U.S. patent application number 14/400473 was filed with the patent office on 2015-05-21 for immobilized cycloaliphatic peptide acyltransferase and preparation method and uses thereof.
The applicant listed for this patent is SHANGHAI TECHWELL BIOPHARMACEUTICAL CO., LTD. Invention is credited to Xiaoming Ji, Shidong Liu, Zhaoli Zhang.
Application Number | 20150140604 14/400473 |
Document ID | / |
Family ID | 49532397 |
Filed Date | 2015-05-21 |
United States Patent
Application |
20150140604 |
Kind Code |
A1 |
Liu; Shidong ; et
al. |
May 21, 2015 |
IMMOBILIZED CYCLOALIPHATIC PEPTIDE ACYLTRANSFERASE AND PREPARATION
METHOD AND USES THEREOF
Abstract
Disclosed in the present invention are an immobilized
cycloaliphatic peptide acyltransferase and a preparation method and
use thereof. The cycloaliphatic peptide acyltransferase is
immobilized on a carrier; the cycloaliphatic peptide
acyltransferase is derived from natural or artificial mutants or
variants thereof, or can be obtained by introducing a foreign
cyclic acyltransferase gene and transforming thereafter; the
material of the carrier is selected from an inorganic carrier or a
polypropylene resin carrier. Also disclosed in the present
invention are the preparation method for the immobilized
cycloaliphatic peptide acyltransferase and uses thereof.
Inventors: |
Liu; Shidong; (Shanghai,
CN) ; Zhang; Zhaoli; (Shanghai, CN) ; Ji;
Xiaoming; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHANGHAI TECHWELL BIOPHARMACEUTICAL CO., LTD |
Shanghai |
|
CN |
|
|
Family ID: |
49532397 |
Appl. No.: |
14/400473 |
Filed: |
May 10, 2013 |
PCT Filed: |
May 10, 2013 |
PCT NO: |
PCT/CN2013/075508 |
371 Date: |
November 11, 2014 |
Current U.S.
Class: |
435/68.1 ;
435/176; 435/180 |
Current CPC
Class: |
C12N 9/86 20130101; C12P
21/02 20130101; C07K 7/56 20130101; C12N 11/14 20130101; C12N 11/08
20130101; C12N 9/80 20130101 |
Class at
Publication: |
435/68.1 ;
435/176; 435/180 |
International
Class: |
C12N 11/14 20060101
C12N011/14; C12N 9/86 20060101 C12N009/86; C12P 21/02 20060101
C12P021/02; C12N 11/08 20060101 C12N011/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2012 |
CN |
201210148392.7 |
Claims
1. An immobilized cycloaliphatic peptide acyltransferase.
2. The immobilized cycloaliphatic peptide acyltransferase according
to claim 1, wherein cycloaliphatic peptide acyltransferase is
immobilized on a carrier; and the carrier is selected from
inorganic carrier, or porous, hydrophilic enzyme carrier.
3. The immobilized cycloaliphatic peptide acyltransferase according
to claim 2, wherein, based on the total weight of the inorganic
carrier, the content of SiO.sub.2 is more than 50 wt %, and the
content of Al.sub.2O.sub.3 is more than 1 wt %.
4. The immobilized cycloaliphatic peptide acyltransferase according
to claim 2, wherein the porous hydrophilic enzyme carrier is
selected from the following: a enzyme-carrier in which
polymethacrylate is used as matrix with bonded epoxide or
containing amino functional groups.
5. The immobilized cycloaliphatic peptide acyltransferase according
to claim 1, wherein the immobilized cycloaliphatic peptide
acyltransferase is used to catalyze deacylation of acyl of R' in
the compound of formula I, thereby obtaining the compound of
formula II, ##STR00012## wherein R.sup.1 is an acyl, R.sup.2 is a
hydroxy or acyloxy, R.sup.3 is a hydrogen or hydroxy, R.sup.4 is a
hydrogen or hydroxy, R.sup.5 is a hydrogen or a hydroxy
sulfonyloxy, and R.sup.6 is a hydrogen or carbamoyl.
6. The immobilized cycloaliphatic peptide acyltransferase according
to claim 1, wherein the compound of formula I is the compound of
formula Ia or Ib or a pharmaceutically acceptable salt thereof:
##STR00013##
7. A method for preparing the immobilized cycloaliphatic peptide
acyltransferase according to claim 1, comprising the following
steps: mixing a solution containing free cycloaliphatic peptide
acyltransferase with a carrier to obtain the immobilized
cycloaliphatic peptide acyltransferase according to claim 1.
8. The method according to claim 7, wherein said cycloaliphatic
peptide acyltransferase is derived from natural or artificial
mutants, or variants thereof and can be obtained through
transformation by introducing foreign genes of cycloaliphatic
peptide acyltransferase.
9. The method according to claim 7, wherein the solution containing
free cycloaliphatic peptide acyltransferase is obtained from the
following manner: culturing a strain, so as to obtain the solution
containing free cycloaliphatic peptide acyltransferase; or
culturing the strain, and then breaking the cell wall of obtained
hyphae, so as to obtain the solution containing free cycloaliphatic
peptide acyltransferase.
10. The method according to claim 9, wherein the strain belongs to
Actinoplanes or Streptomyces.
11. The method according to claim 7, wherein the carrier is
selected from inorganic carriers, or porous, hydrophilic enzyme
carriers.
12. The method according to claim 7, wherein the mixing ratio of
the free cycloaliphatic peptide acyltransferase to the carrier is
10-1000 unites (u), preferably 20-600 units (u) of enzyme vs one
gram of carrier.
13. The method according to claim 7, wherein the pH value of the
solution containing free cycloaliphatic peptide acyltransferase is
4-9.
14. The method according to claim 7, wherein the temperature for
mixing the solution containing free cycloaliphatic peptide
acyltransferase with the carrier is 0-80.degree. C.; preferably,
20-35.degree. C.
15. The method according to claim 7, wherein after mixing the
solution containing free cycloaliphatic peptide acyltransferase
with the carrier, the free cycloaliphatic peptide acyltransferase
is separated from the cycloaliphatic peptide acyltransferase
binding to the carrier, so as to obtain the immobilized
cycloaliphatic peptide acyltransferase.
16. A method for preparing the compound of formula II, comprising
the following steps: (a) mixing the compound of formula I with a
buffer solution, so as to obtain solution 1; (b) mixing solution 1
with the immobilized cycloaliphatic peptide acyltransferase
according to claim 1 for conducting deacylation reaction, so as to
give the compound of formula II; ##STR00014## wherein R.sup.1 is an
acyl, R.sup.2 is a hydroxy or acyloxy, R.sup.3 is a hydrogen or
hydroxy, R.sup.4 is a hydrogen or hydroxy, R.sup.5 is a hydrogen or
a hydroxy sulfonyloxy, and R.sup.6 is a hydrogen or carbamoyl.
17. The method according to claim 16, wherein the ratio of the
immobilized cycloaliphatic peptide acyltransferase to the compound
of formula I is 0.01-10 u/g.
18. The method according to claim 16, wherein the pH value of the
buffer in step (a) is 4-9.
19. The method according to claim 16, wherein the buffer in step
(a) is selected from one or more of the group consisting of sodium
citrate buffer, potassium dihydrogen phosphate-disodium hydrogen
phosphate buffer and Tris-HCl buffer.
20. The method according to claim 16, wherein the temperature for
deacylation reaction in step (b) is 20-70.degree. C.
21. The method according to claim 16, wherein, in step (b), upon
deacylation, the immobilized cycloaliphatic peptide acyltransferase
is separated from the product-containing reaction solution to give
the compound of formula II.
22. The method according to claim 21, wherein the method for
separating the immobilized cycloaliphatic peptide acyltransferase
from the product-containing reaction solution includes filtration
or centrifugation.
Description
TECHNICAL FIELD
[0001] The present invention relates to enzyme immobilization,
particularly to an immobilized cycloaliphatic peptide
acyltransferase and preparation method and uses thereof.
BACKGROUND
[0002] Echinocandins, as a new class of antifungal agents, exhibit
good effects in the treatment of infections caused by Candida or
Aspergillus. Caspofungin, Micafungin and Anidulafungin are the
representatives of such medicaments. Echinocandins inhibit fungus
by suppressing the formation of 1,3-.beta. glycosidic bond, so as
to reduce the harm to human body, and reduce the side effects while
remaining high efficiency. Therefore, they are safer in use than
traditional antifungal agents.
[0003] FK463 (Micafungin) is the compound of formula [Ie], which is
obtained by cutting the side-chain of FR901379 as precursor
(compound of Formula Ia), thus forming FR179642 (compound of
Formula [Ic]), and then by synthesis method.
[0004] Anidulafungin is the compound of formula [If], which is
obtained by cutting the side-chain of echinocandin B as precursor
(compound of Formula Ib), thus forming compound of Formula [Id],
and then by synthesis method.
[0005] It has been reported that enzymes deacetylating the
side-chain of cycloaliphatic peptides, such as FR901379,
echinocandin B, Ciclosporin A and the like, belong to the enzymes
produced by Actinoplanes utahensis IFO-13244, A. utahensis
NRRL-12052. In WO97/32975, enzymes produced by bacteria belonging
to Streptomyces (Streptomyces anulatus 4811 strain, Streptomyces
anulatus 8703 strain, Streptomyces sp. 6907 strain) have been
reported. Additionally, in WO97/47738, enzymes produced by
Oidiodendron tenuissimum IFO 6797 strain, Oidiodendron echinulatum
IFO 31963 strain, Oidiodendron truncatum IFO 9951 strain,
Oidiodendron truncatum IFO 31812 strain, Oidiodendron sp. 30084
strain, Verticillium sp. 30085 strain have been reported.
[0006] The acyltransferase can be found in the cultured mycelium
and filtrate. Generally, the bioactive substance can be recovered
by the following process: the cultured broth is filtrated or
centrifuged, so as to obtain mycelium and filtrate, and the
obtained mycelium and filtrate are separated and purified by
conventional methods, such as high concentration salt solution
extraction, conventional solvent extraction, concentration under
reduced pressure, lyophilization, pH adjustment, anion exchange
resin or cation exchange resin, crystallization, recrystallization
and the like, thereby obtaining the acyltransferase. In CN
1161462C, a deacylation method using the enzyme has been disclosed,
wherein culture was used to transform cycloaliphatic peptides, such
as FR901379, echinocandin B and the like. However, the drawback of
the method is that there is organic solvent present in the
transformation system, it is difficult to post-treat the product,
conversion speed is low, acyltransferase can not be reused, and the
purity of conversion product is low, thus increasing the difficulty
to purify the deacylated product.
[0007] Therefore, people try to look for a method for fixing the
enzyme, so as to recover, and purify the acyltransferase, and
improve the utilization ratio of the enzyme.
SUMMARY OF THE INVENTION
[0008] One object of the present invention is to provide an
immobilized cycloaliphatic peptide acyltransferase.
[0009] In the present invention, a preparation method for the
immobilized cycloaliphatic peptide acyltransferase is also
provided.
[0010] A further object of the present invention is to provide use
of such immobilized cycloaliphatic peptide acyltransferase.
[0011] In the first aspect of the present invention, an immobilized
cycloaliphatic peptide acyltransferase is provided. The
cycloaliphatic peptide acyltransferase is immobilized on a carrier;
said cycloaliphatic peptide acyltransferase is derived from natural
or artificial mutants, or variants thereof and can be obtained
through transformation by introducing foreign genes of
cycloaliphatic peptide acyltransferase; and the carrier is selected
from inorganic carrier, or porous, hydrophilic enzyme carrier.
[0012] The immobilized cycloaliphatic peptide acyltransferase is
used to catalyze deacylation of acyl of R' in the compound of
formula I, thereby obtaining the compound of formula II,
##STR00001##
[0013] Wherein R.sup.1 is an acyl, R.sup.2 is a hydroxy or acyloxy,
R.sup.3 is a hydrogen or hydroxy, R.sup.4 is a hydrogen or hydroxy,
R.sup.5 is a hydrogen or a hydroxy sulfonyloxy, and R.sup.6 is a
hydrogen or carbamoyl.
[0014] In a preferred embodiment, the compound or a
pharmaceutically acceptable salt thereof which can be deacylated
through catalysis is represented by formula Ia or Ib:
##STR00002##
[0015] In a preferred embodiment, the carrier is selected from the
following inorganic carrier: an inorganic carrier, wherein, based
on the total weight of the inorganic carrier, the content of
SiO.sub.2 is more than 50 wt %, and the content of Al.sub.2O.sub.3
is more than 1 wt %; preferably, the inorganic carrier selected
from: catalyst carrier CELITE, Expanded perlite, diatomaceous
earth, kaolin, or porous glass.
[0016] In another preferred embodiment, the carrier is selected
from the following porous hydrophilic enzyme-carrier: a porous
hydrophilic enzyme-carrier in which polymethacrylate is used as
matrix with bonded epoxide or containing amino functional groups;
preferably selected from: Relizyme EP403, SEPABEADS EC-EP, Relizyme
HA403 or SEPABEADS EC-HA.
[0017] In a preferred embodiment, in the immobilized cycloaliphatic
peptide acyltransferase, 10-1000 unites (u), preferably 20-600
units (u) of cycloaliphatic peptide acyltransferase is immobilized
on one gram of the carrier.
[0018] In another preferred embodiment, the immobilized
cycloaliphatic peptide acyltransferase is immobilized on an
inorganic carrier, and 10-100 unites (u), preferably 20-80 units
(u) of cycloaliphatic peptide acyltransferase is immobilized on one
gram of the carrier.
[0019] In another preferred embodiment, the immobilized
cycloaliphatic peptide acyltransferase is immobilized on a porous
hydrophilic enzyme-carrier, for example, an enzyme-carrier in which
polymethacrylate is used as matrix with bonded epoxide or
containing amino functional groups, and 100-1000 unites (u),
preferably 120-600 units (u) of cycloaliphatic peptide
acyltransferase is immobilized on one gram of the carrier.
[0020] In the second aspect of the present invention, a method for
preparing the immobilized cycloaliphatic peptide acyltransferase of
the present invention is provided, comprising the following
steps:
[0021] mixing a solution containing free cycloaliphatic peptide
acyltransferase with a carrier for adsorption and immobilization to
obtain the immobilized cycloaliphatic peptide acyltransferase
provided by the present invention as said above.
[0022] In the above preparation method, said cycloaliphatic peptide
acyltransferase is derived from natural or artificial mutants, or
variants and can be obtained through transformation by introducing
foreign genes of cycloaliphatic peptide acyltransferase.
[0023] In the above preparation method, the strain is from
Actinoplanes or Streptomyces.
[0024] In the above preparation method, the solution containing
free cycloaliphatic peptide acyltransferase is obtained from the
following manner: culturing the strain, so as to obtain the
solution containing free cycloaliphatic peptide acyltransferase; or
culturing the strain, and then breaking the cell wall of obtained
hyphae, so as to obtain the solution containing free cycloaliphatic
peptide acyltransferase; or purifying the solution containing free
cycloaliphatic peptide acyltransferase and removing impurity
protein, so as to obtain the pure solution containing free
cycloaliphatic peptide acyltransferase.
[0025] In the above preparation method, the carrier is selected
from inorganic carriers, or porous, hydrophilic enzyme
carriers.
[0026] In the above preparation method, the carrier is selected
from the following inorganic carrier: an inorganic carrier,
wherein, based on the total weight of the inorganic carrier, the
content of SiO.sub.2 is more than 50 wt %, and the content of
Al.sub.2O.sub.3 is more than 1 wt %; preferably, the inorganic
carrier selected from: catalyst carrier CELITE, Expanded perlite,
diatomaceous earth, kaolin, or porous glass.
[0027] In the above preparation method, the porous hydrophilic
enzyme carrier is selected from the enzyme-carrier in which
polymethacrylate is used as matrix with bonded epoxide or
containing amino functional groups; preferably selected from:
Relizyme EP403, SEPABEADS EC-EP, Relizyme HA403 or SEPABEADS
EC-HA.
[0028] In the above preparation method, the mixing ratio of the
free cycloaliphatic peptide acyltransferase to the carrier is
10-1000 unites (u), preferably 20-600 units (u) of enzyme vs one
gram of carrier.
[0029] In the above preparation method, the mixing ratio of the
free cycloaliphatic peptide acyltransferase to the inorganic
carrier is 10-100 unites (u), preferably 20-80 units (u) of enzyme
vs one gram of carrier.
[0030] In the above preparation method, the mixing ratio of the
free cycloaliphatic peptide acyltransferase to the porous
hydrophilic enzyme carrier is 100-1000 unites (u), preferably
120-600 units (u) of enzyme vs one gram of carrier.
[0031] In the above preparation method, the pH value of the
solution containing free cycloaliphatic peptide acyltransferase is
4-9; preferably, 6-7.
[0032] In the above preparation method, the temperature for mixing
the solution containing free cycloaliphatic peptide acyltransferase
with the carrier is 0-80.degree. C.; preferably, 20-35.degree. C.;
most preferably, 20-25.degree. C.
[0033] In the above preparation method, after mixing the solution
containing free cycloaliphatic peptide acyltransferase with the
carrier, the free cycloaliphatic peptide acyltransferase is
separated from the cycloaliphatic peptide acyltransferase binding
to the carrier, so as to obtain the immobilized cycloaliphatic
peptide acyltransferase.
[0034] In the third aspect of the present invention, a method for
preparing the compound of formula II is provided, comprising the
following steps:
[0035] (a) mixing the compound of formula I with a buffer solution,
so as to obtain solution 1;
[0036] (b) mixing solution 1 with the immobilized cycloaliphatic
peptide acyltransferase as said above provided by the present
invention for conducting deacylation reaction, so as to give the
compound of formula II;
##STR00003##
[0037] Wherein R.sup.1 is an acyl, R.sup.2 is a hydroxy or acyloxy,
R.sup.3 is a hydrogen or hydroxy, R.sup.4 is a hydrogen or hydroxy,
R.sup.5 is a hydrogen or a hydroxy sulfonyloxy, and R.sup.6 is a
hydrogen or carbamoyl.
[0038] In another preferred embodiment, the ratio of the
immobilized cycloaliphatic peptide acyltransferase to the compound
of formula I is 0.01-10 u/g.
[0039] In another preferred embodiment, the pH value of the buffer
in step (a) is 4-9, preferably 6-7; and the buffer in step (a) is
selected from one or more of the group consisting of sodium citrate
buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate
buffer and Tris-HCl buffer.
[0040] In another preferred embodiment, the temperature for
deacylation reaction in step (b) is 20-70.degree. C.; preferably
40-50.degree. C.
[0041] In another preferred embodiment, in step (b), upon
deacylation, the immobilized cycloaliphatic peptide acyltransferase
is separated from the product-containing reaction solution to give
the compound of formula II; preferably, the method for separating
the immobilized cycloaliphatic peptide acyltransferase from the
product-containing reaction solution includes filtration or
centrifugation.
[0042] Accordingly, a method for immobilizing cycloaliphatic
peptide acyltransferase is provided, thereby recovering, purifying
the acyltransferase, and improving the utilization of the
enzyme.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is HPLC pattern of deacylated product obtained by
using the immobilized enzyme (vi) in Example 10.
[0044] FIG. 2 is HPLC pattern of deacylated product obtained by
using the free enzyme liquid in Comparative Example 1.
MODE FOR CARRYING OUT THE INVENTION
[0045] After extensive researches, the inventors found an
industrialized production method for immobilized cycloaliphatic
peptide acyltransferase and a production method for deacylating the
"amide group" on the side chain of cycloaliphatic peptides by using
the immobilized enzyme preparations to form "amino group". The
method is advanced, simple, and easy to operate, and the deacylated
product will possess high purity.
[0046] Moreover, the present invention relates to the
immobilization method for acyltransferase for deacylating the acyl
side chain of FR901379 (Patent No. CN 1051757A) produced by
Colephoma sp. F-11899 (FERM BP-2635), echinocandin B (U.S. Pat. No.
4,288,549) produced by Aspergillus nidulans Nrrl 11440 and the
like, and the deacylation method by using the immobilized
enzyme.
[0047] Structural formulae of compounds mentioned in BACKGROUND and
the present invention are listed in the following table:
TABLE-US-00001 ##STR00004## I ##STR00005## II ##STR00006## Ia
(FR901379) ##STR00007## Ib (echinocandin B) ##STR00008## Ic
(FR179642) ##STR00009## Id ##STR00010## Ie (FK463(Sodium
Micafungin) ##STR00011## If (Anidulafungin)
[0048] wherein R.sup.1 is an acyl, R.sup.2 is a hydroxy or acyloxy,
R.sup.3 is a hydrogen or hydroxy, R.sup.4 is a hydrogen or hydroxy,
R.sup.5 is a hydrogen or a hydroxy sulfonyloxy, and R.sup.6 is a
hydrogen or carbamoyl.
[0049] The cycloaliphatic peptide acyltransferase according to the
present invention is immobilized, and the preparation method
thereof includes the following steps:
[0050] a. preparing a cycloaliphatic peptide acyltransferase
liquid;
[0051] b. mixing the cycloaliphatic peptide acyltransferase liquid
with a carrier based on certain ratio, so as to immobilize the
cycloaliphatic peptide acyltransferase on the carrier; and
[0052] c. separating the cycloaliphatic peptide acyltransferase
liquid from the carrier, so as to obtain the immobilized
cycloaliphatic peptide acyltransferase.
[0053] It is not necessary to define the source of cycloaliphatic
peptide acyltransferase used in the present invention, and the
cycloaliphatic peptide acyltransferase derived from natural or
artificial mutants, or variants thereof and obtained through
transformation by introducing foreign genes of cycloaliphatic
peptide acyltransferase can be included in the present
invention.
[0054] The carrier is selected from an inorganic carrier or porous
hydrophilic carrier. The advantage of inorganic carrier is that the
enzyme won't be inactivated and the enzyme adsorbed on the carrier
is active, however, the drawback thereof is that the specific
activity of enzyme is low, and the amount of adsorbed enzyme per
unit of carrier is far below that on porous hydrophilic carrier.
The porous hydrophilic carrier can adsorb more enzyme, however, a
portion of enzyme will be inactivated, that is, only some of the
enzyme adsorbed on the carrier will be active, so that the recovery
is low.
[0055] The inorganic carrier can be hydrophobic carrier, such as
catalyst carrier CELITE [Chemical composition of which is: 87%
SiO2, 0.9% CaO, 6.1% Al2O3, 1.6% Fe2O3, 1.6% Na2O+K2O], expanded
perlite [chemical composition of which is: SiO2 (70-75%), CaO
(0.1-2.0%), Al2O3 (12-16%), Na2O (1.0-5.0%), Fe2O3 (0.1-1.5%), K2O
(1.0-5.0%)], diatomaceous earth, kaolin, or a porous glass, etc.,
the carriers commonly used in immobilization, and based on the
total weight of the inorganic carrier, the SiO2 content is more
than 50 wt %, Al2O3 content is more than 1 wt %; and the inorganic
carrier is preferably selected from: catalyst carrier CELITE,
expanded perlite, diatomaceous earth, kaolin, or a porous glass;
most preferably, catalyst carrier CELITE, expanded perlite.
[0056] The porous hydrophilic enzyme carrier can be an
enzyme-carrier in which polymethacrylate is used as matrix with
bonded epoxide or containing amino functional groups. For example,
the enzyme carrier with epoxide as functional group includes
Relizyme EP403, SEPABEADS EC-EP. The enzyme carrier with
hexylenediamine as functional group includes Relizyme HA403. And
The enzyme carrier with hexamethyleneimine as functional group
includes SEPABEADS EC-HA.
[0057] During the immobilization process by using inorganic
carriers, the ratio of enzyme to carrier is 10-100 u of enzyme per
gram of carrier; preferably, 20-80 u of enzyme/g of carrier. When
the enzyme amount is lower than 10 u/g of carrier, the activity of
immobilized enzyme is too low to conducting enzymatic reaction,
however, when the enzyme amount is higher than 100 u/g of carrier,
the immobilization efficiency of enzyme is too low, so that some of
enzyme is in free state and the enzyme activity will lost upon
using for one time.
[0058] During the immobilization process by using porous
hydrophilic carrier, the ratio of enzyme to carrier is 100-1000 u
of enzyme per gram of carrier; preferably, 120-600 u of enzyme/g of
carrier. In particular, during the immobilization process by using
porous hydrophilic carriers, the enzyme liquid is preferably
purified, and the lower content of impurity protein in the enzyme
liquid will facilitate immobilization.
[0059] Immobilization conditions for using inorganic carrier are:
the activity of cycloaliphatic peptide acyltransferase in the crude
enzyme liquid obtained as above is tested by HPLC; carriers in
solid state are added based on certain ratio of enzyme/carrier; at
0-80.degree. C., the resulting system is agitated at pH 4-9 over
0.5 hour; the system is thoroughly washed, filtered, and dried at
low temperature, so as to obtain immobilized cycloaliphatic peptide
acyltransferase and then stored at 0-5.degree. C.
[0060] Immobilization conditions for using porous hydrophilic
carrier with epoxide as functional group are: the activity of
cycloaliphatic peptide acyltransferase in the purified enzyme
liquid obtained as above is tested by HPLC; enzyme carriers with
epoxide as functional group are added based on certain ratio of
enzyme/carrier; at 0-80.degree. C., the resulting system is
agitated at pH 4-9 over 24 hours; the system is thoroughly washed,
filtered, and dried at low temperature, so as to obtain immobilized
cycloaliphatic peptide acyltransferase and then stored at
0-5.degree. C.
[0061] Immobilization conditions for using porous hydrophilic
carrier with amino as functional group are: enzyme carriers are
pre-activated with glutaraldehyde; upon activation, the carriers
are thoroughly washed to remove residual glutaraldehyde; the
activity of cycloaliphatic peptide acyltransferase in the purified
enzyme liquid obtained as above is tested by HPLC; the enzyme
carriers pre-activated with glutaraldehyde are added based on
certain ratio of enzyme/carrier; at 0-80.degree. C., the resulting
system is agitated at pH 4-9 over 24 hours; the system is
thoroughly washed, filtered, and dried at low temperature, so as to
obtain immobilized cycloaliphatic peptide acyltransferase and then
stored at 0-5.degree. C.
[0062] In the present invention, the unit of enzyme activity as
said above is defined as: at 40.degree. C., the amount of enzyme
for producing 1 .mu.mol of product in 1 hour is defined as 1 u.
Steps for testing the enzyme activity are: 17.5 ml of crude enzyme
liquid, 5 ml of potassium dihydrogen phosphate buffer (0.25 mol/L,
pH 6.0) containing FR901379 and 2.5 ml of methanol are taken
respectively; reaction is conducted in a water-bath at 40.degree.
C. for 1 hour; the reaction system is suitably diluted with
deionized water, filtered with nylon membrane (0.22 um); the
concentration of product is tested by HPLC.
[0063] Alternatively, in the present invention, the unit of enzyme
activity as said above is defined as: at 40.degree. C., the amount
of enzyme for producing 1 .mu.mol of product in 1 hour is defined
as 1 u. Steps for testing the enzyme activity are: 17.5 ml of crude
enzyme liquid, 2.5 ml of solution of echinocandin B in DMSO (100
mg/ml) are taken respectively; 5.0 ml of KCl (1.2 M) and
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer (0.5 M) is added into
the mixture of the crude enzyme-containing solution and DMSO
solution; reaction is conducted in a water-bath at 40.degree. C.
for 1 hour; the reaction system is suitably diluted with methanol,
filtered with nylon membrane (0.22 um); the concentration of
product is tested by HPLC.
[0064] In a preferred embodiment of the present invention, the
preparation of cycloaliphatic peptide acyltransferase in step a
includes the following steps: culturing the strain, and then
breaking the cell wall of obtained hyphae, so as to obtain the
cycloaliphatic peptide acyltransferase liquid.
[0065] The strain refers to an excellent strain capable of
secreting cycloaliphatic peptide acyltransferase belonging to
Actinomyces or Streptomyces, mainly including Actinoplanes
utahensis IFO-13244, A. utahensis NRRL-12052. In WO97/32975,
enzymes produced by bacteria belonging to Streptomyces
(Streptomyces anulatus 4811 strain, Streptomyces anulatus 8703
strain, Streptomyces sp. 6907 strain) have been reported.
Additionally, in WO97/47738, enzymes produced by Oidiodendron
tenuissimum IFO 6797 strain, Oidiodendron echinulatum IFO 31963
strain, Oidiodendron truncatum IFO 9951 strain, Oidiodendron
truncatum IFO 31812 strain, Oidiodendron sp. 30084 strain,
Verticillium sp. 30085 strain have been reported.
[0066] Enzymes produced by Actinoplanes utahensis IFO-13244, A.
utahensis NRRL-12052 and Streptomyces sp. 6907 strain are
preferred.
[0067] The medium for culturing the strain includes the following
substances: sucrose 10%, soy peptone 0.1-0.1%, K.sub.2HPO.sub.4
0.1-0.2%, KH.sub.2PO.sub.4 0.01-0.1%, MgSO.sub.4.7H.sub.2O
0.01-0.05%. The strain is cultured at 25-36.degree. C., preferably
30.degree. C., with ventilation being 1-2 vvm, stirring speed being
200-800 r/min. After 3-5 days of culture, great amount of mycelium
can be obtained.
[0068] The cell wall of mycelium obtained as said above can firstly
be broken to collect intracellular enzymes in bacterial cells, so
that better cycloaliphatic peptide acyltransferase can be obtained.
The cell wall of mycelium can be broken by known methods, such as
extraction by high concentration solution of salt, sonication,
mechanical crushing, lysozyme method, etc, so that the
cycloaliphatic peptide acyltransferase can be transferred out of
the cell. And then the enzyme liquid can be separated from mycelium
by filtration or centrifugation, so as to obtain a crude enzyme
liquid.
[0069] For obtaining better immobilized enzymes, the cycloaliphatic
peptide acyltransferase liquid can be purified firstly: used
inorganic carriers are added into the crude enzyme liquid as
obtained above at certain ratio to adsorb free enzymes; the
inorganic carriers with adsorbed enzyme are separated from the
enzyme liquid; the carriers are thoroughly washed; and the
cycloaliphatic peptide acyltransferase is desorbed from the
inorganic carriers by using salt solution with high concentration,
so as to obtain an enzyme liquid with higher purity.
[0070] Ultrafiltration, ion-exchange resin method can be used to
purify cycloaliphatic peptide acyltransferase as disclosed in
Membrane-associate echinocandin B deacylase of Actinoplanes
utahensis: purification, characterization, heterologous cloning and
enzymatic deacylation reaction. By such method, an enzyme liquid
with higher purity can be obtained, however, the recovery of enzyme
for this method is low.
[0071] In the present invention, deacylation is performed by using
immobilized enzymes, comprising the following steps:
[0072] A. preparing solution 1 containing cycloaliphatic peptide by
adding buffer solution;
[0073] B. adding the immobilized cycloaliphatic peptide
acyltransferase into solution 1 for conducting deacylation
reaction;
[0074] C. separating the immobilized cycloaliphatic peptide
acyltransferase from the reaction solution containing product.
[0075] Wherein the ratio of immobilized cycloaliphatic peptide
acyltransferase to cycloaliphatic peptide is 0.01-10 u/g;
preferably, 0.1-5 u/g.
[0076] In step A, pH of the buffer solution is controlled at 4-9;
preferably, 5-7; more preferably, about 6.0. The buffer solution is
0.5 M sodium citrate buffer, 0.5 M
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer, Tris-HCl buffer or the
mixture thereof; preferably, 0.5 M
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer.
[0077] In step B, the temperature for deacylation is controlled at
about 20-70.degree. C.; preferably, about 30-50.degree. C.
[0078] In step C, the method for separating the immobilized enzyme
from the reaction solution containing products includes filtration
or centrifugation.
[0079] After the enzyme is immobilized on suitable carriers,
deacylation can be readily conducted in a continuous stirred tank
reactor, instead of in batch.
[0080] In the present invention, said "cyclic lipopeptide compound"
or "cycloaliphatic peptide" refers to a substance of
ring-containing peptide, wherein the side chain of the ring
contains "acylamino", while other side chains can be contained in
such substance.
[0081] Representative for such cyclic lipopeptide compound is
FR901379 or echinocandin B, which is known as possessing antifungal
activities.
[0082] The immobilized cycloaliphatic peptide acyltransferase of
the invention can deacylate "amide group" on side chain of
cycloaliphatic peptide to form "amino group". In particular, the
enzyme can deacylate palmitoyl side chain of FR901379 or a salt
thereof or acyl side chain of the compound of formula I, so as to
form a cyclic peptide of formula II.
[0083] As used herein, "pharmaceutically acceptable salt"
preferably includes: metal salt such as alkali metal salts (such as
sodium salt, potassium salt), alkaline earth metal salt (such as
calcium salt, magnesium salt, etc.), ammonium salt, salt formed
with organic bases (e.g., trimethylamine salt, triethylamine salt,
pyridine salt, picoline salt, dicyclohexylamine salt,
N,N,-dibenzylethylenediamine salt, diisopropylethylamine salt,
etc.), etc., an organic acid addition salt (e.g. formate, acetate,
trifluoroacetate, maleate, tartrate, methanesulfonate,
benzenesulfonate, toluenesulfonate, etc.), an inorganic acid
addition salt (such as hydrochloride, hydrobromide, hydroiodide,
sulfate, phosphate, etc.), salt formed with an amino acid (e.g.
arginine, aspartic acid, glutamic acid, etc.), and the like.
[0084] The features of the present invention mentioned above, or
the features mentioned in the examples, can be optionally combined.
Any feature disclosed in the present specification can be used in
combination with any other features, and each feature disclosed in
the specification can be replaced with alternative feature which
can serve an identical, equivalent, or similar purpose. Therefore,
the features disclosed herein are only general exemplary examples
of the equivalent or similar features, unless specifically
indicated otherwise.
[0085] The advantages of the invention mainly include:
[0086] 1. According to the immobilization method for cycloaliphatic
peptide acyltransferase provided in the present invention, the
enzyme can be repeatedly used, thereby improving the utilization
rate of cycloaliphatic peptide acyltransferase, reducing production
cost and facilitating industrial production.
[0087] 2. The purity of the deacylated product of the present
invention is significantly improved.
[0088] The invention will be further illustrated with reference to
the following specific examples. It is to be understood that these
examples are only intended to illustrate the invention, but not to
limit the scope of the invention. For the experimental methods in
the following examples without particular conditions, they are
performed under routine conditions or as instructed by the
manufacturer. Unless otherwise specified, all percentages, ratios,
proportions or parts are by weight.
[0089] The unit of the weight/volume percentages in the invention
is well known to the skilled in the art, for example, the weight of
a solute in a 100 mL solution.
[0090] Unless otherwise defined, all scientific and technical terms
used herein have the same meaning as commonly understood by the
skilled in the art. Furthermore, any process or material similar or
equivalent to those described herein can be used in the process of
the present invention. The preferred embodiments and materials
described herein are merely provided for illustration.
[0091] HPLC detection method for Compound II in the following
Examples are described as follows:
[0092] Samples are analyzed on Waters analytical HPLC system.
Reverse-phase HPLC analysis is used to determine FR179642,
Echinocandin B nuclear materials and the like. PLATISIL ODS column
(particle size 5 .mu.m, 4.6 mmi.d.times.250 cm) is used for reverse
phase analysis, and remained at 30.degree. C. 3% acetonitrile/0.5%
sodium dihydrogen phosphate is used as the mobile phase, flow rate
is 1 ml/min, and UV detection is performed at 210 nm.
Example 1
Preparation of Acyltransferase a Produced by Actinoplanes utahensis
IFO-13244 Strain
[0093] According to the fermentation method in U.S. Pat. No.
5,376,634, Actinoplanes utahensis IFO-13244 strain was cultured to
obtain culture containing mycelia (150 L). 0.01 M KH.sub.2PO.sub.4
buffer was added, pH value was adjusted to 6.0, 1.0 M KCl was
added, and the resulting system was agitated at low temperature
over 20 hours for extraction. The resulting mixture was filtered
through a Buchner funnel to collect filtrate containing
acyltransferase (116 L), namely free enzyme liquid (a). The
activity of enzyme in the collected filtrate was detected as
2.3.times.10.sup.5 u by HPLC.
Example 2
Preparation of Acyltransferase b Produced by A. utahensis
NRRL-12052 Strain
[0094] According to the fermentation method in U.S. Pat. No.
4,320,053, Actinoplanes utahensis NRRL-12052 strain was cultured to
obtain culture containing mycelia (160 L). 0.01 M KH.sub.2PO.sub.4
buffer was added, pH value was adjusted to 6.0, 1.0 M KCl was
added, and the resulting system was agitated at low temperature
over 20 hours for extraction. The resulting mixture was filtered
through a Buchner funnel to collect filtrate containing
acyltransferase (121 L), namely free enzyme liquid (b). The
activity of enzyme in the collected filtrate was detected as
2.1.times.10.sup.5 u by HPLC.
Example 3
Preparation of Acyltransferase c Produced by Streptomyces sp. 6907
Strain
[0095] According to the fermentation method in WO97/32975,
Streptomyces sp. 6907 strain was cultured to obtain culture
containing mycelia (160 L). 0.01 M KH.sub.2PO.sub.4 buffer was
added, pH value was adjusted to 6.0, 1.0 M KCl was added, and the
resulting system was agitated at low temperature over 20 hours for
extraction. The resulting mixture was filtered through a Buchner
funnel to collect filtrate containing acyltransferase (124 L),
namely free enzyme liquid (c). The activity of enzyme in the
collected filtrate was detected as 2.9.times.10.sup.5 u by
HPLC.
Example 4
Purification of Acyltransferase b
[0096] According to the method described in Membrane-associate
echinocandin B deacylase of Actinoplanes utahensis: purification,
characterization, heterologous cloning and enzymatic deacylation
reaction, the crude enzyme liquid obtained in Example 2 was
purified to give pure acyltransferase liquid (3.0 L), namely free
enzyme liquid (d). The activity of enzyme in the collected enzyme
liquid was detected as 0.5.times.10.sup.5 u by HPLC.
Example 5
Immobilization of Acyltransferase on Inorganic Carrier
[0097] For immobilization, 50 L of free enzyme liquid (a), (b), (c)
was taken respectively, and then 1.5 kg of expanded perlite was
added respectively. The resulting system was agitated over 1 hour
for adsorption. The immobilized acyltransferase, i.e., immobilized
enzyme (i), (ii) and (iii), was collected by filtration and then
washed with 0.01 M KH.sub.2PO.sub.4 buffer (pH 6.0) for 3 times,
and dried at room temperature for several hours. The immobilized
enzyme was stored at 4.degree. C. until use.
TABLE-US-00002 TABLE 1 Adsorption ratio of expanded perlite as
carrier for different free enzyme liquids Residual Specific Immobi-
Adsorp- enzyme activity lized acyltrans- tion activity (u/g wet
enzyme ferase source ratio (%) u/50 L carrier) I Free Actinoplanes
96.1 290 21 enzyme utahensis liquid (a) IFO-13244 strain Ii Free
Actinoplanes 94.3 520 18 enzyme utahensis liquid (b) NRRL-12052
strain Iii Free Streptomyces 98.0 175 25 enzyme sp. 6907 liquid (c)
strain
Example 6
Immobilization of Acyltransferase on Porous Hydrophilic Enzyme
Carrier with Epoxide as Functional Group
[0098] For immobilization, two portions of 0.8 L of pure
acyltransferase liquid (d) prepared in Example 4 were taken
respectively, and 59.2 g of KCl and 0.01 mol/L KH.sub.2PO.sub.4
buffer (pH 7.0) were added respectively. And then 30 g of Relizyme
EP403 and 30 g of SEPABEADS EC-EP were added respectively. The
resulting systems were agitated at 25.degree. C. over 24 hour. The
immobilized acyltransferase, i.e., immobilized enzyme (iv), (v),
was collected by filtration. The immobilized enzyme was washed with
0.01 M KH.sub.2PO.sub.4 buffer (pH 7.0) for 3 times, and dried at
room temperature for several hours. The immobilized enzyme was
stored at 4.degree. C. until use.
TABLE-US-00003 TABLE 2 Immobilization of free enzyme liquid on
porous hydrophilic enzyme carrier with epoxide Specific Residual
Immobi- activity enzyme lized (u/g wet activity Recovery enzyme
Enzyme carrier carrier) (u/0.8 L) rate (%) iv Relizyme EP403 120 80
27.2 v SEPABEADS EC-EP 212 50 48.2
Example 7
Immobilization of Acyltransferase on Porous Hydrophilic Enzyme
Carrier with Amino Group as Functional Group
[0099] For immobilization, the carrier was pre-activated as
follows: 8 g of Relizyme HA403 and 8 g of SEPABEADS EC-HA were
taken respectively, and 90 ml of 2% glutaraldehyde solution and
0.02 mol/L K.sub.2HPO.sub.4 buffer (pH 8.0) were added. The
resulting system was agitated at 20-25.degree. C. for 1 hour.
Supernatant was removed, and the resins were washed with pure
water. Two portions of 0.4 L of pure acyltransferase liquid (d)
prepared in Example 4 were taken respectively, and two portions of
activated carriers were added respectively. The resulting systems
were agitated at 20-25.degree. C. over 20 hour. The immobilized
acyltransferase, namely immobilized enzyme (vi), (vii), was
collected by filtration. The immobilized enzyme was washed with
0.01 M KH.sub.2PO.sub.4 buffer (pH 7.0) for 3 times, and dried at
room temperature for several hours. The immobilized enzyme was
stored at 4.degree. C. until use.
TABLE-US-00004 TABLE 3 Immobilization of free enzyme liquid on
porous hydrophilic enzyme carrier of amino type Specific Residual
Immobi- activity enzyme lized (u/g wet activity Recovery enzyme
Enzyme carrier carrier) (u/0.4 L) rate (%) (vi) Relizyme HA403 600
30 72.7 (vii) SEPABEADS EC-HA 380 10 46.0
Example 8
Deacylation in Batches by Immobilized Enzyme
[0100] 2 L of buffer (0.2 M KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4
buffer; pH 6.0) and 15 g of immobilized enzyme (i), immobilized
enzyme (ii), or immobilized enzyme (iii) were added into 2 L of
FR901379 aqueous solution (20 mg/ml, HPLC purity of 79.2%; 20 g of
FR901379, 16.7 mmol). Deacylation reaction was conducted at
40.degree. C. for 4 hours. The production and purity of FR179642
were detected by HPLC.
TABLE-US-00005 TABLE 4 Conversion rate of FR901379 and purity of
FR179642 after conducting deacylation for 4 hours by using
immobilized enzymes Immobilized Conversion Purity of enzyme rate X
(%) FR179642 (%) Immobilized 94.6 95.2 enzyme (i) Immobilized 95.2
94.7 enzyme (ii) Immobilized 98.7 95.9 enzyme (iii)
Example 9
Deacylation in Batches by Immobilized Enzyme
[0101] 2 L of buffer (0.2 M KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4
buffer; pH 6.0) and 15 g of immobilized enzyme (iv), immobilized
enzyme (v), immobilized enzyme (vi) or immobilized enzyme (vii)
were added into 2 L of FR901379 aqueous solution (20 mg/ml, HPLC
purity of 79.2%; 20 g of FR901379, 16.7 mmol). Deacylation reaction
was conducted at 40.degree. C. for 1 hour. The production and
purity of FR179642 were detected by HPLC.
TABLE-US-00006 TABLE 5 Conversion rate of FR901379 and purity of
FR179642 after conducting deacylation for 1 hours by using
immobilized enzymes Immobilized Conversion Purity of enzyme rate X
(%) FR179642 (%) Immobilized 92.1 94.8 enzyme (iv) Immobilized 93.7
94.7 enzyme (v) Immobilized 94.7 95.1 enzyme (vi) Immobilized 94.5
94.2 enzyme (vii)
Comparative Example 1
Experiment in Batches by Free Enzyme Liquid
[0102] According to deacylation method in WO97/32975, 100 ml of
buffer (0.2 M KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer; pH 6.0)
and 100 ml of methanol were added into 100 ml of FR901379 aqueous
solution (100 mg/ml, HPLC purity of 79.2%; 10 g of FR901379, 38.35
mmol). And then 700 ml of free enzyme liquid (c) was added.
Deacylation reaction was conducted at 40.degree. C. for 7 hours
with a conversion rate of 71.1%. The purity of FR17964 was detected
as 75.7% by HPLC. HPLC analytical patterns can be found in FIG. 2
and table 10.
[0103] Upon conversion, almost no enzyme activity can be detected
in the liquid Almost all of enzyme was inactivated during
conversion.
TABLE-US-00007 TABLE 10 Retention Peak Peak Peak time Area Height
No. (Min) (mV*s) (mV) % Area 1 5.682 142308 14951 8.09 2 6.290
40524 3337 2.30 3 6.719 4818 463 0.27 4 7.151 65642 4673 3.73 5
8.141 20293 1597 1.15 6 8.525 25462 2034 1.45 7 9.218 1332042 60431
75.72 8 11.126 21882 1041 1.24 9 13.031 8234 516 0.47 10 14.805
20289 961 1.15 11 18.268 16222 421 0.92 12 23.238 61491 2382
3.50
[0104] It can be seen from the above Examples and Comparative
Example 1 that, compared with deacylation using free enzyme liquid,
deacylation using immobilized enzyme possesses significant
advantages. The conversion rate and purity of product have been
greatly improved.
Example 10
Continuous Deacylation in Continuous Stirred Tank Reactor
[0105] At 45.degree. C., continuous deacylation from FR901379 to
FR179642 was performed in a 50 L continuous stirred tank reactor.
0.3 Kg of immobilized enzyme (vi), 20 L of FR901379 aqueous
solution (20 mg/ml, HPLC purity of 79.2%), and 20 L of buffer (0.2
M KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer; pH 6.0) were added.
After 3 hours, the conversion rate of substrate achieved 95.99%.
HPLC analytical patterns can be found in FIG. 1 and table 6.
TABLE-US-00008 TABLE 6 Retension Peak Peak Peak time Area Height
No. (Min) (mV*s) (mV) % Area 1 5.148 6472 590 0.32 2 5.679 3661 467
0.18 3 6.412 29895 2111 1.48 4 9.223 1940814 90329 95.99 5 11.161
8269 386 0.41 6 18.341 24274 694 1.20 7 23.249 8466 334 0.42
[0106] The immobilized acyltransferase can remain stable and active
for at least 30 hours, therefore, it can be used for more than 5
times during production.
[0107] Compared with Comparative Example 1, it can be seen from
Example 10 that immobilized enzyme can be repeatedly used, so that
the usage rate can be greatly improved and the damage and pollution
to environment can be reduced.
Example 11
Comparison of Immobilization Between Different Carriers for Free
Enzyme and of Deacylation Between Immobilized Enzymes
[0108] For immobilization, 5 L of free enzyme (c) was taken, and
then 0.15 kg of expanded perlite, CELITE, active carbon (SiO.sub.2
content is lower than 0%), molecular sieve, and porous glass were
added respectively. The resulting systems were agitated at
30.degree. C. over 1 hour. The immobilized acyltransferase, namely
immobilized enzyme (viii), (ix), (x), (xi), (xii), was collected by
filtration. The immobilized enzyme was washed with 0.01 M
KH.sub.2PO.sub.4 buffer (pH 6.0) for 3 times, and dried at room
temperature for several hours. The immobilized enzyme was stored at
4.degree. C. until use.
[0109] Then, 2 L of buffer (0.2 M
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer; pH 6.0) and 15 g of
immobilized enzyme (viii), (ix), (x), (xi), (xii) were added into 2
L of FR901379 aqueous solution (20 mg/ml, HPLC purity of 79.2%; 20
g of FR901379, 16.7 mmol) respectively. Deacylation reaction was
conducted at 40.degree. C. for 4 hours. The production and purity
of FR179642 were detected by HPLC.
TABLE-US-00009 TABLE 7 Comparison of adsorption rate between 3
carriers for free enzyme and of deacylation between immobilized
enzymes Adsorption Conversion Purity of Carrier rate (%) rate X (%)
FR179642 (%) Expanded perlite (viii) 98.0 98.7 94.9 CELITE (ix)
99.1 95.2 95.8 Active carbon (x) 2.6 1.2 Molecular sieve (xi) 0.5
0.1 Porous glass (xii) 1.0 0.2
Example 12
Comparison of Carriers Under Different Ratios for Immobilization
Between Free Enzyme and of Deacylation Between Immobilized
Enzymes
[0110] For immobilization, 7 portions of 5 L of free enzyme liquid
(c) were taken, the enzyme activity of which was detected as
1.17.times.10.sup.4 U by HPLC. And then, 0.05 kg, 0.12 kg, 0.15 kg,
0.24 kg, 0.58 kg, 1.17 kg, and 1.75 kg, of CELITE were added
respectively. The resulting systems were agitated at 30.degree. C.
over 1 hour for adsorption. The immobilized acyltransferase was
collected by filtration. The immobilized enzyme was washed with
0.01 M KH.sub.2PO.sub.4 buffer (pH 6.0) for 3 times, and the enzyme
activity in filtrate was detected by HPLC. The immobilized enzyme
was dried at room temperature for several hours, and stored at
4.degree. C. until use.
[0111] Then, 1 L of buffer (0.2 M
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer; pH 6.0) and 75 g of
immobilized enzymes as above prepared were added into 1 L of
FR901379 aqueous solution (20 mg/ml, HPLC purity of 79.2%; 10 g of
FR901379, 8.35 mmol) respectively. Deacylation reaction was
conducted at 40.degree. C. for 4 hours. The production and purity
of FR179642 were detected by HPLC.
TABLE-US-00010 TABLE 8 Comparison of adsorption rate between
carriers for free enzyme under different ratios and of deacylation
between immobilized enzymes Ratio of enzyme Adsorption Conversion
to carrier(U/g) rate (%) rate X (%) 243/1 46.1 60.3 97.5/1 82.0
99.1 About 80.0/1 98.1 97.2 About 50/1.sup. 98.5 95.7 20.0/1 99.5
93.9 .sup. 10/1 98.2 87.8 .sup. 15/1 98.1 41.2
Example 13
Comparison of Immobilization of Carriers for Free Enzyme Under
Different pH
[0112] For immobilization, 5 portions of 5 L of free enzyme liquid
(a) were taken, the enzyme activity of which was detected as
1.0.times.10.sup.4 U by HPLC. And then, 0.15 kg of CELITE was added
respectively. pH was adjusted to 3.5, 4.0, 6.0, 9.0, 9.5 by using 2
mol/l HCl or 2 mol/l NaOH respectively. The resulting systems were
agitated at 25.degree. C. over 1 hour for adsorption. The
immobilized acyltransferase was collected by filtration. The
immobilized enzyme was dried at room temperature for several hours,
and stored at 4.degree. C. until use.
[0113] Then, 1 L of buffer (0.2 M
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer; pH 6.0) and 75 g of
immobilized enzymes as above prepared were added into 1 L of
FR901379 aqueous solution (20 mg/ml, HPLC purity of 79.2%; 10 g of
FR901379, 8.35 mmol) respectively. Deacylation reaction was
conducted at 40.degree. C. for 4 hours. The production of FR179642
was detected by HPLC.
TABLE-US-00011 TABLE 9 Comparison of immobilization of carriers for
free enzyme under different pH Conversion Immobilization pH rate X
(%) 3.5 8.6 4.0 89.1 6.0 96.2 9.0 95.9 9.5 61.5
Example 14
[0114] 500 ml of KCl (1.2 M) and
KH.sub.2PO.sub.4--Na.sub.2HPO.sub.4 buffer (0.5 M, pH 7.0) was
added into 100 mL of echinocandin B solution in DMSO (100 mg/ml, 10
g of Echinocandin B, 9.43 mmol). And then 25 g of immobilized
enzyme (ii) was added. Deacylation reaction was conducted at
50.degree. C. for 2 hours. The conversion rate was detected
85.6%.
[0115] The above examples are merely the preferred examples for the
present invention, and such examples cannot be used to limit the
scope of the invention. The substantial technical contents
according to the present invention are broadly defined in the
claims. And any entities or methods accomplished by others should
be considered as the equivalents and fall within the scope as
defined by the claims, if said entities or methods are the same as
those defined by the claims.
* * * * *