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Process Biochemistry 50 (2015) 230–236

Contents lists available at ScienceDirect

Process Biochemistry

jo u r n al homep age: www.elsev ier .com/ locate /procbio

nsight into microwave assisted immobilized Candida antarctica lipase catalyzed kinetic resolution of RS-(±)-ketorolac

omnath D. Shinde, Ganapati D. Yadav ∗

epartment of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, India

r t i c l e i n f o

rticle history:eceived 2 October 2014eceived in revised form1 November 2014ccepted 11 December 2014vailable online 20 December 2014

a b s t r a c t

Microwave assisted biocatalytic transformation is a green process for chiral drug resolution. Differentimmobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipase Amano AS andLipase AYS amino were screened for the kinetic resolution of RS-(±)-ketorolac under microwave irradia-tion. To study kinetics and mechanism, the effect of different parameters on the conversion, initial ratesand enantiomeric excess of substrate were studied. Under microwave synergism, an increase in initialrates up to 1.5-fold was observed. Novozym 435 effectively catalyzed the enantioselective esterification

eywords:sterificationicrowave irradiation

nzyme catalysisetorolacinetics

of RS-(±)-ketorolac, showing excellent conversion (50%) and enantiomeric excess (>99%) in 3 h at 50 ◦Cand 300 rpm. Based on initial rate and progress curve data, the reaction was found to follow the PingPong bi–bi mechanism with inhibition by n-octanol.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Recently, the exploitation of enzymes as catalysts in chemicalynthesis has been much in evidence [1–6]. Interesting applica-ions of enzymatic reactions have been discovered in synthesisnd purification of chiral compounds [7–10]. Enzymes have gainedotential applications for biotransformations [11,12] and biosen-ors [13–15]. Chiral building blocks play a key role in synthesisf pharmaceutical drugs and drug discovery process. Racemic chi-al acids and alcohols produced in synthesis of pharmaceuticalrugs can be resolved through enantioselective esterification bysing lipases [16]. Thus, enzymatic biotransformation has received

great importance [17]. Lipases and esterases are most widely usediocatalysts in non-aqueous media because of their high recycla-ility in immobilized form, being inexpensive with broad substratepecificity and they do not require cofactors to carry out the reac-ions [18,19].

Various strategies have been adopted to enhance the per-

ormance of biocatalysts [20–24]. Microwave irradiation is anpproach for process intensification in a variety of reactions.icrowave irradiated synthesis in organic solvents using both

∗ Corresponding author. Tel.: +91 22 3361 1001/1111/2222;ax: +91 22 3361 1002/1020.

E-mail addresses: dr.sdshinde@gmail.com (S.D. Shinde), gdyadav@yahoo.com,d.yadav@ictmumbai.edu.in (G.D. Yadav).

ttp://dx.doi.org/10.1016/j.procbio.2014.12.005359-5113/© 2014 Elsevier Ltd. All rights reserved.

chemical and biocatalysis has received significance in green pro-cess development. Under microwave irradiation, high yields andreaction selectivity can be achieved at reduced time of reac-tion [16,25–36]. Microwave irradiation results in an instantaneouslocalized superheating which is achieved due to dipole rotation orionic conduction [37–41].

Ketorolac [(±)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2-a]pyrrole-1-carboxylic acid] is a nonsteroidal anti-inflammatorydrug (NSAID) with one stereogenic carbon centre [42]. It hasgained significant therapeutic importance due to its potent anti-inflammatory and moderate analgesic activities. The single-doseefficacy of ketorolac is reportedly greater than that of many otheranti-inflammatory drugs such as aspirin, naproxen, indomethacin,ibuprofen, morphine, meperidine and pentazocine [43,44]. Mech-anism of action involves inhibition of prostaglandin synthesisthrough cyclooxygenase inhibitory action. Recently, stereochem-istry of drugs is given prime importance in all phases of drugdiscovery and use of pure enantiomers is needed to avoid anyside effects of other inactive enantiomeric form of drug. It isnow commonly accepted that therapeutically active enantiomerof ketorolac is the (−)- or (S)-form while (R)-isomer is inactive[43]. In fact, (R)-isomer shows adverse side effects includingrenal impairment, gastrointestinal bleeding and platelet inhibi-

tion with altered haemostasis. Thus, pure (S)-enantiomers werestereoselectively synthesized by hydrolysis in aqueous media,or esterification in organic media catalyzed by enzymes or bymicroorganisms [41,45–47]. Unfortunately, results to date using

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S.D. Shinde, G.D. Yadav / Proce

nzymatic biotransformation required longer reaction time tochieve desired enantiomeric excess of (S)-(−) form of ketorolac.ence it was thought worthwhile to utilize microwave assistednzyme catalysis to improve reaction rate in chiral drug resolution.e report here the synergistic effect of microwave irradiation

n kinetic resolution of RS-(±)-ketorolac via esterification usingmmobilized lipase.

Kinetic mechanisms for lipase were reported to proceed viaing Pong bi–bi mechanism, ternary complex ordered bi–bi mech-nism or ternary complex random bi–bi mechanism [12,16,47,48].owever, the kinetic modelling of microwave assisted Candidantarctica lipase B catalyzed resolution of RS-(±)-ketorolac hasot yet been reported. Detailed studies on effect of speed of agi-ation, various solvents, temperature, concentration of substratend catalyst concentration were performed to elucidate kinetics ofS-(±)-ketorolac resolution.

. Experimental

.1. Enzyme and chemicals

Lipozyme RM IM, Lipozyme TL IM and Novozym 435 werebtained as free gift samples from Novo Nordisk, Denmark. LipaseYS Amano was a gift sample from Amano Enzyme Inc., Japan.ipozyme RM IM is Rhizomucor miehei lipase immobilized onnionic exchange resin (activity of 30 U g−1, based on tristearinssay) whereas Lipozyme TL IM is Thermomyces lanuginosus immo-ilized on silica. Novozym 435 is C. antarctica lipase B (CALB)

mmobilized on a macroporous polyacrylic resin beads (bead size.3–0.6 mm, bulk density 0.430 g cm−3, water content 3%, activityf 7000 PLU g−1). Lipase AYS Amano is Candida rugosa lipase in formf lyophilized powder (activity 30,000 U g−1).

Chemicals used in the study were AR grade, purchased fromeputed firms. Ketorolac tromethamine was obtained as a giftample from Centaur Pharma, Goa, India. Toluene, n-heptane, diiso-ropyl ether, diisobutyl ether, tetrahydrofuran, 1,4-dioxan andcetonitrile were purchased from SD. Fine Chemicals Pvt. Ltd.,umbai, India. Solvents used for HPLC analysis were obtained from

homas Baker, Mumbai.

.2. Analytical method

The enantiomeric separation of RS-(±)-ketorolac was estab-ished on chiral stationary phase HPLC (Fig. S1). Knauer K-2501PLC-system using Chiralpak IA column (0.46 mm × 250 mm;

�m, Daicel Chemical Industries, Japan) was used fornalysis under the following conditions: mobile phase, n-exane/isopropyl alcohol/trifluoroacetic acid (90:10:0.1, v/v);ow rate, 1.0 mL min−1; and column temperature, 25 ◦C; wave-

ength, 310 nm. The enantiomeric excess for substrate (ee(S))nd product (ee(P)); conversion, C = ee(S)/(ee(S) + ee(P)) and enan-iomeric ratio, E = (ln (1 − C (1 + ee(P)))/(ln (1 − C (1 − ee(P))) wereetermined from their respective areas using HPLC. The initialates were calculated by multiple point method and it was deter-ined from the plot of concentration versus time. Progress of the

nzymatic resolution was monitored by periodic withdrawal ofamples.

.3. Removal of tromethamine from ketorolac tromethamine

Ketorolac was obtained from its tromethamine salt using a pre-ipitation method. Following addition of 1 N HCl drop by drop into

ketorolac tromethamine solution, ketorolac was precipitated. Theollected precipitate was purified by extraction with ethyl acetate,ollowed by evaporation to dryness. The purity of ketorolac wasonfirmed by melting point measurement and HPLC analysis. The

chemistry 50 (2015) 230–236 231

obtained product was measured to have a melting point of 155 ◦C,practically the same as reported in literature with 100% HPLCpurity.

2.4. Experimental set-up

2.4.1. Conventional heatingThe experimental set up used for conventional heating studies

was the same as reported earlier [31]. A typical reaction procedurefor lipase catalyzed enantioselective resolution of RS-(±)-ketorolaccontained 0.117 mmol of RS-(±)-ketorolac and 1.17 mmol of n-octanol, diluted to 15 mL with solvent. Reaction mixture wasagitated at 50 ◦C for 15 min at a speed of 300 rpm. Reaction wasinitiated by adding known fixed quantity of lipase. Samples werecollected at regular intervals, filtered to eliminate particulate mat-ter, if any, and analyzed by HPLC to determine the conversion andee.

2.4.2. Microwave reactorMicrowave reactor (Discover, CEM-SP 1245 model) set-up used

in the study was the same as reported earlier [31] (Fig. S2). TheCEM Discover microwave reactor could be used to carry out reac-tions up to microwave power of 300 W. The experiments wereconducted at constant temperature. A constant microwave irradia-tion was provided (30–40 W). Experimental conditions maintainedfor microwave irradiation studies were the same as mentioned forconventional heating, unless otherwise stated.

2.5. Enzyme kinetics

Different parameters were studied to elucidate the kinetics oflipase catalyzed enantioselective resolution of RS-(±)-ketorolac(Scheme 1). Concentrations of reactants were systematically var-ied over a wide range to study their effects on rate of reactionusing 30 mg Novozym 435. Concentration of n-octanol (B) waschanged from 0.117 to 2.34 mmol at various constant concen-trations of RS-(±)-ketorolac (A) (0.078–0.351 mmol). In anotherset, concentration of RS-(±)-ketorolac (A) was changed from0.078 to 0.351 mmol at a different constant concentration of n-octanol (0.117–2.34 mmol). The quantified data obtained for 20%conversion was used to calculate initial rates of reaction. Only(R)-ketorolac ester was obtained during the initial period, whileconversion of (S) ketorolac was not seen for up to 20% conversionand hence initial rate for (S) ketorolac was considered to be zero.

3. Results and discussion

3.1. Effect of various biocatalysts

Different supported lipases were evaluated for enantioselec-tive resolution of RS-(±)-ketorolac. The conversion and ee variednoticeably for different lipases (Fig. 1). Lipozyme RM IM, LipozymeTL IM, Lipase Amano AS, and Novozym 435 showed conversionof 4, 6, 5 and 39%, respectively, whereas very little activity wasobtained with Lipase AYS Amano. The objective to study these dif-ferent enzymes was to discern if any significant activation couldbe attained owing to microwave irradiation, regardless of theirwell-known applications. Novozym 435 showed the highest con-

version with 99% ee(P) and >200E value. This is attributed to higherconformational flexibility of Novozym 435 towards R-isomer thanS-isomer compared to other enzymes studied. Novozym 435 beingthe most efficient biocatalyst was used in further experiments.

232 S.D. Shinde, G.D. Yadav / Process Biochemistry 50 (2015) 230–236

Scheme 1. Microwave assisted Novozym 435 catalyzed kinetic resolution of ketorolac.

Fig. 1. Effect of various biocatalyst (reaction condition: (RS)-ketorolac, 0.117 mmol;n-octanol, 1.17 mmol; solvent, 1,4-dioxan up to 15 mL; temperature, 50 ◦C; catalyst,2 mg mL−1; speed of agitation, 300 rpm; reaction time, 3 h).

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0 30 60 90 120 150 180 210 240 27 0 30 0 330

ee (s

) (%

), ee

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%)

Con

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ion

(%)

Time (min )

Conversi on

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ig. 2. Effect of different solvent (reaction condition: (RS)-ketorolac, 0.117 mmol;-octanol, 1.17 mmol; solvent up to 15 mL; temperature, 50 ◦C; Novozym 435,

mg mL−1; speed of agitation, 300 rpm; reaction time, 3 h).

.2. Effect of various solvents

Different solvents such as n-heptane, toluene, diisobutyl ether,iisopropyl ether, tetrahydrofuran, acetonitrile and 1,4-dioxaneere used to study their effect on conversion and enantioselec-

ivity. Nature of solvent has great impact on activity of enzymehich requires essential water activity for maintaining the native,

atalytically active enzyme conformation in the organic solvent.o obtain maximum ee, proper solvent is required for enzyme toield enantioselective conversion of substrate (Fig. 2). The solventsnder study with log P value in the range of 4 to −1.1 exhibitedood enantioselectivity (E > 200) for R-isomer conversion. Acetoni-rile showed less enantioselectivity (E, 167) compared to otherolvents. This indicates good structural stability of catalytic triadf immobilized Novozym 435 under different solvents resulted in

o unaltered selectivity. The varied conversion and hence enan-iomeric excess under different solvents is attributed to the abilityf solvent to maintain necessary water layer for enzyme activity.or non-aqueous enzymatic transformations, non-polar solvents

Fig. 3. Effect of reaction time (reaction condition: (RS)-ketorolac, 0.117 mmol; n-octanol, 1.17 mmol; solvent, diisopropyl ether up to 15 mL; temperature, 50 ◦C;Novozym 435, 2 mg mL−1; speed of agitation, 300 rpm).

are more favoured against polar solvents. Because polar solventscause striping of the necessary water layer around the enzyme,it results in reduced enzyme activity. Higher enzyme activity wasachieved with non-polar solvents while polar solvents such as ace-tonitrile and tetrahydrofuran showed less conversion as well asenantioselectivity. Diisopropyl ether with log P value of 1.9 showedthe optimal conversion and enantioselectivity compared with oth-ers. Hence, diisopropyl ether was used for further studies.

3.3. Effect of reaction time

The effect of reaction time was investigated to study its effecton conversion and enantioselectivity. A good conversion and enan-tioselectivity was observed at reduced time of reaction undermicrowave irradiation as compared to findings reported elsewherefor ketorolac resolution [42,45,46]. Kim et al. [42] reported 55%ketorolac conversion with 98% ee(S) and >99% ee(P) in 30 h. Undermicrowave irradiation, we observed 50% conversion with >99% ee(S)and ee(P) in 3 h (Fig. 3). As reaction is continued beyond 3 h, therewas no significant change in enantioselectivity of lipase towardssubstrate, which clearly suggest an unaltered enantioselectivity atlonger duration of time under microwave irradiation. Hence, reac-tion time of 3 h was chosen as optimum.

3.4. Effect of speed of agitation

Optimal speed of agitation and enzyme loading of optimumparticle size can be used to minimize external mass transfer andinternal diffusion limitations. Hence, effect of speed of agitationwas studied in the range of 100–400 rpm using Novozym 435 anddiisopropyl ether as solvent (Fig. 4). An increase in conversion from30 to 50% was found when speed of agitation was increased from

100 to 300 rpm. Whereas the ee(P) and E obtained was always above99% and 200, respectively. On the other hand, there was no signifi-cant increase in rate and conversion at 300 and 400 rpm. At higherspeed of agitation, the biocatalyst particles were thrown out of the

S.D. Shinde, G.D. Yadav / Process Biochemistry 50 (2015) 230–236 233

Fig. 4. Effect of speed of agitation (reaction condition: (RS)-ketorolac, 0.117 mmol;n-octanol, 1.17 mmol; solvent, diisopropyl ether up to 15 mL; temperature, 50 ◦C;Novozym 435, 2 mg mL−1; speed of agitation, 100–400 rpm; reaction time, 3 h).

Fig. 5. Effect of different alcohol (reaction condition: (RS)-ketorolac, 0.117 mmol;alcohol, 1.17 mmol; solvent, diisopropyl ether up to 15 mL; temperature, 50 ◦C;N −1

rea

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Fig. 6. Effect of catalyst loading (reaction condition: (RS)-ketorolac, 0.117 mmol;n-octanol, 1.17 mmol; solvent, diisopropyl ether up to 15 mL; temperature, 50 ◦C;Novozym 435, 1–6 mg mL−1; speed of agitation, 300 rpm; reaction time, 3 h).

Fig. 7. Effect of mole ratio of n-octanol concentration (reaction condition: (RS)-

ratio of 1:10 of RS-(±)-ketorolac/n-octanol was 50% with 98% ee(S)and 99.99% ee(P). Hence, the molar ratio of 1:10 was considered as

ovozym 435, 2 mg mL ; speed of agitation, 300 rpm; reaction time, 3 h).

eaction medium on the reactor wall, which resulted in reducedffective biocatalyst loading. Thus, further studies were carried outt an optimum speed of 300 rpm.

.5. Effect of various alcohols

Esterification of RS-(±)-ketorolac was studied with variousrimary and secondary alcohols such as n-butanol, n-pentanol,-hexanol, n-heptanol, n-octanol, n-decanol, isopropyl alcoholnd isobutyl alcohol under otherwise similar conditions (Fig. 5).onversions obtained with primary alcohols such as n-butanol, n-entanol, n-hexanol, n-heptanol were 65%, 63%, 56% and 54% andnantioselectivity as 91%, 86%, 91% and 89%, respectively. Whereassterification with secondary alcohols such as isobutyl alcohol andsopropyl alcohol showed lower conversions of 15% and 2% withnantiomeric excess of substrate (ee(S)) as 93% and 89%, respec-ively. Primary alcohols such as n-octanol and n-decanol exhibitedood conversions of 50% and 41% and enantiomeric excess ofroduct (ee(P)) as 99.99% and 97%, respectively. Reduced enan-ioselectivity was observed with secondary alcohols (E < 151) asompared to primary alcohols (E ≥ 183). This is attributed to thenhanced stearic hindrance due to branched alkyl chain resulting

n to low stereo preference towards the substrate.

ketorolac, 0.117 mmol; n-octanol, 1:1–1:20 mmol; solvent, diisopropyl ether up to15 mL; temperature, 50 ◦C; Novozym 435, 2 mg mL−1; speed of agitation, 300 rpm;reaction time, 3 h).

3.6. Effect of catalyst amount

Novozym 435 loading was varied from 1 to 6 mg mL−1 to studyits effect on enantioselective resolution of RS-(±)-ketorolac at con-stant molar ratios of substrates (Fig. 6). The conversion increasedlinearly with an increase in enzyme loading up to 4 mg mL−1

beyond which no significant increase in the conversion was foundwhich clearly shows that active sites were much higher than theneeded and the rate was limited by the external mass transfer.Enzyme loading of 2 mg mL−1 under specified conditions showed50% conversion, ee(P) > 99% and ee(S) 98% and hence it was used infurther experiments.

3.7. Effect of mole ratio of n-octanol

Enantioselective resolution of RS-(±)-ketorolac was studied atdifferent concentrations of n-octanol, keeping the concentrationof RS-(±)-ketorolac constant using diisopropyl ether as solvent.Linear increase in conversion with increased mole ratio of RS-(±)-ketorolac/n-octanol from 1:1 to 1:15 was observed (Fig. 7). Optimalperformance was obtained at a mole ratio 1:10. As the mole ratioincreased beyond 1:15, a marginal decrease in the conversion andenantioselectivity was observed which can be attributed to forma-tion of inhibitory complex with n-octanol. The conversion at mole

the optimum ratio.

234 S.D. Shinde, G.D. Yadav / Process Biochemistry 50 (2015) 230–236

0

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ee (s

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), ee

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%)

Con

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ion

(%)

Temper ature (°C)

Convers ion (%) MConversion (%) Cee(s) Mee(p) Mee(s) Cee(p) C

Fig. 8. Effect of temperature (conventional (C) versus microwave (M) heating)(reaction condition: (RS)-ketorolac, 0.117 mmol; n-octanol, 1.17 mmol; solvent,diisopropyl ether up to 15 mL; temperature, 50 ◦C; Novozym 435, 2 mg mL−1; speedof agitation, 300 rpm; reaction time, 3 h).

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e (p

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Con

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(%)

Catalyst reusability

Conversion (%) ee(p) ee(s)

1/T (1/K ) × 10

Fig. 9. Arrhenius plot (ln r versus 1/T).

.8. Effect of temperature

The effect of temperature was investigated in the range of0–60 ◦C by comparing the enantioselective resolution of RS-(±)-etorolac under conventional and microwave heating (Fig. 8).ith an increase in temperature in range of 30–60 ◦C undericrowave irradiation the initial rate increased from 4.67 × 10−1

o 21.33 × 10−1 mol L−1 min−1 kg−1 of enzyme and the conversionncreased from 23 to 53%. This is attributed to the momentum pro-ided by microwave energy to overcome energy barrier and thushe reaction is completed more quickly than conventional heating.he change in temperature has not shown much effect on enantio-electivity in both microwave irradiation and conventional heating.he activation energy values were obtained by the Arrhenius plotFig. 9) as 10.29 and 9.02 kcal mol−1 under microwave and con-entional heating respectively which are close suggesting that theicrowaves provide enhancement in collision frequency.

.9. Synergism between microwave irradiation and lipaseatalysis

To study synergistic effect of microwave irradiation withnzyme catalysis on enantioselective resolution of RS-(±)-etorolac, reactions were carried out under conventional as well asicrowave heating. It was found that under microwave irradiation,

he reaction rate improved up to1.5-fold and shorter period of timeas needed to achieve the higher conversion and enantiomeric

xcess (>99%) compared with that under conventional heating. Thiseflects that the effect may not be purely thermal and microwave

Fig. 10. Effect of catalyst reusability (Reaction Condition: (RS)-Ketorolac, 0.117mmol; n-octanol, 1.17 mmol; solvent, diisopropyl ether up to 15 ml; temperature,50◦C; Novozym 435, 2 mg/mL; speed of agitation, 300 rpm; reaction time, 3h).

absorbing character of the feed was contributing to the faster reac-tion rate. In the reaction mixture, RS-(±)-ketorolac may be the goodmicrowave absorbing material and its dipole may be reorientingquickly under microwave irradiation making the functional groupmore active at the interface of RS-(±)-ketorolac and n-octanol. Itis also possible that enzyme may behave slightly differently andbecome more active, because conformational change in enzymecan facilitate the substrate to approach active site of enzyme moreeasily under microwave irradiation than that under conventionalheating. The control experiment in the absence of Novozym 435did not show any conversion and only microwave irradiation with-out enzyme did not initiate the reaction. Thus, there is a definitesynergism between enzyme catalysis and microwave irradiation.

3.10. Effect of catalyst reusability

The reusability of Novozym 435 was studied under optimizedprocess parameter conditions to examine the stability and recycla-bility of the enzyme. The enzyme after the first use was filtered,washed with diisopropyl ether after each use, dried at room tem-perature and reused for further studies. Similar steps were followedfor each further reusability studies. After filtration, any small lossin catalyst was made up and it was reused. Marginal decrease inconversion (∼3%) was observed after three cycles of use for enan-tioselective resolution of RS-(±)-ketorolac (Fig. 10). There was noenzyme activity loss due to leaching, which was confirmed by noobserved activity of enzyme in the reaction medium after filteringthe catalyst. This is acceptable.

3.11. Kinetic model based on initial rate measurements

Based on initial rates (V) obtained under specified condi-tions, a kinetic model for Novozym 435 catalyzed enantioselectiveresolution of RS-(±)-ketorolac was proposed. Initial rates for (R)-ketorolac conversions were determined systematically from thelinear section of the conversion plot using 2 mg mL−1 of Novozym435. With increase in the n-octanol (B) concentration from 0117to 2.34 mmol at different fixed RS-(±)-ketorolac (A) concentration(0.078–0.351 mmol), initial rate was increased and attained themaximum at the critical concentration. Reaction rate decreasedwith further increase in concentration of B and thus the n-octanolinhibition was significant. Thus, it is believed that n-octanol at

high concentrations forms dead end inhibitory complex withthe lipase. However, there was no inhibition observed by RS-(±)-ketorolac (A) at the concentrations used (0.078–0.351 mmol).The Lineweaver–Burk plot of 1/initial rate (L min mol−1) versus

S.D. Shinde, G.D. Yadav / Process Biochemistry 50 (2015) 230–236 235

0

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ig. 11. Lineweaver–Burk plot of 1/initial rate (L min mol−1) versus 1/[n-octanol]L mol−1) at different constant ketorolac concentration.

/[n-octanol] (L mol−1) at different constant RS-(±)-ketorolac con-entration gives parallel lines (Fig. 11). This suggests a Ping-Pongechanism, also called a double-displacement reaction. The action

f lipase is a ping-pong reaction because the binding of the ketoro-ac and n-octanol causes the lipase to switch back and forth betweenwo states. A sequential mechanism can be ruled out as there waso common intersection.

Hence Ping-Pong bi–bi mechanism with n-octanol inhibition isostulated. These assumptions are used to design a reaction mech-nism that is depicted in Cleland’s notation, as shown below.

By analogy to the classical mechanism of esterification byipases, it is assumed that RS-(±)-ketorolac [A] first binds to theree enzyme [E] and forms a non-covalent enzyme–substrate com-lex [EA], which releases the first product, water [P] and E′ modifiednzyme. The second substrate, n-octanol [B] binds to E′ to give com-lex [E′B] and gives the product (+)-R-ketorolac ester [Q] and freenzyme [E]. Along with this, B also forms the dead-end complexEiB] by binding to the free enzyme [E].

The rate equation is as follows:

= vmax[A][B]

KmB[A] + KmA[B](

1 + [B]KiB

)+ [A][B]

here [A] and [B] are the initial concentrations of RS-(±)-ketorolacnd n-octanol, respectively; KmA is Michaelis constant for RS-(±)-etorolac; KmB is Michaelis constant for n-octanol; KiB is inhibitiononstant for n-octanol; V and Vmax are initial rate and maximumate of the reaction, respectively.

The initial rate data were used to determine the kineticarameters of above mechanism by non-linear regression anal-sis using the software package Polymath 6.0 and their values

ere estimated. The values for kinetic parameters obtained

rom nonlinear regression analysis were found to be Vmax

f 1.97 × 10−1 mol L−1 min; KmA of 5.5 × 10−3 mol L−1; KmB of.01 × 10−2 mol L−1 and KiB of 4.13 × 10−2 mol L−1. The rates of

rexpe rimental (mol/L.min)

Fig. 12. Parity plot (experimental rate versus simulated rate).

reaction were simulated using above equation to verify the pro-posed kinetic model. The best fit of plot between stimulated rateand experimental rate, suggests that the proposed mechanism wasvalid for this reaction (Fig. 12).

4. Conclusions

Enantioselective resolution of RS-(±)-ketorolac was studiedunder microwave heating using various immobilized lipases among

which Novozym 435 was the most active. Effect of variousparameters such as speed of agitation, solvent, catalyst loading,temperature and substrate ratio was studied. A kinetic modelwas proposed by collecting both the initial rate data and con-centration time profiles for the enantioselective resolution ofRS-(±)-ketorolac. The Ping-Pong bi–bi mechanism with inhibitionby n-octanol substrate provides support for the mechanism whichwas found to fit the data well for microwave irradiated enzymecatalysis.

Acknowledgements

GDY received support from R.T. Mody Distinguished ProfessorEndowment and J. C. Bose National Fellowship of Department ofScience and Technology, Government of India. SDS received SRFfrom UGC under its Meritorious Fellowship BSR programme (CASin Chemical Engineering Department). The authors thank NovoNordisk, Denmark and Amano, Japan for the gifts of enzymes.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.procbio.2014.12.005.

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36 S.D. Shinde, G.D. Yadav / Proce

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