The encapsulation of pepsin by electrostatically controlled diffusion from solution into thermally evaporated fatty amine films is described and the catalytic activity of the immobilized enzyme on hemoglobin is investigated

Dash, Chandravanu

Gole, Anand

Rao, Mala

Sastry, Murali

English

Publications of the IAS Fellows

Encapsulation and biocatalytic activity of the enzyme pepsin in fatty lipid films
by selective electrostatic interactions
Anand Gole,a Chandravanu Dash,b Mala Rao,*b and Murali Sastry*a
a Materials Chemistry Division, National Chemical Laboratory, Pune-411 008, India. E-mail: sastry@ems.ncl.res.in 
b Biochemical Sciences Division, National Chemical Laboratory, Pune-411 008, India. 
E-mail: malarao@dalton.ncl.res.in
Received (in Cambridge, UK) 29th November 1999, Accepted 19th January 2000
The encapsulation of pepsin by electrostatically controlled
diffusion from solution into thermally evaporated fatty
amine films is described and the catalytic activity of the
immobilized enzyme on hemoglobin is investigated. 
The entrapment of proteins in different inert matrices with the
aim of protecting the proteins against microbial degradation,
hydrolysis, autoproteolysis, deamidation, etc.; retention of the
native protein structure and accessibility of the encapsulated
proteins to cofactors, substrates and redox agents is a problem of
current interest especially where application in biosensors/
biocatalysis is sought. Proteins have been immobilized in
phospholipid bilayers,1 on self-assembled monolayers
(SAMs),2 in silicate sol–gels,3 in polymer matrices,4 in
Langmuir–Blodgett films,5 within the galleries of a-zirconium
phosphates6 as well as polymer microspheres.7 Developing on
our earlier work on the spontaneous self-organization of fatty
acid salts8 and electrostatic assembly of colloidal nano-
particles,9,10 we show here, that the proteolytic enzyme pepsin
(which occurs in the gastric juice of all mammals), can be
encapsulated via electrostatic interaction in thermally evapo-
rated fatty lipid matrices (octadecylamine, ODA) by simple
immersion of the lipid film in the protein solution under
extremely mild preparation conditions (Schematic 1, inset of
Fig. 1). The encapsulated enzyme showed good biocatalytic
activity using hemoglobin as the substrate. The biocatalytic
activity was determined by estimating the amount of acid-
soluble tyrosine and tryptophan residues released by reaction of
the encapsulated pepsin on hemoglobin.
Pepsin (molecular weight = 37400; pI ≈ 1 where pI
represents the isoelectric point)11 was obtained from Sigma
Chemicals and used as received. A 1026 M solution of pepsin
was prepared in glycine–HCl buffer (0.05 M, pH 3), close to the
pH value at which pepsin exhibits maximum catalytic activity.
250 and 1000 Å thick ODA (Aldrich) films were deposited by
thermal evaporation on gold coated AT cut quartz crystals (for
quartz crystal microgravimetry (QCM) measurements) and
Si(111) substrates (for FTIR and activity measurements) in an
Edwards E306A chamber. Fig. 1 shows a plot of the QCM mass
uptake recorded ex situ from a 250 Å thick ODA film as a
function of time of immersion in the pepsin solution. Details of
the QCM measurement procedure may be obtained from our
earlier reports.9,10 It can be seen that the diffusion of pepsin into
the lipid matrix is extremely rapid with maximum protein
uptake being accomplished within 10 min of immersion. The
small time scale for encapsulation of the enzyme in the lipid
matrix is a particularly attractive feature of this approach and
considerably improves upon the days to weeks timeframe
required in other techniques for synthesis of such bio-
composites.3,6 At pH 3, the ODA matrix is positively charged
(pKB of ODA = 10.5) while the pepsin molecules are
negatively charged (pI of pepsin ≈ 1.0 ) thereby leading to
attractive electrostatic interactions and a rapid diffusion of the
proteins into the lipid matrix (see inset of Fig. 1). The maximum
mass loading in the film (Fig. 1) is measured to be ca. 3900 ng
cm22 of the film yielding a pepsin concentration of 6.3 3 1013
molecules per cm2 of the ODA–pepsin composite film.
It is well known that proteins spontaneously concentrate at
the phase boundaries12 and simple surface adsorption of pepsin
on the ODA film surface must be ruled out. Contact angle
measurements of a sessile water drop (1 ml, Rame Hart 100
goniometer used) were carried out on a 250 Å thick ODA
film on Si(111) substrates after immersion in 1026 M pepsin
solution at pH 3 for 60 min and careful washing of the film. The
measurements carried out at different points on the film surface
yielded a mean value of 90° which is very close to the contact
angle of 100° recorded for the as-deposited ODA film. The
contact angle recorded for the pepsin–ODA composite film is
much higher than the values of 20 and 15° measured for the bare
Si(111) substrate surface and a pepsin film formed on Si by
evaporating a drop of the pepsin solution respectively, indicat-
ing clearly that the pepsin molecules are not adsorbed on the
surface of ODA but within the lipid film.
Fig. 2 shows the FTIR spectra recorded from a 1000 Å thick
as-deposited ODA film (curve 1), the ODA film after
immersion in 1026 M pepsin solution kept at pH 3 for 60 min
(curve 2) and the pepsin–ODA nanocomposite film after testing
the catalytic activity of the pepsin–ODA film by immersion in
hemoglobin solution (curve 3, as discussed subsequently). A
number of vibrational modes can be observed for the three
films. The amide I band occurs at 1647 cm21 for the pepsin–
ODA nanocomposite film (feature A, curve 2, Fig. 2) and the
film after reaction with hemoglobin (curve 3, Fig. 2). Whereas
a small feature at this wavenumber does occur in the as-
deposited ODA film, the intensity of this band increases in films
2 and 3 clearly showing that it originates in the pepsin
molecules. The position of this band is close to that reported for
native proteins in earlier reports6,13 and indicates that the
Fig. 1 QCM mass uptake curve measured ex situ as a function of time of
immersion of 250 Å thick ODA films in 1026 M pepsin solution at pH 3.
The schematic in the figure illustrates the procedure adopted for synthesis
of the pepsin–ODA composite and the probable microscopic structure of the
film and enzyme encapsulation.
This journal is © The Royal Society of Chemistry 2000
Chem. Commun., 2000, 297–298 297
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secondary structure of the enzyme in the ODA environment is
unperturbed. The amide II band, which occurs at 1521 cm21
(feature C, Fig. 2), can also clearly be seen for the pepsin–ODA
composite film as well as the film after the activity test with
hemoglobin (curves 2 and 3 respectively). This band also
indicates that the secondary structure of the protein is
maintained in the encapsulated form.13 Small differences are
observed in the amide II bands for the pepsin–ODA films before
and after reaction with hemoglobin. This may be due to a
contribution of hemoglobin molecules in the ODA matrix in the
digested/undigested form and suggests a possible mechanism
for the action of encapsulated pepsin on the heme-protein.
However, further work is required before an unequivocal
statement can be made on this observation. The origin of the
band at 1564 cm21 (feature B, Fig. 2) is as yet not fully
understood but it clearly arises from the ODA matrix and its
intensity is reduced on complexation of the ODA molecules
with pepsin.
The biochemical activity of the enzyme encapsulated in the
lipid matrix was determined by reaction with a solution of
hemoglobin (5 mg ml21) prepared in glycine–HCl buffer (0.05
M, pH 3.0) and by incubating the reaction mixture at 37 °C for
30 min. Pepsin digests hemoglobin and yields acid soluble
products which are readily detected by their strong UV
signatures at 280 nm.14 Quartz substrates of known dimensions
coated with 250 Å thick ODA films were immersed in pepsin
solution for 1 h until the pepsin density in the films reached
equilibrium values (6.3 3 1013 pepsin molecules cm22 of film,
see QCM studies). These films were immersed in 1 ml of
hemoglobin solution (5 mg ml21) and incubated at 37 °C for 60
min and then withdrawn. The reaction was quenched by
addition of an equal volume of 1.7 M perchloric acid to the
reaction mixture and the precipitate removed by centrifugation.
The supernatant containing the acid soluble tyrosine and
tryptophan residues was analysed using UV–VIS spectroscopy
and the optical absorbance at 280 nm determined (Shimadzu-
6201 PC spectrophotometer operated at a resolution of 1 nm).
For comparison, the enzymatic activity of pepsin in solution
was determined in a similar fashion. The values obtained from
these activity measurements are given in Table 1. It is observed
that the activity of pepsin in the ODA matrix is slightly less than
that of the enzyme in solution. This may be a consequence of the
orientation of the enzyme in the lipid matrix limiting the
accessibility of the substrate hemoglobin to the active enzyme
sites. Another possibility is that all the enzyme molecules do not
participate in the biocatalysis in the first run. That this is so is
indicated by the fact that one of the films showed enzymatic
activity during three successive runs, albeit considerably
reduced each time (Table 1).
The different measurements on the pepsin–ODA composite
films clearly establish the following. The electrostatically
controlled diffusion of the enzyme molecules from the aqueous
phase into the lipid matrix may be accomplished under
conditions close to that where the enzyme shows maximum
activity by suitable choice of the lipid (either cationic or
anionic). The enzyme molecules are encapsulated within the
ODA matrix without significant distortion to the native
structure. The elasticity of the bilayers may be primarily
responsible for this and enables the matrix to adopt the contours
of the enzyme molecule (Schematic, inset of Fig. 1). The
reasonably fast time-scales for the synthesis of the enzyme–
lipid composites and the enzyme–friendly encapsulation condi-
tions are a major improvement over other techniques currently
being investigated.1,3,4,6
Two of us (A. G. and C. D.) thank the Council for Scientific
and Industrial Research (CSIR), Government of India, for
financial assistance.
Notes and references
1 I. Hamachi, A. Fujita and T. Kunitke, J. Am. Chem. Soc., 1994, 116,
8811.
2 J. Fang and C. M. Knobler, Langmuir, 1996, 12, 1368.
3 I. Gill and A. Balleseros, J. Am. Chem. Soc., 1998, 120, 8587.
4 K. Yoshinaga, K. Kondo and A. Kondo, Polym. J., 1995, 27, 98.
5 A. Boussaad, L. Dziri, R. Arechabaleta, N. J. Tao and R. M. Leblanc,
Langmuir, 1998, 14, 6215.
6 C. V. Kumar and G. L. McLendon, Chem. Mater., 1997, 9, 863.
7 F. Karen, K. Griebenow, L. Hsieh, A. M. Klibanov and R. Langer,
J. Controlled Release, 1999, 58, 357. 
8 P. Ganguly, S. Pal, M. Sastry and M. N. Shashikala, Langmuir, 1995,
11, 1078.
9 M. Sastry, V. Patil and S. R. Sainkar, J. Phys. Chem. B., 1998, 102,
1404.
10 V. Patil, R. B. Malvankar and M. Sastry, Langmuir, 1999, 15, 8197.
11 The Enzymes: Vol. 4 ed. by P. D. Boyer, H. Lardy and K. Myrback,
Academic Press, New York, 1960, ch. 4.
12 L. Razumovsky and S. Damodaran, Langmuir, 1999, 15, 1392.
13 A. Dong, P. Huang and W. S. Caughey, Biochemistry, 1992, 31, 182.
14 M. Anson, J. Gen. Physiol., 22, 79, 1938.
Communication a909385k
Fig. 2 FTIR spectrum recorded from a 1000 Å thick ODA film before (curve
1) and after immersion for 60 min in pepsin solution at pH 3 (curve 2). The
spectrum measured from the pepsin–ODA film after assay with hemoglobin
is also shown (curve 3). Features A–C are discussed in the text.
Table 1 Comparison of enzymatic activity of pepsin in solution and ODA
entrapped pepsin using hemoglobin as the substrate
System
Amount of
pepsin/mg
Activitya/
units
Activity/
units mg21
Pepsin 2.0 7.5 3.75
Pepsin–ODA (film 1) 2.7 9 3.33
Pepsin–ODA (film 2, run 1) 3.6 10 2.77
Pepsin–ODA (film 2, run 2) 3.6 1.2 0.333
Pepsin–ODA (film 2, run 3) 3.6 0.5 0.136
a One unit of enzyme will produce a change in absorbance at 280 nm of
0.001 min21 at pH 3.0 and 37 °C measured as PCA soluble products using
hemoglobin as the substrate (ref. 14).
298 Chem. Commun., 2000, 297–298
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Encapsulation and biocatalytic activity of the enzyme pepsin in fatty lipid films by selective electrostatic interactions

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Encapsulation and biocatalytic activity of the enzyme pepsin in fatty lipid films by selective electrostatic interactions

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