U.S. patent application number 10/277253 was filed with the patent office on 2003-03-06 for fat reforming method with use of activated enzyme and activated enzyme deactivating method.
This patent application is currently assigned to Japan as Represented by Dir. of National Food Res. Inst., Ministry of Agri. , Forestry and Fisheries. Invention is credited to Ichikawa, Sosaku, Maruyama, Tatsuo, Nabetani, Hiroshi, Nakajima, Mitsutoshi, Seki, Minoru.
Application Number | 20030044952 10/277253 |
Document ID | / |
Family ID | 13673621 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030044952 |
Kind Code |
A1 |
Nakajima, Mitsutoshi ; et
al. |
March 6, 2003 |
Fat reforming method with use of activated enzyme and activated
enzyme deactivating method
Abstract
A fat reforming method using an enzyme which has been activated
by adding the enzyme to a system containing water and fat phases,
activating the enzyme by a function of a boundary surface/layer
between the water and fat phases, and removing the water and fat
phases through freeze drying. Tetradecane may be used as the fat
phase. Just after adding tetradecane into a water phase in which
lipase is dissolved, though existing in water phase as shown in
FIG. 4(a), the lipase moves towards the boundary surface since it
has a tendency to gather around the boundary surface between the
water and fat phases. The lipase moved to the boundary surface has
hydrophobic portion at the side of the fat phase and a hydrophilic
portion of the lipase at the side of the water phase, as shown in
FIG. 4(b), and at the same time the lid covering the activating
portion is opened by the function of the boundary surface. This
condition is maintained even after the system is freeze-dried to
remove the water and fat phases and the activated enzyme may be
subsequently used to reform fat, and may be deactivated.
Inventors: |
Nakajima, Mitsutoshi;
(Ibaraki, JP) ; Nabetani, Hiroshi; (Ibaraki,
JP) ; Ichikawa, Sosaku; (Ibaraki, JP) ; Seki,
Minoru; (Tokyo, JP) ; Maruyama, Tatsuo;
(Ibaraki, JP) |
Correspondence
Address: |
CARRIER BLACKMAN AND ASSOCIATES
24101 NOVI ROAD
SUITE 100
NOVI
MI
48375
|
Assignee: |
Japan as Represented by Dir. of
National Food Res. Inst., Ministry of Agri. , Forestry and
Fisheries
1-2, Kannondai 2-chome
Ibaraki
JP
|
Family ID: |
13673621 |
Appl. No.: |
10/277253 |
Filed: |
October 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10277253 |
Oct 21, 2002 |
|
|
|
09415924 |
Oct 12, 1999 |
|
|
|
Current U.S.
Class: |
435/198 ;
435/271 |
Current CPC
Class: |
C12N 9/20 20130101 |
Class at
Publication: |
435/198 ;
435/271 |
International
Class: |
C12N 009/20; C12S
003/18 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 1999 |
JP |
11-078861 |
Claims
What is claimed is:
1. A fat reforming method with use of activated enzyme, comprising
the steps of: adding enzyme to water and fat phases; activating the
enzyme by a function of a fat-and-water boundary surface between
the fat and water phases; removing the water and fat phases while
maintaining an activated condition of the enzyme; and contacting
said activated enzyme with fat in one of a non-aqueous system and a
nano-aqueous system.
2. A method for deactivating an activated enzyme, wherein said
activated enzyme is dispersed and stirred into a buffer solution in
which there exists no boundary surface between fat and water.
3. A method for deactivating an activated enzyme as described in
claim 2, wherein said activated enzyme is produced by: adding
enzyme to water and fat phases; activating the enzyme by a function
of a fat-and-water boundary surface between fat and water phases;
and removing the water and fat phases while maintaining an
activated condition of the enzyme.
4. A fat reforming method as defined in claim 1, wherein said
activated enzyme contains no other reforming material
therewith.
5. A fat reforming method as defined in claim 1, wherein said
enzyme comprises lipase and said fat phase comprises
tetradecane.
6. A method for deactivating an activated enzyme as defined in
claim 2, wherein said enzyme comprises lipase and said fat phase
comprises tetradecane.
7. A fat reforming method as defined in claim 1, wherein said
enzyme adding step involves dissolving the enzyme in the water
phase to form a buffer solution and then contacting the fat phase
thereto.
8. A fat reforming method as defined in claim 1, wherein said
removing step is conducted by freeze drying.
9. A fat reforming method as defined in claim 1, wherein said water
phase is maintained in a vicinity of neutrality.
10. A method for deactivating an activated enzyme as defined in
claim 2, wherein said enzyme adding step involves dissolving the
enzyme in the water phase to form a buffer solution and then
contacting the fat phase thereto.
11. A method for deactivating an activated enzyme as defined in
claim 2, wherein said removing step is conducted by freeze
drying.
12. A method for deactivating an activated enzyme as defined in
claim 2, wherein said water phase is maintained in a vicinity of
neutrality.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to an activating method of an
enzyme such as lipase, etc., to reaction of an enzyme including
reformation of fat with the use of the enzyme which is activated by
this method, and further to a method of deactivating the enzyme
once it is activated.
[0003] 2. Description of Related Art
[0004] Conventionally, the production of many useful products has
been achieved with use of enzymes. In particular, lipase was widely
used for reforming fat so as to produce edible oil, soap, glycerin,
dairy or milk products, etc., because of the versatility
thereof.
[0005] Today where resources, energy, and ecological problems are
discussed daily on a global scale, many expectations are applied to
efficient and safe production of important materials (i.e., fatty
acids, etc.) in the chemical industry, as well as to
high-performance materials to which much attention is paid in the
fields of pharmaceuticals and foodstuffs, with use of the enzyme
which can be represented by lipase.
[0006] As a method for obtaining a target material with use of the
enzyme, there can be listed a hydrolysis reaction with use of
lipase, transesterification, and ester synthesis or composing
reaction, etc.
[0007] As a different method for reforming those fats,
conventionally, an organic synthetic method using high temperature
is mainly used or practiced. However, such method cannot be applied
to materials which are unstable at high temperatures, and it is
impossible to reproduce the target material with high efficiency
due to poor enatioselectivity. Further, with the enzyme method, but
not with the organic synthetic method, since a residual activating
material which may have toxicity or may cause a side reaction is
used for increasing the activity of the enzyme, a problem arises in
particular in the field of foodstuffs.
[0008] Therefore, production of the useful materials by catalytic
reaction with use of an enzyme with high efficiency and with safety
is considered.
[0009] For example, in a case of the catalytic reaction with use of
lipase, it was reported that it proceeds with the useful
enatioselectivity where 1-phenylethylelaurate (R-soma) is
synthesized on a basis of 1-phenylethanol and lauric acid, as the
ester synthesis between fatty acid and asymmetric alcohol.
[0010] Also, aside from the enatioselectivity, if the lipase having
selectivity in a reacting portion, in particular participating in
ester bonding between a first order (1st order) and a third order
(3rd order) of triglyceride is used, it is possible to put the
target fatty acid into the positions of only the first and the
third orders. However, in a case of a chemical method using an
inorganic catalyst, there is no such selectivity, therefore the
fatty acid is put in at random.
[0011] However, most enzymes, such as lipase, show only a
hydrolysis reaction in water solution, and will not proceed to the
reaction if a powder thereof is simply dispersed into an organic
solvent by itself.
[0012] Then, for conducting the transesterification and the ester
composing reaction, which are industrially valuable, with high
efficiency in a non-aqueous or nano-aqueous system or condition,
there are proposed various methods, including a method in which the
enzyme is fixed onto a carrier, a method in which the surface of
the enzyme is made hydrophobic with a covalent bond, and a method
in which the enzyme is modified with a surfactant or fatty
acid.
[0013] For fixing the enzyme onto the carrier, there is already
known a method in which the enzyme is physically absorbed onto the
surface of an inorganic carrier, such as alumina particles, and
there is also known a method in which the enzyme is fixed with the
covalent bond onto the surface of latex particles or silica
particles.
[0014] For making the surface of the enzyme hydrophobic, there is
already known a method in which polyethylene glycol is bonded on
the surface of the enzyme with the covalent bond, and there is also
known a method in which the enzyme is made soluble into an organic
solvent.
[0015] Further, for modifying the lipase (i.e., the enzyme) with
the fatty acid or the surfactant, there is known a method in which
the enzyme is bonded with the fatty acid or the surfactant through
an interaction which seems to be electrostatic, hydrophilic or
hydrophobic.
[0016] With the method of fixing the enzyme onto the carrier,
however, it is impossible to conduct the transesterification or the
ester composing reaction with high efficiency. Further, there
results a solid enzyme which cannot be applied to the production of
foodstuffs.
[0017] With the method of making the surface of an enzyme
hydrophobic, however, the enzyme is easily deactivated during the
process of making it hydrophobic, and the activity which can be
actually obtained therefrom is low. The chemical (s) used for
making the surface of the enzyme hydrophobic cannot be used for
producing the foodstuffs, thereby restricting the utilization of
the enzyme itself.
[0018] With the method of modifying the enzyme with the fatty acid
and/or the surfactant, the enzyme shows interesterification
activity under the condition where it forms a compound with the
fatty acid and/or the surfactant. However, the enzyme itself does
not show the activity. In other words, since it is impossible to
remove the modification(s) of the fatty acid and/or the surfactant
from a reaction system, there is a probability that the fatty acid
and/or the surfactant may participate in some kind reaction so that
the modification is mixed or added into a product or a secondary
product.
SUMMARY OF THE INVENTION
[0019] It is considered that lipase has an active site and a "lid"
covering the active site, and it is low in interesterification
activity under the condition where the lid is closed as shown in
FIG. 1(a), while high in interesterification activity under the
condition where the lid is open as shown in FIG. 1(b).
[0020] The lipase is considered to close the lid thereof in water
solution so that it is in the condition where a substrate (i.e.,
water) cannot enter into the active site. However, in a water
solution mixed with fat, it is considered that the lipase gathering
on a boundary surface between water and fat opens the lid thereof
so as to accelerate the hydrolysis reaction. By the way, the above
consideration can be confirmed by the fact that hydrolysis is
increased rapidly by a fat concentration exceeding a level of
saturation of fat.
[0021] As mentioned previously, the reaction will not occur even if
the powder of lipase is dispersed into the organic solvent.
However, the lipase being modified with the fatty acid shows the
activity of the enzyme in the organic solvent.
[0022] This is inferred by the inventors, as shown in FIG. 2,
because the hydrophobic portion of the lipase enters into double
layers of fat being approximately 5 nm in the thickness thereof,
while the hydrophilic portion thereof forms a compound protruding
from the double layers of fat.
[0023] According to the present invention, upon the above
inference, it is considered that the enzyme should show the
activity thereof if it is kept with the lid open in the non-aqueous
or nano-aqueous system, where no boundary surface exists with a
water phase, thereby accomplishing the present invention.
[0024] Namely, according to the present invention, there is
provided a method for producing activated enzyme, comprising the
following steps:
[0025] adding enzyme to water and fat phases of a multi-phase
system;
[0026] activating the enzyme due to a function of a fat-and-water
boundary surface between the water and fat phases; and
[0027] removing the water and fat phases while maintaining an
activated condition of the enzyme.
[0028] As a concrete method for adding the enzyme into the
two-phase system of the water phase and the fat phase, there can be
considered a method of adding the fat phase to a water solution
into which the enzyme has been dissolved in advance.
[0029] And, as a means for removing the fat phase and the water
phase while keeping the activating condition of the enzyme,
freeze-drying (or lyophilization) is appropriate.
[0030] Further, a concrete example of the enzyme can be listed as
lipase, and as the fat phase can be listed a volatile one, such as
tetradecane.
[0031] Further, when activating, a pH of the water phase (buffer
solution) is preferably maintained in a vicinity of neutrality, and
addition amount of said tetradecane is to be from 1% to 10% of the
volume of the buffer solution.
[0032] By contacting the activated enzyme obtained from the method
according to the present invention described above with fat in
non-aqueous system or nano-aqueous system, reforming can be
conducted, such as transesterification reaction, ester composing
reaction, etc.
[0033] Further, in a method for inactivating an activated enzyme,
according to the present invention, the activated enzyme, which is
activated by means of the above method or the other method(s), is
dispersed and stirred into a buffer solution in which there exists
no boundary surface between fat and water.
BRIEF DESCRIPTION OF DRAWINGS
[0034] FIG. 1(a) is a block diagram of showing lipase being in a
not-active condition, while FIG. 1(b) shows it being in the active
condition;
[0035] FIG. 2 is a block diagram of lipase compound being modified
with fatty acid;
[0036] FIG. 3 is a graph showing a relationship between a
concentration of product through transesterification between
stearic acid and tripalmitin, with respect to time;
[0037] FIGS. 4(a) and (b) are views showing behavior of the lipase
in a two-phase system;
[0038] FIG. 5 is a graph showing a relationship between
interesterification activity of lipase and the carbon number of a
straight chain saturated hydrocarbon constituting a fat phase;
[0039] FIG. 6 is a graph showing a relationship between
interesterification activity of lipase and pH of buffer solution;
and
[0040] FIG. 7 is a graph showing a relationship between relative
hydrolysis activity of lipase and pH.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] Hereinafter, detailed explanation of the embodiments
according to the present invention will be given by referring to
the attached drawings. As used herein, the language "fat phase" is
used interchangeably with "organic phase" and "organic
solvents".
[0042] Lipase (20 mg, originated from Rhizopus japonicus) is
dissolved into buffer solution (5 ml), and into the obtained water
solution is added tetradecan C.sub.14H.sub.30 as fatty phase.
[0043] After stirring at 40.degree. C. for one (1) hour under the
condition of two-phases of the tetradecan C.sub.14H.sub.30 and the
buffer solution, the lipase is freeze-dried, whereby water and fat
are removed therefrom.
[0044] With use of such a lipase as obtained in this manner,
transesterification reaction is conducted between stearic acid and
tripalmitin in hexane.
[0045] In the graph of FIG. 3, there is shown a relationship
between elapsed time of the transesterification reaction between
stearic acid and tripalmitin and product formed thereby.
[0046] As is apparent from FIG. 3, it can be seen that the lipase
which has not shown catalytic activity in organic solvent until now
begins to show the catalytic activity remarkably.
[0047] On the other hand, if the powdered lipase without this
operation is used under the same condition, such a reaction will
not proceed at all. Namely, it is apparent that the lipase can show
the activity in the organic solvent, when it is freeze-dried from
the buffer solution under the existence of the tetradecan.
[0048] As the reason of showing the activity in the organic solvent
in this manner, it can be considered that, just after adding the
tetradecan into water phase into which the lipase is dissolved, as
shown in FIG. 4(a), although there exists the lipase in the water
phase, if there is a boundary surface between the water phase and
the fat phase, the lipase moves towards the boundary surface since
it has a tendency to gather around the boundary surface. The
lipase, having moved to the boundary surface, has a hydrophobic
portion at the side of the fat phase while having a hydrophilic
portion at the side of the water phase, as shown in FIG. 4(b), and
at the same time the lid covering over the activating portion is
opened by the function of the boundary surface. It is also
considered that this condition is maintained even if the lipase is
freeze-dried.
[0049] (Kinds of Fat Phase)
[0050] With use of various straight chain saturated hydrocarbons in
place of the tetradecan, the activation of the lipase is tried
under the same conditions as in the above. The result is shown in
FIG. 5. The straight chain saturated hydrocarbons include hexane,
octane, decane, dodecane, hexadecane, and octadecane other than the
tetradecan.
[0051] As is apparent from FIG. 5, the highest activity is shown in
a case where the tetradecan is used as the fat phase. This is
because much of the lipase has a substrate specificity, and the
substrate specificity is mainly determined by the lid of the
lipase. Therefore, there exists the fat phase being suitable for
opening the lid of the lipase, and it can be said that the
tetradecan is the fat phase being most suitable among those
hydrocarbons.
[0052] (Influences of pH)
[0053] The result of testing of the influence on the activation of
the lipase by the pH of the buffer solution when treating a contact
process on the boundary surface between fat and water is indicated
on the graph in FIG. 6. In this graph, the buffer solution shows
the highest activity in a case where the buffer solution is in the
vicinity of neutrality (pH 7).
[0054] On the other hand, the testing result of a relationship
between relative hydrolysis activity of native crude lipase and the
pH is indicated on the graph in FIG. 7.
[0055] The graphs shown in FIGS. 6 and 7 are similar to each other.
This seems to indicate that a dissociation condition of remaining
amino acid radicals at pH 7 is necessary for the lipase to show the
catalytic activity.
[0056] (Amount of Fat Phase)
[0057] The amount of tetradecan with respect to the buffer solution
(5 ml) is changed in a range from 1% (50 .mu.L) to 10% (500 .mu.L).
As the result, there can be obtained catalytic activity being same
as or similar to the case where the addition amount mentioned above
is 5% (250 .mu.L).
[0058] (Inactivation and Reactivation)
[0059] If the activation of the lipase by the contact processing on
the boundary surface between fat and water mentioned above is
caused by the fact that the lipase opens the lid, it is considered
that the lid can also be closed, and the following experimentation
was conducted.
[0060] First of all, treating the contact processing on the
boundary surface between fat and water by the tetradecane, the
activated lipase which has been freeze-dried is added to water so
as to be dispersed in it again (since there remains original salt
therein), and it is stirred at 4.degree. c. for 24 hours.
Thereafter, the activity of the dispersed lipase, which is obtained
by freeze-drying, in the organic solvent is investigated. As the
result, it is determines that the lipase has returned back to the
catalytic activity before the activation thereof.
[0061] Namely, it comes to be clear that the activated lipase can
be easily deactivated by treating it in the buffer solution without
the boundary surface between fat and water therein.
[0062] In this manner, if it can be deactivated temporarily and
with ease, the enzyme can be deactivated freely when it should not
participate the reaction during a series of synthetic reactions.
Further, by controlling the deactivation and the re-activation of
the enzyme, it is possible to proceed with the reactions in
multi-steps or stages in the same reactor.
[0063] In the present embodiment, the enzyme is explained as
lipase, however, for example, amylase and protease can also be
activated on the boundary surface between water phase and fat
phase.
[0064] As is explained in the above, according to the present
invention, it is possible to obtain the activated enzyme (i.e.,
lipase) with a simple operation.
[0065] Further, since it is possible to obtain the enzyme activated
individually without being reformed with other materials, no
undesirable reaction caused by the reforming materials will occur
nor will it be necessary to remove such reforming materials.
[0066] Although there have been described what are the present
embodiments of the invention, it will be understood that variations
and modifications may be made thereto without departing from the
gist, spirit, or essence of the invention. The scope of the
invention is indicated be the appended claims.
* * * * *