36604405-Lipase-catalyst-biodiesel

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Investigation of lipases from various Carica papaya varietiesfor hydrolysis of olive oil and kinetic resolution of

(R,S)-profen 2,2,2-trifluoroethyl thioesters

I-Son Ng a, Shau-Wei Tsai b,*a Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan

bInstitute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan Tao-Yuan 33302, Taiwan

Received 17 May 2005; received in revised form 28 September 2005; accepted 6 October 2005

Abstract

With olive oil hydrolysis in aqueous solutions and hydrolytic resolution of ( R,S)-profen 2,2,2-trifluoroethyl thioesters in water-saturated

isooctane as the model systems, the lipolysis and enantioselective hydrolysis activities of four partially purified Carica papaya lipases of different

plant variety and geography location of cultures were compared to select pCPL-Indo from Indonesia as the best lipase preparation. For lipolysis, an

optimal pH of 8.5 for all lipase preparations was found. Yet, pCPL-Indo possessed the highest activity at pH ranged from 7 to 10. For the kinetic

resolution, the thermodynamic analysis implied that pCPL-Indo has changed the conformation at 60 8C and the enantiomer discrimination was

dominated by DDH. The kinetic analysis also indicated that the enantiomeric discrimination was mainly due to the difference of k2S and k2R in the

acylation step. Agreements between experimental time-course conversions XS and best-fitted results were illustrated by considering effects of

product inhibition and enzyme deactivation.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Carica papaya lipases; Lipolysis; Hydrolytic resolution; (R,S)-Profen 2,2,2-trifluoroethyl thioesters

1. Introduction

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) have been

widely applied as versatile biocatalysts for the lipids conversion

and kinetic resolution of a variety of racemates [12]. Although

industrial lipases are produced mainly from animals or

microorganisms, Carica papaya lipase stored in the crude

papain and produced from C. papaya latex is now available in

large quantities such that an extensive use in pilot or large-scale

application for lipids bioconversion is possible [34].

Recently, we discovered that a crude papain referred as the

crude C. papaya lipase (CPL), as a product from Sri Lanka,possessed high enantioselectivity for the kinetic resolution of

(R,S)-naproxen 2,2,2-trifluoroethyl thioester and ester in water-

saturated organic solvents, giving the desired (S)-naproxen as

an important non-steroidal anti-inflammatory drug [56]. As

the lipase activity is located in the non-water-soluble aggregate

of papaya latex, improvements of enzyme activity, stereo-

selectivity and thermal stability were furthermore reported

when CPL was partially purified in deionized water to remove

the water-soluble contaminants [7,8]. Since CPL may be

regarded as a waste in producing the refined papain and

chymopapain from crude papain, the cheap raw material and

low production cost is obviously beneficial for the industrial

application of pCPL as an efficient biocatalyst.

The plant variety, the geography location of cultures and

even different processing conditions from various sources may

affect the biocatalytic activities ofC. papaya lipase stored in the

crude papain. In this work, we compared the lipolysis and

enantioselective hydrolysis activities of pCPL with otherlipases prepared from commercially available and freshly

collected crude preparations of papain. These investigations

were performed in order to have a better chemical character-

ization of these enzymes, to determine the relationship between

the different catalytic activities of partially purified lipases, and

to select the best preparation for lipolysis and kinetic resolution

of racemates.

The hydrolysis of olive oil in aqueous solutions by the pH-

stat method was first employed for comparing the lipolysis

activity. The kinetic resolution of several 2,2,2-trifluoroethyl

www.elsevier.com/locate/procbioProcess Biochemistry 41 (2006) 540546

* Corresponding author. Tel.: +886 3 2118800x3415; fax: +886 3 2118668.

E-mail address: [email protected] (S.-W. Tsai).

1359-5113/$ see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2005.10.011


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thioesters of (R,S)-2-arylpropionic acids (i.e. (R,S)-profens) in

water-saturated isooctane at different temperature was then

investigated (Scheme 1). Finally, the thermodynamic and

kinetic analysis by considering product inhibition and enzyme

deactivation was carried out to simulate the time-course

conversions of (S)-naproxen thioester.

2. Materials and methods

2.1. Materials

(S)-Naproxen ((S)-2-(6-methoxyl-2-naphthyl)propionic acid), (R,S)-feno-

profen ((R,S)-2-(3-phenoylphenyl)propionic acid) calcium salt, (R,S)-ketopro-

fen ((R,S)-2-(3-benzoylphenyl)propionic acid), (S)- and (R,S)-ibuprofen ((R,S)-

4-isobutyl-2-methylphenylacetic acid), (R,S)-flurbiprofen ((R,S)-2-fluoro-2-

methyl-4-biphenylacetic acid), crude papain (product code P3375, a cystine

protease of 2.1 units/mg, product from Sri Lanka) and phenyl dichloropho-

sphate were purchasedfromSigma(St. Louis, MO). Other crude preparations of

papain were kindly donated from Biacsoft Technologies (Surabaya, Indonesia)

and Javely Biological Products (Nanning, China). We also prepared fresh crude

papain by first tapping green fruits of female papaya planted in the campus,

collecting exuded latex and then lyophilized. Other chemicals of analytical

grade were commercially available as follows: 2,2,2-trifluoroethanethiol from

Aldrich (Milwaukee, WI); isooctane, sodium chloride, chloroform and 1,2-

dimethoxyethane from Tedia (Fairfield, OH); anhydrous pyridine from Riedel-

deHaen (Seelze, Germany).All (R,S)-profen 2,2,2-trifluoroethyl thioesterswere

synthesized and characterized according to reference [7].

2.2. Preparation of partially purified papaya lipases

To 1.35 g of the crude papain from different varieties was added 15 mL

deionized water at 4 8C with gentle stirring for 30 min. The resultant solution

was centrifuged to remove the supernatant. The above procedures were repeated

once more. The remaining precipitate was then collected and lyophilized at

40 8C and 100 mmHg for 4 h, giving about 15% (w/w) recovery based on theinitial crude preparation. Notations pCPL, pCPL-China, pCPL-Indo and pCPL-

Taiwan were referred as the partially purified lipases prepared from the crude

papain produced in Sri Lanka, China, Indonesia and Taiwan, respectively.

2.3. Analysis

The pH-stat method in a Mettler DL-25 titrator (Mettler-Toledo, Switzer-

land) was employed for measuring lipase activity in aqueous solution. The

substrate solution was prepared by stirring 20 mL olive oil and 10 g gum arabic

in 200 mL deionized water. To 15 mL of the substrate solution incubated at40 8C was added 1 mL deionized water containing 5 mg of the partially purified

papaya lipase. The pH of the resultant solution was adjusted from 7 to 10 by

using phosphate buffers and then titrated by using 82 mM NaOH solution. One

unit (U) of the lipase activity was defined as the amount of enzyme required to

release 1 mmol fatty acid/min under the defined assay condition. The back-

ground hydrolysis experiment without adding the lipase at the specific reaction

condition was carried out and deducted from that with the enzyme. Similar

measurements were carried out at pH 8.5 in the temperature ranging from 35 to

60 8C. More experiments for studyingenzyme thermal stability were performed

by storing the enzyme solution in a specified temperature for 2 h and then

measured the lipase activity at pH 8.5 and 40 8C.

Thehydrolysisof (R,S)-profen thioesters in water-saturatedorganic solvents

were monitored by using HPLC equipped with a chiral column (Chiralcel OD,

Daicel Chemical Industries, Japan) capable of separating the internal standard

of 2-nitrotoluene, (R)- and (S)-thioesters, (R)- and (S)-profens. The mobilephase was a mixture of n-hexane, isopropanol and acetic acid at a flow rate of

1 mL/min. UV detection at 270 nm was used for quantification at the column

temperature of 25 8C. Detailed analytic conditions for each enantiomer were

given in Table S1 of the Supporting Materials in reference [7].

2.4. Kinetic resolution of (R,S)-profen 2,2,2-trifluoroethyl thioesters

To 135 mg of each lipase preparation was added 10 mL water-saturated

isooctane containing 1 mM (R,S)-naproxen 2,2,2-trifluoroethyl thioester at a

specified temperature. The resultant solution was stirred with a magnetic stirrer.

Samples wereremoved andinjectedonto theabove HPLC systemat different time

intervals for analysis. From the time-course conversions, the initial rate for each

enantiomer and hence the enantiomeric ratio (i.e. Evalue defined as the ratio of

initialratesfor both substrates) canbe estimated.Similar experimentswerecarried

out by using other (R,S)-profen 2,2,2-trifluoroethyl thioesters as the substrate.More experiments were performed at 45 and 60 8C for 10 mL water-

saturated isooctane containing 135 mg pCPL (or pCPL-Indo) and (R,S)-

naproxen 2,2,2-trifluoroethyl thioester of concentrations varied from 0.5 to

16.0 mM. The kinetic constants for each enantiomer can be estimated from the

variation of initial rate with initial substrate concentration. Similar experiments

were carriedout at 60 8C forstudyingthe product inhibition, where1 mM(R,S)-

naproxen 2,2,2-trifluoroethyl thioester and (S)-naproxen of concentrations

varied from 0.25 to 1.0 mM were employed.

3. Model development

As the hydrolysis product 2,2,2-trifluoroethanethiol of low

boiling point is a good leaving group, an irreversible Michaelis

I.-S. Ng, S.-W. Tsai/ Process Biochemistry 41 (2006) 540546 541

Nomenclature

eep enantiomeric excess for (S)-naproxen, (XS XR)/(XS + XR)

E enantiomeric ratio, i.e. the ratio of initial rates for

both substrates or as k2SKMR/k2RKMS

(Et) lipase concentration (mg/mL)kd deactivation constant (h

1)

KMR, KMS MichaelisMenten constants for (R)- and (S)-

thioester (mM)

K2R, K2S kinetic constant for (R)- and (S)-thioester

(mmol/(g h))

KP inhibition constant for (S)-naproxen (mM)

pCPL partially purified Carica papaya lipase from Sri

Lanka

pCPL-China partially purified Carica papaya lipase from

China

pCPL-Indo partially purified Carica papaya lipase from

Indonesia

pCPL-Taiwan partially purified Carica papaya lipase

from Taiwan

(PS) (S)-naproxen concentration (mM)

(SR), (SS) (R)- and (S)-thioester concentration (mM)

(SR)o, (SS)o initial (R)- and (S)-thioester concentration

(mM)

T temperature (K)

VR, VS initial rates of (R)- and (S)-substrates (mM/h)

XR, XS conversions of (R)- and (S)-thioester, i.e.

[1 (SR)/(SR)o] and [1 (SS)/(SS)o], respectivelyDDG difference in activation free energy between

transient states of (S)- and (R)-thioesters (kJ/mol)

DDH difference in activation enthalpy between tran-sient states of (S)- and (R)-thioesters (kJ/mol)

DDS difference in activation entropy between the tran-

sient states of (S)- and (R)-thioesters (J/(mol K))


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Menten kinetics can be employed for modeling the lipase-

catalyzed hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl

thioester in water-saturated isooctane. By furthermore assum-

ing that (S)-naproxen acts as an inhibitor, the rate equations for

both enantiomers are expressed as

VSdSS

dt

K2SSSEt=KMS1 SS=KMS SR=KMR PS=KP

(1)

VR dSR

dt

k2RSREt=KMR1 SS=KMS SR=KMR PS=KP

(2)

Notations (Et), (PS), (SR) and (SS) denote the concentrations of

enzyme, (S)-naproxen, (R)- and (S)-thioester, respectively.

Moreover, k2R, KMR, k2S, KMS and KP are the kinetic constants

in MichaelisMenten kinetics and inhibition constant, respec-

tively. Since both pCPL and pCPL-Indo are highly enantiose-

lective for the (S)-thioester, one may neglect (SS)/KMS in

Eq. (2), but not in Eq. (1), when estimating k2R and KMR.

By assuming an irreversible first-order deactivation for the

lipase, (PS) = [(SS)o (SS)] and (SR) = (SS)o in Eq. (1) due to

the high enzyme enantioselectivity, an analytical solution for(S)-thioester conversion XSderived from Eq. (1) is expressed as

1

SSoKP1 SSo=KMR

ln1 XA

SSoXS

KP1 SSo=KMR

k2AEtoexpkdt 1

kdKMS1 SSo=KMR(3)

Therefore, the deactivation constant kd can be estimated from

Eq. (3) and the experimental time-course data of XS.

4. Results and discussion

4.1. Comparison of lipolysis

Fig. 1 illustrated the bell shape of lipase specific activity

varied with pH at 40 8C by using olive oil as the substrate,

where the maximum activity at pH 8.5 for each enzyme

preparation was obtained. Similar result of optimal lipase

activity at pH 8.0 and 55 8C with tributyrin as the substrate has

been reported when using the particulate fraction of crude

papain as the biocatalyst [9]. The highest specific activity of40.9 U/mg for pCPL-Indo in comparison with 26.7 U/mg for

pCPL, 12.0 U/mg for pCPL-China and 9.1 U/mg for pCPL-

Taiwan was estimated from Fig. 1. Change of pH to 10 or 7.0

I.-S. Ng, S.-W. Tsai / Process Biochemistry 41 (2006) 540546542

Scheme 1.

Fig. 1. Effects of pH on lipase specific activity for the hydrolysis of olive oil in

aqueous solution at 40 8C for: pCPL-Indo (*), pCPL (*), pCPL-China (!)

and pCPL-Taiwan (5).


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resulted in the sharp reduction of specific activity for pCPL-

Indo and pCPL but not for pCPL-China and pCPL-Taiwan. This

implied that C. papaya lipases of different sources may have

different enzyme conformations and hence ionization states at a

specified pH. Indeed, the curve of lipolytic activity shown in

Fig. 1 differed substantially depending on the plant variety and

geography location of cultures. Yet, pCPL-Indo always

maintained the highest activity at pH ranged from 7 to 10.

The specific activity varied with temperature at pH 8.5 wasdemonstrated in Fig. 2(A) where a maximum occurred between

40 and 45 8C for pCPL-Indo and 45 to 50 8C for pCPL. Similar

results of 50 8C at pH 8.5 for CPL and 55 8C at pH 8.0 for the

particulate fraction of crude papain were reported when

employing tributyrin as the substrate [9,10]. Fig. 2(B) demon-

strated the enzyme thermal stability at pH 8.5. In general, pCPL-

Indo was more thermally stable than pCPL, yet similar residual

activities around 20% for bothlipases wereshown as temperature

wasgreaterthan50 8C. Based on the highspecificactivity, pCPL-

Indo was selected as the best lipase for the lipolysis of olive oil.

4.2. Comparison of kinetic resolution

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-

trifluoroethyl thioesters in water-saturated isooctane as the

model system, Table 1 indicated that pCPL was the most active

in obtaining the highest conversion XS at 45 8C. No correlation

between the lipolytic activity for olive oil and hydrolytic

activity for (S)-naproxen thioester was observed. Similarly, no

relationship between the proteolytic and lipolytic activities for

the crude papaya latex from different plant variety has been

found [10]. However, all lipase preparations possessed good to

excellent enantioselectivity, with pCPL-Indo to be the most

enantioselective.Increase of temperature to 60 8C resulted in an enhancement

of pCPL activity for (R)- and (S)-naproxen 2,2,2-trifluoroethyl

thioester (Fig. 3(A and B)), yet with the reduction of E value

from 173 to 67 (Table 2). Similarly, the Evalue decreased from

650 (or >200) to 183 (or 158) for pCPL-Indo when increasingthe temperature from 45 to 60 8C (or 65 8C). It stressed that in

comparison with pCPL-Indo, pCPL possessed higher initial

rate for (S)-naproxen 2,2,2-trifluoroethyl thioester at 45 8C

(Fig. 4(A)), but opposite at 60 8C (Fig. 4(B)). This implied that

pCPL-Indo might have changed the conformation at 60 8C,

which was elucidated latter.

By changing the substrate to other (R,S)-profen 2,2,2-trifluoroethyl thioesters, pCPL-Indo in general showed lower

activity for the (S)-substrate compared with pCPL at 45 8C

(Fig. 3(A)), but opposite at higher temperature (Fig. 3(B)). Yet

except for (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester, both

lipases possessed similar E values for a specific racemic

substrate when temperature increased. In order to elucidate this

interesting behavior, the thermodynamic and kinetic analysis in

water-saturated isooctane containing (R,S)-naproxen 2,2,2-

trifluoroethyl thioester for both lipases was performed.

4.3. Thermodynamic analysis

The thermodynamic analysis has been proposed toinvestigate effects of solvent type and mixture, acyl donor

and acceptor, lipase type and mutant on the temperature

dependence of E value in lipase-catalyzed kinetic resolutions


Fig. 2. (A) Specific activity and (B) residual activity varied with temperaturefor the hydrolysis of olive oil in aqueous solution at pH 8.5: for pCPL-Indo (*)

and pCPL(*).

Table 2

Effect of lipase varieties and temperature on E value for hydrolysis of 1 mM

(R,S)-profen 2,2,2-trifluoroethyl thioesters

Lipases (8C) Naproxen Fenoprofen Ketoprofen Flurbiprofen Ibuprofen

pCPL (45) 173 25 30 18 14

pCPL-Indo

(45)

>200 28 23 12 8

pCPL (60) 67 45 20 12 8pCPL-Indo (65) 158 19 9 8 n.d.

Conditions: 13.5 mg/mL lipase in water-saturated isooctane; n.d. as not

determined.

Table 1

Comparison of specific initial rates, E value, conversions and eep for various lipase varieties

Lipases VS/(Et) 104 (mmol/(g h)) VR/(Et) 10

6 (mmol/(g h)) E Time (h) XS (%) XR (%) eep (%)

pCPL 9.11 5.26 173 120 88.5 2.52 94.5

pCPL-Taiwan 5.74 9.04 64 126 65.6 3.43 90.1

pCPL-Indo 4.35 0.67 >200 124 66.8 0.24 99.3pCPL-China 0.73 0.75 97 124 10.0 0.25 95.1

Conditions: hydrolysis of 1 mM of (R,S)-naproxen 2,2,2-trifluoroethyl thioester by using 13.5 mg/mL lipase in water-saturated isooctane at 458C.


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[2,1115]. The difference in activation free energy DDG for the

transient states of fast-reacting enantiomer, i.e. (S)-naproxen,(S)-flurbiprofen, (S)-ibuprofen, (R)-fenoprofen or (R)-ketopro-

fen thioester, and slow-reacting enantiomer, i.e. (R)-naproxen,

(R)-flurbiprofen, (R)-ibuprofen, (S)-fenoprofen or (S)-ketopro-

fen thioester, can be separated into the differences in activation

enthalpy (DDH) and activation entropy (DDS). Therefore, a

clear elucidation on whether the enantiomer discrimination tobe either enthalpy-driven or entropy-driven or both equally

important is reached.

The variations of logarithm of initial rates versus the inverse

of absolute temperature for pCPL and pCPL-Indo were


Fig. 3. Initial rates of fast-reacting enantiomer of various (R,S)-profen 2,2,2-

trifluoroethyl thioesters: (A)for pCPL-Indo (emptybar) andpCPL(filled bar) at

45 8C and (B) for pCPL-Indo (empty bar) at 65 8C and pCPL (filled bar) at

60 8C. Notations: Nap, Feno, Keto, Flu and Ibu represent 2,2,2-trifluoroethyl

thioesters of (R,S)-naproxen, (R,S)-fenoprofen, (R,S)-ketoprofen, (R,S)-flurbi-

profen and (R,S)-ibuprofen, respectively.

Fig. 4. (A) Variations of initial ln(VR) (! and 5) and ln(VS) (* and*) withinverse of absolute temperature for pCPL-Indo (empty) and pCPL (filled). (B)

Variation of ln(E) with inverse of absolute temperature for pCPL-Indo (*) and

pCPL (*). Condition: hydrolytic resolution of 1 mM (R,S)-naproxen 2,2,2-

trifluoroethyl thioester in water-saturated isooctane.

Table 3

Kinetic constants for hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl thioester in water-saturated isooctane at 45 and 60 8C for pCPL-Indo and pCPL

Lipase (8C) k2S 103 (mmol/(g h)) k2R 10

4 (mmol/(g h)) KMS (mM) KMR (mM) KP (mM) kd 102 (h1)

pCPL (45) 6.33 3.20 2.68 19.9 0.80 0.43

pCPL-Indo (45) 2.75 0.07 1.43 2.77 0.50 0.39

pCPL (60) 16.79 3.00 5.72 6.67 1.53 0.69

pCPL-Indo (60) 47.21 4.00 3.66 6.74 1.66 2.68


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illustrated in Fig. 4(A). The Arrhenius relationship for either

(R)- or (S)-thioester was observed, implying that pCPL was still

stable at 80 8C. These behaviors were valid for pCPL-Indo, yet

an obvious change of the slope of Arrhenius relationship at

60 8C was illustrated. This implied that pCPL-Indo might have

changed the conformation, yet more experiments to confirm

this deduction by using purified papaya lipases as the

biocatalyst were needed.

Inspections ofFig. 4(B) revealed that pCPL-Indo was moreenantioselective, which was mainly due to the lower initial VRat temperature less than 60 8C and the higher VS at the higher

temperature. The relationship RTln(E) = DDH+ TDDS wasemployed to estimate DDH and DDS from Fig. 4(B) for both

lipases. The results for pCPL were DDH= 41.75 kJ/mol andDDS= 89.12 J/(mol K), and those for pCPL-Indo asDDH= 73.52 kJ/mol and DDS= 177.2 J/(mol K) at tem-perature ranged from 45 to 60 8C as well as DDH= 41.63 kJ/mol and DDS= 81.02 J/(mol K) at temperature ranged from60 to 80 8C, respectively. The large difference of activation

enthalpy between the transient states of both enantiomers at

these two temperature ranges implied that the enzyme

conformation for pCPL-Indo did change at 60 8C.A good linear relationship of DDS= 26.87 + 2.951 DDH

(r2 = 0.979) has been reported previously, no matter what

combination of lipase sources, solvents, hydrolysis for (R,S)-

profen 2,2,2-trifluoroethyl ester and thioester or esterification

for (R,S)-naproxen and 2-(4-chloro-phenoxy)propionic acid

was made [16]. This linear enthalpyentropy compensation

relationship was modified as DDS= 26.85 + 3.028 DDH

(r2 = 0.981) when data for pCPL-Indo were added. From the

variation of DDH and DDS for pCPL-Indo and pCPL, it

concluded that both activation enthalpy and activation entropy

were important for the enantiomer discrimination, yet the

former was dominating in the temperature range investigated.

4.4. Kinetic analysis

Fig. 5(A and B) illustrated the initial V1S

varied with (PS)

and initial rates changed with the substrate concentration,

respectively, for pCPL-Indo at 60 8C and other conditions (not

given here). The kinetic constants were then estimated form

Eqs. (1) and (2), and represented in Table 3. In general, the

enzyme enantioselectivity for both lipases was mainly due to

the difference ofk2S and k2R, i.e. the formation and breaking oftransient states for both substrates in the acylation step. In

comparison with pCPL, the lower initial rate VS for pCPL-Indo

at 45 8C and vice versa at higher temperature (Fig. 4(A)) was

attributed to the great enhancement of k2S. Moreover, by

comparing KP and KMS, each lipase preparation possessed

higher affinity for (S)-naproxen in comparison with (S)-

naproxen thioester.

The enzyme deactivation constants represented in Table 3

for both lipases at different temperature were furthermore

estimated from the time-course conversions XS and Eq. (3).

Agreements between the time-course conversions XS and best-

fitted results were illustrated in Fig. 6.

5. Conclusions

With olive oil hydrolysis in aqueous solutions as the

model system, the lipolysis activities for four partially

purified lipases prepared from the crude papain of various

varieties were first compared. An optimal pH of 8.5 at 40 8C

for all lipase preparations was found, yet pCPL-Indo from

Indonesia possessed the highest specific activity at pH ranged

from 7 to 10. Maximum enzyme activities between 40 and

45 8C for pCPL-Indo and between 45 and 50 8C for pCPL

from Sri Lanka were obtained. Moreover, the former

demonstrated better enzyme thermal stability as temperature


Fig. 5. (A) Variation of initial V1S with(PS) and(B) variations of initial VR (&)and VS (5) with initial substrate concentration with (SR)o or (SS)o at 60 8C forpCPL-Indo. () Best-fitted results.

Fig. 6. Time-course conversions ofXS: for pCPL-Indo at 60 8C (5), pCPL at

60 8C (!), pCPL-Indo at 45 8C (*) and pCPL at 45 8C (*). () Best-fittedresults.


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was less than 50 8C and vice versa at temperature greater than

50 8C.

With the hydrolytic resolution of (R,S)-naproxen 2,2,2-

trifluoroethyl thioester in water-saturated isooctane as the

model system, pCPL and pCPL-Indo possessed the highest

lipase activity and enantioselectivity for the (S)-thioester at

45 8C, respectively. Yet, pCPL-Indo was superior to pCPL at

the temperature greater than 55 8C. Very similar performances

for both lipase preparations were found when other (R,S)-

profen 2,2,2-trifluoroethyl thioesters were used as substrates.

The thermodynamic analysis indicated that the enantiomer

discrimination was driven byDDHandDDS, yet the former was

dominating for both lipase preparations. From the variation of

DDH with temperature, pCPL-Indo might change the con-

formation at 60 8C.

The kinetic analysis for pCPL and pCPL-Indo indicated that

the enantiomeric discrimination was mainly due to the

difference of k2S and k2R in the acylation step. Moreover, k2Sbut not KMS possessed more influence on the initial rate VS

when comparing the lipase activity at different temperature andlipase preparation. Agreements between the time-course

conversions XS and best-fitted results for pCPL-Indo were

obtained when the product inhibition and enzyme deactivation

were considered. Based on the enzyme performance of activity

and enantioselectivity, pCPL-Indo was selected as the best

lipase preparation.

Acknowledgement

The financial support of NSC 93-2214-E-006-008 from

National Science Council is appreciated.

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