pong bi-bi mechanism with dead endalcohol inhibition Kinetic parameters were obtained by using nonlinear regressionThis process is green clean and easily scalableas compared to the chemical process

1 Introduction

Enantiomeric pure chemicals are needed for synthesis ofoptically active compounds that have significant marketvalues across a variety of industries [1] among which chiralsecondary alcohols have several applications in pharmaceu-tical and chemical industries such as chiral auxiliaries andthey can be easily derivatizedwith different functional groups[2] In comparison with classical chemical methods such aspreferential crystallization diastereomerization chromato-graphic separation and asymmetric reduction by chiralmetaloxides biocatalytic processes are broadly accepted as goodoptions to prepare optically pure secondary alcohols [3]Biocatalysts score over chemical processes because they oftenpossess high stereo- chemo- and regio-selectivity Biocat-alytic systems can be operated at mild operating conditionsthat reduce byproduct formations and are recyclable andeasily adoptable in nonaqueous and neoteric solvents Biocat-alytic processes are reported as simple and cost effective forthe synthesis of single enantiomeric compounds Howeverthe major drawback of these processes is the fact that they

are slow in nature and need to be intensified in order to meetindustrial requirements [4ndash6]

Among various biocatalytic paths lipase catalyzed kineticresolution of racemic mixtures have been favored for prepar-ing the optically active secondary alcohols through hydrolysisand transesterification reactions Lipases have inherent abilityto accept a broad range of substances and do not requireexpensive cofactors like NAD(P)H Lipases are readily avail-able from animal plant and microbial sources and remainactive in both organic and neoteric solvents like ionic liquidsand supercritical carbon dioxide (scCO

2) However alcohol

dehydrogenases catalyzed asymmetric reduction requiresexpensive cofactors for its catalytic activity and is also lessadoptable to nonaqueous media [7 8]

In recent years microwave irradiation a green and cleanalternative energy source has been employed to enhancethe reaction rates and selectivities for organic synthesis aswell as materials production [9 10] In the conventionalheating the rate of heat transfer from external heating sourceto reaction system depends on the thermal conductivity ofreaction vessel which might lead to higher temperature at

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 482678 9 pageshttpdxdoiorg1011552014482678

2 BioMed Research International

OH

CH +O

ONovozym 435

O

H

Vinyl acetate1-Phenylpropagyl alcoholAcetaldehyde

O

CH

O

OH

CH

+(R)-1-Phenylpropagyl acetate

(S)-1-Phenylpropagyl alcohol

n-hexane MW

CH3

Scheme 1 Lipase catalysed transesterification of (RS)-1-phenylpropagyl alcohol with vinyl acetate under microwave irradiation

vessel surface than the reaction mixture Hence it requireslonger equilibrium time and is difficult to control On thecontrary microwave irradiation results in efficient internalheating by direct coupling of microwave energy with polarmolecules such as solvents reagents or catalysts in thevessel Microwaves pass through the wall of reactor and areconverted into heat directly in bulk mass of material in thevessel It is irrespective of the thermal conductivity of vesselmaterial The temperature gradient observed in microwaveheating is exactly opposite to that in conventional heatingThus it reduces the reaction time [11] The synergism withmicrowave irradiation is found to enhance the reaction rate inenzymatic reactions [12]The rate of lipase catalyzed reactionis enhanced to several folds as compared to conventionalheating and the lipase is quite stable under microwaveirradiation [13 14]

Optically active 1-phenyl-2-propyn-1-o1 and its substi-tuted derivatives are important precursors for synthesis ofenantiomeric pure natural compounds and biologically activemolecules such as eicosanoids and macrolides antibiotics[15] They can also be used as starting materials for stereos-elective synthesis of polyhydroxylated compounds allenesbenzo[b]furan (1-benzofuran) and derivatives [16 17] Thereare a few studies reported on the synthesis of the single enan-tiomer of 1-phenyl-2-propyn-1-o1 by asymmetric hydrolysisby using microbes asymmetric chemical catalysis or lipasecatalyzed kinetic resolution under conventional heating [18ndash23] All these processes require either longer reaction timeor high enzyme loading and there is no information onkinetics of the reaction which is required for reactor designand scale-up In the current work microwave assisted lipasecatalyzed transesterification of (RS)-1-phenyl-2-propyn-1-o1was undertaken with vinyl acetate in nonaqueous media(Scheme 1) The effect of various parameters such as differentcommercially available immobilized lipases acyl donorssolvents agitation speed temperature catalyst loading andacyl donor concentration was studied systematically by vary-ing one parameter at a time Finally the mechanism wasproposed and kinetics were developed All results are noveland useful to scale up this process

2 Materials and Methods

21 Enzymes The following enzymes were received as giftsamples fromMsNovozymes AS (Bagsvaerd Denmark) (i)Novozym 435 Lipase B from Candida antarctica supportedon a macroporous acrylic resin with a water content of 1-2 (ww) and enzyme activity 10000 PLUg (ii) LipozymeRM-IM Lipase from Rhizomucor miehei supported on amacroporous anion exchange resin with a water content of2-3 (ww) and enzyme activity 6 BAUg and (iii) LipozymeTL IM Lipase fromThermomyces lanuginosus supported onporous silica granulates with water content 1-2 and enzymeactivity 175 IUg

22 Chemicals Hexane toluene cyclohexane isopropylether vinyl acetate and other analytical and HPLC gradereagents were purchased from Ms SD Fine ChemicalsPvt Ltd Mumbai India (plusmn)-1-Phenyl-2-propyn-1-ol vinylbutyrate and vinyl laurate were purchased from Sigma-Aldrich India Pvt Ltd Bangalore India All chemicalsand enzymes were used without any further modifica-tionpurification

23 General Experimental Setup

231 Conventional Heating The experimental setup con-sisted of a 3 cm id mechanically agitated glass reactor of50 cm3 capacity equipped with four baffles and a six-bladedturbine impeller The entire reactor assembly was immersedin a thermostatic water bath which was maintained ata predetermined temperature with an accuracy of plusmn1∘CA typical reaction mixture consisted of 0001mol racemicalcohol and 0001mol vinyl ester diluted to 15 cm3 with n-hexane as a solvent The reaction mass was agitated at 60∘Cfor 15min at a speed of 300 rpm and then 10mg of enzymewas added to initiate the reaction

232 Microwave Heating The studies were carried outin a commercial microwave reactor assembly (Discover

BioMed Research International 3

CEM-SP1245 model CEM Corp Matthews NC USA) atthe desired temperature The reactor was a 100mL capac-ity 45 cm id cylindrical glass vessel with a provision formechanical stirring A standard six-blade-pitched turbineimpeller of 15 cm diameter was used for agitation Howeverthe actual reactor volume exposed to the microwave irradi-ation was 45mL with 55 cm height The temperature in thereactor was computer controlled The quantities of reactantand enzyme used for microwave reaction procedure wereidentical to those used for conventional heating

24 Analysis Reaction progress and enantiomeric excess(ee) were monitored by periodic withdrawal of clear liquidsamples from the reaction mixture which were analyzedby high performance liquid chromatography (HPLC) (1260infinity series Agilent technologies CAUSA) equippedwithchiralpak- IB analytical column (250 times 46mm ID) (DaicelCorporation Japan and particle size 5120583m) Samples (10 120583L)were injected via autosampler The mobile phase consistedof n-hexane and isopropyl alcohol (90 10) and the flow ratewas maintained at 1mLsdotminminus1 A DAD detector was used at awavelength of 220 nm Retention time of (S) (R)-1-phenyl-2-propyn-1-ol and (R)-ester were 76min 65min and 44minrespectively

The enantioselectivity ratio (119864) and conversion (119888 )were calculated from the enantiomeric excess of the substrate(ee119904 ) and product (ee

119901 ) based on the following

119864 =

ln [(1 minus 119888) (1 minus ee119904)]

ln [(1 minus 119888) (1 + ee119904)]

(1)

where

119888 =ee119904

ee119904+ ee119901

ee119904=

119861(S) minus 119861(R)

119861(S) + 119861(R)

ee119901=

119876(S) minus 119876(R)

119876(S) + 119876(R)

(2)

where 119861(R) 119861(S)119876(R) and119876(S) denote area under the curve of

(R)-1-phenyl-2-propyn-1-ol (S)-1-phenyl-2-propyn-1-ol andtheir corresponding esters respectively

3 Results and Discussion

31 Comparison of Microwave Irradiation and ConventionalHeating In order to compare the effect of microwave irra-diation and conventional heating the enzymatic resolutionof (plusmn)-1-phenyl-2-propyn-1-ol was performed in both modesusing vinyl acetate as the model acyl donor and identicalreaction conditions It was observed that the reaction ratewas increased about 128-fold from 116 times 10minus5molsdotdmminus3sdotsminus1to 149times 10minus5molsdotdmminus3sdotsminus1 undermicrowave irradiationThe119864 and ee

119904under microwave irradiation increased by 161- and

127-fold respectively The final conversion of 4878 with 119864and ee

119904of 334 and 9325 respectively was obtained within

0

10

20

30

40

50

60

70

80

90

100

OA BA EA IPA MA VA VB VLAcyl donor

ees

or co

nver

sion

()

Conversionees

Figure 1 Effect of acyl donor (Reaction condition 1-phenyl-2-propyn-1-olmdash0001mol acyl donormdash0001mol n-hexane upto 15 cm3 speed of agitationmdash300 rpm catalyst loadingmdash10mgtemperaturemdash60∘C OAmdashOctyl acetate BAmdashButyl acetate IPAmdashIsopropyl acetate EAmdashEthyl acetate MAmdashMethyl acetate VAmdashVinyl acetate VBmdashVinyl butyrate and VLmdashVinyl laurate)

2 h under microwave irradiation While in conventionalheating a conversion of 4285 with 119864 and ee

119904of 207

and 7333 respectively was obtained Under microwaveirradiation the ions or dipole of the reactants align in theapplied electric field As the applied field oscillates the dipoleor ion field tries to realign itself with the alternating electricfield and in the process energy is released in the form ofheat through molecule collisions and dielectric loss [24] Thequantity of heat released is directly related to the ability ofthe matrix to align itself with the frequency of the appliedfield Microwave irradiation leads to efficient in situ heatingresulting in uniform distribution of temperature throughoutthe reaction mass It is thus likely that minor conformationalalterations in enzyme structure can also improve access ofreactant to the active site as compared to conventionalheating [25] A control experiment in the absence of enzymedid not show any conversion which indicated that theremust be a definite synergism between enzyme catalysis andmicrowave irradiation Thus all further studies were carriedout using microwave irradiation

32 Effect of Different Catalysts In order to compare theactivities of three different commercially available immo-bilized lipases such as Novozym 435 (Candida antarcticalipase B) Lipozyme RMIM (Rhizomucor miehei lipase)and Lipozyme TLIM (Thermomyces lanuginosus lipase) forresolution of (plusmn)-1-phenyl-2-propyn-1-ol under microwaveirradiation transesterification reaction was performed usingvinyl acetate as the model acyl donor under similar condi-tions (Figure 1) It was observed that Novozym 435 showedhighest activity among the different catalyst employed in this


0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

Di-isopropyl ethern-hexane

CyclohexaneToluene

Figure 2 Effect of solvent (Reaction condition 1-phenyl-2-propyn-1-olmdash0001 vinyl acetatemdash0001mol solvent up to 15 cm3 speed ofagitationmdash300 rpm catalyst loadingmdash10mg temperaturemdash60∘C)

reaction With Novozym 435 4878 conversion and 933ee119904were obtained While Lipozyme RMIM and Lipozyme

TLIM gave conversions of only 637 and 44 respectivelyIt is reported that both Lipozyme RM IM and LipozymeTL IM are mainly used to interesterify bulk molecules suchas fatty acid derivatives and they have less activity in theresolution of racemic molecules [26] Hence further studywas carried out using Novozym 435 as the catalyst

33 Effect of Acyl Donors Acyl donors have the ability tochange the enzyme activity and selectivity in nonaqueousmedium [27 28] Effect of different acyl donors was studiedby using 1 1 mole ratio of racemic alcohol to acyl donorwith 10mg enzyme loading at 60∘C in n-hexane as solventfor 2 h The liquid phase volume was made up to 15 cm3under microwave irradiation (Figure 2) The acyl donorswere methyl acetate (MA) ethyl acetate (EA) isopropylacetate (IPA) butyl acetate (BA) octyl acetate (OA) vinylacetate (VA) vinyl butyrate (VB) and vinyl laurate (VL)It was observed that both enantioselectivity and conversionincreased with increase in alcoholic carbon chain length ofalkyl ester from methyl to isopropyl acetate whereas furtherincrease in alcoholic chain length from C4 to C8 led todecrease in conversion and enantioselectivity of reactionAs compared to alkyl esters vinyl esters gave good conver-sion and enantioselectivity Vinyl alcohol produced duringreaction is instantaneously and irreversibly tautomerized toacetaldehyde which escapes due to its low boiling point andthus there is no inhibition by the coproduct vinyl alcoholIncreasing the chain length of vinyl esters has not shown anysignificant differences in conversion and enantioselectivityof reaction Maximum conversion (4878) and enantiose-lectivity (9325) were obtained with vinyl acetate vis-a-vis

other vinyl esters Thus further studies were conducted byusing vinyl acetate as the acyl donor

34 Effect of Reaction Media A proper selection of organicmedia is necessary for any enzymatic reaction It has beenwell reported that organic solvents have the ability to alterthe enzyme conformational structure that leads to changesin their activity and also regio- and stereo-selectivity Thusproper selection of solvent is the most important parameterfor lipase catalyzed reactions [29] A number of experimentswere performed to study the effect of solvents such ashexane (log119875 = 36) cyclohexane (log119875 = 32) toluene(log119875 = 25) and diisopropyl ether (log119875 = 2) undersimilar conditions (Figure 2) Highest conversion (4878)and enantiomeric excess (ee

119904= 9325) were observed

when n-hexane was employed as the solvent whereas lowconversion (2245) and enantiomeric excess (ee

119904= 2896)

were observed in di-isopropyl ether which has the lowestlog119875 value among the solvents used in this study On thecontrary conversions of 4537 and 4193 were obtainedwith cyclohexane and toluene respectively It has beenreported that a tiny water layer around the enzyme particleis necessary to maintain its structure and activity Solventshaving high log119875 are hydrophobic in nature but do not stripthewater layer around enzyme particle and its structure is notaltered [30] Thus they show high enzyme activity Furtherwork was carried out using n-hexane as the solvent

35 Effect of Speed of Agitation In immobilized enzymecatalyzed reactions the reactants have to transport from bulkmixture into enzyme active site in the porous particle throughconvection and diffusion processes [31] The magnitudes ofexternal mass transfer resistance and intraparticle diffusionlimitation play a crucial role in these processes Externalmasstransfer resistance can be overcome by choosing appropriatespeed of agitation A number of experiments were performedin the range of 100 to 400 rpm by taking 0001mol racemicalcohol and vinyl acetate each and 10mg Novozym 435 madeup to 15 cm3 with n-hexane at 60∘C (Figure 3) Both theconversion and rate of reaction increased with an increase ofagitation speed from 100 to 300 rpm There was a marginalchange in conversion and rate of reaction at 400 rpm Thisindicated that there was no external mass transfer limitationabove 300 rpm The external mass transfer and intraparticlediffusion rates were further evaluated by comparing the timeconstants for reaction (119905

119903) and diffusion (119905

119889) using theoretical

calculations These are defined as follows 119905119903= 1198620119903(1198620) and

119905119889= 119863S(119896SL)

2 if 119905119903≫ 119905119889means that the reaction was

not mass transfer controlled [32] Both 1198620and 119903(119862

0) were

determined experimentally and their values were obtained as006667molsdotdmminus3 and 14916 times 10minus5molsdotdmminus3sdotsminus1 Diffusiv-ity of racemic alcohol at 60∘C was calculated using Wilke-Chang equation as 51063 times 10minus5 cm2 sminus1 [33] The averagediameter of the support particle was taken as 006 cm sincethe particle size ranged between 003 and 009 cm The valueof mass transfer coefficient of liquid phase was calculatedby using Sherwood number of 2 to be 05301 cmsdotsminus1 Fromthese values the calculated time constants for reaction and


0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

100RPM200RPM

300RPM400RPM

Figure 3 Effect of agitation speed (Reaction condition 1-phenyl-2-propyn-1-olmdash0001 vinyl acetatemdash0001mol n-hexane up to15 cm3 speed of agitation 100ndash400 rpm catalyst loadingmdash10mgtemperaturemdash60∘C)

diffusion were 4469 times 103 s and 1817 times 10minus4 s The timeconstant for the reaction was much higher than diffusionindicating that there was no external mass transfer resistance

Further it is necessary to rule out the intraparticlediffusion limitation It could be done by comparing the rateof substrate diffusion per unit interfacial area (119896SL1198620) withthe reaction rate per unit area (120593119903

0119886) where 120593 is the phase

volume ratio and ldquo119886rdquo is the interfacial area per unit volumeof organic phase [32] Assuming the particle was spherical120593119886 = 119877

1199013 where 119877

119901is the radius of the particle It was

found that the value of 119896SL1198620 = 00353molsdotcmminus2sdotsminus1 and1205931199030119886 = 1492 times 10

minus7molsdotcmminus2sdotsminus1 It indicated that therate of substrate diffusion per unit interfacial area was muchhigher than the reaction rate per unit area This suggestedthat there was no intraparticle diffusion limitation Thusthe reaction was controlled by intrinsic enzyme kineticsTherefore further experiments were performed at a speed of300 rpm

36 Effect of Temperature It has been reported in mostof the literature that temperature plays a critical role inany enzyme catalyzed reaction It will have an effect onthe enzyme activity enantioselectivity and rate of reactionbecause temperature has pronounced effect on viscositysolubility and diffusivity of reactants and products Howeverat high temperature the changes in conformation of proteinstructure will affect their activity and selectivity [34] Severalexperiments were performed in the range of 40 to 70∘Cunder similar conditions (Figure 4) It was observed that bothconversion and rate of reaction increased with increase intemperature up to 60∘C beyondwhich there was no change inconversionThe enzyme particlesmay either get denatured or

0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

40∘C50∘C

60∘C70∘C

Figure 4 Effect of temperature (Reaction condition 1-phenyl-2-propyn-1-olmdash0001 vinyl acetatemdash0001mol n-hexane upto 15 cm3 speed of agitation 300 rpm catalyst loadingmdash10mgtemperaturemdash40ndash70∘C)

intraparticle diffusion resistance sets in Further Arrheniusplot was obtained by plotting the ln (initial rate) versus 1119879for both modes of heating and activation energies obtainedfor microwave and conventional heating were 1739 kJsdotmolminus1and 1593 kJsdotmolminus1 respectively These values are similar tothose reported for most of the enzymatic reactions [35] Inthe case of enantioselectivity it was observed that the 119864value increases as temperature increased from 40 to 70∘CIt has been reported that enantioselectivity is dependenton reaction temperature and is controlled by enthalpy andentropy of reaction [36] The following equations were usedto relate these terms

ln119864 = minusΔ119877minus119878Δ119867Dagger

119877

sdot1

119879

+Δ119877minus119878Δ119878Dagger

119877

Δ119877minus119878Δ119866Dagger= minus119877119879 ln (119864)

(3)

The enthalpy and entropy values were calculated by plottingthe ln(119864) values against the reciprocal reaction temperature(Figure 5) The values of enthalpy and entropy obtainedwere 9406 kJsdotmolminus1 and 7593 Jsdotmolminus1sdotKminus1 respectively Thedifference in transition state energy of both enantiomers(Δ119877minus119878Δ119866Dagger) is minus1588 kJmolminus1 From these values it was

observed that the entropy value was much higher thanenthalpy of system and it favors the maximum enantioselec-tivity at high temperature [37] Since 119864 value was found tobe the highest at 60∘C while maintaining a higher enzymeactivity 60∘Cwas selected as the optimum temperatureThusfurther experiments were conducted at this temperature

37 Effect of Enzyme Loading To select the appropriateamount of enzyme loading several experiments were per-formed in the range from 5 to 15mg enzyme loading under


54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)

Figure 5 Graphical determination of Δ119877minus119878Δ119867Dagger and Δ

119877minus119878Δ119878Dagger

0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg

Figure 6 Effect of enzyme loading (Reaction condition 1-phenyl-2-propyn-1-olmdash0001 vinyl acetatemdash0001mol n-hexane up to15 cm3 speed of agitation 300 rpm catalyst loadingmdash5ndash15mgtemperaturemdash60∘C)

similar conditions The conversion and rate of reactionincreased with increase in enzyme loading up to 10mgabove this loading value there were no substantial changesin conversion and rate (Figure 6) This would suggest thatfurther addition of enzyme has no effect and it indicated thatexternal mass transfer had limited the rate [29] Further weobserved that the initial rate increased linearly with enzymeloading up to 10mg which clearly indicated that the reactionwas kinetically controlled Thus further experiments werecarried out at 10mg as optimum enzyme loading

0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4

Figure 7 Effect of acyl donor concentration (Reaction condition1-phenyl-2-propyn-1-olmdash0001 vinyl acetatemdash0001ndash0004mol n-hexane up to 15 cm3 speed of agitation 300 rpm catalyst loadingmdash10mg temperaturemdash60∘C)

38 Effect of Acyl Donor Concentration A number of exper-iments were carried out to analyze the effect of acyl donorconcentration on the rate and conversion in the resolutionof (plusmn)-1-phenyl-2-propyn-1-ol at 60∘C with 10mg of enzymeloading in n-hexane The concentration of (plusmn)-1-phenyl-2-propyn-1-ol was kept constant (0001mol) and vinyl acetateconcentration varied from 0001 to 0004mol the mix-ture volume was kept constant at 15 cm3 by adjusting theamount of n-hexane addition It was found that there wereinsignificant changes in conversion and rate of reaction withincrease concentration of vinyl acetate (Figure 7) It might bepossible that at high concentration the interaction betweenacyl donor and enzyme active site increases which leads toreversible competition with alcohol at the active site Thusfurther reactions were conducted with 0001 mole of vinylacetate

39 Catalyst Reusability After completion of each run thecatalyst particles were filtered using a membrane filter Mul-tiple washes were given to the catalyst with fresh solvent (n-hexane) dried at room temperature for 12 h and reused Itwas found that there was a marginal decrease in conversionfrom 4878 to 4522 after three reuses which was due to lossof enzyme during filtration (sim2-3) and drying Nomake-upquantity was added Thus the enzyme was reusable Whenthemake-up quantity was added almost the same conversionwas obtained

310 KineticModel Several mechanisms have been proposedfor lipase catalyzed reactions (eg ordered bi-bi mechanismping-pong bi-bi mechanism) However ping-pong bi-bimechanismwas well adopted formost of the lipases catalyzed


0

200

400

600

800

1000

1200

1400

0 5 10 15 20 25 30 351vinyl acetate concentration (molminus1middotdm3)

1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M

Figure 8 Lineweaver-Burk plot (Reaction condition 1-phenyl-2-propyn-1-olmdash00333ndash01333M vinyl acetatemdash00333ndash01333M n-hexane up to 15 cm3 speed of agitation 300 rpm catalyst loadingmdash10mg temperaturemdash60∘C)

reactions The first step of this mechanism is formation ofan acyl-enzyme intermediate and the product is releasedbetween additions of substrates [38] In the ordered bi-bimechanism the enzyme forms a ternary complex with bothsubstrates and subsequently releases the products [26] Thusit was desirable to investigate the mechanism for enantiose-lective transesterification of (plusmn)-1-phenyl-2-propyn-1-ol withvinyl acetate For determination of initial rates a number ofexperiments were carried out by changing the concentrationsof both alcohol (00005ndash0002M) and vinyl acetate (00005ndash0002M) over a wide range under similar conditions Initialrates were calculated systematically from the linear portion ofthe concentration-time profiles The Lineweaver-Burk plotsof reciprocal rate versus reciprocal concentration of vinylacetate were made (Figure 8) From the plot it is observedthat there are no crossings of lines which rules out thepossibility of ternary mechanism At low concentrations ofsubstrates the slope of the lines is not influenced by theconcentration of the fixed substrate This is indicative of amechanism that requires the dissociation of one productbefore the association of the second substrate to the enzyme-substrate complexThe curved shape at higher concentrationsof 1-phenyl-2-propyn-1-ol can be used as an indication forformation of enzyme dead end complex with alcohol Theproposed mechanism is as follows

119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)

Table 1Values of kinetic parameters for transesterification reaction

Kinetic parameter Value119881119898(molsdotdmminus3sdotsminus1) 2508 times 10

minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861

(molsdotdmminus3) 08863119870119894(molsdotdmminus3) 000265

Inhibition steps by 1-phenyl-2-propyn-1-ol

119864 + 119861

119896119894

999448999471 119864119894119861 (5)

From these observations a ping-pong bi-bi mechanism withdead end alcohol inhibition was postulated These assump-tions are used to develop a reaction mechanism which isdepicted in Clelandrsquos notation as shown in Figure 10

By analogy to the classical mechanism of lipase catalysisit is assumed that vinyl acetate (119860) binds first to the freeenzyme (119864) and forms a noncovalent enzyme acetate complex(119864119860) which releases the first product aldehyde (119875) and (1198641015840)modified enzyme The second substrate alcohol (119861) reactswith activated enzyme (1198641015840) to give the complex (1198641015840119861) whichgives the product ester (119876) and free enzyme (119864) Along withthis alcohol (119861) also forms the dead end complex (119864

119894119861) by

binding to free enzyme (119864) Here 119861 is the R-isomerThe kinetic representing this model for initial reaction

rate can be expressed as

V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)

whereas V is the rate of reaction V119898the maximum rate of

reaction [119860] the initial concentration of vinyl acetate [119861] theinitial concentration of (R)-1-phenyl-2-propyn-1-ol and119870

119898119860

the Michaelis constant for vinyl acetate as (S)-enantiomerof alcohol remains unreacted during the reaction changesin concentration of (S)-enantiomer is neglected 119870

119898119861the

Michaelis constant for (R)-1-phenyl-2-propyn-1-ol and119870119894the

inhibition constant for (R)-1-phenyl-2-propyn-1-ol The ini-tial rate data were used to determine the kinetic parameters ofabove mechanism by nonlinear regression analysis using thesoftware package Polymath 51 (Table 1) The initial rates ofreaction for different reactant concentration were simulatedusing above equation to verify the proposed kinetic modelThe parity plot between simulated rate and experimental ratesuggested that the proposed mechanism was valid for thisreaction (Figure 9)

4 Conclusion

In this work lipase catalyzed kinetic resolution of 1-phenyl-2-propyn-1-ol using vinyl acetate as acyl donor was studiedundermicrowave irradiation Among the employed catalystsNovozym 435 was found to be the most effective cata-lyst in n-hexane as solvent There was synergism betweenmicrowave irradiation and lipase Both conversion and rateof reaction were increased under microwave as comparedto conventional heating Microwave irradiation leads to


0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)

Experimental rate (molmiddotdmminus3middotsminus1)

y = 10225x

R2 = 09854

Figure 9 Parity plot of experimental versus simulated rates

E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10

increase the affinity of the substrate toward the active site oflipase Further microwaves enhance the collision frequencyof reactant molecules that leads to increase in entropy ofthe system The effect of various parameters was studied onconversion and rate of reactionThemaximum conversion of4878 was obtained in 2 h using 10mg enzyme loading withequimolar concentration of alcohol and ester at 60∘C undermicrowave irradiation From the progress curve analysis itwas found that the reaction followed the ping-pong bi-bimechanism with dead end inhibition of alcohol The kineticparameters were refined by using nonlinear regressionTherewas a good fit between experimental and simulated ratesThe preparation of chiral secondary alcohols using lipasecatalyzed kinetic resolution is mild and clean as compared tochemical process

Nomenclature

119860 Initial concentration of vinyl acetatemolsdotdmminus3

119861 Initial concentration of(R)-1-phenyl-2-propyn-1-ol molsdotdmminus3

119864 Free enzyme119864119860 Enzyme-acyl complex with 1198601198641015840119861 Modified enzyme-alcohol complex119864119894119861 Dead end enzyme-alcohol complex119870119894 Inhibition constant for

(R)-1-phenyl-2-propyn-1-ol molsdotdmminus3119870119898119860

Michaelis constant for vinyl acetatemolsdotdmminus3

119870119898119861

Michaelis constant for(R)-1-phenyl-2-propyn-1-ol molsdotdmminus3

119875 Acetaldehyde119876 EsterV Rate of reaction molsdotdmminus3sdotsminus1V119898 Maximum rate of reaction molsdotdmminus3sdotsminus1

Δ119877minus119878Δ119867Dagger Differential activation enthalpy

Δ119877minus119878Δ119878Dagger Differential activation entropy

Δ119877minus119878Δ119866Dagger Difference in transition state energy of

both enantiomers119879 Temperature (K)119877 Gas constant (Jsdotmolminus1sdotKminus1)119864 Enantiomeric ratioee119904 Enantiomeric excess for substrate

ee119901 Enantiomeric excess for product

119888 Conversion

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

SaravananDevendran received JRF fromUGCunder itsMer-itorious Fellowship BSR programme (CAS in Food Engineer-ing and Technology) Ganapati D Yadav received supportfrom R T Mody Distinguished Professor Endowment and JC Bose National Fellowship of Department of Science andTechnology Government of India The authors are thankfulto Novo Nordisk Denmark for providing lipases as giftsample

References

[1] R A Sheldon Chirotechnology Industrial Synthesis of OpticallyActive Compounds Marcel Dekker New York NY USA 1993

[2] R N Patel ldquoBiocatalytic synthesis of chiral pharmaceuticalintermediatesrdquo Food Technology and Biotechnology vol 42 no4 pp 305ndash325 2004

[3] A Ghanem and H Y Aboul-Enein ldquoLipase-mediated chiralresolution of racemates in organic solventsrdquo Tetrahedron vol15 no 21 pp 3331ndash3351 2004

[4] G D Yadav and K M Devi ldquoEnzymatic synthesis of perlauricacid using Novozym 435rdquo Biochemical Engineering Journal vol10 no 2 pp 93ndash101 2002

[5] G D Yadav and S Devendran ldquoLipase catalyzed synthesisof cinnamyl acetate via transesterification in non-aqueousmediumrdquo Process Biochemistry vol 47 no 3 pp 496ndash502 2012

[6] G D Yadav A D Sajgure and S B Dhoot ldquoEnzyme catalysisin fine chemical and pharmacuetical industriesrdquo in EnzymeMixtures and Complex Biosynthesis S K Bhattacharya Ed pp79ndash108 Landes Biosciences Austin Tex USA 2007

[7] J Durand E Teuma andMGomez ldquoIonic liquids as amediumfor enantioselective catalysisrdquo Comptes Rendus Chimie vol 10no 3 pp 152ndash177 2007

[8] R A Sheldon ldquoGreen solvents for sustainable organic synthesisstate of the artrdquoGreen Chemistry vol 7 no 5 pp 267ndash278 2005


[9] B LHayesMicrowave Synthesis Chemistry at the Speed of LightCEM Matthews NC USA 2002

[10] A Loupy Ed Microwaves in Organic Synthesis Wiley-VCHWeinheim Germany 2002

[11] G D Yadav and P R Sowbna ldquoModeling of microwave irra-diated liquid-liquid-liquid (MILLL) phase transfer catalyzedgreen synthesis of benzyl thiocyanaterdquo Chemical EngineeringJournal vol 179 pp 221ndash230 2012

[12] D Yu H Wu A Zhang et al ldquoMicrowave irradiation-assistedisomerization of glucose to fructose by immobilized glucoseisomeraserdquo Process Biochemistry vol 46 no 2 pp 599ndash6032011

[13] B Rejasse S Lamare M-D Legoy and T Besson ldquoStabilityimprovement of immobilized Candida antarctica lipase B inan organic medium under microwave radiationrdquo Organic ampBiomolecular Chemistry vol 2 no 7 pp 1086ndash1089 2004

[14] G D Yadav and P S Lathi ldquoSynergism betweenmicrowave andenzyme catalysis in intensification of reactions and selectivitiestransesterification ofmethyl acetoacetatewith alcoholsrdquo Journalof Molecular Catalysis A vol 223 no 1-2 pp 51ndash56 2004

[15] W R Roush and R J Sciotti ldquoEnantioselective total synthesis of(minus)-chlorothricoliderdquo Journal of the American Chemical Societyvol 116 no 14 pp 6457ndash6458 1994

[16] X Ariza J Garcia Y Georges and M Vicente ldquo1-phenylprop-2-ynyl acetate a useful building block for the stereoselectiveconstruction of polyhydroxylated chainsrdquo Organic Letters vol8 no 20 pp 4501ndash4504 2006

[17] S Peng LWang and JWang ldquoIron-catalyzed ene-type propar-gylation of diarylethylenes with propargyl alcoholsrdquo Organic ampBiomolecular Chemistry vol 10 no 2 pp 225ndash228 2012

[18] B I Glanzer K Faber and H Griengl ldquoEnantioselectivehydrolyses by bakerrsquos yeastmdashIII microbial resolution of alkynylesters using lyophilized yeastrdquo Tetrahedron vol 43 no 24 pp5791ndash5796 1987

[19] S-K Kang T Yamaguchi S-J Pyun Y-T Lee and T-GBaik ldquoPalladium-catalyzed arylation of 120572-allenic alcohols withhypervalent iodonium salts synthesis of epoxides and diolcyclic carbonatesrdquo Tetrahedron Letters vol 39 no 15 pp 2127ndash2130 1998

[20] CWaldinger M Schneider M Botta F Corelli and V SummaldquoAryl propargylic alcohols of high enantiomeric purity via lipasecatalyzed resolutionsrdquo Tetrahedron vol 7 no 5 pp 1485ndash14881996

[21] D Xu Z Li and S Ma ldquoNovozym-435-catalyzed enzymaticseparation of racemic propargylic alcohols A facile route tooptically active terminal aryl propargylic alcoholsrdquo TetrahedronLetters vol 44 no 33 pp 6343ndash6346 2003

[22] C Raminelli J V Comasseto L H Andrade and A L MPorto ldquoKinetic resolution of propargylic and allylic alcohols byCandida antarctica lipase (Novozyme 435)rdquoTetrahedron vol 15no 19 pp 3117ndash3122 2004

[23] P Chen and X Zhu ldquoKinetic resolution of propargylic alcoholsvia stereoselective acylation catalyzed by lipase PS-30rdquo Journalof Molecular Catalysis B vol 97 pp 184ndash188 2013

[24] G D Yadav and S Devendran ldquoLipase catalyzed kineticresolution of (plusmn)-1-(1-naphthyl) ethanol under microwave irra-diationrdquo Journal of Molecular Catalysis B vol 81 pp 58ndash652012

[25] P Mazo L Rios D Estenoz and M Sponton ldquoSelf-esterification of partially maleated castor oil using conventionalandmicrowave heatingrdquoChemical Engineering Journal vol 185-186 pp 347ndash351 2012

[26] G D Yadav and P Sivakumar ldquoEnzyme-catalysed opticalresolution ofmandelic acid viaRS(∓)-methylmandelate in non-aqueous mediardquo Biochemical Engineering Journal vol 19 no 2pp 101ndash107 2004

[27] T Ema SMaeno Y Takaya T Sakai andMUtaka ldquoSignificanteffect of acyl groups on enantioselectivity in lipase-catalyzedtransesterificationsrdquo Tetrahedron Asymmetry vol 7 no 3 pp625ndash628 1996

[28] TMiyazawa S Kurita SUeji T Yamada and S Kuwata ldquoReso-lution of mandelic acids by lipase-catalysed transesterificationsin organic media inversion of enantioselectivity mediatedby the acyl donorrdquo Journal of the Chemical Society PerkinTransactions 1 no 18 pp 2253ndash2255 1992

[29] K Nakamura Y Takebe T Kitayama and A Ohno ldquoEffectof solvent structure on enantioselectivity of lipase-catalyzedtransesterificationrdquoTetrahedron Letters vol 32 no 37 pp 4941ndash4944 1991

[30] L A S Gorman and J S Dordick ldquoOrganic solvents strip wateroff enzymesrdquo Biotechnology and Bioengineering vol 39 no 4pp 392ndash397 1992

[31] G D Yadav and A H Trivedi ldquoKinetic modeling ofimmobilized-lipase catalyzed transesterification of n-octanolwith vinyl acetate in non-aqueous mediardquo Enzyme and Micro-bial Technology vol 32 no 7 pp 783ndash789 2003

[32] R H Perry and D W Green Eds Perryrsquos Chemical EngineersrsquoHandbook McGraw-Hill New York NY USA 1984

[33] C R Wilke and P Chang ldquoCorrelation of diffusion coefficientsin dilute solutionsrdquo AIChE Journal vol 1 no 2 pp 264ndash2701955

[34] Y Yasufuku and S-I Ueji ldquoEffect of temperature on lipase-catalyzed esterification in organic solventrdquo Biotechnology Let-ters vol 17 no 12 pp 1311ndash1316 1995

[35] I H Segel Enzyme Kinetics Behavior and Analysis ofRapid Equilibrium and Steady State Enzyme Systems Wiley-Interscience New York NY USA 1975

[36] R S Phillips ldquoTemperature effects on stereochemistry ofenzymatic reactionsrdquo Enzyme andMicrobial Technology vol 14no 5 pp 417ndash419 1992

[37] J Ottosson J C Rotticci-Mulder D Rotticci and K HultldquoRational design of enantioselective enzymes requires consid-erations of entropyrdquo Protein Science vol 10 no 9 pp 1769ndash17742001

[38] S H Krishna and N G Karanth ldquoLipase-catalyzed synthesis ofisoamyl butyrate a kinetic studyrdquo Biochimica et Biophysica Actavol 1547 no 2 pp 262ndash267 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


OH

CH +O

ONovozym 435

O

H

Vinyl acetate1-Phenylpropagyl alcoholAcetaldehyde

O

CH

O

OH

CH

+(R)-1-Phenylpropagyl acetate

(S)-1-Phenylpropagyl alcohol

n-hexane MW

CH3

Scheme 1 Lipase catalysed transesterification of (RS)-1-phenylpropagyl alcohol with vinyl acetate under microwave irradiation

vessel surface than the reaction mixture Hence it requireslonger equilibrium time and is difficult to control On thecontrary microwave irradiation results in efficient internalheating by direct coupling of microwave energy with polarmolecules such as solvents reagents or catalysts in thevessel Microwaves pass through the wall of reactor and areconverted into heat directly in bulk mass of material in thevessel It is irrespective of the thermal conductivity of vesselmaterial The temperature gradient observed in microwaveheating is exactly opposite to that in conventional heatingThus it reduces the reaction time [11] The synergism withmicrowave irradiation is found to enhance the reaction rate inenzymatic reactions [12]The rate of lipase catalyzed reactionis enhanced to several folds as compared to conventionalheating and the lipase is quite stable under microwaveirradiation [13 14]

Optically active 1-phenyl-2-propyn-1-o1 and its substi-tuted derivatives are important precursors for synthesis ofenantiomeric pure natural compounds and biologically activemolecules such as eicosanoids and macrolides antibiotics[15] They can also be used as starting materials for stereos-elective synthesis of polyhydroxylated compounds allenesbenzo[b]furan (1-benzofuran) and derivatives [16 17] Thereare a few studies reported on the synthesis of the single enan-tiomer of 1-phenyl-2-propyn-1-o1 by asymmetric hydrolysisby using microbes asymmetric chemical catalysis or lipasecatalyzed kinetic resolution under conventional heating [18ndash23] All these processes require either longer reaction timeor high enzyme loading and there is no information onkinetics of the reaction which is required for reactor designand scale-up In the current work microwave assisted lipasecatalyzed transesterification of (RS)-1-phenyl-2-propyn-1-o1was undertaken with vinyl acetate in nonaqueous media(Scheme 1) The effect of various parameters such as differentcommercially available immobilized lipases acyl donorssolvents agitation speed temperature catalyst loading andacyl donor concentration was studied systematically by vary-ing one parameter at a time Finally the mechanism wasproposed and kinetics were developed All results are noveland useful to scale up this process

2 Materials and Methods

21 Enzymes The following enzymes were received as giftsamples fromMsNovozymes AS (Bagsvaerd Denmark) (i)Novozym 435 Lipase B from Candida antarctica supportedon a macroporous acrylic resin with a water content of 1-2 (ww) and enzyme activity 10000 PLUg (ii) LipozymeRM-IM Lipase from Rhizomucor miehei supported on amacroporous anion exchange resin with a water content of2-3 (ww) and enzyme activity 6 BAUg and (iii) LipozymeTL IM Lipase fromThermomyces lanuginosus supported onporous silica granulates with water content 1-2 and enzymeactivity 175 IUg

22 Chemicals Hexane toluene cyclohexane isopropylether vinyl acetate and other analytical and HPLC gradereagents were purchased from Ms SD Fine ChemicalsPvt Ltd Mumbai India (plusmn)-1-Phenyl-2-propyn-1-ol vinylbutyrate and vinyl laurate were purchased from Sigma-Aldrich India Pvt Ltd Bangalore India All chemicalsand enzymes were used without any further modifica-tionpurification

23 General Experimental Setup

231 Conventional Heating The experimental setup con-sisted of a 3 cm id mechanically agitated glass reactor of50 cm3 capacity equipped with four baffles and a six-bladedturbine impeller The entire reactor assembly was immersedin a thermostatic water bath which was maintained ata predetermined temperature with an accuracy of plusmn1∘CA typical reaction mixture consisted of 0001mol racemicalcohol and 0001mol vinyl ester diluted to 15 cm3 with n-hexane as a solvent The reaction mass was agitated at 60∘Cfor 15min at a speed of 300 rpm and then 10mg of enzymewas added to initiate the reaction

232 Microwave Heating The studies were carried outin a commercial microwave reactor assembly (Discover






119864 =

ln [(1 minus 119888) (1 minus ee119904)]

ln [(1 minus 119888) (1 + ee119904)]

(1)

where

119888 =ee119904

ee119904+ ee119901

ee119904=

119861(S) minus 119861(R)

119861(S) + 119861(R)

ee119901=

119876(S) minus 119876(R)

119876(S) + 119876(R)

(2)








0

10

20

30

40

50

60

70

80

90

100


ees

or co

nver

sion

()

Conversionees



119904of 207




0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)


CyclohexaneToluene









119904= 2896)






119905119889= 119863S(119896SL)



0) were



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

100RPM200RPM

300RPM400RPM






1199013 where 119877





0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

40∘C50∘C

60∘C70∘C




119877

sdot1

119879


119877


(3)





54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4






0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






119864 =

ln [(1 minus 119888) (1 minus ee119904)]

ln [(1 minus 119888) (1 + ee119904)]

(1)

where

119888 =ee119904

ee119904+ ee119901

ee119904=

119861(S) minus 119861(R)

119861(S) + 119861(R)

ee119901=

119876(S) minus 119876(R)

119876(S) + 119876(R)

(2)








0

10

20

30

40

50

60

70

80

90

100


ees

or co

nver

sion

()

Conversionees



119904of 207




0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)


CyclohexaneToluene









119904= 2896)






119905119889= 119863S(119896SL)



0) were



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

100RPM200RPM

300RPM400RPM






1199013 where 119877





0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

40∘C50∘C

60∘C70∘C




119877

sdot1

119879


119877


(3)





54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4






0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)


CyclohexaneToluene









119904= 2896)






119905119889= 119863S(119896SL)



0) were



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

100RPM200RPM

300RPM400RPM






1199013 where 119877





0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

40∘C50∘C

60∘C70∘C




119877

sdot1

119879


119877


(3)





54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4






0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

100RPM200RPM

300RPM400RPM






1199013 where 119877





0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

40∘C50∘C

60∘C70∘C




119877

sdot1

119879


119877


(3)





54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4






0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


54

55

56

57

58

59

6

61

00027 00028 00029 0003 00031 00032 00033

y = minus1131446x + 9133

R2 = 0944

1T (Kminus1)

ln(E)



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

5mg75mg10mg

125mg15mg



0

5

10

15

20

25

30

35

40

45

50

0 15 30 45 60 75 90 105 120 135

Con

vers

ion

()

Time (min)

1 11 2

1 31 4






0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

200

400

600

800

1000

1200

1400


1in

itial

rate

(mol

minus1middotd

m3middots)

00333M00666M

00999M01333M



119864 + 119860

1198961

999448999471

119896minus1

119864119860

119864119860

1198962

999448999471

119896minus2

119875 + 1198641015840

1198641015840+ 119861

1198963

999448999471

119896minus3

1198641015840119861

1198641015840119861

1198964

999448999471

119896minus4

119864119876

119864119876

119870119901

997888997888997888997888997888rarr 119876 + 119864

(4)



minus2

119870119898119860

(molsdotdmminus3) 00019119870119898119861



119864 + 119861

119896119894

999448999471 119864119894119861 (5)



119894119861) by



V =V119898 [119860] [119861]

119870119898119861 [119860] + 119870119898119860 [119861] (1 + ([119861] 119870119894)) + [119860] [119861]

(6)



119898119860


119898119861the



4 Conclusion



0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

00005

0001

00015

0002

00025

0003

00035

0 00005 0001 00015 0002 00025 0003 00035

Sim

ulat

ed ra

te (m

olmiddotd

mminus3middotsminus

1)


y = 10225x

R2 = 09854


E

A

B

EA

P

B

Q

EE998400 E998400B

EiB

Figure 10


Nomenclature






119870119898119861








119888 Conversion



Acknowledgment


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article microwave assisted enzymatic kinetic...

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