Note: Descriptions are shown in the official language in which they were submitted.
-l~ 131862~
TITLE OF THE INVENTION
Preparation of immobilized lipases and their
use in the synthesis of glycerides.
FIELD OF THE INVENTION
-
The present invention relates to a dry porous
macromolecular matrix comprising a cross-linked mixture
of an inert protein, a lipase enzyme and a
cross-linking agent. This matrix is particularly
suitable or adapted to catalyze the reaction of fatty
acids such as oleic acid and glycerol to produce mono-,
di- and tri-olein.
BACKGROUND OF THE IN~ENTION
The use of immobilized lipase preparations for
the transesterification of fats is well known. For
example, in UK Patent 1,577,933, an immobilized lipase
suitable for transesterification is attached on an
indifferent particulate carrier which may be
diatomaceous earth or alumina and which exhibits a very
high surface area. It has also been proposed to
prepare an immobilized lipase preparation for
interesterification with n-hexane as a solvent
comprising lipase and a strong anion exchange resin
(see European Journal of Applied Microbiology and
Biotechnology, no. 14, p. 1-5 (1982)). E.P.O.
application No. 0069599 discloses a lipase enzyme
suported on a carrier such as Celite for continuous
interesterification in a column.
-2- 131~62~
In Biotechnology and Bioengineering, vol. XV,
p. 359-375, 1973, G. Broun et al, methods of
cross-linking enzyme molecules inside a matrix with or
without an inactive protein are described. More
specifically, the method comprises the cross-linking of
an enzyme molecule with an inactive protein, such as
albumin, in the presence of a bifunctional
cross-linking agent without any preformed matrix.
In practice, this article teaches the mixing
of albumin, glutaraldehyde and an enzyme in a phosphate
buffer, freezing the solution obtained at -30C and
allowing the frozen mass to warm at 4 C. After
standing for 4 hours at 4C, the spongelike proteinic
copolymer is thoroughly rinsed, lyophilized and ground.
It is then suitable for suspending in a mixed solution
of substrate or for pouring into a column with a flux
of substrate solution flowing through it. Examples
using glucose oxidase, urease, trypsin and catalase are
i given.
Unfortunately, it has been found that when any
of the enzymes used in the G. Broun et al disclosure is
replaced by lipase, there is no obtention of a solid
matrix. It is however possible to obtain the desired
porous matrix by increasing the amount of bifunctional
cross-linking agent but in this situation, the lipase
enzyme loses its activity after lyophilization.
Therefore, a method making the use of active
immobilized lipase possible would be highly desirable.
~3~ 1 3~ 8~2
SUMMARY OF THE INVENTION
In accordance with the present invention,
there is provided a dry porous macromolecular matrix
which comprises a cross-linked mixture of 69.9% to
86.6% by weight of an inert protein, 0.4% to 10.2% by
weight of a lipase enzyme and a cross-linking agent,
the amount of cross-linking agent being from about
11.7% to 23~ by weight.
Also in accordance with the present invention,
there is provided an improvement in the process for
preparing the dry porous macromolecular matrix of the
present invention whereby a reaction mixture of the
lipase enzyme, inert protein, cross-linking agent and
buffer i.s frozen at a temperature ranging from -20C to
-195C and allowed to thaw in distilled water at a
temperature ranging from 4C to 25C after which the
matrix is rinsed with water and acetone. The matrix is
then vigorously wrung and dried at room temperature for
a period of about 12 hours. The matrix of the present
invention may be used for the catalysis of fatty acids
such as oleic acid with glycerol into compounds such as
mono-, di- and tri-olein.
IN THE DRAWINGS
Figure 1 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reactlon
products when concentrated lipase obtained from
~4~ 1~18624
Rhizopus arrhizus and immobilized using glutaraldehyde
at a concentration of 1.125% is used.
Figures 2 to 5 represent successive eyeles of
consumption of oleic acid through its enzymatic
reaetion with lipase and glycerol and the corresponding
appearance of reaction products when immobilized
eoncentrated lipase obtained from Rhizopus arrhizus is
reused.
Figure 6 represents the eonsumption of oleie
aeid through its enzymatie reaetion with lipase and
glycerol and the corresponding appearanee of reaetion
produets when a solution of free lipase obtained from
Rhizopus arrhizus is used.
Figures 7 to 11 represent the first and
sueeessive eyeles of eonsumption of oleie aeid through
its enzymatie reaetion with lipase and glyeerol and the
eorresponding appearanee of reaetion produets when
eoneentrated lipase obtained from Rhizopus delemar and
immobilized using glutaraldehyde at a eoneentration of
1.125% is reused.
Figures 12 and 13 represent two sueeessive
eyeles of eonsumption of oleie acid through its
enzymatie reaction with lipase and glycerol and the
corresponding appearanee of reaction produets when
eoneentrated lipase obtained from Candida eylindraeea
and immobilized using glutaraldehyde at a concentration
of 1.125~ is used.
~5~ 1318624
Figure 14 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reaction
products when concentrated lipase obtained from
Rhizopus arrhizus and immobilized using glutaraldehyde
at a concentration of 0.75% is used.
Figure 15 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reaction
products when concentrated lipase obtained from
Rhizopus arrhizus and immobilized using glutaraldehyde
at a concentration of 1.5% is used.
Figure 16 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reaction
products when concentrated lipase obtained from
Rhizopus delemar and immobilized using glutaraldehyde
at a concentration of 0.75~ is used.
Figure 17 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reaction
products when concentrated lipase ob+ained from
Rhizopus delemar and immobilized using glutaraldehyde
at a concentration of 1.5~ is used.
Flgure 18 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glyceroi and the corresponding appearance of reaction
-6~ 862~
products when concentrated lipase obtained from Candida
cylindracea and immobilized using glutaraldehyde at a
concentration of 0.75~ is used.
Figure 19 represents the consumption of oleic
acid through its enzymatic reaction with lipase and
glycerol and the corresponding appearance of reaction
products when concentrated 'ipase obtained from Candida
cylindracea and immobilized using qlutaraldehyde at a
concentration of 1.5~ is used.
Figure 20 represents the consumption of oleic
acid through its enzymatic reaction with methanol and
the corresponding appearance of reaction products when
concentrated lipase obtained from Rhizopus arrhizus and
immobilized using glutaraldehyde at a concentration of
1.125% is used.
Figure 21 represents the consumption of oleic
acid through its enzymatic reaction with ethanol and
the corresponding appearance of reaction products when
concentrated lipase obtained from Rhizopus arrhizus and
immobilized using glutaraldehyde at a concentration of
1.125~ is used.
DETAILED DESCRIPTION OF THE INVENTION
THE MACROMOLECULAR MATRIX
The dry porous macromolecular matrix of the
present invention is a cross-linked mixture of an inert
carrier, a lipase enzyme and a cross-linking agent,
preferably a bifunctional cross-linking agent. More
~7~ 1 31~
specifically, the matrix contains from 69.9% to 86.6%
of inert protein, 0.4% to 10.2% by weight of lipase
enzyme and 11.7 to 23~ by weight of cross-linking
agent. The matrix can be used as such in
esterification procedures or can be ground to a fine
powder.
THE INERT CARRIER
The inert support is used as an enzyme
carrier. It may be in the form of powder, fibers or the
l~ like although it is preferable that the carrier be in
the form of granules in view of the continuous use of
the resulting enzyme preparation. It is usually
preferred to use inert proteins which are, in the
context of the present invention, very suitable inert
supports.
As inert protein, human and animal albumin may
be used, for example, bovine albumin, fibrinogen,
hemoglobin and the like. The amount of inert protein
in the initial mixture is 5% by weight. In the final
macromoiecular matrix, the quantity of inert protein
may range from 69.9~ to 86.6% by weight.
THE ENZYME
The lipase enzyme used for immobilization
purposes in accordance with the present invention may
be selected from lipases originating from Candida
cylindracea, Mucor miehei, Rhizopus arrhizus, Rhizopus
.
delemar and the like. These enzymes are bonded to an
13~8624
inert protein carrier through a cross~linking agent.
The mixture rati~s of the enzyme to the carrier may
vary from 6.3:1200 to 157.1:1200.
THE CR~SS-LINKING AGENT
The cross-linking agent is used to bound the
enzyme onto the inert carrier. Although it is possible
to employ various types of cross-linking agents, it has
been found that glutaraldehyde in quantities ranging
between 11.7~ and 23% by weight is preferred.
THE PROCESS
In order to disperse, adsorb or bond the
enzyme or the enzyme-containing material in or to the
carrier, for example, the enzyme or the
enzyme-containing material is dissolved in a buffer
solution and the resulting solution is admixed with the
carrier.
Thus, in a buffered solution, there is added
5% by weight of an inert carrier, preferably an inert
protein, 0.75% to 1.5% by weight of a cross-linking
agent and 50 to 440 ul of lipase enzyme diluted in the
buffer.
After allowing the mixture to stand at room
temperature for a period of from 5 to 1~ minutes to
allow prereticulation or cross-linking between the
2~ inert carrier and the enzyme via the cross-linking
agent, the mixture is then frozen at a temperature
ranging from -20 C to -195 C until ready to use. When
~31862~
the matrix is to be used in a catalytic reaction, the
frozen mixture is thawed slowly up to a temperature
that may vary from 4C to 25C in distilled water,
thereby yielding a solid matrix. After thorough
washing with water to remove any unreacted compound and
any free enzyme, the matrix is blotted dry on filter
paper and shaked in acetone for about two hours to
eliminate any water in the core of the matrix. The
matrix is then allowed to dry on filter paper at room
temperature for a period of 3 to 24 hours. Preferably,
the reaction mixture is to be frozen at -80C, thawed
at 4C and allowed to dry for 12 hours. It is to be
noted that the drying step is essential to the process
of the present invention since the presence of water
prevents the catalytic reaction of fatty acids with
glycerol. The final product obtained is a porous
macromolecular matrix.
REACTION SUBSTRATES
Typical examples of fatty substrate used in
the context of the present invention is a mixture of
fatty acids and short alcohols such as methanol,
ethanol, glycerol and the like. Furthermore, the
substrate may include other fatty esters on which a
lipolytic enzyme can act, such as monoglycerides,
diglycerides, methyl esters, ethyl esters or the like.
Also fatty acids having C3 to C22 carbon atoms may be
used as substrates either alone or together with these
fatty esters.
-lo- 131862~
The substrate should be a liquid at the
reaction temperature, and no organic solvent should be
used. The reaction medium is composed of only the
required substrates for the reaction. Preferably, the
most desirable substrate to be used in the context of
the present invention is a mixture of oleic acid and
glycerol in stoichiometric proportions.
CATALYTIC REACTION
The catalytic reaction of the present
invention through which fatty acids and alcohols, such
as oleic acids and glycerol, are preferably transformed
into mono-, di- and tri-olein is carried out by
continuously or repeatedly contacting the enzyme or the
enzyme preparation with a supply of the fatty acids and
glycerol.
The enzymatic reaction of the present
invention can be carried out in a batchwise operation
or in a continuous operation using for example a
fluidized bed of the enzyme preparation or a column
packed with the enzyme preparation.
Generally, a mixture containing between 0.5 to
10 g of oleic acid and between 0.0S43 to 1.086 g of
glycerol and from 50 to 440 ~1 of lipase immobilized
in the previously described matrix is strongly agitated
at a temperature ranging between 25C and 60C for a
time ranging from 10 to 1000 hours. From time to ~ime,
samples of the reaction mixture are analyzed in order
131862~
to measure the amount of substrate still in solution
and to analyze the various reaction products.
With the immobilization technique of the
present invention, it is possible to use the
immobilized enzyme for up to four successive cycles
before observing a decline in enzymatic activity.
The following examples are disclosed in order
to illustrate rather than limit the scope of the
present invention.
Example 1
Immobilization of lipase originating from Rhizopus
arrhizus onto bovine albumin.
~1 (222 units, 0.63 mg protein) of a
commercially available concentrated lipase originated
from Rhizopus arrhizus (Sigma no. L-4384) was diluted
in 390 ~ of phosphate buffer, 0.02 M, p~ 6.72. To the
resulting solution was added l ml of phosphate buffer
0.02 M, pH 6.72, 0.6 ml of bovine albumin 20% and 0.36
ml of glutaraldehyde 7.5%. The final mixture
demonstrated a final albumin concentration of 5~ and a
final glutaraldehyde concentration of 1.125% with a
final volume of 2.4 ml. The mixture is then allowed to
stand at room temperature for 10 minutes in order to
permit prereticulation or crosslinking between the
albumin and the lipase via glutaraldehyde. The
resulting mixture is then frozen at -80~. When it is
-12- 13 1 ~ 62 ~
desired to use the enzyme bound macromolecular matrix,
the mixture is slowly thawed up to a temperature of 4C
in distilled water. The resultin~ matrix is then
thoroughly rinsed, vigorously wrung out, washed with
acetone and dried on filter paper at room temperature
for 12 hours. The final matrix demonstrated a final
albumin concentration of 81.3~ and a final
glutaraldehyde concentration 18.3%.
Example 2
The procedure described in Example 1 was
repeated in order to obtain a final glutaraldehyde
concentration of 0.75% in the mixture which
necessitated the use of 0.36 ml of glutaraldehyde 5% in
the initial reaction mixture. After drying, the
albumin concentration was 86.6~ and the glutaraldehyde
concentration 13~.
Example 3
The procedure described in Example 1 was
repeated in order to obtain a final glutaraldehyde
concentration of 1.5% in the mixture which necessitated
the use of 0.36 ml of glutaraldehyde 10~ in the initial
reaction mixture. After drying, the albumine
concentration was 76.6% and the glutaraldehyde
concentration 23%.
-13-
1318624
Example 4
Immobilization of lipase originating from Rhizopus
delemar onto bovine albumin.
q'he procedure described in Example 1 was
repeated using 440 ~ (1571.4 units, 15.71 mg protein)
of a commercially available concentrated lipase
originated from Rhizopus delemar instead of lipase
originated from Rhizopus arrhizus. The procedure of
Example 1 was repeated in order to obtain a final
glutaraldehyde concentration of 0~75% in the mixture
which necessitated the use of 0.36 ml of glutaraldehyde
5% in the initial reaction mixture. After drying, the
albumin concentration was 78.1% and the glutaraldehyde
concentration 11.7~.
Example 5
The procedure described in Example 4 was
repeated in order to obtain a final glutaraldehyde
concentration of 1.125~ in the mixture which
necessitated the use of 0.36 ml of glutaraldehyde 7.5~
in the initial reaction mixture. After drying, the
albumin concentration was 73.8~ and the glutaraldehyde
concentration 16 ~ 6~o .
Example 6
The procedure described in Example 4 was
repeated in order to obtain a final glutaraldehyde
-14- 1 3i8624
concentration of 1.5% in the mjxture, which
necessitated the use of 0.36 ml of glutaraldehyde 10~
in the initial reaction mixture. After drying, the
albumin concentration was 69.9% and the glutaraldehyde
5 concentr~tion 21%.
Example 7
Immobilization of lipase originating from Candida
cylindracea onto bovine albumin.
The procedure described in Example 1 was
repeated using 440 ~1 (1571.4 units, 3.96 mg protein)
of a commercially available concentrated lipase
originated from Candida cylindracea instead of lipase
originated from Rhizopus arrhizus. The procedure of
Example 1 was repeated in order tG obtain a final
glutaraldehyde concentration of 0.75% in the mixture
which necessitated the use of 0.36 ml of glutaraldehyde
5% in the initial reaction mixture. After drying, the
albumin concentration was 84.5% and the glutaraldehyde
concentration 12.7%.
Example 8
The procedure described in Example 7 was
repeated in order tO obtain a final glutaraldehyde
concentration of 1.5% in the mixture which necessitated
the use of 0.36 ml of glutaraldehyde 10% in the initial
reaction mixture. After drying, the albumin
~ ~I5~ ~3~862~
concentration was 75% and the glutaraldehyde
concentration 22.5~.
Example 9
The procedure described in Example 7 was
repeated in order to obtain a final glutaraldehyde
concentration of 1.125% in the mixture which
necessitated the use of 0.36 ml of glutaraldehyde 7.5%
in the initial reaction mixture. After drying, the
albumin concentration was 79.5% and the glutaraldehyde
concentration 17.9%.
Example 10
Transformation of oleic acid into mono-, di- and
tri-olein.
0.5 g of oleic acid and 0.05 g of glycerol
were reacted with the support prepared in Example 1 by
strongly agitating the reaction mixture on an
Eppendorf mixer at 34 C. Figures 1 to 5 represent the
successive cycles through which oleic acid
progressively reacted to yield the various reaction
- products. The results are expressed as the percentage
of the number of moles of oleic acid present in the
initial reaction medium. Between each reaction cycle,
the enzymatic support was vigorously washed with
acetone for 24 hours and dehydrated at room temperature
using air and filter paper.
16- 1318624
As it can be seen from Figures 1 to 5, a
decrease in enzymatic activity is observed after the
fourth reaction cycle although the enzyme is still
active right up to the seventh cycle.
s
Example 11
The procedure described in Example 10 was
repeated but 50 ~1 of a solution of free lipase
obtained from Rhizopus arrhizus were used instead of
the support of Example 1. Results are shown in Figure
6.
Example 12
The procedure described in Example 10 was
repeated using the support of Example 2 as the source
of lipase. Results are shown in Figure 14.
Example 13
The procedure described in Example 10 was
repeated using the support of Example 3 as the source
of lipase. Results are shown in Figure 15.
Example 14
The procedure described in Example 10 was
repeated using the support of Example 4 as the source
of lipase. Results are shown in Figure 16.
` _l7_ 1318~2~
Example 15
The procedure described in Example 10 was
repeated using the support of Example 6 as the source
of lipase. Results are shown in Figure 17.
Exa~ple 16
The procedure described in Example 10 was
repeated using the support of Example 7 as the source
of lipase. Results are shown in Figure 18.
Example 17
The procedure described in Example 10 was
repeated using the support of Example 8 as the source
of lipase. Results are shown in Figure 19.
Example 18
The procedure described in Example 10 was
repeated using the support of Example 5 as the source
of lipase. Figures 7 to 11 represent the successive
cycles of glycerides synthesis when reusing the same
support.
Example 19
The procedure described in Example 10 was
repeated using the support of Example 9 as the source
of lipase. Figures 12 and 13 represent the successive
cycles of glycerides synthesis when reusing the same
support twice.
~318~2~
Exa~ple 20
Transformation of oleic acid and methanol into oleic
acid methyl ester.
0.5 g of oleic acid and 72 ~1 of methanol
were reacted with the support prepared in Example 1 by
strongly agitating the reaction mixture on an
Eppendorf mixer at 36 C. Figure 20 represents the
appearance of oleic acid methyl ester and the
consumption of oleic acid. After 358 hours of
reaction, 36 ~1 of methanol was added.
Example 21
The procedure described in Example 21 was
repeated using 103 ~1 of ethanol instead of methanol.
Figure 21 represents the appearance of oleic acid ethyl
ester and the consumption of oleic acid. After 358
hours of reaction, 52 ~1 of ethanol was added.
