Indian Journal of Biochemistry & Biophysics Vol. 37, June 2000, pp. 171-177

, -..

'- 'Reverse micelles as water property control system to investigate the hydration, superactivity and kinetics of invertase

_Anjana {s ingh and R S ubey* -

( Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 00 , India '--

, (. ,'" S u c.. .. -

Received 26 October 1999; revised 7 February 2000 . C rl:' L,. t •

'"'-~

C Invertase, entrapped in hydrated reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (Aerosol-OT, AOT) in isooctane showed 5.8 fold enhancement in its activity in comparison to the activity of invertase in bulk aqueOus medium. Fluorescence spectra of the enzyme was examined as a function of W" (molar ratio of water to surfactant) to monitor overall conformational changes associated with polarity changes due to the presence of tryptophan in these proteins. The degree of hydration required in terms of W" for maximum activity of invertase was from 8 to 18. The pH/activity profile of the enzyme was bell shaped in the aqueous system with optimum pH of 5.0, whereas, within reverse micelles, maximum activity was near alkaline pH and the pH/activity profile approached a sigmoid shape. The activity increased within reverse micelles in presence of I to 400 roM NaCI whereas under similar NaCI concentration in the aqueous medium almost static level of enzyme activity was observed . In presence of MgCI 2 the activity of invertase decreased in aqueous medium, whereas the activity was enhanced within reverse micelles. Under 50-1000 ~ Pb(N03)2 almost similar level of invertase activity was observed in both aqueous medium and within reverse micelles. In presence of Cd(N03h (1000 J..lM) there was 38 per cent increase in enzyme activity within reverse micelles but there was no alteration in enzyme activi~ in aqueous medium~ ~ S" '-1 .

Replacement of water by organic solvents and their catalytic activity and substrate specificit/o-'4 .

hydrophobic media is normally accompanied by Protein hydration plays a critical role in the catalytic either a total denaturation of the enzymes or dramatic activity and conformational stability of enzymes9

, 15-17.

decrease of their catalytic activity and also the loss of The enzyme molecule requires a small hydration layer their substrate specificity'. This fact impedes the that acts as the primary component of the enzymic application of enzymes in several biotechnological environment This layer acts as a buffer between the processes where hydrophobic media are required2

-5

. enzyme surface and the bulk reaction medium. The By entrapping water-soluble enzymes into the degree of hydration, W 0, of reverse micelles, defined aqueous pockets of reverse micelles formed by as [H20]/[AOT], can be determined very precisely . surfactants in hydrophobic medium '.6-8, the enzyme Different experimental approaches have been used can be protected from the unfavourable action of to determine the extent of hydration required by an organic solvents . Reverse micelles are small aqueous enzyme molecule to express its optimum catalytic droplets surrounded and stabilized by a monolayer of activity and to maintain its maximum conformational surfactant molecules and dispersed in a water stabili ty '5. These approaches require a system in immiscible organic solvent. The reverse micelles which not only the amount of water molecules can be formed in organic solvents by surfactants form a very precisely controlled but also a system in which thermodynamically stable, optically transparent the enzyme conformation/activity relationship can be system in which the core radius of internal water followed by a very sensitive technique. One very pools varies from one to a few tenths of a nanometer. promising approach to investigate the properties of The water present in the water pool of reverse enzymes in a water restricted environment is based on micelles mimic the structural and dynamic properties enzyme entrapment within the reverse micelles9

.

of water bound to biomolecules confined in biological In the present investigation we have studied the cells and membranes9

. It has been shown that the hydrolysis of sucrose by invertase in aqueous and enzymes solubil ized inside the reverse micelles retain reverse micellar system using the surfactant AOT and

the organic solvent isooctane under different *Author for correspondence conditions of [H20]/[AOT] molar ratio, pH and salt Fax : 91 -542-3 17074; E-mail: rsdubey@ banaras.ernet.in ions. A detailed analysis of the results has been made

172 INDIAN J BIOCHEM BIOPHYS, VOL. 37, JUNE 2000

which is expected to help in understanding the specific activity of invertase in apolar solvents under controlled water environment for its enhanced activity using reverse micellar system.

Material and Methods

Assay of invertase in aqueous system The activity of invertase was assayed according to Gajjar et al.'4. The assay mixture in a total volume of I ml contained 0.2 ml of 50 mM sodium acetate buffer (PH 4.5), 0.79 ml of sucrose solution of appropriate strength (0.05 to 0.7 M) and 10 III pure yeast invertase (Sigma, 22.7 unit mr'). The mixture

was incubated for 30 min at 35°C, reaction stopped by adding 0.5 ml of 0.1 M Na2HP04 solution, followed by boiling in a water bath for 10 min. Reducing sugars produced were estimated by Nelson­Somogyi method' s. For this purpose, I ml of alkaline copper reagent was added to 0.5 ml of reaction mixture and boiled in a water bath for 20 min, cooled under tap water fo llowed by addition of I ml arsenomolybdate colour reagent. The volume was made up to 12.5 ml by adding 10 ml disti lled water and absorbance was recorded at 560 nm. Glucose served as standard. One unit (IU) of invertase is that amount which catalyzes the formation of I Ilmole of product per minute under above mentioned conditions. Specific activity of invertase is expressed as Ilg of reducing sugars produced min-' mg"' protein.

Entrapment and assay of invertase in the reverse micelles

Invertase solution 0.1 mg mr' (Sigma, 22.7 unit mr') was prepared in 50 mM sodium acetate buffer, pH 4.5 . To 3 ml of isooctane was added 0.13 g of purified AOT (100 mM)'9 and dissolved properly, followed by addition of 10 III of sucrose of desired strength (0.05 to 0 .7 M), 10 III enzyme and 30 III buffer. [The desired W 0 value was reached by the addition of a previously determined volume of 50 mM acetate buffer, pH 4.5]. This resulted in entrapment of substrate and enzyme within reverse micelles at W 0

9.0. The conditions for the assay of invertase activity were same while doing experiments in aqueous medium and within the reverse micelles . The reaction mixture was incubated for 30 min at 35°C. After incubation, aqueous extraction was done by addi ng 4.5 ml of double distilled water with stirring and reaction was immediate ly terminated by addition of

0.5 ml of 0.1 M Na2HP04 followed by boiling in a water bath for 10 min as described by Oser' 8.

Fluorescence measurements Fluorescence measurements were recorded on

Perkin-Elmer LS-5B fluorometer at 25°C. The tryptophan steady-state fluorescence spectra of invertase at different Wo were measured usmg an excitation wavelength of 290 nm.

Effect of pH on the activity of enzymes in aqueous medium and within reverse micellar water pool

Sodium acetate buffer (50 mM) of different pH was used for the determination of invertase activity within reverse micelles at Woof 9.

Effect of salts and heavy metals on activity of invertase

The effects of NaCl, MgCb, Cd(NO})2 and Pb (N03h on the activity of invertase within the reverse micellar system as well as in aqueous system were studied. Different concentrations of salts were prepared in 50 mM sodium acetate buffer, pH 4.5. Salt solutions were entrapped within the reverse micelles (Wo=9.0).

Protein estimation Protein content was estimated in all the samples by

120 . b · the method of Lowry et a. usmg ovme serum albumin (BSA, Sigma) as standard.

Determination of maximum velocity (VlIlll.rJ and Michaelis constant (KlIl )

Vmax and Km were determined from the Lineweaver­Burk plots and checked by Corni sh Bowden plot2

' .

Results Activity of enzyme entrapped within reverse micelles

The data on Vm•1X and Km of puri fied invertase are presented in Table I. The activity of invertase within reverse micelles increased by almost 6-fold and its

Table I-Vma. and Km of invertase in normal aqueous medium and after entrapment in reverse mi celles formed by AOT in isooctane. [Data represent mean ± S.D. based on three independent determinations].

Aqueous medium Reverse micelles

VOla. (J..lmole min· l) 0.126 ± 0.015

Km (M) 0.200 ± 0.029

Vma.IKm 0.63

0. 741 ± 0.096

0. 110 ± 0.015

6.74

SINGH & DUBEY : REVERSE MICELLES AS WATER PROPERTY CONTROL SYSTEM TO INVESTIGATE INVERTASE J 73

catalytic efficiency (VrnaxlKrJ increased by II-fold when compared to the activity observed in the normal aqueous system. The Lineweaver-Burk plot is shown in Fig. 1.

25

20 (1)

-- 15 c

> 'E - III .... CI)

'0 E 10 • 2:

-10 -8 -6 -4 -2 0 2 4 6 8 10 12

1/(S] (Mr'

Fig. I-Lineweaver- Burk plot for invertase (22.7 units mr ') catalyzed reaction in aqueous system without surfactant , (I ) and after entrapment in the reverse micelles, (2) formed by AOT in isooctane. [Experiments were performed at 35°C using 50 mM sodium acetate bu ffer, pH 4.5].

16 >. .... ,... .- c

. ~ .- 14 u Q)

0 CIS ... U 0-t;:-' 0 '00 12 ~E tIl_

Q) C til . -CIS E t 10 Q) eo > ::t

..5 '-'

8

0 2 4

EffectofWo

The dependence of specific activity and Km on Wo in 0.1 M AOTlisooctane reverse micelles for invertase is shown in Figs 2 and 3. It is observed that specific activityIWo profile is dependent on the water content in the system (Fig. 2). At Wo of 1.8, the enzyme had high activity. On increasing Wo upto 5, specific activity decreased to a minimum value. Above Wo of 5, the specific activity increased and reached a plateau at Wo of 8 and beyond. Km decreased gradually from Wo 2-6 and there was no significant change in Kmon further increase in WOo

0.32

0.28

0.24

~ 0.20 .J

0.16

0.12

0.08

0 2 4 6 8 10 12

w.

Fig. 3-Dependence of Km on W" fo r invertase catalyzed hydrolysis of sucrose in 0.1 M AOTlisooctane reverse mi cell ar system [Values are mean ± S.D. based on three independent determinations]

6 8 10 12

Wo

Fig. 2-Dependence of Vnla, on W" for in vertase catalysed hydrolysis of sucrose in 0.1 M AOT/i sooctane reverse micell ar system. [Values are mean ± S.D. based on three independent determinati ons]

174 INDIAN J BIOCHEM BIOPHYS. VOL. 37. JUNE 2000

Tryptophan has been regarded as a natural fluorescence sensItive indicator of the microenvironment in proteins9

.22

. Using tryptophan as a probe. the fluorescence spectrum of invertase was examined as a function of Wo to monitor overall conformational changes associated with polarity changes (Fig. 4). Tryptophan fluorescence at low Wo values i.e. at Wo of 0.6 (Fig. 4, curve I), is blue shifted in comparison to that in aqueous solution (Fig. 4, curve 2) reflecting a decrease in the average polarity near the emitting tryptophan residues. On increasing Wo to 5.4, the emission spectrum increased to an even slightly more red shifted value than observed in solution . With further increase in Wo upto 16.2 there was no variation from the value in solution. When the fluorescent effects on the protein was compared using tryptophan derivative i.e. L-tryptophanamide hydrochloride In micellar medium, a continuous red shift was observed with increase of WOo Experiments were performed upto Wo 23.3.

Effect of pH The velocity of the reaction catalyzed by invertase

at 6 different pH in aqueous system as well as in reverse micelles is shown in Fig. 5. A shift in optimum pH of the reaction towards a much higher pH was observed in the reverse micelles when compared to normal aqueous system. In aqueous medium, the pH/specific activity profile was bell shaped whereas in reverse micellar medium it was

.f' I. 0 C .. ~~0.8 .. ~

u .~ c :: .. :::I

~ CO.6 ... .. o :.. :J .~

~"f! ~ -!!.0.4 . ~ .. E ~ 0 .2 z

300 320 340 360 380 400 Wavelength (nlll)

Fig. 4-Effect of WI) on the fluorescence spectra of invertase C I 0 mg mrl ) at different W" values [curve I. C---------) W" = 0.6; curve 2. C--). bulk water; curve 3. (-.-.-.-.-) W" = 5.4; curve 4. (- ..... -). W" = 16.2 [Excitat ion wavelength was 290 nm]

sigmoid. From pH 7.0 to 8.0 a sharp increase in invertase activity was observed in the reverse micelles, whereas a sharp decrease in enzyme activity was noted in normal aqueous medium.

Effect of salts Table 2 shows the effect of NaCI and MgCh on the

activities of invertase in aqueous system and after entrapment within reverse micelles. Increasing concentration of NaCl or MgCl2 beyond I mM upto 400 mM in reverse micelles caused a significant Increase in the activity of invertase, whereas in aqueous medium even I mM MgCIz caused about

18 ,----------------,

.... >-0

;t:::: ..--

~x U "......

c:( .~ 02 1;::0 'u 0.. Q) 0.0;­C/)Ol Q) E Vl_ <U 'c t .­Q) E ~ Ol

- ::1. '-'

16

14

12

10

8

6·

3 4 5 G 7 !3

pH

Fig. 5-Effect of pH on the activity of invertase at 35°C in normal aqueous medium. ( .-. ); and within the reverse micell es formed by AOT in isooctane. (x-x)

Table 2-Effect of NaCI and MgCI2 on the activity of invertase (mg min·1 mg' l protein) in aqueous system and after entrapment within reverse micelles formed by AOT and isooctane at WI) 9.0. [50 mM sodium acetate buffer of pH 4.5 was used. Values are mean based on three independent observations] .

Concentration NaCI MgCI 2 of salts Aqueous Reverse Aqueous Reverse (mM) system micelles system micell es

0 22.73 133.33 22.73 133.33

I 34.69 153.78 4.55 174.99

50 28.43 164.37 2.84 145.83

100 21.68 163 .85 4.06 183.33

200 23.44 214.38 4.64 158.33

400 37.38 273.70 4.38 156.48

SINGH & DUBEY: REVERSE MICELLES AS WATER PROPERTY CONTROL SYSTEM TO INVESTIGATE INVERTASE 175

Table 3-Effect of Pb(N03h and Cd(N03h on the activity of invertase (mg min-I mg-I protein) in aqueous system and after entrapment within reverse micelles formed by AOT and isooctane Wu 9.0. [50 mM sodium acetate buffer of pH 4.5 was used. Values are mean based on three independent observations.]

Concentration Pb(NO,h Cd(NO,)~

of salts Aqueous Reverse Aqueous Reverse (11 M) system micelles system micelles

0 22.73 133.33 22.73 133.33

50 20.41 143.33 19.68 163 .79

100 19.94 123.33 18.69 147.37

250 20.41 106.67 19.68 194.38

500 22.03 123.33 20.98 187.83

1000 21 .65 129.99 20.34 183.48

82% inhibition in the enzyme actIvIty. Increasing concentration of NaCI in the aqueous medium caused no significant change in the activity of the enzyme.

When increasing concentrations (0-1 000 ~ of heavy metal salts were incorporated within reverse micelles Cd(NOJh caused increase in invertase activity whereas with Pb(NOJh upto 1000 flM there was no significant change in the activity of enzyme (Table 3). In aqueous medium 50-1000 flM Cd(NOJ) 2

caused almost no alteration in invertase activity, whereas within reverse micelles there was significant increase in enzyme activity.

Discussion Increase in enzyme activity indicates that the

reverse micellar aqueous cavities protect the enzyme against denaturation by the surrounding organic solvents. It is likely that the net charge distribution inside the aqueous cavities, the net charge on enzyme molecule and that on substrate molecule could affect the interaction between the enzyme and the substrate . 'd h . II 1221-25 A . d InSI e t e reverse mIce es ,. , major a vantage of this technique is that the enzyme is dispersed in a thermodynamically stable liquid solution which is capable of solubilizing polar, apolar and interfacially acti ve substrates 7.26,27.

Madamwar et at. 28 reported that the activity of invertase can be enhanced by 2-3 fold when entrapped within reverse micelles formed by sodium lauryl sulfate and sodium tauroglycocholate in organic solvents . Gajjar et al.14 showed that invertase activity from Brewer's yeast was enhanced by 1.33 fold when the cells were entrapped within reverse micelles formed by CPCI-n hexane. In this communication we report increase in invertase

actIVIty within reverse micelles formed by AOT­isooctane. Besides the ease of formation of AOT­isooctane reverse micelles, the activity of invertase increased by almost 6-fold and the catalytic efficiency of the enzyme (as measured by the ratio VrnaxlKm) increased by II-fold when the reaction was performed within the aqueous pool of the reverse micelles in comparison to the reaction performed in aqueous medium.

Our observation that the tryptophan fluorescence spectrum of invertase at Wo of 0 .6 is blue shifted in comparison to that in bulk water indicates that the invertase in reverse micelles with 0.6 Wo is in an apolar environment. Consequently, it appears that there is a structural change in the protein that either decreases the exposure of the tryptophan to the solvent or removes the polar groups within the protein away from the tryptophan side chain. In the absence of a hydration shell, the protein could be, to a great extent frozen and remain inactive9

. A small increase in water content to Wo of 1.8 possibly, leads to increase in the enzyme polarity as well as the active site polarity and the enzyme displays a high catalytic activity.

Proteins can exist in a number of main conformations and subconformations depending on the physicochemical parameters of the environment9

.

Enzyme alone as well as some ligand-enzyme complexes exist in a number of different conformational states and substates9

• Our results imply that substrate binding possibly induces change in enzyme conformation rendering it active at very low W 0 values. It is known that protein exists in different conformational substates as a consequence of water-protein interactions, Further increase in water content of the micellar system can be expected to transfer new water molecules to the negatively charged surfactant heads aRd the enzyme molecule may become even more rigid (at Wo 2.7). The tryptophan fluorescence spectrum shows a red shift at Wo of 5.4 which is even higher than that observed in solution (Fig. 4, curve 3), This indicates that the average polarity around the enzyme molecule is higher than in water. This situation may occur when the protein globule is surrounded by water molecules tightly bound to the surfactants of reverse micelles (Wo - 3) . In AOT reverse micelles at Wo > 3, an intermediate unstable state of water with unfavourable enthalpy is formed between strongly bound and bulk water, which is completely formed at

176 INDIAN J B10CHEM B10PHYS, VOL. 37, JUNE 2000

Wo of approximately 5. Under these conditions, the enzyme displays minimum activity (Fig. 2). Thus depending on the water content of the system, the protein-water structure is trapped in the local subs tate minimum, which may slightly differ in free energy but is separated by high barriers, so that the lifetime of the substates is relatively long9

• Due to these high barriers the protein-water system does not move along the reaction coordinate upon increasing the water content of the system thus keeping the enzyme irreversibly inactivated. Our observations support the

I· 29·31 ear ler reports .

The microenvironment of proteins in reverse micelles simulates the microenvironment of proteins in vivo, i.e. in cellular organelles and biological membranes due to controlled water environments32

.

AOT used for the preparation of reverse micelles is a biological surfactant. As yeast invertase shows optimum activity in acidic pHD, experiments were conducted to determine its pH optimum in the pH range 3-8. The pH optima of the enzyme shifted towards alkaline range within reverse micelles, suggesting that alkaline pH is more favourable for catalysis within the reverse micelles. Our observation is similar to the pH profile of tyrosinase activity in reverse micelles formed by AOT in isooctane which showed increase of activity from pH 4.0 to 6.0 beyond which upto pH 9.0 the activity remained constant, establishing a broad plateau34

. When the kinetics of alkaline phosphatase catalyzed hydrolysis of p-nitrophenyl phospha~e was studied in aqueous buffered as well as in reverse micellar buffered medium formed by AOT and decane, the pH optimum observed was 10.2 in the reverse micellar system34

.

Lipases from Humicola lanuginosa (HIL) and Rhizomucor miehei (Rml) when solubilized in AOT­stabilized water-in-oil microemulsions in n-heptane and p-nitrophenyl butyrate and the hydrolytic activity was measured as a function of buffer pH, a sigmoidal curve was observed with the highest activities at alkaline pHs. The pH-activity profile suggests that enzyme undergoes conformational changes with change of pH in the aqueous microenvironment of reverse micelles that possibly favours more conformationally active enzymic form for increased activity.

In presence of inorganic salts like NaCl, MgCh and heavy metal salts like Cd(N03h and Pb(N03)2 the activity of invertase in aqueous and in reverse micellar systems, was different NaCI, MgCh and

Cd(N03h enhanced the actlVlty in reverse micelles but not in the aqueous medium suggesting that the effects of ions on enzyme activities in bulk water systems are different from the effects on enzymes in a restricted water environment.

Acknowledgement One of the us (A S) is grateful to University Grants

Commissions, New Delhi for the award of a NET senior research fellowship. We are also grateful to Dr M V Jagannadham, Molecular Biology Unit for providing instrumentation facilities.

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