Dramatic hyperactivation of lipase of Thermomyces lanuginosa by a cationic surfactant: Fixation of the hyperactivated form by adsorption on sulfopropyl-sepharose

Article abstract of DOI:10.1016/j.molcatb.2015.08.015

Soluble lipase from Thermomyces lanuginosa (TLL) was dramatically hyper-activated by a cationic surfactant, cetyltrimethylammonium bromide (CTAB). The greatest hyperactivation (above 340-fold) was observed with 0.005% CTAB. In addition to that, very high

Full text of DOI:10.1016/j.molcatb.2015.08.015

Accepted Manuscript
Title: DRAMATIC HYPERACTIVATION OF LIPASE OF
Thermomyces lanuginosa BY A CATIONIC SURFACTANT:
FIXATION OF THE HYPERACTIVATED FORM BY
ADSORPTION ON SULFOPROPYL-SEPHAROSE
Author: Sonia Moreno-Perez Nesrine Ghattas Marco Filice
Jose M. Guisan Gloria Fernandez-Lorente
PII:
S1381-1177(15)30053-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.08.015
MOLCAB 3224
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
16-6-2015
28-7-2015
19-8-2015
Please cite this article as: Sonia Moreno-Perez, Nesrine Ghattas, Marco Filice,
Jose M.Guisan, Gloria Fernandez-Lorente, DRAMATIC HYPERACTIVATION
OF LIPASE OF Thermomyces lanuginosa BY A CATIONIC SURFACTANT:
FIXATION OF THE HYPERACTIVATED FORM BY ADSORPTION ON
SULFOPROPYL-SEPHAROSE, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2015.08.015
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DRAMATIC HYPERACTIVATION OF LIPASE OF Thermomyces lanuginosa BY A
CATIONIC SURFACTANT: FIXATION OF THE HYPERACTIVATED FORM BY
ADSORPTION ON SULFOPROPYL-SEPHAROSE.
Sonia Moreno-Perez1,2, Nesrine Ghattas3, Marco Filice1, Jose M. Guisan1,* &
Gloria Fernandez-Lorente2,*
1.- Instituto de Catalisis. CSIC. Campus de la UAM. 28049 Madrid. Spain
2.- Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM
28049 Madrid. Spain.
3.- Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB),
National Institute of Applied Sciences and Technology, University of Carthage,
B. P. 676, 1080, Tunis Cedex, Tunisie
Corresponding authors:
Prof. José M. Guisan (jmguisan@icp.csic.es)
Dr. Gloria Fernandez-Lorrente (gflorente@icp.csic.es)

Graphical abstract
HIGHLIGHTS
-
-
-
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An extraordinary hyperactivation (340-fold) of a lipase (from Thermomyces
lanuginosa. TLL) by very low concentrations of a cationic surfactant (0.005%)
A high hyperactivation of the lipase (TLL) in the presence of “high surfactant
concentrations” (100 fold)
The use of the cluster of a number of surfactant molecules attached to the active
center of the lipase to adsorb the hyperactivated TLL on a cation – exchanger
Such Immobilized and hyperactivated TLL derivatives have been never reported :
(80-fold hyperactivated for small substrates and 40-fold hyperactivated for large
substrates )
ABSTRACT
Soluble lipase from Thermomyces lanuginosa (TLL) was dramatically hyper-activated by a
cationic surfactant, cetyltrimethylammonium bromide (CTAB). The greatest hyperactivation
(above 340-fold) was observed with 0.005 % CTAB. In addition to that, very high
hyperactivation was also observed with much higher concentrations of surfactant (100-fold
in the presence of 0.3 % CTAB).

Without surfactant or with very low surfactant concentrations, TLL was not adsorbed to the
cationic exchangers. However, in the presence of high concentrations of surfactant (0.3 %),
the lipase was completely and strongly adsorbed on sulfopropyl-Sepharose. The adsorbed
enzyme remained hyperactivated (80-fold more active than the soluble enzyme) after
elimination of the excess of surfactant. Complete desorption of the hyperactivated TLL
from the cationic exchanger is only achieved at 2 M NaCl.
The same level of hyperactivation was observed for the hydrolysis of a large substrate: fish
oil. The release of EPA (eicosapentaenoic acid) was 80-fold more rapid with hyperactivated
TLL derivatives than with TLL very mildly immobilized on CNBr activated agarose.
Hyperactivated derivatives were very stable at 25 ᴼC and 37 ᴼC . Full activity was preserved
after 1 week.
KEYWORDS: novel hyperactivated lipase derivatives, hydrolysis of small synthetic substrates,
hydrolysis of fish oil, immobilization of hyperactivated TLL
INTRODUCTION
In aqueous media, an inactive closed form (the large hydrophobic active center is
inaccessible to the medium) and an open and active form (the large hydrophobic active
center is exposed to the medium) of soluble or mildly immobilized lipases exist in
equilibrium. At first glance, the closed form is more stable, but a small percentage of
molecules should exhibit their open form (1, 2, 3, 4). In fact, soluble lipases and those

immobilized on porous supports (which cannot be hyperactivated by interactions with air
bubbles, other proteins, etc.) (5) exhibit catalytic activity in aqueous media. However, the
stabilization of the open form of all lipase molecules could dramatically increase their
catalytic activity (6,7).
Many soluble or immobilized lipases are hyperactivated by surfactants; even their selectivity
properties may be altered (8,9,10). The hydrophobic moiety of the surfactant covers the
hydrophobic active center, and the hydrophilic moiety becomes exposed to the medium,
making the hyperactivated open form much more stable in aqueous medium.
The dramatic hyperactivation of lipases by very low concentrations of ionic or non-ionic
surfactants is very interesting because these very low concentrations of surfactants can be
added to the reaction media (10). On the other hand, the hyperactivation of soluble lipases
by high concentrations of ionic surfactants could be even more interesting. The active
center, now covered by a number of surfactant molecules, could become adsorbed and
stabilized in the open form on different ion exchangers. The hydrophobic moiety of the
molecules of surfactant may remain adsorbed to the hydrophobic active center, and the
cationic moiety may become adsorbed to the cation exchanger. In these conditions the
surfactant may become a “connector” between the hyperactivated lipase and the cation-
exchanger. Then a novel protocol for immobilization and fixation of hyperactivated
structures of lipases could be developed.
In this paper, the hyperactivation of the lipase from Thermomyces lanuginosa (TLL) by a
cationic surfactant (cetyltrimethylammonium bromide (CTAB)) was studied. The
preservation of an interesting hyperactivation in the presence of high concentrations of
surfactants was also evaluated. The hyperactivated enzyme in the presence of high

concentrations of surfactant was adsorbed on a strong cation-exchanger (sulfopropyl-
Sepharose). The binding strength between the hyperactivated enzyme and the cation
exchanger was studied. The fixation of the hyperactivated form of TLL, after washing of the
excess of detergent, was also evaluated. The behavior of these new TLL derivatives in the
hydrolysis of a large substrate (fish oil) was also studied (11) in order to test the general
applicability of this new hyperactivation-immobilization protocol.
MATERIALS AND METHODS
Materials
Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), sodium borohydride (NaBH4)
cetyl trimethyl ammonium bromide (CTAB), p-nitrophenyl butyrate (p-NPB) were obtained
from Sigma Chemical Co. (St. Louis, Mo. USA). Octyl SepharoseTM, Sulfo-propyl Sepharose,
CNBr activated Sepharose 4B (CNBr-Sepharose) and inert Sepharose (Sepharose 4BCL) were
purchased from GE Healthcare (Uppsala, Sweden). The lipase from Themomyces lanuginosa

was kindly donated by Novozymes (TL-100) (Bagsvaed, Denmark). Sardine oil was a kind gift
from Biotecnología Aplicada - (BTSA) (Madrid, Spain). The other reagents and solvents that
were used were of analytical or HPLC grade.
Standard lipase activity assay
The analysis of the activities of the soluble lipases and their immobilized preparations were
performed in a quartz reaction cell (cuvette) provided with magnetic stirring. The reaction
was analyzed spectrophotometrically by measuring the increase in absorbance at 348 nm
( = 5150 M-1 cm-1) produced by the release of p-nitrophenol (pNP) from the hydrolysis of
0.4 mM pNPB in 25 mM sodium phosphate buffer at pH 7, 1% acetonitrile and 25°C. To
initialize the reaction, 0.05-0.2 mL of lipase solution (blank or supernatant) or suspension
was added to 2.5 mL of substrate solution (11). Enzymatic activity is given as μmol of pNP
produced per minute per mg of enzyme (IU) under the conditions described above. At 348
nm the absorbance of p-nitrophenol is lower than at 420 nm but it is completely
independent of pH and hence very small variations in the pH of the assay have no influence
on the measured activity.
Hyperactivation study of the soluble TLL lipase in the presence of CTAB.
A solution of 20 mL of NaH2PO4 10 mM pH 7 buffer with 0.2 mL of crude TLL extract (14
mgprot/mL) was prepared, and its enzymatic activity was measured with the standard assay
described above. The hyperactivation of the soluble TLL lipase was calculated by measuring
its activity using the enzymatic assay solution described above containing different growing
concentrations of cetyltrimethylammonium bromide (CTAB)

Adsorption of hyperactivated TLL lipase onto sulfopropyl-Sepharose in the presence of
CTAB
0.2 mL of crude TLL extract (14 mgprot/mL) was added to 20 mL of 10 mM NaH2PO4 (pH 7)
buffer solution at room temperature. After that, different concentrations of
cetyltrimethylammonium bromide (CTAB) were added. After 10 min, 2 g of previously
equilibrated anionic exchanger (Sulfopropyl Sepharose (SP)) (14) were added. The
immobilization suspension was kept with mechanical stirring at room temperature. A similar
suspension was prepared under identical conditions but using inert Sepharose. The
immobilization course was determined by comparing the lipase activity of the supernatant
and the suspension. The highest loading capacity was of 20 mg of hyperactivated enzyme
per wet gram of support but in general the derivatives were prepared with 0.1 mg of protein
/wet g in order to prevent diffusional limitations.
Immobilization of TLL on CNBr-activated Sepharose
TLL was immobilized CNBr-Sepharose support as previously described (12).. The
immobilization yield was 30% of the input protein. The immobilization was followed by the
enzymatic activity assay described above. In general, immobilization of enzymes under these
conditions will occur via just one covalent attachment. These immobilized preparations
result in a good model of the enzyme properties in the absence of intermolecular
phenomena (13, 14). Lipases have been shown to exhibit a strong tendency to form lipase-
lipase aggregates and lipase-protein aggregates in crude extracts, making it very complex to
study isolated lipase molecules in soluble form (7,8).

Hyperactivation study of TLL lipase mildly immobilized on CNBr-activated Sepharose in the
presence of CTAB.
A suspension of 20 mL of NaH2PO4 10 mM pH 7 buffer with 2 grams of immobilized TLL was
prepared, and its enzymatic activity was measured with the standard assay described above.
The hyperactivation of the immobilized TLL lipase was calculated by measuring its activity
using the enzymatic assay solution described above containing different growing
concentrations of cetyltrimethylammonium bromide (CTAB)
Desorption studies of TLL-CTAB immobilized onto an anionic exchanger
To study the desorption of TLL-CTAB immobilized onto an anionic exchanger, 0.1 g of (TTL-
CTAB)-SP derivative was re-suspended in 10 mL of NaH2PO4 10 mM at pH 7. Increasing
concentrations of NaCl were added, and the suspension was mechanically stirred for 1 h at
25°C. The desorption-course was monitored by comparing the enzymatic activity of the
supernatant and suspension.
Hydrolysis of sardine oil by immobilized TLL
The hydrolysis of sardine oil was performed in an organic-aqueous two-phase system (15).
The released EPA was partitioned into the organic phase. UV-HPLC chromatograms of
aliquots of the organic phase of the reaction mixture were compared to the corresponding
calibration curves; hence, the rates of hydrolysis expressed as rate of release of EPA could be
calculated. Samples of the organic phase were analyzed by HPLC as previously described
(15)

Stability of TLL derivatives
Different TLL derivatives were incubated in 5 mM sodium phosphate buffer at pH 7 and 25°C
or 37 °C . Samples were periodically withdrawn using a pipette with a cut tip, and they were
vigorously stirred to create a homogenous biocatalyst suspension. The remaining activities
were determined as described for the pNPB assay.
Experimental errors:
All experiments were carried out in triplicate and the mean values were calculated. The
standard error was never over 5%.
RESULTS AND DISCUSSION
Hyperactivation of soluble and immobilized TLL with ionic surfactants
TLL is dramatically hyperactivated in the presence of cationic surfactants (CTA) (Figure 1).
Hyperactivation is very high in the presence of CTAB, and a maximum of 350-fold was
achieved with very low surfactant concentrations (0.005 %). In addition, good
hyperactivation values are maintained even at high surfactant concentrations (a 100-fold
increase of activity was observed in 0.3% of CTA). Similar hyperactivation values were
observed with very mildly covalently immobilized derivatives on CNBr-activated Sepharose
(a 150-fold hyperactivation was observed in the presence of 0.005% CTAB), which means
that dramatic hyperactivations are mainly intramolecular processes, yielding new active
centers of TLL with highly improved catalytic properties (Figure 2). In the presence of

surfactants immobilized TLL only can undergo an intramolecular process but the soluble
enzyme (higher hyperactivation) can also undergo dissociation of aggregates as commented
above. Specific activity of pure soluble enzyme was 7.1 Units /mg and this activity is the
same for the very mildly immobilized on CNBr-activated Sepharose derivatives. In the
presence of low concentration of CTAB (0.005%) the activity of soluble enzyme increases up
to 2407 Units /mg of pure enzyme. Similar hyperactivation is observed for the very mildly
immobilized derivative. Immobilization of lipases or other enzymes on CNBr activated
Sepharose at neutral pH takes place via the N terminus of the enzyme (pK = 7.5) and not via
Lys residues on its surface (pK=10.5). In addition to that, neutral pHs, short immobilization
times and low temperatures dramatically reduce the possibilities of multipoint covalent
immobilization. In general these immobilized derivatives exhibit the same activity-stability
than pure and very diluted enzyme solutions. However these derivatives can be prepared by
using impure enzyme extracts, they can be used in organic cosolvents and they can be
stirred without problems of enzyme aggregations that can mask the real properties of
soluble enzymes. In addition to that immobilization of lipases in the presence of surfactants
avoids dimerization and allows the one-point immobilization of monomers of lipase. Then,
the most suitable blank to test he properties of different lipase derivatives is the one-point
derivative immobilized on CNBr-activated Sepharose. This blank is much more accurate than
soluble lipases.
The active centers of lipases are very hydrophobic but the rest of the lipase surface is mostly
hydrophilic. Otherwise, lipases in solution will trend to form complex aggregates. However,
soluble lipases usually form dimeric structures as a consequence of adsorption of two active
centers of two lipase molecules. These aggregates are disrupted in the presence of

surfactants ( 16 ). In addition to that, surfactants promote hyperactivation of lipases,
promote the desorption of lipases from hydrophobic supports, etc. Then, the selective
adsorption of surfactants on the active center of lipases seems to be main mechanism of
interaction between surfactants and lipases (see Figure 2).
Adsorption of hyperactivated TLL on SP-Sepharose. Activity towards p-nitrophenyl
butyrate.
In the absence of surfactant or in the presence of very low concentrations of CTAB (eg.,
0.05%) TLL is only minimally adsorbed on sulfopropyl-agarose (SP-Sepharose). However, at a
high surfactant concentration (0.3% TX), 100% of the hyperactivated enzyme is adsorbed.
After washing the immobilized derivative to eliminate the free surfactant, the TLL derivative
remains hyperactivated (600 U/ mg of immobilized enzyme and this is 83-fold more active
than the one-point immobilized derivative) (Table 2). The cluster of surfactant molecules
adsorbed between the enzyme and the support seems to be very stable. It is likely that the
hydrophobic moiety of the surfactant remains adsorbed to the hydrophobic active center
and the cationic moiety remains strongly adsorbed to the cation exchanger in the presence
of high concentrations of surfactants (Figure 3). In the presence of lower concentrations of
surfactant (eg, 0.005%) the hyperactivation of TLL is higher but the number of surfactant
molecules on the active center of the lipase may be too low to allow a strong adsorption on
cationic exchangers.
Desorption of hyperactivated TLL from SP-Sepharose.

The hyperactivated enzyme becomes very strongly adsorbed to the exchanger. In fact, only a
minimal amount of enzyme is desorbed away from the support in the presence of 1 M NaCl.
Desorption is complete at 2 M NaCl (Figure 4). This adsorption is much stronger than the
adsorption of standard proteins on ionic exchangers (e.g., TLL on Sepharose Q). It seems
that the surfactant cluster covering the active center of TLL contains a number of surfactant
molecules that are very close together and that are strongly adsorbed to the support.
Hydrolysis of fish oil by hyperactivated TLL derivatives.
The hydrolysis of fish oil was measured in order to test the ability of the hyperactivated
derivatives to act on large substrates. A very easy identification of EPA (via UV detection) can
be accurate enough.
The release of omega-3 fatty acids was studied. The catalytic activity (measured as the rate
of release of EPA) was excellent, even better than that of TLL adsorbed on octyl-agarose.
The differences between both hyperactivated derivatives are lower than with pNPB, perhaps
due to surfactant molecule hindering the access of large molecules of fish oil to the catalytic
site.
Stability of TLL derivatives under optimal reaction conditions

Derivatives are very stable at 25 ᴼC and 37 ᴼC . In all cases full activity is preserved after 1
week. At higher temperatures, the hyperactivated derivatives become very unstable. It
seems that the complex enzyme-surfactant support may be destroyed.
CONCLUSIONS
In this paper, an extraordinary hyperactivation of TLL promoted by a cationic surfactant is
described (a 340 hyperactivation factor). This value may be over-estimated because it was
measured on a commercial preparation of TLL. Some lipase-lipase or lipase-protein
aggregations may promote a too high hyperactivation value. A more accurate value was
obtained for one-point immobilized derivative where only intramolecular processes are
possible. This new lower value is again extraordinary (150-fold hyperactivation). This
hyperactivation is even higher than other very interesting hyperactivation previously
described (17): small unilamelar vesicles of anionic phospholipids (SUV), such as 1-
palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG), provide an interface where
Thermomyces lanuginosa lipase (TLL) binds and adopts a catalytically active conformation
for the hydrolysis of substrate partitioned in the interface, such as tributyrin or p-
nitrophenylbutyrate, with an increase in catalytic rate of more than 100-fold. The
hyperactivation of immobilized TLL by vry low concentrations of solvents can be very useful
to perform interesting selective biotransformations in the presence of these very low cost-
effective surfactant concentrations.
In addition to its extraordinary value, the
hyperactivation here reported is even observed at high concentrations of surfactant and it
permits the fixation of the hyperactivated form of TLL by adsorption on SP-Sepharose. In
this way, new immobilized TLL derivatives were obtained that were 80-fold more active than

one-point covalently immobilized ones and 4-fold more active than the open form of TLL
directly adsorbed on hydrophobic supports. Such a hyperactivated derivatives have not
been reported in literature.
These new derivatives are also very active in the modification of large substrates (hydrolysis
of fish oil). They are also 40-fold more active than one-point covalently immobilized
derivatives. These new derivatives are able to hydrolyze fish oil with a good catalytic activity
(430 IU per gram of support) (see Table 4). In addition to this, the derivatives are very stable
under optimal reaction conditions. They exhibit 100% activity after incubation for a week.
Acknowledgments
This work was sponsored by the Spanish Ministry of Economy and Innovation (project AGL-
2009-07526 and BIO-2012-36861). We gratefully recognize the Spanish Ministry of Science
and Innovation for the “Ramón y Cajal” contract for Dr. Fernandez-Lorente and The Ministry
of Science from Tunisia for supporting Nesrine Gattas. We also like to thank Novozymes and
Ramiro Martinez for the generous gift of Lipolase. Soledad Peña (from BTSA) is also
recognized by the donation of fish oil.
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Figure 1.- Hyperactivation of soluble TLL in the presence of ionic surfactants.
Activity of the enzyme in the absence of surfactant=1. Squares: hyperactivation of the
enzyme in the presence of different concentrations of CTAB;

Figure 2.- Desorption of TLL from sulfopropyl-Sepharose.
Experiments were carried out as described in the Methods. Squares: desorption of TLL
adsorbed (in the presence of 0.3% CTAB) on Sulfopropyl-Sepharose.
Table 1.- Hiperactivation and Adsorption of different TLL preparations.
Experiments were performed as desribed in Methods. Activty 1 is taken as the activity of
soluble TLL in the absence of surfactant.
TLL PREPARATION
Activity of
different lipase SP-Sepharose
preparations
yield, %
Adsorption on
Activity after
adsorption on SP-
Sepharose
Soluble in the absence of
detergent
1
-
-
Soluble in the presence of
CTAB 0.005%
339x
100x
15
nd
83x
Soluble in the presence of
CTAB 0.3%
100

Table 2.- Hydrolysis of pNPB and fish oil (release of omega-3 EPA) catalyzed by different
TLL derivatives.
CNBr-S: one-point covalently immobilized derivative via very mild covalent immobilization
on CNBr-activated Sepharose. OS: TLL adsorbed on octyl-Sepharose. SP-CTAB. TLL
hyperactivated with 0.3 % of CTA and adsorbed on Sulfopropyl-Sepharose.
Activity is given as micromols of EPA releases per hour and per gram of catalyst. Each gram
of catalyst contains 25 mg of hyperactivated TLL. Experiments were performed as described
in Methods.
The data of hydrolysis of pNPB are reported taken as 100 % the activity of the mildly
immobilized enzyme (a one-point covalently immobilized TLL) and hence the activities of the
other derivatives directly represent the hyperactivation of immobilized TLL
Hydrolysis of
Hydrolysis of fish oil
Reference
pNPB
UI /g
13.8
150
BrCN-S
OS
100
2000
8500
[15]
[15]
SP-CTAB
436
This manuscript
Reference 15: Fernández-Lorente G, Betancor L, Carrascosa AV, Guisan JM. Release of omega-3
fatty acids by the hydrolysis of fish oil catalyzed by lipases immobilized on hydrophobic supports. J
Am Oil Chem. Soc. 2011;88:1173-1178.