Lipase Catalysis of Reactions in Mixed Micelles - ACS Symposium

Chapter 12

Lipase Catalysis of Reactions in Mixed Micelles Donn N . Rubingh and Mark Bauer Downloaded by STANFORD UNIV GREEN LIBR on August 10, 2012 | http://pubs.acs.org Publication Date: September 8, 1992 | doi: 10.1021/bk-1992-0501.ch012

Corporate Research Division, The Procter & Gamble Company, Cincinnati, OH 45239

Enzyme catalyzed reactions on micellized substrates are much less well understood than for soluble substrates. We develop expressions for the lipase catalyzed rate of hydrolysis of surfactant substrates comicellized with non-hydrolyzable surfactant molecules. The equations contain a term involving inhibition by the non-hydrolyzable surfactant molecule as well as an interfacial activation parameter which may be a function of composition. Under appropriate limiting conditions the well known Michaelis-Menten kinetic expression is obtained except that the concentration of the substrate is that in the micelle. We find a number of surfactant esters and mixtures of surfactant esters show an abrupt change in rate of lipase catalyzed hydrolysis at the cmc. This suggests that both single component and mixed micelles are capable of activating lipase catalysis of ester hydrolysis. The concentration dependence of the rate of hydrolysis of surfactant esters in single component micelles is described by a Michaelis-Menten type expression allowing values of the kinetic parameters, k and Km*, to be obtained. In mixtures of hydrolyzable and non-hydrolyzable surfactants both competitive inhibition and exclusion (changes in interfacial activation) can affect the hydrolysis rate depending on surfactant structure. cat

Enzymes are biological catalysts that greatly accelerate the rate of various chemical reactions. For example, the protease BPN' which catalyzes internal peptide bond hydrolysis in proteins, can accelerate the rate of reactions on certain substrates up to 10 (1). The mechanisms of many enzyme-substrate reactions are well characterized, particularly when the substrate is a water soluble compound(2). Many excellent 9

0097-6156/92/0501-0210$06.00/0 © 1992 American Chemical Society

In Mixed Surfactant Systems; Holland, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by STANFORD UNIV GREEN LIBR on August 10, 2012 | http://pubs.acs.org Publication Date: September 8, 1992 | doi: 10.1021/bk-1992-0501.ch012

12.

RUBINGH & BAUER

Lipase Catalysis of Reactions in Mixed Micelles

books andreviewsare available for the mathematical description of enzyme kinetics for soluble substrates(5)(4). A wide variety of enzymes, broadly divided into various classes depending on the nature of the reaction they catalyze, are found in nature. Well known examples include proteases for amide bond hydrolysis, esterases for ester bond hydrolysis, and cellulases for cellulose hydrolysis. Some enzymes function on soluble substrates; however, many function on insoluble substrates or, stated differently, at the interface between the insoluble substrate particle and the aqueous solution containing the enzyme. Cellulases and lipases are classes of enzymes where this is true. The mechanisms and mathematical descriptions of enzyme kinetics for insoluble substrate hydrolysis are less well defined than for soluble substrate hydrolysis. Lipases are active against insoluble esters, particularly insoluble fats or triglycerides^). Lipases share with phospholipases an interesting feature in that they arerelativelyinactive until brought in contact with an interface. This phenomenon often called interfacial activation is characterized by an abrupt increase in activity upon crossing a solubility boundary (ie. at the first appearance of an interface) and was first clearly demonstrated by Sarda and Desnuelle(6) for porcine pancreatic lipase. In the past few years the molecular basis for interfacial activation is beginning to be understood. It has been shown by X-ray diffraction that the catalytic triad of Mucor mehei lipase is geometrically identical to serine proteases; however, it is covered by a "flap" which prevents access to the active site(7). Subsequent structural studies on this enzyme have shown that the "flap" is opened when an inhibitor is bound in the active site exposing a hydrophobic patch(5). The implication is that interfacial activation involves a conformational change, upon adsorption to a sufficiently hydrophobic interface, and this change allows entry of the substrate to be hydrolyzed. Micelle forming materials have been recognized as possible substrates for lipases and phospholipases(5). Most studies appear to have been carried out on phospholipases(9) which share with lipases the ability to be interfacially activated, but differ from lipases in other importantrespects(iO).We have found that a number of surfactant esters, when used as lipase substrates, exhibit a rate discontinuity upon crossing the critical micelle concentration (cmc) (Figure 1). This is similar to the solubility boundary behavior observed by Sarda and Desnuelle. The possibility of using ester linked surfactants as substrates in lipase assays appears not to be widely recognized even though they offer some advantages over emulsions since micellar solutions are thermodynamically stable and reproducibly prepared. The fact that micelles can activate lipases gave impetus to the idea of using mixed micelles as models to understand the behavior of lipases at oil-water interfaces in systems containing surfactants. The hydrolysis of triglycerides is significantly reduced in the presence of surfactants which limits the efficacy of lipases in certain applications. The mechanism for this reduction in catalytic effectiveness of lipases is not clear. Possibilities include competition between detergent and substrate for the active site, exclusion of the lipase from the substrate by the presence of surfactant at the interface or changed interfacial properties such as increased viscosity due to the surfactant.


211


212

MIXED SURFACTANT SYSTEMS

We present a theoretical treatment of the enzyme catalyzed hydrolysis of a substrate in a mixed micelle which explicitly recognizes both the competition between the detergent and substrate for the active site as well as the need for interfacial activation. In principle, the two effects can be separated by appropriate application of the model and may shed some light on the more realistic situation where the surfactant and hydrolysis products compete with lipase for the oil-water interface or with substrate for the active site of the enzyme. Surfactant esters of decanoic acid with pentaoxyethylene and sodium phenol sulfonate were used to verify the proposed description of micellar kinetics. Mixtures of these surfactant substrates with the non-hydrolyzing surfactants C10E5 and C10E9 were studied in an attempt to find the relative importance of competitive inhibition and mechanisms interfering with the activation process to the rate loss in the presence of surfactant. Experimental Materials. The substrate CJQOBS (Sodium p-Decanoyloxybenzenesulfonate) (Figure 2) was prepared by acylating disodium p-phenol sulfonate with decanoyl chloride in toluene. The product was recrystallized twice from 87:13 methanol/water. The structure and purity were verified by NMR. The substrate C10E5 ester (pentaoxyethylene glycol monodecanoate) (Figure 2) was prepared by acylating pentaoxyethylene glycol (E5) with decanoyl chloride (CJQ) in the presence of pyridine. The product was purified by preparative HPLC (Waters Piep500) using 95:5 hexane/isopropanol. Purity was verified by TLC. The detergent ether (pentaoxyethylene glycol monodecyl ether) was prepared by NaOH catalyzed alkylation of pentaoxyethylene glycol (excess) with bromodecane. The product was recovered by vacuum distillation and the purity was verified by GC. The detergent C10E9 ether (nonaoxyethylene glycol monodecyl ether) was prepared from C10E4CI (C10E4 + S O C I 2 ) and pentaoxyethylene glycol in the presence of NaOH. The product wasrecrystallizedfromhexane and the purity was verified by GC. Lipase was purchased from NOVO under the trade name "Lipolase". Lipolase is a 31.5kD single domain glycosylated lipase from Humicola Lanuginosa. The commercial sample was further purified by HPLC (Waters 625) over a mono-Q anion exchange column. Purity was verified by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Methods. Critical Micellar Concentration (cmc) data were obtained by the duNuoy Ring Method of surface tension measurement(ii). C10E5 ester hydrolysis rates were measured by pH stat(72) on a Brinkmann titrimeter system (Metrohm model 665 Dosimat, 614 Impulsomat, and 632 pH meter) at pH 9.0 in 50mM NaCl, lOmM CaCl2 and 25°C. Rates were measured as moles OH" ions/minute necessary to maintain the pH at 9.0. The number of moles of OH" added correlates with the number of moles of bonds hydrolyzed by the enzyme. Data acquisition was accomplished by Laboratory Technologies Corporation's "Labtech Acquire" software package. CXQOBS hydrolysis rates were also measured on the pH stat



12.


RUBINGH & BAUER

C1QOBS

(mM)

Figure 1. Rate of C OBS hydrolysis by 8ppm lipolase in lOOmM NaCl, pH 9.0 as a function of substrate concentration. The cmc, indicated by arrow, was determined by surface tension measurements. 10

q OBS: 0

CH -(CH ) 3

2

SQ

8

Na

3

C E (ester): 10

5

O C H -(CH ) -C-CMCH C H -0) -H 3

2

8

2

a

B

Figure 2. Structural formulas of the micellizing substrates ester.

CIQOBS

and


C10E5

213

214


system described above in lOOmM NaCl pH 9.0 and on a Beckmann DU-70 spectrophotometer at 252nm in Tris [Tris(hydroxymethyl)aminomethane] buffer at pH 9.0, lOOmM NaCl and 25°C.


Theoretical Development A few treatments for the kinetics of hydrolysis of substrate incorporated into a mixed micelle exist(i5)(I4). These have primarily addressed the action of phospholipase on phospholipids incorporated into micelles. One of these differs from the present in that competitive binding between surfactant and substrate (the phospholipid) is not considered(ii). The other, althoughrecognizingthe possibility, assumes a specific adsorption isotherm (Langmuir) for the enzyme to the surface(W). Neither addresses the solution properties of the substrate or detergent, particularly as itrelatesto the individual cmcs, and how these influence the composition of the mixed micelle. This is aresultof the fact that they were developed to describe insoluble amphiphilic compounds (ie. phospholipids) rather than the soluble substrates and detergents considered here. Our derivation follows the development of Ransac et. al.(i5) which describes the effect of water soluble inhibitors on enzyme action at an oil-water interface. In the present treatment, however, the mixed micelle rather than a mixed interface of substrate and inhibitor (or detergent) provides the activating "phase". The relevant process we need to consider in this case is the adsorption of the lipase to the micelle resulting in enzyme activation as described by equation 1. k

p

E + M
E*M

(1)

*d Here E*M denotes the activated lipase adsorbed to a micelle. To simplify notation, we will use E* for E*M as the activated enzyme at the micelle surface. We specify the rate constant for the formation of activated enzyme as kp, since it isrelatedto the rate of penetration of the enzyme into a sufficiently hydrophobic environment for activation to occur. The rate constant for desorptionfromthe micelle is k^. The activated lipase molecule has the potential to bind in its active site either the surfactant without an ester linkage (hereafter designated detergent) or the surfactant substrate. The binding of detergent is described by equation 2. Since the detergent cannot be hydrolyzed, it acts as a competitive inhibitor for the binding of substrate. The ratio of the forward andreversebinding reaction is .

E* + D ^

> E*D

(2)

The reaction described in equation 2 is postulated to occur between a detergent in the micelle which has been responsible for activation as opposed to a monomelic detergent molecule. In Mixed Surfactant Systems; Holland, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

12. RUBINGH & BAUER


215

The binding of substrate and subsequent reaction to form products is described in equation 3. The species E*S is analogous to the Michaelis-Menten complex for soluble substrate kinetics although here it is postulated to occur between the substrate co-micellized with detergent in the mixed micelle to which the activated lipase is adsorbed. The products of the reaction are fatty acid and alcohol and are indicated by P in equation 3. ki Downloaded by STANFORD UNIV GREEN LIBR on August 10, 2012 | http://pubs.acs.org Publication Date: September 8, 1992 | doi: 10.1021/bk-1992-0501.ch012

E*

+

H 0 2

S^Z±E*S-^->E* + P

(3)

k-i To avoid unnecessary complexity, a more detailed mechanistic description, such as the formation of an acyl enzyme with subsequent deacylation by reaction with water, which is known to occur with proteases and quite probably with lipases as well is omitted. However, it is well known that the general form of the resulting equations are similar although the definition of the various constants differs(76). The equations for the initial rate ofreactionfor substrates in a mixed micelle -dfSI are solved in the standard way. The initial rate of substrate disappearance ( ^ ) is given by equation 4. v =^ 0

= WE*S]

(4)

In the steady state approximation the rate of change of concentration of the Michaelis-Menten complex is equal to zero so that: = k![S][E*] - (k.! + W t E ' S ] = 0

(5)

Additionally, the conservation of enzyme mass implies that: [ E J = [E] + [E*S]

+ [E*M] + [E*D]

(6)

where [EQ] is the total enzyme concentration and [E] is the free enzyme concentration. Using the steady state approximation for all enzyme intermediates allows one to obtainrelationshipsamong the various species and to solve for [E S] in terms of the equilibrium constants in equations 1-3 and the substrate concentration [S] and the total enzyme concentration [ E ] . After a certain amount of algebra one obtains the equation for the initial rate of substrate disappearance in the mixed micelle of substrate and detergent shown in equation 7. This can be thought of as a micellar Michaelis-Menten equation. 0


216


xVmax v

=

( 7 )

° ^r*—TTS—sr* 7 — + (c-cmc)

/ J +—r(l-x) +x (c-cmc) K D

Here x is the mole fraction of substrate in the mixed micelle, c is the total concentration (substrate + surfactant), V is the maximal velocity and is equal to kcatf o]» * ^ critical micelle concentration of the mixture, and R(x) is equal to m a x


E

cmc

s

e

where M is the molecular weight of the micelle at composition x. It should be noted that both the penetration and desorption rate constants as well as the micelle molecular weight can depend on the composition x. This is why the parameter R is explicitly written as a function of composition. The physical interpretation of this parameter involves the ability of the enzyme to penetrate the micelle and be activated. The other parameters are K * which is the micellar Michaelis-Menten m

constant and is given by K

m

= —r

similar to its definition for soluble

substrates and K * , the micellar detergent inhibition constant, which is given by D

K

°

=

k_ A number of limiting cases of equation 7 are of interest. The first is when there is only one surfactant that is also a substrate. In this case x=l and equation 7 reduces to: 2

,v

^max

Q

v = ~~Z K,m (c-cmc)^ (c-cmc)'' * 0

+

(8)

+

This equation is similar to the standard Michaelis-Menten equation except that it contains the interfacial penetration factor R(x) and the concentration scale begins at zero micelle concentration as opposed to zero substrate concentration as is the case for soluble substrates. Equation 8 can be rewritten in a way that demonstrates this by assuming R(x)«l, and can therefore be dropped, and defining the substrate concentration as [S] = c-cmc. Equation 8 becomes under these conditions: V

max[S1

/a\

v =— I (9) ° K * + [S] The effect of the interfacial penetration parameter is shown in Figure 3. At low substrate concentrations finite values of R cause significant deviations from the standard Michaelis-Menten rate (R=0). These differences disappear, however, as substrate concentrations increase and the curves approach the same asymptote, n

m

namely V

m a x

.


12.


RUBINGH & BAUER

211

A second case of interest is for mixed systems with concentrations of detergent and substrate well above the cmc. In this case the micelle mole fraction (x) and bulk mole fraction (a) are essentially the same since almost all the surfactant is micellized and the term . ^ c

is necessarily negligible. Under these conditions

c

equation 7 reduces to:


a

v =— i ;

^max

/

—H

0

i

m

(10)

- + —i(l-a) +a

(c-cmc)

K *

V

'

D

A familiar form of equation 10 can be obtained by multiplying both the numerator and denominator by (c-cmc) which is the concentration of micellized substrate and detergent and recognizing that a(c-cmc) = [S ] and (l-a)(c-cmc) = [D ] where [S ] and [D ] are the concentration of substrate and detergent molecules in the mixed micelle environment. Rearranging results in equation 11 which is the well known expression for the rate in the presence of a competitive inhibitor^). m

m

m

m

^maxt^m]

v =" ft

IPJ"

t

1+

(11)

1

The effect of competitive inhibition by a detergent as a function of micelle composition, assuming constant penetrability, is shown in Figure 4. When the m

binding of substrate and detergent are equal, — j = 1, a linear decrease in rate with K

D

mole fraction of detergent in the mixed micelle is observed . If detergent binding is Km

very much favored over substrate, — j > 1, then small amounts of detergent will K

D

rapidly decrease the rate while the converse will be true if substrate binds preferentially over the detergent to the active site. Some of the most interesting cases may occur when the substrate and the detergent are strongly nonideal. In this case the cmc and the composition of the mixed micelle are controlled by the individual cmcs of the materials and their interactions within the micelle. Mixed surfactant theories(i 7)(18) provide the required data for micelle mole fraction, x, and the mixed cmc to apply equation 7. Results Figures 1 and 5 give typical responses for the rate of micellizing esters as a function of total concentration. A common feature of all the systems studied is a rapid increase in rate as one crosses the cmc. This correspondence between rate discontinuity and cmc is shown explicitly for the C10E5 ester in Figure 5 using the


218


1000

.-tf

800

-


600--

400--

O

•

R=0 R=1x10""

4

200

0 0.0

—H 0.4

0.2

0.6

1.0

0.8

c - c m c (mM) Figure 3. Effect of the exclusion parameter on the concentration dependence of rate. From equation 8 with V = 1000 and K * = 0.1 mM, R=0 is the normal Michaelis-Menten behavior. m a x

K

0

m>D*- -

0.2

m

1

0.4

0.6

0.8

1.0

mole fraction of substrate in mixed micelle

Figure 4. Hydrolysis rates for different values of — f r o m equation 7 with L

(c-cmc)

J

(c-cmc)

' '


12.


RUBINGH & BAUER

219


standard surface tension-concentration method to determine the cmc. In addition, we have observed this behavior with PEG-monoleate(a commercial ester detergent from Stepan) and C9OBS (Data not shown). The maximum in rate at concentrations below the cmc exhibited in Figure 5 for the C10E5 ester was not observed for the other esters; however, detailed studies in this region were not made. Analysis of the data on pure surfactant systems can be made by rearranging equation 8 in a double-reciprocal or, Lineweaver-Burk, format. This gives the following expression: K

1^1 v

o~

v

max

+

V

m* L max

K

1

(c-cmc)

J +

m*

V

R

1

r

L m a x

l 2

n

\

2

J

( c-cmc)

Note that if the last term can be ignored then equation 12 reduces to the normal Lineweaver-Burk expression with the substrate concentration displacedfromzero by the cmc. When the linear and quadratic fits were made to the data in Figure 5 a slightly better fit was seen for the quadratic equation; however, the differences were not significant. The results of the twofitsin terms of kinetic parameters are shown in Table I. Table I. The kinetic constants for both pure surfactants including the quadratic fit for C10E5 ester

Substrate

Fit

C10E5 ester

Km (mM)

linear quadratic linear

C10E5 ester

C OBS 10

3.9 2.1 2.3

R

^cat (sec ) -1

550 414 3

....

1.3 X lO"

4

—-

The data for CIQOBS in Figure 1 was best fit by the linear form of equation 12. The kinetic constants for both pure surfactant esters are shown in Table I. Although the K * values are approximately equal, large differences are seen in k ^ . One significant difference between the two systems is the presence of C a in the C10E5 ester system. Separate experiments have shown dramatic increases in initial velocity in the C10E5 ester system upon addition of C a . Unfortunately, CJQOBS precipitates with added C a making comparison of the two substrates with identical C a concentrations impossible. m

+2

+2

+2

+2


220


We turn our attention now to mixtures where one of the components is a substrate and the second is the detergent component. The first system we examine is a mixture of C10E5 ester and C10E5 ether. The cmc data for pure surfactants and a 1:1 mixture are shown in Table II. The value of the mixed cmc, 1.0 x 10 M, is in excellent agreement with the calculated value from the ideal mixing equation(29) and the measured values of the pure components. Thus, this system behaves ideally.


-3

Table II. Comparison of the theoretical to the experimental cmc for a 1:1 mixture of C E ester:C E ether 1 0

5

10

5

EstenEther Ratio

CMC (mM)

Ideal CMC (mM)

1:0 1:1 0:1

1.30 1.00 0.82

1.01

—

Figure 6 shows the rate of hydrolysis as a function of molefractionC10E5 ester substrate when the total concentration is fixed at 4.23 x 10" M. From the pure K R compound data we can estimate a value of the parameter -[1 + ~ -] as (c-cmc) (c-cmc) K 0.09 and fit the data using equation 9. The best fit is obtained with a — j of 0.9. The 2

m

r

r

m

K

D

physical meaning of this result is that the substrate is marginally favored over the detergent in the competition for the active site (refer to Figure 4). The cmcs from surface tension and the breakpoint from hydrolysis rate vs. concentration data for mixtures of CJQOBS and C10E9 are shown in Figure 7. Once again excellent agreement between the two discontinuities is observed except in the case of mole fraction CIQOBS = 0.25 where the breakpoint is most difficult to determine. The implication of the data is that mixed micelles of substrate and detergent are still capable of activating the lipase for hydrolysis. Figure 7 also gives the best theoretical fit from the regular solution model of mixed micellization to the experimental cmc data(77). The interaction parameter (p) which best describes the data is -0.9. This is considerably less than -4, typical for many mixtures of an anionic with a nonionic surfactant(20). These cmcs were determined in 0.1 M NaCl which is expected to reduce electrostatic repulsions and therefore offset some of the favorable enthalpy of mixing from the nonionic surfactant. Even considering the electrolyte effect, the interaction, as measured by p, is smaller than expected and may also reflect a difference between aromatic vs. aliphatic (or ester linked vs non-ester linked) anionic surfactants in mixtures with alkyl ethoxylate nonionic surfactants. In Mixed Surfactant Systems; Holland, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


12. RUBINGH & BAUER

0.01


0.1

1

10

100

CjgEg Concentration (mM)

Figure 5. CMC and rate discontinuity comparison for C10E5 ester in 50mM NaCl, lOmM C a C l 2 at pH 9.0. Surface tension data (O), rate data Enzyme concentration = 0.02ppm.

mole fraction C ^ E g ester

Figure 6. Rate vs. mole fraction for C10E5 ester:CioE5 ether mixtures. Points are experimental data and the line is the fit using equation 10. In Mixed Surfactant Systems; Holland, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

221

222


In Figure 8 we show data for the rate of CJQOBS hydrolysis with added C10E9. As before the experiment is run at a total concentration much higher than the cmc. Contrary to the ideal mixture, however, the theoretical fit is not particularly •WJT

good. At low mole ratios of CJQEÇ a value of — * = 4 fits the data but at higher


K

D

molefractionsa value of 12 is required. A plausible interpretation of the data within the present model is that the exclusion parameter R(x) is strongly dependent upon composition and penetration becomes increasingly difficult at high mole fraction of C10E9 in the mixed micelle. In fact, this compositional dependence must be very strong to offset the high concentrations which were used to minimize this contribution to the rate loss in these experiments where composition was deliberately varied. Another way to approach the question of how the activity is lost in a mixed system containing both a detergent inhibitor and substrate is to fix the ratio of detergent and substrate in the mixed micelle and vary the total concentration. This is not generally possible by simply fixing the bulk mole fraction since the micelle mole fraction will change as a function of total concentration near the cmc as is well known from from mixed micelle theories(i7). However, at concentrations well above the cmc, particularly in this case where the individual cmcs are nearly equal, this effect becomes negligible. We show the result of such experiments in figure 9 where the concentration is varied in a range well above the cmc so that the micelle molefractionisreasonablyconstant. Inversion of equation 7 would suggest that at fixed χ and high total concentrations a linear relationship would exist between — and VQ

*

and the slopes

C*CUIC

of these lines would vary inversely with mole fraction. Thus at χ = 0.5 the slope would be twice that of die pure CJQOBS. The rate itself could be more strongly affected due to the competitive inhibition, but this effect would be constant at constant χ or, stated differently, would exhibit itself in the intercept. Examination of the slope of the lines in Figure 9 shows that they vary much more rapidly than 1/mole fraction. The ratio of the slope at molefractionof 0.5 to molefraction1 is 23 rather than 2; while at molefraction0.25 this ratio is 105 rather than 4. Both the failure of equation 8 to describe the data in figure 8 and the higher than expected slopes in Figure 9 are consistent with a decreasing ability of the enzyme to be activated by the micelle as the molefractionof the C^QEC increases. Using the data in Figure 9, we can estimate the exclusion parameter as a function of composition and insert this value into equation 7 to obtain the rate at different compositions. Theresultof this is plotted in Figure 10. The agreement between theory and experiment is significantly improved although there is still systematic deviation between theory and experiment. Discussion We have shown that a theoretical model similar to that presented by Ransac et. al.(25) for interfaces can be applied to mixed micelles containing substrate and detergent molecules. The model describes the single substrate case quite accurately.


12.


RUBINGH & BAUER

φ χ= CMC by surface tension


Δ » Rate discontinuity

Δ

0.2

+-

0.4

0.8

0.6

1.0

bulk m o l e f r a c t i o n

Figure 7. CMC and rate discontinuity vs. mole fraction for CioOBSrCioEp mixtures. CMC determined from surface tension concentration plot. Rate discontinuities determinedfromrate vs. concentration plot. The line is theoretical for theregularsolution model with β = -0.9.

3.0 Bulk Molar Concentration - 1.14 χ 10" M 2

2.5 + / \

I

2.0 +

I Ο

S

1

5

K

i

m

7K

D

* =

,4 Ε 0.5 +

0.01

0.0

•

0.1

0.2

0.3

K

D · ·

9

?

,

,

0.4

0.5

0.6

0.7

m

VK

1 0.8

D

* = 12 1 0.9

1.0

mole fraction C Q O B S 1

Figure 8. Rate vs. mole fraction for

CIQOBSÎCIQEÇ

mixtures. Experiment vs.

theory for the values of — j indicated (using equation 10). D K


223


224


-2

0

2 4 1 / c - c m c (mM)

6

8

10

1

Figure 9. Doublereciprocalplot for different (fixed) values of the composition, αrepresentsthe mole fraction of substrate.

3.0

0.0

0.2

0.4

0.6

0.8

1.0

m o l e f r a c t i o n of C ^ g O B S

Figure 10. Rate vs. mole fraction for CjoOBStC^oEç mixtures. Line is best m

theoretical fit using equation 7. The points are experimental data with — * = 4 K

and R(x) as described in text. In Mixed Surfactant Systems; Holland, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

D


12.

RUBINGH & BAUER


225

In the mixed surfactant case the model works as expected for an ideal mixture of surfactants. The model also provides a reasonable description of the more complicated case of C I Q O B S with C10E9 where mixing is nonideal and more dramatic competition and exclusion/activation effects might be expected. Our analysis suggests that the observed rate loss upon adding CIQEQ to C J Q O B S is a result of both competitive inhibition and exclusion effects. Although the nature of the physical phenomena responsible for differences in exclusion remain to be elucidated, the ability to separate loss in activity due to exclusion from that due to classical inhibition will allow progress to be made in that direction. Hopefully the model presented here will provide a framework for such separation to be made. The products of the hydrolysis reaction of these simple esters, namely fatty acid and alcohol, can themselves be surface active and co-micellize with substrate and surfactant once formed. In the study presented here the decanoate ion would be expected to partition between the micelle and solution while the alcohol, from either substrate, probably resides in the aqueous phase. By measuring and analyzing only initial rates, where little product is present, it is hoped that this added complexity is successfully avoided; however, the limitation of the current approach to initial velocities of enzyme catalyzed reactions in mixed micelles should be explicitly recognized. Acknowledgments We would like to thank Jim Thompson and Larry Sickman for the synthesis, purification and characterization of both the surfactant substrates and the nonhydrolyzable surfactants and Cathy Oppenheimer and Debbie Thaman for the characterization of NOVO's lipolase. Literature Cited 1. Carter, P. and Wells, J.A.; Nature, 322, 564 (1988). 2. Walsh, C. Enzymatic Reaction Mechanisms, W. H. Freeman and Co, San Fransico, (1979). 3. Segal, I.H. Enzyme Kinetics, John Wiley and Sons, NY (1975). 4. Cornish-Bowden, Α., Principles of Enzyme Kinetics, Butterworths, London (1975). 5. Brockman, H.L. in Lipases, Borgstrom B. and Brockman, H. L. Eds., Elsevier, Amsterdam, p4 (1984) 6. Sarda, L., and Desnuelle, P., Biochim. and Biophvs. Acta., 30, 513 (1958). 7. Brady, L., et. al., Nature, 343.767 (1990) 8. Brzozowski, A. M. Nature, 351, 491 (1991). 9. Dennis, Ε. Α., in The Enzymes,Boyer, P., Ed. 3rd edition, vol. 16, Lipid Enzymology, Academic Press, NY, p307 (1983). 10. Scott, D. L., Science, 250, 1541 (1990). 11. Davies, J. T. and Rideal, Ε. K. in Interfacial Phonomena, Academic Press, NY, p42 (1963). 12. Jacobsen, C. F., Leonis, J., Linderstrom-Lang, K., Ottensen, M. in Methods in Biochemical Analysis, Vol. IV, Interscience Publishers Inc., NY, p171 (1957).


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250,


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Lipase Catalysis of Reactions in Mixed Micelles - ACS Symposium

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