U.S. patent application number 15/288190 was filed with the patent office on 2017-04-13 for enzyme-immobilized porous membrane and preparation method of antibiotics using the same.
The applicant listed for this patent is GWANGJU INSTITUTE OF SCIENCE AND TECHNOLOGY. Invention is credited to Ji-woong Park, Sunoh Shin.
Application Number | 20170101660 15/288190 |
Document ID | / |
Family ID | 58498814 |
Filed Date | 2017-04-13 |
United States Patent
Application |
20170101660 |
Kind Code |
A1 |
Park; Ji-woong ; et
al. |
April 13, 2017 |
ENZYME-IMMOBILIZED POROUS MEMBRANE AND PREPARATION METHOD OF
ANTIBIOTICS USING THE SAME
Abstract
The present disclosure relates to an enzyme-immobilized porous
membrane and a preparation method of antibiotics using the same,
and more specifically, to an enzyme-immobilized porous membrane
prepared by immobilizing a specific enzyme through dead-end
filtration, and a preparation method of antibiotics with a high
yield using the enzyme-immobilized porous membrane. According to
various exemplary embodiments of the present disclosure, the enzyme
capable of promoting the synthesis reaction of the antibiotic
substance is able to be stably immobilized in the porous membrane
by passing the solution of enzyme through the membrane. In
addition, it is possible to provide antibiotics with a high yield
by preparing the antibiotics by passing the reactant solution
through the enzyme-immobilized porous membrane.
Inventors: |
Park; Ji-woong; (Gwangju,
KR) ; Shin; Sunoh; (Gwangju, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GWANGJU INSTITUTE OF SCIENCE AND TECHNOLOGY |
Gwangju |
|
KR |
|
|
Family ID: |
58498814 |
Appl. No.: |
15/288190 |
Filed: |
October 7, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62238145 |
Oct 7, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/84 20130101; C12Y
305/01011 20130101; C12N 11/08 20130101; C12P 37/04 20130101 |
International
Class: |
C12P 37/04 20060101
C12P037/04; A61K 31/545 20060101 A61K031/545; A61K 31/43 20060101
A61K031/43; C12N 9/84 20060101 C12N009/84; C12N 11/14 20060101
C12N011/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2016 |
KR |
10-2016-0068897 |
Claims
1. An enzyme-immobilized porous membrane in which an enzyme
promoting a synthesis reaction of an antibiotic substance is
immobilized, wherein the porous membrane is three-dimensionally
interconnected by pores, the porous membrane forms a
three-dimensional network by polymerizing a first monomer and a
second monomer each having two to four functional groups, the
functional group of the first monomer is an amino group, the
functional group of the second monomer is an isocyanate group, an
acyl halide group or an ester group, the first monomer and/or the
second monomer has four functional groups, and the enzyme is at
least one selected from the group consisting of penicillin G
acylase, penicillin V acylase, and cephalosporin C acylase.
2. The enzyme-immobilized porous membrane according to claim 1,
wherein the antibiotic substance is a penicillin-based substance,
or a cephalosporin-based substance.
3. A preparation method of antibiotics comprising: (B) permeating a
derivative solution of an antibiotic substance through the
enzyme-immobilized porous membrane according to claim 1.
4. The preparation method according to claim 3, wherein the
derivative of the antibiotic substance includes a first derivative
and a second derivative, the first derivative and the second
derivative are mixed at a molar (M) ratio of 1:1 to 3, the first
derivative and the second derivative are different from each other,
and are each independently at least one selected from the group
consisting of 6-aminopenicillanic acid, p-hydroxyphenylglycine
methyl ester, 7-aminodesacetoxycephalosporanic acid, and
phenylglycine.
5. The preparation method according to claim 4, wherein the first
derivative is the 6-aminopenicillanic acid, and the second
derivative is the p-hydroxyphenylglycine methyl ester.
6. The preparation method according to claim 3, further comprising:
(A) preparing the derivative solution of the antibiotic substance,
wherein step (A) includes: (a-1) preparing a first derivative
solution; (a-2) preparing a second derivative solution; and (a-3)
mixing the first derivative solution with the second derivative
solution.
7. The preparation method according to claim 3, wherein the
permeating of step (B) is performed by dead-end filtration.
8. The preparation method according to claim 3, wherein step (B) is
performed at a pressure of 2 to 7 bar in a nitrogen atmosphere.
9. The preparation method according to claim 3, wherein the
antibiotic substance is a penicillin-based substance, or a
cephalosporin-based substance.
10. A preparation method of antibiotics comprising: (B) permeating
a derivative solution of an antibiotic substance through the
enzyme-immobilized porous membrane according to claim 2.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/238,145, filed on Oct. 7, 2015, and Korean
Patent Application No. 10-2016-0068897, filed on Jun. 2, 2016 in
the KIPO (Korean Intellectual Property Office), both of which are
incorporated herein by reference in their entireties.
BACKGROUND
[0002] 1. Technical Field
[0003] The present disclosure relates to an enzyme-immobilized
porous membrane and a preparation method of antibiotics using the
same, and more specifically, to an enzyme-immobilized porous
membrane prepared by immobilizing a specific enzyme through
filtration, and a preparation method of antibiotics with a high
yield using the enzyme-immobilized porous membrane.
[0004] 2. Description of the Related Art
[0005] Enzymes are generally useful for various reactions due to
high steric and chemical selectivity, and have been used as a
catalyst to promote a reaction rate under a mild reaction
condition. However, in general, since the enzymes have a high cost,
it is economically difficult to be used in an industrially large
amount. In addition, most of the enzymes have a limitation in being
used for an organic chemical reaction since they are not dissolved
in organic solvents. Accordingly, a number of researches to
immobilize the enzymes have been conducted to enhance activity and
stability of the enzymes and reuse the enzymes.
[0006] There are three methods to immobilize the enzyme in a
polymer membrane. The first method is to adsorb the enzyme on a
surface of the polymer membrane, the second method is to attach the
enzyme to the polymer membrane via a covalent bond by modifying the
enzyme, and the third method is an entrapping method in which the
enzyme is physically entrapped in pores of the polymer
membrane.
[0007] Since the method of adsorbing the enzyme on the surface of
the membrane via a non-covalent bond is the easiest and simplest, a
number of researches into the adsorption method have been
conducted, but the adsorption method is disadvantageous in that the
enzyme may be easily leached away, and has relatively low
stability.
[0008] In order to solve these disadvantages, there is an attempt
to immobilize the enzyme in a porous membrane, and the
immobilization method is effective to improve an immobilization
rate and an immobilization maintenance rate of the enzyme and is
effective in view of economical aspect since the immobilization
process is relatively simple.
[0009] Penicillin-based antibiotics are .beta.-lactam-based
antibiotics produced by blue mold (Penicillium notatum), etc., and
may be synthesized from Penicillium notatum and Penicillium
chrysogenum that are called the blue mold.
[0010] In the related art, the antibiotics are synthesized by
treating a derivative in a solution state through step-by-step
reactions. However, this preparation method causes side reactions
such as hydrolysis reaction and thus the final antibiotic substance
is produced in a remarkably low yield.
[0011] Therefore, according to the present disclosure, the
enzyme-immobilized porous membrane is used to stably immobilize the
enzyme capable of promoting reactivity of the antibiotic substance
in the porous membrane, thereby improving the reactivity, such that
the antibiotic that is a final substance may have an improved
yield.
SUMMARY
[0012] It is an aspect of the present disclosure to provide a
porous membrane in which an enzyme capable of promoting a synthesis
reaction of an antibiotic substance is stably immobilized by using
dead-end filtration.
[0013] In addition, it is another aspect of the present disclosure
to provide a preparation method of antibiotics with a high yield by
using the enzyme-immobilized porous membrane.
[0014] The present disclosure is not limited to the above aspect
and other aspects of the present disclosure will be clearly
understood by those skilled in the art from the following
description.
[0015] In accordance with one aspect of the present disclosure,
there is provided an enzyme-immobilized porous membrane in which an
enzyme promoting a synthesis reaction of an antibiotic substance is
immobilized, wherein the porous membrane is three-dimensionally
interconnected by pores, the porous membrane forms a three
dimensional network by polymerizing a first monomer and a second
monomer each having two to four functional groups, the functional
group of the first monomer is an amino group, the functional group
of the second monomer is an isocyanate group, an acyl halide group
or an ester group, the first monomer and/or the second monomer has
four functional groups, and the enzyme is at least one selected
from the group consisting of penicillin G acylase, penicillin V
acylase, and cephalosporin C acylase.
[0016] In accordance with another aspect of the present disclosure,
a preparation method of antibiotics includes: (B) permeating a
derivative solution of an antibiotic substance through the
enzyme-immobilized porous membrane as described above.
[0017] According to various exemplary embodiments of the present
disclosure, the enzyme capable of promoting the synthesis reaction
of the antibiotic substance is able to be stably immobilized in the
porous membrane by using the dead-end filtration.
[0018] Further, it is possible to provide the antibiotics with a
high yield by preparing the antibiotics through the dead-end
filtration using the enzyme-immobilized porous membrane.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 illustrates a dead-end cell filtration system for
immobilizing an enzyme according to an exemplary embodiment of the
present disclosure.
[0020] FIG. 2 is a diagram illustrating a preparation method in
which the enzyme is immobilized in the porous membrane according to
the exemplary embodiment of the present disclosure.
[0021] FIG. 3 is a graph illustrating results obtained by adsorbing
nitrogen onto or desorbing nitrogen from a PGA-immobilized porous
membrane according to Example 1 to measure an adsorption degree or
a desorption degree.
[0022] FIG. 4 is a graph illustrating results obtained by measuring
a size of pores formed on the PGA-immobilized porous membrane
according to Example 1.
[0023] FIG. 5A is an image illustrating a cross section of the
PGA-immobilized porous membrane according to Example 1, and FIG. 5B
is an image illustrating results obtained by elemental mapping on
the PGA-immobilized porous membrane according to Example 1 using an
energy-dispersive X-ray spectrometer.
[0024] FIG. 6 is a graph illustrating results obtained by measuring
a yield of the antibiotic prepared by Example 2.
DETAILED DESCRIPTION
[0025] Hereinafter, various aspects and exemplary embodiments of
the present disclosure will be described in detail.
[0026] According to one aspect of the present disclosure, there is
provided an enzyme-immobilized porous membrane in which an enzyme
promoting a synthesis reaction of an antibiotic substance is
immobilized, wherein the porous membrane is three-dimensionally
interconnected by pores, the porous membrane forms a
three-dimensional network by polymerizing a first monomer and a
second monomer each having two to four functional groups, the
functional group of the first monomer is an amino group, the
functional group of the second monomer is an isocyanate group, an
acyl halide group or an ester group, at least one of the first
monomer or the second monomer has four functional groups, and the
enzyme is at least one selected from the group consisting of
penicillin G acylase, penicillin V acylase, and cephalosporin C
acylase.
[0027] The enzymes are characterized by promoting a synthesis
reaction of an antibiotic substance, and among them, the penicillin
G acylase is an enzyme capable of promoting reactions of Reaction
Schemes 1 and 2 below:
##STR00001##
[0028] Firstly, as shown in Reaction Scheme 1 above, carboxylic
acid and 6-aminopenicillanic acid (6-APA) may be produced by
promoting a hydrolysis reaction of an amide group.
[0029] Secondly, as shown in Reaction Scheme 2 below, a reaction of
synthesizing penicillin-based amoxicillin which is a
beta-lactam-based antibiotic may be promoted, and specifically, the
penicillin may be synthesized by promoting a reaction of an ester
group of p-hydroxyphenylglycine methyl ester (PHPGME) and an amine
group of 6-APA to form an amide group:
##STR00002##
[0030] The penicillin G acylase enzyme may act as a catalyst
effective for promoting reactivity in synthesizing the
penicillin-based antibiotic, but has a problem in that stability
thereof is degraded due to the hydrolysis as shown in Reaction
Scheme 1 above.
[0031] Accordingly, in the present disclosure, the enzyme is
immobilized in the porous membrane to prevent decomposition or
leaching of the enzyme, such that stability of the enzyme may be
secured.
[0032] In addition, antibiotics with a high yield may be prepared
only by a relatively simple process in which a derivative of the
antibiotic substance is permeated through the porous membrane in
which the enzyme having secured stability is immobilized.
Specifically, the enzyme-immobilized porous membrane described
above in the present disclosure includes the porous membrane
forming a three-dimensional network and an enzyme that is trapped
in the pores of the porous membrane. The porous membrane may
include the pores having a size of 5 to 100 nm while forming a
three-dimensionally cross-linked monolith, and the pores may be
connected to each other, such that the enzyme immobilized in the
porous membrane is able to be in contact with reaction substrates
in all directions, and a solution may be easily spread, which
prevent a problem that substance transport is degraded due to the
enzyme blocking the pores.
[0033] In addition, in order for the enzyme to be immobilized in
the porous membrane, it is generally known that the size of the
pore is 20 to 50 nm (Membrane-Based Synthesis of Nanomaterials,
Charles R. Martin), and the porous membrane of the present
disclosure is characterized by immobilizing various sizes of
enzymes since it has various ranges of nano pores having a size of
5 to 100 nm as well as pores having a size in micro range.
[0034] The porous membrane of the present disclosure may be
obtained by mixing an organic sol with a polymer solution to obtain
a micro porous membrane, the organic sol consisting of an organic
network structure in which the first monomer having an amino group
is polymerized with the second monomer having an isocyanate group,
an acyl halide group or an ester group that is the functional group
polymerizable with the amino group, and removing the polymer from
the micro porous membrane by using water.
[0035] According to an exemplary embodiment of the present
disclosure, the first monomer may have two to four amino groups,
and the second monomer may have two to four functional groups
selected from the group consisting of the isocyanate group, the
acyl halide group, and the ester group. The first monomer having
two to four amino groups may be C.sub.1-C.sub.100 aliphatic
compound substituted with two to four amino groups or C6-C.sub.100
aromatic compound substituted with two to four amino groups.
[0036] The second monomer having two to four isocyanate groups, the
two to four acyl halide groups, or the two to four ester groups may
be C.sub.1-C.sub.100 aliphatic compound substituted with two to
four isocyanate groups, the two to four acyl halide groups, or the
two to four ester groups or C6-C.sub.100 aromatic compound
substituted with two to four isocyanate groups, the two to four
acyl halide groups, or the two to four ester groups.
[0037] As an example, the first monomer and the second monomer may
be compounds represented by Chemical Formulas 1 to 9 below:
##STR00003##
[0038] in Chemical Formulas 1 to 9 above, R is an amino group, an
isocyanate group, an acyl halide group or an ester group.
[0039] In addition, according to an exemplary embodiment of the
present disclosure, the first monomer and the second monomer may be
a compound represented by Chemical
[0040] Formula 10 below:
##STR00004##
[0041] in Chemical Formula 10 above, R is an amino group, an
isocyanate group, an acyl halide group or an ester group, and n is
0 or 1.
[0042] The first monomer and the second monomer may be polymerized
by a nucleophilic addition or substitution reaction between the
amino group of the first monomer and the isocyanate group, the acyl
halide group or the ester group of the second monomer, and polymers
to be produced may cause additional nucleophilic addition or
substitution reaction by non reacted negative (-), positive (+)
functional groups to generate a crosslinking reaction between the
polymers. As a result, the monomer having the four functional
groups may form a kind of crosslinking point as a tetrahedral
structure, and may form the three-dimensional organic network
structure linked by a strong covalent bond on the basis of the
crosslinking point.
[0043] Specifically, the organic network structure formed by the
polymerization reaction between the first monomer and the second
monomer may be three-dimensionally polymerized and cross-linked to
have a number of fine pores and a large specific surface area, and
to have excellent chemical resistance, heat resistance, and
durability by a high crosslinking rate and the strong covalent
bond.
[0044] In addition, the monomer having two to four amino groups may
be, for example, tetrakis(4-aminophenyl)methane (TAPM), p-phenylene
diamine (PDA), or 4,4'-oxydianiline (ODA), but these examples
thereof are not limited thereto.
[0045] Further, the monomer having two to four isocyanate groups
may be, for example, p-phenylene diisocyanate (PDI), hexamethylene
diisocyanate (HDI), or tetrakis(4-isocyanatophenyl) methane (TIPM),
but these examples thereof are not limited thereto.
[0046] According to an exemplary embodiment of the present
disclosure, the porous membrane may be formed by polymerizing a
monomer represented by Chemical Formula 11 below and the monomer
having two isocyanate groups:
##STR00005##
[0047] in Chemical Formula 11 above, X is a carbon atom or a
silicon atom.
[0048] Further, according to another exemplary embodiment of the
present disclosure, the porous membrane may be formed by
polymerizing the monomer having two amino groups and a monomer
represented by Chemical Formula 12 below:
##STR00006##
[0049] in Chemical Formula 12 above, X is a carbon atom or a
silicon atom.
[0050] The porous membrane may have a flat sheet structure or a
hollow fiber membrane structure.
[0051] In addition, the porous membrane may have a single layered
structure or a plurality of layered structure.
[0052] According to another aspect of the present disclosure, there
is provided a preparation method of antibiotics including: (B)
permeating a derivative solution of an antibiotic substance through
the enzyme-immobilized porous membrane.
[0053] The preparation method preferably further includes step (A)
of preparing the derivative solution of the antibiotic
substance.
[0054] According to the related art, the derivative of the
antibiotic substance is prepared in a solution state, and is
treated with step-by-step reactions, thereby preparing the
antibiotics. The preparation method of the antibiotics through the
step-by-step reactions has problems in that the finally produced
antibiotic substance has a remarkably low yield, and a large amount
of impurities are caused due to the hydrolysis reaction or the
addition reaction of the enzyme.
[0055] Accordingly, according to the present disclosure, stability
of the enzyme is firstly secured by immobilizing the enzyme in the
porous membrane, and then, the antibiotics with a high yield are
prepared only by a simple process of permeating the derivative of
the antibiotic substance through the enzyme-immobilized porous
membrane.
[0056] Specifically, step (A) is a step of preparing the derivative
solution of the antibiotic substance capable of preparing the
antibiotic substance.
[0057] The derivative of the antibiotic substance preferably
includes the first derivative and the second derivative, and the
first derivative and the second derivative may be different from
each other, and the first derivative or the second derivative may
be at least one selected from the group consisting of
6-aminopenicillanic acid (6-APA), p-hydroxyphenylglycine methyl
ester (PHPGME), 7-aminodesacetoxycephalosporanic acid, and
phenylglycine.
[0058] The step (A) may include (a-1) preparing a first derivative
solution; (a-2) preparing a second derivative solution; and (a-3)
mixing the first derivative solution with the second derivative
solution.
[0059] The step (a-1) is a step of preparing the first derivative
solution, wherein the first derivative solution preferably has a
concentration of 1 to 20 mM by adding a solvent to the first
derivative. When the concentration of the first derivative solution
is less than 1 mM, it is not preferred since the concentration is
too thin, reactivity with the enzyme may be degraded, and when the
concentration thereof is more than 20 mM, an amount of the reaction
substance is increased as compared to an amount of the enzyme.
[0060] The step (a-2) is a step of preparing the second derivative
solution, wherein the second derivative solution preferably has a
concentration of 1 to 20 mM by adding a solvent to the second
derivative, which is similar to the step (a-1).
[0061] The solvent is preferably distilled water, but the present
disclosure is not limited thereto.
[0062] The step (a-3) is a step of mixing the first derivative
solution with the second derivative solution, wherein the first
derivative and the second derivative are preferably mixed at a
molar (M) ratio of 1:1 to 3. When the molar ratio is out of the
above-described range, it is not preferred since reactivity may be
degraded.
[0063] In particular, it is confirmed that when the first
derivative is 6-aminopenicillanic acid, the second derivative is
p-hydroxyphenylglycine methyl ester, and the 6-amino-penicillanic
acid is mixed with the p-hydroxyphenylglycine methyl ester at a
molar (M) ratio of 1:2, a penicillin-based antibiotic to be
synthesized has the most effective yield as about 70%, and when the
molar ratio is out of the above-described range, the yield is
rapidly reduced.
[0064] The step (B) is a step of permeating the derivative solution
of the antibiotic substance through the enzyme-immobilized porous
membrane.
[0065] Here, the derivative solution of the antibiotic substance to
be added preferably has a content of 0.8 to 10 parts by weight
relative to 100 parts by weight of the porous membrane.
[0066] The permeating step is preferably performed by applying a
pressure of 1 to 10 bar in a nitrogen atmosphere through dead-end
filtration, cross flow filtration, or a complex manner thereof.
[0067] In particular, it is confirmed that when 1 to 5 parts by
weight of the derivative solution of the antibiotic substance is
permeated through the dead-end filtration at a pressure of 5 bar in
a nitrogen atmosphere, the amount of impurities contained in the
antibiotic which is the final product is rapidly reduced.
[0068] Hereinafter, the present disclosure will be described in
detail with reference to the following Examples, etc. Therefore, it
should be understood that the foregoing embodiments are provided
for illustrative purposes only and are not to be construed in any
way as limiting the present disclosure. In addition, as long as a
person skilled in the art practices the present disclosure based on
the disclosed description of the present disclosure including the
following examples, it is obvious that the present disclosure may
be easily practiced by the person skilled in the art even though
testing results are not specifically provided, and it is natural
that various modifications and changes are included in the
accompanying claims.
[0069] In addition, experimental results below are only
representative experimental results of Examples and Comparative
Examples of the present disclosure, and respective effects of
various embodiments of the present disclosure that are not
presented explicitly below are described in detail in corresponding
sections.
Preparation Example
Preparation of Porous Membrane
[0070] (1) Preparation of (TAPM+HDI/PEG) nano composite film
[0071] Tetrakis(4-aminophenyl) methane (TAPM) (MW: 382.50) was
dissolved in DMF(N,N-dimethylformide) to prepare an organic
solution having a concentration of 4 wt/vol %, and
1,4-hexamethylene diisocyanate (HDI) (MW: 168.19) was dissolved in
DMF to prepare an organic solution having a concentration of 4
wt/vol %. Next, the tetrakis(4-aminophenyl) methane solution was
slowly added to the 1,4-hexamethylene diisocyanate solution, and
mixed with each other. The mixed solution was reacted at room
temperature in a nitrogen atmosphere for 72 hours, to obtain a
mixed solution in a sol-phase.
[0072] Poly ethylene glycol (PEG) having a concentration of 60 wt %
was added to the mixed solvent, followed by sufficient stirring.
The obtained mixture was applied to a glass plate at 50.degree. C.
for 1 hour, at 80.degree. C. for 2 hours, and at 100.degree. C. for
3 hours, followed by drying and curing, to finally synthesize a
nano composite film of an organic molecular network (TAPM+HDI) and
PEG.
[0073] (2) Preparation of TAPM+HDI Porous Membrane--Removal of
PEG
[0074] The synthesized membrane was cooled at room temperature, and
precipitated in water to be separated from a substrate. The
membrane was stirred in water for about one week to remove the
water-soluble polymer, polyethylene glycol (PEG), thereby preparing
a porous membrane having nano pores.
Example 1
Preparation of PGA-Immobilized Porous Membrane
[0075] The porous membrane of Preparation Example for immobilizing
the enzyme was put into a lower portion 20 of a dead-end cell
filtration system as illustrated in FIG. 1. Then, a penicillin G
acylase (PGA) solution having a concentration of 0.4 w/v% was put
into an upper portion (inlet, 10) of the dead-end cell filtration
system, and stirred. Next, a pressure of 5 bar was applied in a
nitrogen atmosphere to prepare a porous membrane in which the
penicillin G acylase (PGA) is immobilized.
Example 2
Preparation of Antibiotic
[0076] First, the PGA-immobilized porous membrane prepared by
Example 1 above was put into the lower portion 20 of the dead-end
cell filtration system. Then, 10 mM p-hydroxyphenylglycine methyl
ester (PHPGME) solution and 10 mM 6-aminopenicillanic acid (6-APA)
solution were prepared, respectively, by using distilled water as a
solvent, and two of the prepared solutions were mixed and put into
the upper portion (inlet, 10) of the dead-end cell filtration
system. Next, a pressure of 5 bar was applied in a nitrogen
atmosphere to permeate the mixed solution of PHPGME and 6-APA
through the PGA-immobilized porous membrane, thereby preparing an
antibiotic.
[0077] (Provided that, the dead-end cell filtration system was used
as the same as that of Example 1 above.)
Test Example 1
Analysis of Pores of Porous Membrane
[0078] In order to confirm a pore size of the PGA-immobilized
porous membrane according to Example 1, nitrogen was added and
adsorbed onto or desorbed from the porous membrane. Results thereof
were illustrated in FIG. 3.
[0079] Referring to FIG. 3, it could be appreciated that an
adsorption and desorption curve of Type 4 isotherm was obtained,
and the pores were well-formed in the porous membrane. The size of
the thus formed pores was measured, and illustrated in FIG. 4.
Referring to FIG. 4, it could be confirmed that the pore size of
the porous membrane had an average diameter of 5 to 30 nm
Test Example 2
Analysis to Whether Enzyme is Immobilized in Porous Membrane
[0080] In order to confirm whether the enzyme was well immobilized
in the PGA-immobilized porous membrane of Example 1, elemental
mapping was performed by using an energy-dispersive X-ray
spectrometer (JOEL JSM-6700 manufactured by Scanning Electron
Microscope), and results thereof were illustrated in FIG. 5A and
FIG. 5B.
[0081] FIG. 5A is an image illustrating a cross section of the
porous membrane according to Example 1.
[0082] The porous membrane is composed of carbon, nitrogen, and
oxygen, and the enzyme is composed of carbon, nitrogen, oxygen and
sulfur, and thus, whether the enzyme is immobilized could be
confirmed by analyzing whether the sulfur element is present.
[0083] Accordingly, as a result obtained by performing the
elemental mapping with regard to the sulfur element, it could be
appreciated that the sulfur elements represented by white dots were
uniformly distributed as illustrated in FIG. 5B. Specifically, it
could be confirmed that the enzyme was uniformly distributed in the
inside of the cross section of the porous membrane.
Test Example 3
Quantitative Analysis of Antibiotic
[0084] In order to quantitatively analyze the antibiotic prepared
by Example 2, the antibiotic substance was separated by using a
Phenomenex Gemini C18 column (150.times.4.6 mm, a particle size of
5 .mu.m) through a high performance liquid chromatography (HPLC),
and was subjected to quantitative analysis by measuring an
adsorption amount of the antibiotic with an UV detector at 225 nm
The conversion amount was calculated by the following Calculation
Formula 1, and results thereof were illustrated in FIG. 6.
Conversion (%)=(experimental amoxicillin amount/theoretical
amoxicillin amount).times.100 [Calculation Formula 11]
[0085] Referring to FIG. 6, it could be confirmed that about 70%
constant conversion was obtained. The result value was
significantly improved as compared to a yield obtained by the
existing preparation method of antibiotics, and reactivity was
improved due to the reaction in which the enzyme immobilized in the
porous membrane was promoted, which indicated that the antibiotics
with a high yield could be prepared.
[0086] Therefore, according to various exemplary embodiments of the
present disclosure, the enzyme capable of promoting the synthesis
reaction of the antibiotic substance is able to be stably
immobilized in the porous membrane by using the dead-end
filtration.
[0087] Further, it is possible to provide the antibiotics with a
high yield by preparing the antibiotics through the dead-end
filtration using the enzyme-immobilized porous membrane.
[0088] Although some embodiments have been disclosed herein, it
should be understood by those skilled in the art that these
embodiments are provided by way of illustration only, and that
various modifications, changes, and alterations can be made without
departing from the spirit and scope of the invention. Therefore, it
should be understood that the foregoing embodiments are provided
for illustrative purposes only and are not to be construed in any
way as limiting the present disclosure.
* * * * *