Note: Descriptions are shown in the official language in which they were submitted.
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SURFACTANT-LIPASE COMPLEX IMMOBILIZED ON
INSOLUBLE MATRIX
Field of the Invention
The present invention relates to an insoluble matrix immobilized
surfactant-coated lipase complex, to a method of preparing same and to the
use of same as a biocatalyst for catalyzing, for example, inter- and/or
trans-esterification of oils and fats in hydrophobic organic media. The novel
procedures include two steps. In the first step, the enzyme is activated by
being coated with a surfactant. In the second step, the enzyme is immobilized
on the matrix of choice. These steps can be executed in any order.
Background of the Invention
Enzymatic modification of the structure and composition of oils and fats is of
great industrial and clinical interest. This process is accomplished by
exploiting regio-specific lipases in inter-esterification and/or
trans-esterification reactions utilizing fats or oils as substrates (Macrea,
A.R.,
1983, J. Am. Oil Chem. Soc. 60: 291-294).
Using an enzymatic process, it is possible to incorporate a desired fatty acyl
group on a specific position of a triacylglycerol molecule, whereas
conventional
chemical inter-esterification does not possess regio-specificity.
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Conventionally, chemical reactions are promoted by sodium metal, sodium
alkoxide or cobalt chloride that catalyze acyl migration among triglyceride
molecules, leading to the production of triglycerides possessing randomly
distributed fatty acyl residues (Erdem-Senatalar, A., Erencek, E. and Erciyes,
A.T., 1995, J. Am. Oil Chem. Soc. 72: 891-894).
In recent years, a number of studies have demonstrated the potential
application of lipases as promising biocatalysts for different esterification
reactions in organic media (Wisdom, R.A., Dunhill, P., and Lilly, M.D., 1987,
Biotechnol. Bioeng. 29: 1081-1085).
Lipases with 1,3-positional specificity principally catalyze hydrolysis of
fats
and oils, to yield free fatty acids and glycerol. However, recent studies have
shown that lipases with 1,3-positional specificity are also capable of
catalyzing
two types of esterification reaction in microaqueous organic media (Quinlan,
P. & Moore, S., 1993, INFORM 4: 580-585). The first of these reactions is an
inter-esterification or acidolysis reaction in which free fatty acids react
with
different iriglycerides to yield new triglyceride molecules. The second type
of
reaction is trans-esterification in which two different triglyceride molecules
react to give new triglyceride molecules (see Figures la-b). In both of these
enzymatic reactions, the sn-2 position of the reacting triglycerides remains
unchanged.
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In general, water concentration plays an important role in determining the
activity of enzymes. It also affects the equilibrium state of the reactions
performed in hydrophobic organic solvents (Valiverty, R.H., Halling, P.J. and
Macrae, A.R., 1993, Biotechnol. Lett. 15: 1133-1138). Since water is vital for
the activity of enzymes in both hydrolysis as well as in synthesis reactions,
as
a compromise between hydrolysis and synthesis of triglycerides, the
concentration of water is lowered so that the occurrence of undesirable
reactions is minimized, but the water available is sufficient for the enzyme
to
remain active.
At high concentrations of water, e.g., above 5 % of solvent weight, lipases
possess preferably their natural hydrolytic activity, therefore, hydrolysis
reaction proceeds. However, at low concentrations of water, e.g., below 1 % of
solvent weight, lipases catalyze the reverse reaction, that is, synthesis.
A typical range of water concentrations needed for promotion of
inter-esterification reaction between different oils in organic media is 1-10
weight percent (wt %) of the hydrophobic organic solvent. This water
concentration can normally facilitate also the hydrolysis reaction thus
producing undesirable partial glycerides (mono- and di-glycerides) in the
range of 10-20 wt % of the initial triglycerides concentration, as byproducts.
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The scope for exploiting the positional specificity of lipases, especially, in
the
food and oleochemical industries for the production of high-valued special
fats
is enormous. For example, cocoa butter substitute, simulated human milk fat
and other structured triglycerides of specific nutritional quality can be
obtained enzymatically by employing lipases with 1,3-positional specificity
(Vulfson, E.N., 1993, Trends Food Sci. Technol. 4: 209-215).
In view of the foregoing, it is recognized that there is a need to develop new
structured triglycerides with both medium-chain and u-3 polyunsaturated
fatty acids that would be devoid of the adverse effects of the naturally
occurring a-3 polyunsaturated fatty acids, or saturated fatty acids. For
example, molecules of MCTs having one of their acyl groups substituted with
an essential long-chain fatty acid would provide the nutritional advantages of
both MCTs and LCTs. This approach is illustrated by the very useful
triglyceride that is formed by incorporating the acyl form of the
polyunsaturated fatty acids, EPA, DHA or a-linolenic acid at the Sn-2 position
of a triglyceride molecule having a medium-chain fatty acyl group at the sn-1
and sn-3 positions (Odle, J. , 1997, J. Nutr. 127: 1061).
The aforementioned polyunsaturated fatty acids incorporated into triglyceride
molecules were shown to have several health benefits with respect to
cardiovascular disease, immune disorders and inflammation, allergies,
diabetes, kidney diseases, depression, brain development and cancer.
Furthermore, medium-chain fatty acids incorporated into the same
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triglyceride molecule are of major importance in some clinical uses,
especially,
for facilitating absorbability and solubilization of cholesterol in blood
serum,
and for providing readily available energy sources for body consumption.
Many different approaches for the use of lipases in organic media have been
attempted in order to activate them and to improve their performance. These
include the use of lipase powder suspended in either microaqueous organic
solvents or in biphasic systems, and native lipases adsorbed on microporous
matrices in fixed- and fluidized-bed reactors (Malcata, et al., 1990, J. Am.
Oil
Chem. Soc. 890-910). Furthermore, lipases have been hosted in reverse
micelles, and in some studies lipases were attached to polyethylene glycol or
hydrophobic residues to increase their solubility and dispersibility in
organic
solvents.
None of the abovementioned approaches was found to be applicable for all
enzymatic systems. However, in many cases, when lipases were treated in
one way or another as described, their performance with respect to activity,
specificity, stability and dispersibility in hydrophobic organic systems was
improved.
In recent studies, the development of surfactant-coated lipase preparations
has been reported (e.g., Basheer. S., Mogi, K. and Nakajima, 1~T.. 1995,
Biotechnol. Bioeng. 45: 187-195). This enzyme modification converts slightly
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active or completely inactive lipases, with respect to esterification of
triglycerides and fatty acids in organic media, into highly active
biocatalysts.
The newly developed surfactant-lipase complexes have been further studied
and used for the inter-esterification reaction in organic solvent systems to
produce structured triglycerides of major importance in medical applications
(Tanaka, Y., Hirano, J. and Funada, T., 1994, J. Am. Oil Chem. Soc. 71:
331-334).
In another approach to the problem, various immobilized-enzyme reactor
systems were used in lipase-catalyzed reactions in microaqueous hydrophobic
organic media (e.g., Basheer, S., Mogi, K., Nakajima, M., 1995, Process.
Biochemistry 30: 531-536). These included fixed- and fluidized-bed reactors,
and a slurry reactor. In the published studies, lipase immobilized onto an
inorganic matrix was used both in a batch reactor system, and in fixed-bed
bioreactor systems. However, the lipases employed were not
surfactant-coated and therefore have the same limitations as free lipase
systems. These limitations include:
1. Difficulties in recovering the enzyme after completion of the process;
2. Rapid loss of activity of the free enzyme in the reaction medium;
3. Problems of recoverability of expensive enzymes;
4. Low synthetic activity of free lipases in organic solvents.
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Neither of the abovementioned strategies has satisfactorily solved the
technical problems encountered in directing trans- and inter-esterification of
fats and oils. It is therefore an object of the invention to provide a lipase
preparation that is capable of catalyzing esterification reactions in fats and
oils with a much greater efficacy than existing methods.
It is another purpose of the invention to provide a lipase preparation that
incorporates both immobilization to a matrix, and treatment by coating with
a surfactant.
It is a further object of the invention to provide such a lipase preparation
that
may be used repeatedly, on an industrial scale with minimal loss of activity.
It is a further object of the invention to provide a method for preparing said
insoluble matrix-immobilized, surfactant-coated lipase complex.
Yet a further purpose of the invention is to provide a process for preparing
structured triacylglycerols, using said insoluble matrix-immobilized,
surfactant-coated lipase complexes.
Other objects and advantages of the invention will become apparent as the
description proceeds.
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Summary of the Invention
It has now been surprisingly found, and this is an object of the invention,
that
the dual modification of crude lipase by (1) coating with a surfactant, and
(2)
immobilization to an insoluble matrix; results in a synergistic improvement in
the efficiency of the enzyme to catalyze trans- and inter-esterification
reactions, when compared to either of these two treatments alone. It has been
further unexpectedly found that it is possible to enhance the catalytic
stability
of said dually modified lipase for esterification reactions, by providing the
enzyme preparation in a granulated form.
The invention is primarily directed to a lipase preparation comprising an
insoluble matrix and a surfactant-coated lipase complex immobilized onto said
insoluble matrix.
The immobilization of the lipase complex onto the insoluble matrix may be
achieved by several different methods. According to a preferred embodiment
of the invention, however, the surfactant-coated lipase complex is covalently,
ionically or physically bound to the insoluble matrix.
The invention encompasses the use of many types of matrix, said matrices
being selected from the group consisting of an inorganic insoluble matrix and
an organic insoluble matrix.
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In a preferred embodiment of the invention, the inorganic insoluble matrix is
selected from the group consisting of alumina, diatomaceous earth, Celite,
calcium carbonate, calcium sulfate, ion-exchange resin, silica gel and
charcoal.
The abovementioned ion-exchange resin may be of any suitable material, but
in a preferred embodiment is selected from the group consisting of Amberlite
and Dowex.
Although any suitable organic insoluble matrix may be use, in a preferred
embodiment of the invention, the organic insoluble matrix is selected from the
group consisting of Eupergit, ethylsulfoxycellulose and aluminium stearate.
In a preferred embodiment, the content of the lipase is 2-20 weight percent of
the surfactant-coated lipase complex. In a still more preferred embodiment,
the content of the lipase is 0.01-1.0 weight percent of the preparation.
The invention provides the above-described lipase preparation, wherein the
surfactant in the surfactant-coated lipase complex includes a fatty acid
conjugated to a hydrophilic moiety. In a preferred embodiment, the fatty acid
is selected from the group consisting of monolaurate, monomyristate,
monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate,
trilaurate, trimyristate, tripalmitate and tristearate. In a preferred
embodiment, the hydrophilic moiety is selected from the group consisting of a
sugar, a phosphate group, a carboxylic group and a hydroxylated organic
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residue. In a more preferred embodiment, the sugar is selected from the
group consisting of sorbitol, sucrose, glucose and lactose. Although the fatty
acid and the hydrophilic moiety may be linked by any suitable type of bond, in
a preferred embodiment, the fatty acid and the hydrophilic moiety are
conjugated via an ester bond.
Although the lipase may be derived or obtained from any convenient source,
in a preferred embodiment, the lipase is derived from a microorganism. Many
different species of both microorganisms and multicellular organisms may be
used as a source of lipase for the lipase preparation of the invention. The
invention, however, is particularly directed to the use of lipase that is
derived
from a species selected from the group consisting of Burhholderia sp., Candida
antractica B, Candida rugosa, Pseudomonas sp., Candida antractica A,
Porcine pancreas lipase, Humicola sp., ll2ucor miehei, Rhizopus javan.,
Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,
Mucor javanicus, Rhizopus sp., Rhizopus japonacus and Candida antractica.
In a further aspect, the invention is directed to a lipase preparation
comprising an insoluble matrix and a surfactant-coated lipase complex
immobilized onto said insoluble matrix, said lipase preparation being provided
in an organic solvent. In a preferred embodiment, the organic solvent is
selected from the group consisting of n-hexane, toluene, iso-octane, n-octane,
benzene, cyclohexane and di-iso-propylether. The invention is further
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directed to the use of said lipase preparation as a catalyst for
esterification,
inter-esterification and trans-esterification of oils and fats and alcoholysis
of
triglycerols and fatty alcohols. In a preferred embodiment, the lipase
preparation is used as a catalyst with 1,3-positional specificity with respect
to
triacylglycerols.
In another aspect, the invention is directed to a lipase preparation as
described above, wherein said preparation is in granulated form.
The invention also provides a lipase preparation, as described hereinabove,
wherein the insoluble matrix has been modified with a fatty acid derivative.
In a further aspect the invention is directed to an enzyme preparation, as
described hereinabove, for use in a reaction environment without the need for
water addition.
The invention also encompasses a method for improving the stability of a
surfactant-coated immobilized lipase complex, comprising granulating same
prior to contacting it with the substrate to be reacted.
In a further aspect, the invention provides a method of preparing an insoluble
matrix-immobilized surfactant-coated lipase complex comprising, in any
desired order, the steps of:
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(a)contacting a lipase in an aqueous medium with a surfactant, at a
concentration and temperature, and for a period of time sufficient
to obtain a coating of said lipase; and
(b) contacting said lipase in an aqueous medium, with an insoluble
matrix, at a concentration, under conditions and for a period of
time sufficient to obtain immobilization of said lipase on said
matrix.
In a preferred embodiment of the abovementioned method, the lipase is first
contacted with the insoluble matrix, and thereafter with the surfactant. In
another preferred embodiment thereof, the lipase is first contacted with the
surfactant, and thereafter with the insoluble matrix.
In a preferred embodiment of the invention, the above-described method
further comprises the separation of the matrix-immobilized surfactant-coated
lipase complex from the aqueous solution in which it was formed. In a still
more preferred embodiment, this method also further comprises the step of
drying said matrix-immobilized surfactant-coated lipase complex. Although
the drying step may be accomplished by any convenient method, in a
preferred embodiment, said drying is effected by freeze drying. In another
preferred embodiment, the matrix-immobilized surfactant-coated lipase
complex is dried to a water content of less than 100 parts per million by
weight.
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In another preferred embodiment, the aqueous solution used in the
above-described method is a buffered-aqueous solution.
In yet another preferred embodiment of the above-described method, the
lipase and surfactant are contacted in the aqueous medium by:
(i) dissolving said surfactant in an organic solvent for obtaining a
dissolved surfactant solution; and
(ii) mixing said lipase and said dissolved surfactant solution in said
aqueous medium.
In another preferred embodiment, the method further comprises sonication of
the aqueous solution.
In yet another preferred embodiment of the method of the invention, the
insoluble matrix is selected from the group consisting of alumina,
diatomaceous earth, Celite, calcium carbonate, calcium sulfate, ion-exchange
resin, silica gel, charcoal, Eupergit, ethylsulfoxycellulose, aluminium
stearate
and fatty acid derivative-treated Celite or other inorganic matrices.
In another preferred embodiment, the surfactant of the method includes a
fatty acid conjugated to a hydrophilic moiety. In a still more preferred
embodiment, said fatty acid is selected from the group consisting of
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monolaurate, monomyristate, monopalmitate, monostearate, dilaurate,
dimyristate, dipalmitate, distearate, trilaurate, trimyristate, tripalmitate
and
tristearate.
In another preferred embodiment of the method of the invention, the
hydrophilic moiety is selected from the group consisting of a sugar and a
phosphate group and a carboxylic group and a polyhydroxylated organic
residue. In a still more preferred embodiment, the sugar is selected from the
group consisting of sorbitol, sucrose, glucose and lactose.
In another preferred embodiment of the method of the invention, the fatty
acid and the hydrophilic moiety are conjugated via an ester bond.
In a preferred embodiment of the method of the invention, the lipase is
derived from an organism. In a more preferred embodiment, the lipase is
derived from a multicellular microorganism. Although the lipase may be
derived from any suitable host, in a preferred embodiment, the lipase is
derived from a species selected from the group consisting of Burhholderia sp.,
Candida antarctica B, Candida rugosa, Pseudomonas sp.,_ Candida antractica
A, Porcine pancreatic lipase, Humicola sp., Mucor miehei, Rhizopus javan.,
Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,
lVlucor iat;anicus. Rhizopzcs sp.. Rhizonus japonict~s and Candida antarctica.
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In another aspect, the invention is directed to a process for preparing
structured triacylglycerols by esterification, acidolysis, trans-
esterification,
inter-esterification or alcoholysis between two substrates comprising
contacting an insoluble matrix-immobilized surfactant-coated lipase complex
with said substrates.
In a preferred embodiment of this process, the matrix-immobilized
surfactant-coated lipase complex is contacted with the substrates in the
presence of an organic solvent.
In another preferred embodiment of this process, at least one of the
substrates
is selected from the group consisting of an oil, a fatty acid, a
triacylglycerol
and a fatty alcohol. Although many different types of oil may be used in this
process, in a preferred embodiment, the oil is selected from the group
consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cotton
seeds
oil, sunflower oil, Nigella satiua oil, canola oil and corn oil. In another
preferred embodiment, the fatty acid is selected from the group consisting of
medium and short-chain fatty acids and their ester derivatives. In a still
more preferred embodiment, the fatty acid is selected from the group
consisting of oleic acid, palmitic acid, linolic acid, linolenic acid, stearic
acid,
arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and their ester
derivatives.
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While the above-described process may be performed in any suitable
receptacle, said process, in a preferred embodiment, is carried out in a tank
reactor or in a fixed-bed reactor.
The invention also encompasses a triacylglycerol prepared according to the
above-described process for use as a cocoa butter substitute, human milk
fat-like triglycerides for special diets, or structured triglycerides for
medical
applications.
Brief Description of the Drawings
The invention herein described, by way of example only, with reference to the
accompanying drawings, wherein:
FIG. la presents an inter-esterification acidolysis reaction catalyzed by
lipase with 1,3-positional specificity. P represents glycerol bound palmitic
acid, C represents glycerol bound capric acid. PA and CA represent free
palmitic and capric acids, respectively.
FIG. 1b presents a trans-esterification reaction catalyzed by lipase with
1,3-positional specificity. P represents glycerol bound palmitic acid and C
represents glycerol bound capric acid.
FIG. 2 depicts the chemistry associated with covalent immobilization of
lipase zo Eupergit C 2~OL followed by coating the covalently immobilized
enzyme with a surfactant.
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FIG. 3 presents inter-esterification reaction profiles of physically
immobilized lipases. Reaction conditions were 50 mg tripalmitin, 35 mg
capric acid and 20 mg surfactant-coated lipase (Saiken 100 - triangles, or
Lilipase A10-FG - squares) immobilized on DE in 10 ml n-hexane. The
reaction system was magnetically stirred and thermostated at 40 ° C.
FIG. 4 presents an Arrhenius plot for the inter-esterification reaction of
tripalmitin and capric acid with DE-physically immobilized surfactant-coated
Lilipase A10-FG.
FIG. 5 is a bar graph showing the functional stability of Lilipase A
IOFG immobilized on Celite and granulated with 2 % starch.
FIG. 6 is a bar graph showing the functional stability of powdered
Lilipase A lOFG modified with sorbitan monostearate and immobilized on
Celite.
FIG. 7 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Celite and
granulated with 2 % starch.
FIG. 8 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Celite and
granulated with ethyl cellulose.
FIG. 9 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Celite and
granulated with 2 °o gum Arabic.
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FIG. 10 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Amberlite
IR.A-900 and granulated with ethyl cellulose.
FIG. 11 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Celite and
granulated with 2 % agarose.
FIG. 12 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Celite and
granulated with 4 % starch.
FIG. 13 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan tristearate, immobilized on Celite and
granulated with 2 % starch.
FIG. 14 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on aluminium
monostearate and granulated with 2 % starch. The graph also shows
comparable results for unmodified enzyme, immobilized on aluminium
monostearate and granulated with 2 % starch.
FIG. l~ is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Amberlite
XAD-16 and granulated with 2 % starch.
FIG. 16 is a bar graph showing the functional stability of Lilipase A
lOFG modified with sorbitan monostearate, immobilized on Amberlite XAD- r
and granulated with 2 % starch.
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Detailed Description of Preferred Embodiments
The present invention relates to a surfactant-coated lipase or phospholipase
complex immobilized on an organic or inorganic insoluble matrix (e.g.,
particulate solid support) which can be used to catalyze inter and
trans-esterification reactions, particularly of oils and fats and alcoholysis
of
fatty-alcohols. The invention also makes provision for preparing the enzyme
preparation in a granulated form that demonstrates increased stability of
activity. Specifically, the present invention can be used for preparing
structured triacylglycerols possessing desired nutritional or biochemical
properties. The present invention is further directed to a method of preparing
an insoluble matrix-immobilized surfactant-coated lipase or phospholipase
complex and of a process of modifying oils and fats using an insoluble
matrix-immobilized surfactant-coated lipase or phospholipase complex.
The principles and operation of the present invention may be better
understood with reference to the drawings and accompanying descriptions.
It is to be understood that the invention is not limited in its application to
the
details of construction and the arrangement of the components set forth in the
following description or illustrated in the drawings. The invention is capable
of other embodiments or of being practiced or carried out in various ways.
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Also, it is to be understood that the phraseology and terminology employed
herein is for the purpose of description and should not be regarded as
limiting.
According to the present invention surfactant-coated lipases or phospholipases
are immobilized onto insoluble matrices by three different methods: (i)
immobilization through hydrophobic (physical) adsorption on inorganic or
organic insoluble matrices; (ii) immobilization through ionic interactions on
various ion exchange resins (polar or apolar matrices); and (iii)
immobilization
through covalent immobilization to insoluble matrix such as Eupergit (organic
matrix).
The immobilized surfactant-coated lipases prepared according to the
procedures described herein were used to catalyze inter-esterification
reactions between triglycerides and fatty acids, one-step alcoholysis
reactions
between triglycerides and fatty alcohols for production of wax esters, and
also
traps-esterification reactions between two different triglyceride molecules or
between two different oils.
The results indicate that coating lipases with a lipid surfactant, such as,
but
not limited to, fatty acid sugar ester types, lead to activation of the
lipases for
use in organic synthesis and in most cases the modification process converts
relatively inactive crude lipases to highly active biocatalysts. To develop an
efficient enzymatic inter/trans-esterification bioreactor from which the
lipase
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enzyme can be easily recovered or used continuously, surfactant-lipase
complexes immobilized on organic and inorganic matrices, were used.
It was found that surfactant-coated lipases immobilized on an organic or
inorganic matrix showed high inter/trans-esterification activity and only
slight activity losses in five consecutive inter-esterification runs using the
same biocatalyst batch. It was further found that granulation of the
matrix-bound, surfactant-treated enzyme considerably enhanced the stability
of the enzyme, permitting its repeated use without substantial loss of
activity.
The immobilized surfactant-lipase complexes prepared according to the
present invention were used for the preparation of structured triglycerides
which have potential applications in medicine and the food industry. The
triglycerides of interest that were synthesized according to the method of the
present invention were produced by inter-esterification of long-chain
triglycerides, such as the hard fraction of palm oil, with short-chain fatty
acids
such as capric acid. Immobilized surfactant-coated lipase catalyzed reactions
yielded predominantly products with 1,3-positional specificity for the
triglycerides of interest. Mono- and di-glycerides were also produced in a
hydrolysis side reaction and their percentage was typically less than 7 weight
percent of the initial triglyceride concentration. The operational stability
of surfactant-lipase complexes immobilized on different solid matrices was
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very high, particularly following the optional granulation step, and no
significant enzyme activity losses were observed.
Inter-esterification reactions using the immobilized surfactant-lipase
complexes were also carried out to obtain fats of special characteristics with
regard to their physical properties. For example, liquid olive oil was
trans-esterified with the hard fraction of palm olefin in order to obtain
blends
of oil for preparation of healthier olive oil based margarines. This enzymatic
process is a useful alternative for chemical oil hydrogenation or
interesterification processes.
Thus, in accordance with the teachings of the present invention there is
provided a lipase preparation which includes an insoluble matrix and a
surfactant-coated lipase complex immobilized onto the insoluble matrix.
As used herein in the specification and in the claims section below the term
"lipase" is not limited to this specific enzyme, but is meant to embrace also
similar enzymes such as phospholipase, proteases and glycosidases. Other
suitable enzymes will be readily apparent to the skilled chemist, and are
therefore not listed herein, for the sake of brevity.
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According to a preferred embodiment the complex is immobilized to the
insoluble matrix via hydrophobic (physical) interaction, ionic interaction or
via
covalent immobilization.
In a preferred embodiment of the invention the insoluble matrix is an
inorganic insoluble matrix, the term "insoluble" referring to its lack of
solubility in both polar (e. g., water) and non-polar (hydrophobic) solvents.
Preferably, the inorganic insoluble matrix according to the present invention
is alumina, aluminium stearate, Celite, calcium carbonate, silica gel,
charcoal,
calcium sulfate, ion-exchange resin, such as, but not limited to, Amberlite
and
Dowex. For physical immobilization, most preferably the inorganic insoluble
matrix employed is diatomaceous earth (DE). For ionic immobilization most
preferably the inorganic insoluble matrix employed is Amberlite and Dowex,
which are strong ion exchangers.
Suitable organic solid matrices according to the present invention include
Eupergit for covalent immobilization and ethylsolfoxycellulose for ion
interaction. Any other suitable organic solid matrix may also be used without
exceeding the scope of the invention.
In another preferred embodiment of the present invention the lipase
represents 2-20, preferably 5-11, weight percent of the surfactant-coated
lipase complex. In yet another preferred embodiment of the present invention
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the lipase represents 0.01-1 weight percent of the preparation. Preferably the
lipase represents about 0.7 weight percent of the preparation.
According to a preferred embodiment of the invention the surfactant employed
is a lipid, which includes a fatty acid conjugated to a hydrophilic moiety.
The
fatty acid is preferably monolaurate, monomvristate, monopalmitate,
monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate,
trimyristate, tripalmitate or tristearate. The hydrophilic moiety is
preferably
a sugar, such as, but not limited to, sorbitol, sucrose, glucose and lactose,
a
phosphate group, a carboxylic group or a polyhydroxylated organic residue.
Typically, the fatty acid and the hydrophilic moiety are conjugated via an
ester bond.
According to another preferred embodiment of the invention the lipase is
derived from a microorganism or a multicellular organism. Species known to
be used for lipase extraction include Burhholderia sp., Candida antarctica B,
Candida rugosa, Pseudomonas sp., Candida antractica A, Porcine pancreas,
Humicola sp., Mucor miehei, Rhizopus javan., Pseudomonas floor, Candida
cylindrcae, Aspergillus niger, Rhizopus oryzae, Mucor javanicus, Rhizopus sp.,
Rhizopus japonicus and Candida antractica.
According to a preferred embodiment of the invention the lipase preparation
maintains lipase catalytic activity in an organic solvent. Lipase catalytic
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activity include hydrolysis, esterification, inter-esterification,
trans-esterification, acidolysis and alcoholysis, preferably with 1,3-
positional
specificity with respect to triacylglycerols. The organic solvent is typically
a
hydrophobic solvent, such as, but not limited to, n-hexane, toluene, iso-
octane,
n-octane, benzene, cyclohexane and di-iso-propylether.
Further according to the present invention there is provided a method of
preparing an insoluble matrix-immobilized surfactant-coated lipase complex.
The method includes the following method steps, wherein in a first step a
lipase, an insoluble matrix and a surfactant are contacted in an aqueous
solution, preferably a buffered solution. Second, conditions (e.g.,
sonication)
are provided for the formation of the matrix-immobilized surfactant-coated
lipase complex. Two alternative schemes are available in this respect. In the
first the lipase is first interacted with the surfactant and only thereafter
the
surfactant-coated lipase is interacted with the matrix. Whereas in the second,
the lipase is first interacted with the matrix and only thereafter the matrix
immobilized lipase is interacted with the surfactant.
According to a preferred embodiment, the method further includes the step of
separating the matrix-immobilized surfactant-coated lipase complex from the
aqueous solution.
According to still another preferred embodiment of the invention the method
further includes the step of drying the matrix-immobilized surfactant-coated
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lipase complex. Drying is preferably effected via freeze drying, fluidization
or
in an oven. Following drying, the matrix-immobilized surfactant-coated lipase
complex preferably includes less than 100, more preferably less than 50, most
preferably less than 20 parts per million water content by weight.
In a preferred embodiment of the method according to the present invention,
contacting the lipase, insoluble matrix and the surfactant within the aqueous
solution is effected by dissolving the surfactant in an organic solvent (e.g.,
ethanol) for obtaining a dissolved surfactant solution, mixing the lipase and
the dissolved surfactant solution (e.g., dropwise) in the aqueous solution;
sonicating the resulting suspension; and adding the insoluble matrix into the
aqueous solution. Alternatively, the lipase is first interacted with the
insoluble matrix and only thereafter with the surfactant.
According to a preferred embodiment, the insoluble matrix of the
above-described lipase preparation is modified with a fatty acid derivative.
This is to permit the immobilization of a hydrophobized lipase on a
hydrophobized carrier, such as aluminium stearate, fatty-acid
derivative-treated Celite and apolar or weak-polar ion-exchange resins, in
order to prepare highly active enzymes.
Further according to the present invention there is provided a process of
preparing structured triacylglycerols by esterification, trans-esterification,
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inter-esterification, acidolysis or alcoholysis between two substrates
effected
by contacting an insoluble matrix-immobilized surfactant-coated lipase
complex with the substrates. Contacting the matrix-immobilized
surfactant-coated lipase complex with the substrates is preferably effected in
the presence of an organic solvent.
In a preferred embodiment, at least one of the substrates is an oil, a fatty
acid
or a triacylglycerol. The oil may be any of the above listed oils. The fatty
acid
is a medium or a short-chain fatty acid or an ester derivative thereof. A
suitable fatty acid is, for example, oleic acid, palmitic acid, linolic acid,
linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid,
docosahexaenoic acid and their ester derivatives.
In a preferred embodiment of the invention contacting the matrix-immobilized
surfactant-coated lipase complex with the substrates is effected within a
reaction reactor, e.g., a tank reactor or a fixed-bed reactor.
Further according to the present invention there is provided a process of
changing the physical properties of oils/fats (e.g., triacylglycerols) by
trans-esterification or inter-esterification between at least two oil/fat
substrates by contacting an insoluble matrix-immobilized surfactant-coated
lipase complex with the substrates. preferably in the presence of an organic
solvent.
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Further according to the present invention there is provided a process of
changing the physical properties of long-chain triglycerides (LCT) and
long-chain fatty alcohols (LCFAL) to produce wax esters by alcoholysis
between at least two such substrates by contacting an insoluble
matrix-immobilized surfactant-coated lipase complex with the substrates,
preferably in the presence of an organic solvent.
According to a preferred embodiment, the matrix-immobilized
surfactant-coated lipase complex represents 2-30 weight percent of the
substrates. In another preferred embodiment the oil/fat substrates are liquid
oils and solid fats. The oil may be any of the above listed oils in a native
or
hydrogenated form.
Further according to the present invention there is provided a triacylglycerol
prepared according to the above process. The triacylglycerol serves an
application such as a cocoa butter substitute, human milk fat-like,
triglycerides for special diets or structured triglycerides for medical
applications.
Yet further according to the present invention there is provided a preparation
which includes a lipase and an organic solvent. The lipase possessing both
esterification (inter- and trans-esterification), acidolysis, alcoholysis and
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hydrolysis catalytic activities with respect to substrates, yielding
esterification
and hydrolysis products, respectively. The hydrolysis products represent less
than about 7, preferably less than about 5, more preferably less than about 3
weight percent of the products.
Examples
Reference is now made to the following examples, which together with the
above descriptions, illustrate the invention in a non-limiting fashion.
Experimental Procedures
Materials
Different crude lipase preparations were tested in this study. Table 1 below
lists commercially available lipase preparations that were employed in this
study, as well as their species source and supplier. All fatty acids and
triglycerides employed in this study were obtained from Fluka (Switzerland)
and, as reported by the supplier, were at least 99 % pure. Olive oil,
sunflower
oil, palm oil, canola oil, corn oil and Nigella sataUa oil were obtained from
local
suppliers in the Galilee area, Israel. Fish oil,
tris(hydroxymethyl)aminomethane and the inorganic matrices used as
supports for the surfactant-coated lipase complexes, including DE, alumina
and silica gel were obtained from Sigma (USA).
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Analytical grade n-hexane and other solvents employed, all of analytical
grade, were from Bio Lab (Israel). Sorbitan fatty acid esters including
sorbitan monolaurate, sorbitan monomyristate, sorbitan monopalmitate and
sorbitan monostearate and sucrose fatty acid esters including mixtures of
mono-, di- and tristearate sucrose esters of variable HLB values were
obtained from Kao Pure Chemicals Ind. (Tokyo, Japan).
Tris(Hydroxymethyl)aminomethane (tris) was from Sigma (USA). Inorganic
and organic matrices used as supports for the modified lipases include
diatomaceous earth (DE), alumina and silica gel; ionic exchange resins were
purchased from Sigma, USA. Eupergit C and Eupergit C 250L
(macroporous, spherical, approximate diameter 150 and 200 Nm, respectively)
were from Rhom (Germany).
Table I
Commercial Source Manufacturer
name
Lili ase A-lOFG Rhizo us ja onicus NR Na ase, Ja an
400 d
Saiken 100 Rhizo us 'a onicus Na ase, Ja an
Li ase EC As er illus ni er Amano, Ja an
Li ase AY Candida ru osa Amano Ja an
Lipase LP Chromobacterium UiscosumAsahi Chem. Ind.
Ja an
~' Li ase PS Pseudomonas ce acia Amano, Ja an
Li ase F Rhizo~s oryzae Amano, Japan
Lipase F EC Rhizopus oryzae Extract
Chemie-Germany
N ewlase F Rhizo us niueus ~ Amano, Ja an
Li ase G Penicillium camembertii Amano, Ja an
~! Li ase A As er illus ni er Amano. Jaban
Lipase M Mucor 'aUanicus
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Lipase modification and immobilization through physical
adsorption
Crude enzyme was first coated with lipid surfactant or other enzyme
activators e.g. gum Arabic or polyethylene glycol. A typical enzyme
modification and immobilization procedure was as follows: crude enzyme
(lipase, phospholipase, protease and glycosidases; protein content
approximately 150mg/L), was dissolved in 1L phosphate or tris buffer
solution with an appropriate pH, and magnetically stirred at 10 °C for
30min. A lipid surfactant or other enzyme activator (0.5g) dissolved in
ethanol (20m1) or other solvents was added dropwise into the stirred
solution. The resulting enzyme solution was sonicated for l5min and then
vigorously stirred at 10 °C for 3 hours. An insoluble organic (20 g
such as
polypropylene, aluminium stearate or chitin) or inorganic matrix (20 g such
as Celite, alumina, silica gel or ceramic support) was added into the stirred
enzyme solution. The solution was magnetically stirred for a further 5 hours
at 10 °C. The precipitate was collected by centrifugation at 12000rpm
(Sorval Centrifuge, model RC-5B) or by filtration, and then was treated by
one of two different methods as follows:
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1. The wet precipitate was lyophilized after freezing overnight at -20
°C. The
formed powder can be directly used for batch enzymatic reactions or
granulated for obtaining particulated modified and immobilized enzyme
with a particle size of 50-1000~.m. The granulation process was performed
using various binding reagents such as starch, methyl or ethyl cellulose,
gums, agarose or other binders. For example, the granulation with starch
was conducted as follows: Starch solution (4 g starch/20m1 water) was
converted to gel at 70 °C. The gel was cooled down to 60 °C and
then
introduced to the modified and immobilized enzyme wet powder. The
mixture was homogenized in a high-speed mixer followed by extruding and
drying at 40-60 °C for 48 hours. The immobilized enzyme was sieved to
obtain particles in the range of 50-1000 ~,m. This particulated enzyme was
used mainly in packed columns.
2. The wet precipitate formed after modification and immobilization was
directly granulated with starch or other binding reagents as described
above.
Enzyme modification and immobilization through ionic adsorption
The above-described modification, immobilization and granulation
procedures were also used in conjunction with ion-exchange resins. The
types of resin used include: strong and week basic anion exchange resins,
strong and weak acidic cationic exchange resins and weak-polar and apolar
ion-exchange resins. Examples of commercially available resins used in the
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experiments (obtained from Sigma, USA) include: Dowex 22, Dowex
lx2-400, Dowex, 2x8-100, cellulose phosphate, Amberlite IRA-95, Amberlite
IRA-200, Amberlite IRA-900, Amberlite XAD-7, Amberlite XAD-16,
DiannonSA-10A, Ectoela cellulose, Sephadex and sulfoxyethylcellulose. A
typical modified immobilized enzyme was prepared according to the
aforementioned procedure.
Enzyme modification and immobilization through covalent binding
Two different immobilization procedures were adopted. According to the first,
the enzyme was primarily coated with a surfactant and then the
lipase-surfactant complex was covalently linked to an Eupergit matrix, which
contains active oxirane groups. To this end, crude lipase (1 gram protein) was
dissolved in 1 liter tris or phosphate buffer pH 5.8. The enzyme solution was
vigorously stirred with a magnetic stirrer at 10 ° C for 30 minutes.
Sorbitan
mono-stearate (0.5 grams) dissolved in 30 ml ethanol were added dropwise to
the stirred enzyme solution. The resulting colloidal enzyme solution was
sonicated for 10 minutes and then stirred for 3 hours at 10 ° C.
Eupergit C or
Eupergit C 250L (125 grams) and 12 ml solution of 5 % hydrogen peroxide
were added into the enzyme solution and the resulting suspension was gently
handshaken for 1 minute, and then incubated for 48 hours at 23 ° C. The
precipitate was filtered, washed with tris or phosphate buffer pH 5.8, and was
freeze-dried overnight.
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According to the second procedure, the lipase was first bound covalently to an
Eupergit matrix and then the bound lipase was coated with a surfactant. The
chemistry involved in this procedure is depicted in Figure 2.
To this end, crude lipase (1 gram protein) was dissolved in 1 liter tris or
phosphate buffer pH 5.8. The enzyme solution was vigorously stirred with a
magnetic stirrer at 10 °C for 30 minutes. Eupergit C or Eupergit C 250L
(125
grams) and 12 ml solution of 5 % hydrogen peroxide were added into the
enzyme solution and the resulting suspension was gently handshaken for 1
minute and then incubated for 48 hours at 23 °C. Sorbitan mono-stearate
(0.5
grams) dissolved in 30 ml ethanol was added dropwise to the suspension
under a gentle shake. The resulting suspension was sonicated for 10 minutes
and incubated at 10 °C for 6 hours. The precipitate was filtered,
washed with
tris or phosphate buffer pH 5.8, and then freeze-dried overnight.
As a control for the activity of the covalently immobilized lipases, the same
immobilization procedures were followed however without adding surfactant
to the enzyme solution.
To this end, crude lipase (1 gram protein), was dissolved in 1 liter tris or
phosphate buffer pH 5.8. The enzyme solution was vigorously stirred with a
magnetic stirrer at 10 °C for 30 minutes. Eupergit C or Eupergit C 250L
(125
grams and 12 ml solution of 5 °% hydrogen peroxide were added into the
enzyme solution and the resulting suspension was gently handshaken for 1
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minute, and then incubated for 48 hours at 23 °C. The precipitate was
filtered, washed with tris or phosphate buffer pH 5.8, and then was
freeze-dried overnight.
Protein determinations according to the Bradford method indicated that all
enzyme preparations prepared according to this method contained 0.9 - 1.5 wt
protein.
Reaction models
Three different reaction models were used to test the activity of the modified
and immobilized enzymes, compared to that of the crude enzymes and of the
immobilized enzymes without modification.
1. Esterification reaction between lauric acid and dodecyl alcohol in an
organic solvent system or in a solvent-free system.
The esterification reaction was initiated by adding 10 mg lipase preparation
to 10 ml n-hexane that contained, typically, 200 mg lauric acid and 186 mg
dodecyl alcohol. The reaction was magnetically stirred at 40° C.
Samples
were periodically withdrawn (50 ~1), filtered with a Millipore filter (0.45
~,m)
and then mixed with a similar volume of n-hexane solution containing
n-hexadecane as an internal standard.
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2. Transesterification reaction between the two triglycerides; tripalmitin
and tristearin in an organic solvent system or in solvent-free system.
The transesterification reaction was initiated by adding 10 mg lipase
preparation to 10 ml n-hexane that contained, typically, 40 mg tripalmitin
and 40 mg tristearin. The reaction was magnetically stirred at 40° C.
Samples were periodically withdrawn (50 ~.1), filtered with a Millipore filter
(0.45 Vim) and then mixed with a similar volume of n-hexane solution
containing n-hexadecane as an internal standard.
3. Alcoholysis reaction between Olive oil and cetyl alcohol in organic solvent
or solvent-free systems.
The alcoholysis reaction was initiated by adding 10 mg lipase preparation to
ml n-hexane that contained, typically, 500 mg olive oil, and 500 mg cetyl
alcohol. The reaction solution was magnetically stirred at 40 ° C.
Samples (50
~l) were periodically removed, filtered with Millipore filters (0.45 Nm) and
then mixed with a similar volume of n-hexane solution containing tridecanoin
as an internal standard.
Unless otherwise indicated, all experiments were conducted under the
above-described conditions. Each esterification reaction was carried out in
duplicate. In all experiments, n-hexane was dried over molecular sieves to
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minimize its water content down to 6 mg/liter. Thus, water concentration in
all reaction systems was less than 30 mg/liter.
Protein content
The protein content of the modified lipases, and the modified and
immobilized lipases was determined by the microkejldahl method.
Enzyme activity in a batch system
The activity of the activated modified enzyme, modified and immobilized
enzyme on insoluble matrix, crude enzymes and immobilized enzymes in
insoluble matrix, was tested using a lml vials containing the substrates.
The vials were shaken at 40 °C and samples were analyzed after
certain
time intervals. Reaction rates were determined at substrate conversions less
than 7% per mg of protein.
Operational stability of modified and immobilized enzyme
The operational stability of the particulated modified and immobilized
enzymes was tested in a jacketed column reactor (0.5cm i.d. and l5cm long)
using the alcoholysis of olive oil and cetyl alcohol in n-hexane as a reaction
model. The enzyme particles were packed in the column and the substrate
solution was recirculated through the packed enzyme. The circulation was
stopped after one hour and the reaction solution was analyzed. After each
run the solution was discarded and the packed immobilized enzyme was
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washed with organic solvent (n-hexane) before charging a fresh substrate
solution. This procedure was repeated 10-20 times.
EXPERIMENTAL RESULTS
Example 1
Protein content of the lipase preparations
The protein content of the different crude lipases, lipases modified with
sorbitan monostearate (SMS), lipases immobilized on Celite and
SMS-modified lipases immobilized on Celite, was measured as described
above. The results are shown in Table II below.
The results show that there is fairly wide variation in protein concentration
between the various preparations. For the surfactant-coated,
matrix-immobilized enzymes, the protein content varied from 0.05 % to 1.12
%, by weight, according to the enzyme used in the preparation. Similar
variation was seen when one enzyme, Lilipase, was treated with different
lipid surfactants or other activating agents, and, optionally, immobilized on
Celite. The results of this investigation are shown in Table III.
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Table II
Lilipase A-10FG
Enzyme Protein content (%)
Lipase crude 4.6
Lipase +SMS 1.62
Lipase +SMS/Celite 0.11
Lipase /Celite 0.1
Liuase M
Enzyme Protein content (%)
Lipase crude 0.96
Lipase +SMS 1.8
Lipase +SMS/Celite 0.12
Lipase /Celite 0.1
Liuase G - Amano 50
Enzyme Protein content (%)
Lipase crude 9.65
Lipase +SMS 1.11
Lipase +SMS/Celite 0.06
Lipase /Celite 0.29
Lipase A- Amano 6
Enzyme Protein content (%)
Lipase crude 21.63
Lipase +SMS 4.85
Lipase +SMS/Celite 0.225
Lipase /Celite 0.2
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Table II continued
Lipase Saiken 100
Enzyme Protein content (%)
Lipase crude 4.84
Lipase +SMS 2.87
Lipase +SMS/Celite 0.06
Lipase /Celite 0.04
Lipase F P-15
Enzyme Protein content (%)
Lipase crude 49.2
Lipase +SMS 9.31
Lipase +SMS/Celite 0.70
Lipase /Celite 0.47
Lipase F -EC
Enzyme Protein content (%)
Lipase crude 49.7
Lipase +SMS 7.2
Lipase +SMS/Celite 1.12
Lipase /Celite 0.2
Lipase EC
Enzvme Protein content (%)
Lipase crude 36.8
Lipase +SMS 6.5
Lipase +SMS/Celite 0.48
Lipase /Celite 0.39
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Table II continued
Lipase PS
Enzvme Protein content (%)
Lipase crude 7.5
Lipase +SMS 0.72
Lipase +SMS/Celite 0.16
Lipase /Celite 0.08
Lipase Newlase F
Enzyme Protein content (%)
Lipase crude 25.1
Lipase +SMS 2.17
Lipase +SMS/Celite 0.24
Lipase /Celite 0.013
Lipase LP
Enzyme Protein content (%)
Lipase crude 6.16
Lipase +SMS 4.6
Lipase +SMS/Celite 0.65
Lipase /Celite 0.21
Lipase AY - Amano 30
Enzyme Protein content (%)
Lipase crude 5.14
Lipase +SMS 2.6
Lipase +SMS/Celite 0.05
Lipase /Celite 0.05
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Table III
Enzyme Protein content (%)
Lilipase+sorbitan monostearate 1.614
Lili ase+sorbitan monostearate/Celite0.112
Lilipase+sucrose ester HLB=5 1.0
Lili ase+sucrose ester HLB=5/Celite0.128
Lilipase+sucrose ester HLB=11 0.15
Lili ase+sucrose ester HLB=11/Celite0.051
Lilipase+sucrose ester HLB=16 0.1
Lili ase+sucrose ester HLB=16/Celite0.04
Lilipase+sorbitan monolaurate no precipitate
Lili ase+sorbitan monolaurate/Celite0.128
Lilipase+sorbitan tristearate 1.436
Lili ase+sorbitan tristearate/Celite0.134
Lilipase+sorbitan trioleate no precipitate
Lili ase+sorbitan trioleate/Celite0.115
Lilipase+monooleate no precipitate
Lili ase+monooleate/Celite 0.140
Lilipase+lecithin no precipitate
Lili ase+lecithin/Celite 0.16
Lilipase+stearic acid 1.65
Lili ase+stearic acid/Celite 0.108
Lilipase+Octadecanoic acid 1.17
Lili ase+Octadecanoic acid/Celite 0.102
Lilipase polyoxyethylene-8-stearate2.35
Lili ase olvox eth lene-8-stearate/Celite0.115
Lilipase +polyethyleneglycol no precipitate
Lili ase + of eth lene 1 col/Celita0.10
Lilipase gum Arabic no precipitate
Lilipase gum Arabic/Celite ~ 0.172
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Example 2
Esterification, transesterification and alcoholvsis activities of the
lipase preparations
A comparison was made of the enzymatic activities of the various lipase
preparations ( prepared as described above). For the purposes of this
comparison, the results (shown in Table I~ are presented as reaction rates
(ri).
Table IV
Lilipase A-lOFG
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(umol/min.mg
(~mol/min.mg n) protein)
protein) (~mol/min.mg
protein)
Crude 0.16 0.01 0
Lipase+SMS 7.43 5.2 2.1
Lipase+SMS/Celite27.4 18.1 7.1
Lipase/Celite 4.5 0.8 0.4
Li>7ase M
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~.mol/min.mg
(~mollmin.mg n) protein)
protein) (~mol/min.mg
_ protein)
Crude 0.2 0 0
Lipase+SMS 6.7 4.8 2.0
Lipase+SMS/Celite22.3 16.4 6.7
Lipase/Celite 3.2 0.1 0.3
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Table IV continued
Lipase PS
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~,mol/min.mg
(~,mol/min.mgn) protein)
protein) (~.mol/min.mg
protein)
Crude 0.3 0 0
Lipase+SMS 5.2 4.3 1.6
Lipase+SMS/Celite18.3 12.5 6.5
Lipase/Celite 4.5 0.4 0.2
Lipase LP
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~,mol/min.mg
(~.mol/min.mgn) protein)
protein) (~.mol/min.mg
protein)
Crude 0.3 0 0
Lipase+SMS 4.9 3.3 1.4
Lipase+SMS/Celite15.5 9.5 2.8
Lipase/Celite 2.3 0.2 0.35 _
Libase EC
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~,mol/min.mg
(~,mol/min.mgn) protein)
protein) (~mol/min.mg
protein)
Crude 0 0 0
Lipase+SMS 0.7 1.9 1.9
Lipase+SMS/Celite1.9 10.3 5.4
Lipase/Celite 0.1 1.1 0.3
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Table IV continued
Lipase AY Amano 30
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~,mol/min.mg
(~,mol/min.mgn) protein)
protein) (~mol/min.mg
_ protein)
Crude 0 0 0
Lipase+SMS 2.2 0.3 0.7
Lipase+SMS/Celite16.3 0.8 1.3
Lipase/Celite 0.9 0.05 0.4
Lipase G
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~mol/min.mg
(~,mol/min.mgn) protein)
protein) (~mol/min.mg
_ protein)
Crude 0 0 0
Lipase+SMS 0.1 0.15 0
Lipase+SMS/Celite1.4 0.4 0
Lipase/Celite 0.1 0 0
Lipase A
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~.mol/min.mg
(~.mol/min.mgn) protein)
protein) (~.mol/min.mg
protein)
Crude 0 0 0
Lipase+SMS 0.9 0.5 0.1
Lipase+SMS/Celite3.0 1.2 0.6
Lipase/Celite 0.3 0.1 0
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Table IV continued
Liuase F-AP15
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~.mol/min.mg
(~.mol/min.mg n) protein)
protein) (~mol/min.mg
protein)
Crude 0.3 0 0
Lipase+SMS 6.7 4.9 1.9
Lipase+SMS/Celite26.4 12.7 5.4
Lipase/Celite 0.8 0.93 .3
Lipase F-EC
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~.mol/min.mg
(~mol/min.mg n) protein)
protein) (~mol/min.mg
protein)
Crude 0.4 0 0
Lipase+SMS 7.1 3.4 1.5
Lipase+SMS/Celite23.5 10.5 5.4
Lipase/Celite 1.9 0.6 0.6
Lipase Saiken 100
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~.mol/min.mg
(~.mol/min.mgn) protein)
protein) (umol/min.mg
protein)
Crude 0.15 0 0
Lipase+SMS 6.9 4.2 2.4
Lipase+SMS/Celite29.3 10.3 8.2
Li~ase/Celite 1 2 3.2 0.4
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Table IV continued
Lipase Newlase F
Enzyme ri ri ri (alcoholysis)
(esterification)(transesterificatio(~,mol/min.mg
(~,mol/min.mgn) protein)
protein) (~.mol/min.mg
protein)
Crude 0 0 0
Lipase+SMS 1.3 1.3 0.4
Lipase+SMS/Celite6.4 6.8 0.5
Lipase/Celite 0.2 0.1 0
These results indicate that in their native form the crude lipases used in
this
study lack measurable esterification or transesterification activity with the
low water concentrations used. In contrast, both modification with the
surfactant sorbitan monostearate and, independently, immobilization onto
Celite, resulted in detectable levels of esterification and
transesterification.
When the enzyme was subjected to both of these treatments, however, the
reaction rates of both the esterification and transesterification reactions
studies increased to much greater levels. From the numerical results
presented in Table IV, it is clear that there is an unexpected synergism
between the two stages of modification, i.e., treatment with surfactant and
immobilization onto the matrix.
Figure 3 presents the conversion of tripalmitin with time when
DE-immobilized surfactant-coated Saiken-100 (triangles) and Lilipase A10-FG
(squares) were used to catalyze the inter-esterification reaction of
tripalmitin
and capric acid. The inter-esterification reaction rates thus measured were
0.096 and 0.104 mmol/min.mg biocatalyst, respectively.
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Example 3
Fatty acid specificity of immobilized surfactant-coated lipase complexes
The specificity of the inorganic matrix-immobilized surfactant-coated lipase
complexes of the present invention toward different fatty acid substrates was
tested by monitoring the inter-esterification of fatty acids of various chain
lengths with tripalmitin. The results are summarized in Table V below.
Reaction conditions were as follows:
Inter-esterification activity of crude lipase preparations was tested in
n-hexane using tripalmitin (50 mg) as a triacylglycerol substance and capric
acid (35 mg) as a medium-chain fatty acid substance. Inter-esterification
activities of 10 mg non-immobilized surfactant-coated lipase (5-10 % protein
content), or of 20 mg of inorganic matrix surfactant-coated lipase (0.2-2
protein content), in n-hexane (10 ml), were examined using the same
substrates.
Table V
Fatty acid Fatty acid chainInter-esterification
length rate
(mmol/min.mg biocat.)
Butyric acid (C4) 0.055
Hexanoic acid (C6) 0.06
Octanoic acid (C8) 0.09
Decanoic acid (C10) 0.104
Lauric acid (C 12) 0.15
Myristic acid (C14) 0.2
Palmitic acid (C16) 0.25
Stearic acid (C18) 0.26
Oleic acid (C18:1) 0.3
Arachidonic (C20) 0.28
acid
Behenic acid (C22) 0.28
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From Table V it can be seen that the immobilized surfactant-coated Lilipase
complexes according to the present invention predominantly catalyzed the
inter-esterification of fatty acids and tripalmitin with 1,3-positional
specificity. The concentration of hydrolysis products did not exceed 5 wt % of
the initial tripalmitin concentration.
It is further noted that the inter-esterification activity of the inorganic
matrix-immobilized surfactant-coated lipase complexes according to the
present invention was affected by the fatty acid chosen to be used as a
substrate. Thus, fatty acids having longer alkyl chains, such as palmitic and
stearic acids, are better substrates for the DE-immobilized surfactant-coated
lipase complexes than fatty acids having shorter alkyl chains.
Example 4
Influence of surfactant choice on inorganic matrix-immobilized
surfactant-coated lipase complexes
Table VI below demonstrates that the type of sorbitan fatty acid ester
selected
for coating the lipase influences the inter-esterification activity of
Celite-immobilized surfactant-coated lipase complexes.
When sorbitan monostearate was used for coating, the inter-esterification
activity of the complex was the highest. However, using a shorter fatty acid
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chain length in the sorbitan ester led to decrease in the activity of the
complex.
Table VI
Lilibase A-lOFG
Enzyme ri ri n
(esterification) (transester (alcoholysi
(mol/min.mg ification)
((mol/min.
protein) (mol/min. mg
mg protein)
protein)
Lilipase crude 0.16 0.01 0
Lilipase/Celite 4.5 0.1 0.4
Lilipase+sorbitan monostearate 7.43 5.75 1.2
Lilipase+sorbitan monostearate/Celite27.4 15.5 2.3
Lilipase+sucrose ester HLB=5 23.6 4.5 3.5
Lilipase+sucrose ester HLB=5/Celite22.4 7.5 5.5
Lilipase+sucrose ester HLB=11 31.1 12.5 6.2
Lilipase+sucrose ester HLB=11/Celite25.3 30 8.2
Lilipase+sucrose ester HLB=16 8.5 2 1.1
Lilipase+sucrose ester HLB=16/Celite16.2 12.5 4.2
Lilipase+sorbitan monolaurate 6.2 4 0.6
Lilipase+sorbitan monolaurate/Celite7.1 11.5 2.1
. Lilipase+sorbitan tristearate 10.1 4 1.9
Lilipase+sorbitan tristearate/Celite22.5 31 7.2
Lilipase+sorbitan trioleate 18.3 7.5 2.6
Lilipase+sorbitan trioleate/Celite27.3 20 9.3
Lilipase+ sorbitan monooleate 6.2 2.5 1.4
Lilipase+monooleate/Celite 8.5 10 4.2
_ 10.2 3.5 1.15
Lilipase+lecithin
Lilipase+lecithin/Celite 16.2 10 3.2
Lilipase+stearic acid 6.3 3.75 1.6
Lilipase+stearic acid/Celite 13.4 14 5.3
Lilipase+pctadecanoic acid 6.2 3 0.81
Lilipase+pctadecanoic acid/Celite14.6 11.5 3.4
Lilipase polyoxyethylene-8-stearate6.5 4.4 2.1
Lilipase 13.5 16 6.2
polyoxvethylene-8-stearate/Celite
Lilipase +polyethylenglycol 5.4 0 0
Lilipase +polyethylenglycol./Celite9.2 0.4 0.4
Lilipase gum Arabic 9.3 2 0.5
Lilipase gum Arabic/Celite 11.1 5 1.1
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Example 5
Physical binding of modified lipase to insoluble matrices
The esterification, transesterification and alcoholysis activity For Lilipase
A-lOFG immobilized on various insoluble matrices was measured, and
compared with the activities of Lilipase A-lOFG modified with sorbitan
monostearate and immobilized on different inorganic matrices. The results
are shown in Table VII.
Table VII
Enzyme/Insoluble ri ri (trans- ri
matrix* (esterification)esterification)(alcoholysis)
(umol/min.mg(~.mol/min.mg(~.mol/min.m
protein) protein) g protein)
Lilipase A10-FG crude0.16 0.01 0
Lilipase + SMS 7.43 5.2 2.1
Lilipase + SMS/Celite27.4 18.1 7.1
(acid-washed)
Lilipase/Celite 4.5 0.8 0.4
(acid-washed)
Lilipase + SMS/Celite25.4 13.5 6.0
(acid-nonwashed)
Lilipase/Celite 3.5 0.5 0.1
(acid-nonwashed)
Lilipase + SMS/fatty*41.3 27.4 17.3
acid-treated Celite
Lilipase/fatty acid-treated19.7 9.5 4.6
Celite
Lilipase+ SMS /Alumina23.2 11.1 5.4
LilipaselAlumina 1.6 0.3 0.4
Lilipase + SMS 45.3 29.1 19.2
/Aluminium
monostearate
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Table VII continued
Lilipase/Aluminium 19.2 9.8 6.1
monostearate
Lilipase + SMS/Silica21.3 10.8 5.7
Lilipase/Silica 3.2 0.4 0.2
Lilipase + SMS/Calcium18.6 12.3 6.1
carbonate
Lilipase/Calcium 2.4 0.4 0.2
carbonate
Lilipase + SMS/Calcium 16.6 11.3 5.6
sulfate
Lilipase/Calcium sulfate 0.3 0.2 - - 0.3
* Fatty acid-(or its derivative) treated Celite (hydrophobized Celite) was
prepared as follows: Celite (100 g) was suspended in phosphate buffer
solution (100 ml) of pH=5.7. A solution of free fatty acid or its derivative
(stearic acid or fatty acid sugar ester) dissolved in ethanol (5g/30m1) was
added dropwise to the vigorously stirred suspension at 70~C. The suspension
was stirred for 2 h, filtered, washed with water and then lyophilized.
The most suitable carriers for the immobilized enzymes used in this study
are aluminum monostearate and fatty acid-treated Celite. As can be seen in
Table VII, nonmodified Lilipase immobilized on Aluminum monostearate or
fatty acid treated-Celite gave relatively good activity in all of the three
reaction models. The results presented in this Table prove that the enzyme
modification with a surfactant in a first step and then immobilizing the
modified enzyme on a fatty acid-treated insoluble matrix leads to a further
increment in the activity of the enzyme. As can be seen in the above Table,
the activity of the fatty acid derivative-modified and immobilized lipase on a
fatty acid derivative-treated insoluble matrix (Aluminum monostearate,
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fatty acid derivative-treated Celite) is much greater than the activity of
lipase immobilized on a fatty acid derivative-treated insoluble matrix
Example 6
The effect of the choice of ion-exchange resin on enzyme activity
The esterification, transesterification and alcoholysis activities of Lilipase
A-lOFG immobilized on various ion-exchange resins, and of the same enzyme
modified with sorbitan monostearate prior to immobilization were compared.
The results of this comparison (expressed as reaction rate, ri) are shown in
Table VIII.
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Table VIII
Lilipase/Ion-exchangeri ri (traps- ri
resin* (esterification)esterification)(alcoholysis)
(~.mol/min.mg(~mol/min.mg (~,mol/min.m
protein) protein) g protein)
Dowex 22 4.9 4.7 1.9
Dowex 22 + SMS 32.2 23.1 6.3
Dowex 1x2-400 6.9 3.5 1.1
Dowex 1x2-400 + 29.6 25.4 6.1
SMS
Dowex2x8-100 5.5 4.7 2.2
Dowex2x8-100 + SMS 21.3 16.2 7.3
Cellulose phosphate3.5 0.2 0.1
Cellulose phosphate6.3 1.5 1.1
+
SMS
Amberlite IRA-95 7.6 0.8 0.9
Amberlite IRA-95 + 26.2 12.5 2.3
SMS
Amberlite IRA-200 9.2 3.1 1.4
Amberlite IRA-200 28.6 18.9 5.3
+
SMS
Amberlite IRA-900 6.1 4.2 2.3
Amberlite IRA-900 37 28.3 8.6
+
SMS
DiannonSA-10A 6.1 2.8 1.2
DiannonSA-l0A + SMS 31 19.5 11.3
Ectoela cellulose 5.4 2.3 1.7
Ectoela cellulose 29.5 12.2 5.7
+ SMS
Sephadex 1.5 0.2 0.1
Sephadex + SMS 2.9 0.9 0.8
Sulfoxyethylcellulose5.6 3.2 2.5
Sulfoxyethylcellulose32.3 17.0 12.6
+
SMS
Amberlite XAD-7 8.3 4.5 3.8
(Weak-polar)
Amberlite XAD-7 + 37.3 29.5 17.5
SMS
Amberlite XAD-16 6.7 6.5 4.3
(apolar)
Amberlite XAD-16 + 33.4 24.4 14.3
SMS
* All ion-exchange resins were purchased from Sigma, USA.
These results demonstrate the dramatic increase in esterification and
traps-esterification activity of the modified enzymes upon immobilization.
Furthermore, it can be seen from Table VIII that ion-exchange resins
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containing hydrophobic groups in their structure behave much better as
carriers for the surfactant-modified enzymes.
Example 7
Biosynthesis of structured tri~lycerides from different oils
usin inorganic matrix-immobilized surfactant-coated lipase
complexes
Table IX below demonstrates the rates of inter-esterification reactions
between different oils and capric acid in n-hexane system and in a solvent
free
system at 60 ° C. The inter-esterification reactions were catalyzed by
DE-immobilized surfactant-Lilipase A10-FG complexes.
DE-immobilized surfactant-Lilipase complexes predominantly catalyzed the
inter-esterification of various oils with capric acid in n-hexane and solvent
free systems. The highest inter-esterification reaction was obtained when
olive oil was employed. Similar reaction rates where obtained when the
inter-esterification reactions were carried-out in a solvent-free system.
Table IX
Source of oil R.R. (mmol/minmg biocat.)R.R. (mmol/minmg biocat.)
in n-hexane in solvent free system
Olive oil 0.52 0.73
Fish oil 0.4 0.53
Sun flower oil 0.3 0.42
Palm oil 0.2 0.31
Canola oil 0.32 0.45
Corn oil 0.24 0.41
lVi~ella saiiua 0.2 - 0.46
oil
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Example 8
Operational stability of modified and immobilized enzymes
The stability of the enzyme preparations over repeated cycles of use was
determined as described in the methods section above. For the purposes of
this investigation, Lilipase A-lOFG was used to catalyze the conversion of
olive oil to a wax ester. Activity was measured in a 10-run experiment, each
run requiring one hour for completion. The level of activity was measured as
percentage conversion of the olive oil. The reaction conditions used were as
follows:
100 ml of n-Hexane containing 20g olive oil and 20g cetyl alcohol recirculated
over an immobilized enzyme packed-bed at a circulation rate of 2.3 ml/min at
40° C. The column used to pack the granulated enzyme was 12 cm long
with
an internal diameter of 0.75 cm. The results of this investigation are shown
in figures 5 - 12.
Fig. 5 shows that the activity of Lilipase A-lOFG immobilized on Celite
(without modification) and granulated with 2% starch was low, and that the
activity decreased with each re-use. Fig. 6 shows that the activity of the
same lipase powder after modification with sorbitan monostearate and then
immobilization on Celite (without granulation) was 9-fold higher than the
activity of the same non-modified lipase. It can be seen from Fig. 2 that
there was sharp activity loss after the first, second and the third runs,
which can be attributed to washing out of the immobilized enzyme. Figs.
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7-16 show that the enzyme activity in organic solvent can be essentially
retained by granulation with different binding reagents such as starch,
ethyl cellulose, gums, starch, etc. The granulation process also facilitates
the
flow in the packed column with enzyme. The binders used in these figures
are as follows:
Fig. 7 Starch 2%
Fig. 8 Ethyl cellulose 2
Fig. 9 Gum Arabic 2
Fig. 10 Ethyl cellulose 2
Fig. 11 Agarose 2%
Fig. 12 Starch 4%
Figs. 13 - 16 Starch 2
Immobilization was performed on Celite in the experiments shown in figures
- 9 and 12 - 13, on Amberlite IRA-900 in the experiments shown in figures
and 11, on aluminium monostearate in figure 14, on Amberlite XA.D-16 in
figure 15 and on Amberlite XAD-7 in figure 16. In all cases where lipase was
modified by surfactant, the surfactant used for this treatment was sorbitan
monostearate, except for the experiment shown in figure 13, where the
surfactant was sorbitan tristearate.
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Example 9
Covalent binding~'of modified and non-modified lipases to Eupergit
Lipases were covalently bound to Eupergit according to the manufacturer's
specification. The covalent binding was carried out the two procedures
described above.
Table X
Lilinase A10-FG
Enzyme form Conversion (%)
Crude Lilipase A10-FG 2
Lilipase on Eupergit 4
Lilipase on Eupergit + SMS 19
Lilipase on Eupergit + SMS after 48h 26
Lipase LP
Enzyme form Conversion (%)
Crude lipase LP 1
Lipase LP on Eupergit 2
Lipase LP on Eupergit + SMS 14
Lipase LP on Eupergit + SMS after 48h 18
Lipase PS
Enzyme form Conversion (%)
Crude lipase PS 1
Lipase PS on Eupergit 2
Lipase PS on Eupergit + SMS 8
Lipase PS on Eupergit + SMS after 48h 12
It can be seen from Table X that different lipases show different
inter-esterification activity when are treated similarly. This result is
ascribed to the different sources of the lipases used. All crude lipases
showed
very low inter-esterification activity under the described conditions while
their activity has slightly increased when they were covalently immobilized on
Eupergit. It is interesting to notice that when lipases were coated with a
surfactant their inter-esterification activity has significantly increased.
The
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highest conversion of tripalmitin to its inter-esterification products with
1,3-positional specificity that was achieved after 2 h reaction, was when the
lipases were first covalently immobilized on Eupergit and then coated with
surfactant. Lilipase A10-FG coated with the surfactant and immobilized on
Eupergit yielded the highest inter-esterification activity within the three
lipases tested in this respect (Table ~.
Example 10
Effect of binders on the enzyme activity.
As previously mentioned, different binders have been used for the
granulation of the modified-immobilized lipases. Table XI shows the average
conversion of olive oil to its wax esters in the first and second runs, using
different binders for granulating Lilipase A10-FG modified with sorbitan
monostearate and immobilized on Celite. The percentage of all binders was
2% dry weight of the granules. In these experiments, the granulated
enzyme was packed in a column and used in 10 runs.
The average conversion of olive oil to its wax esters in the first and second
runs using Lilipase A lOFG modified with sorbitan monostearate and
immobilized on Celite and then granulated with different binders (2%).
Reaction conditions: 100m1 of n-Hexane containing 2g olive oil and 2g cetyl
alcohol recirculated over an immobilized enzyme packed-bed at a circulation
rate of 2.5 ml/min and at 40°C. Column dimensions: 12 cm long, 0.75 cm
i.d.
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Table XI
Binder Conversion (%)
Starch 92
Ethyl cellulose 44.6
Methyl cellulose 38.1
Agarose 16.8
Gelatin Inactive
Polyvinylpyrrollodone Inactive
Gum Arabic 49.1
Gum Xan 33.2
Gum Karaya 49.5
Gum Tragacanth 20.1
Gum Locas 33.6
From the above table it may be concluded that starch, Gum Arabic and Gum
Karaya are the most effective binders of those tested in this study, that
yielded active biocatalysts.
Although the invention has been described in conjunction with specific
embodiments thereof, it is evident that many alternatives, modifications and
variations will be apparent to those skilled in the art. Accordingly, it is
intended to embrace all such alternatives, modifications and variations that
fall within the spirit and broad scope of the appended claims.
