U.S. patent application number 12/180780 was filed with the patent office on 2009-01-08 for novel fructosyl peptide oxidase.
This patent application is currently assigned to Kikkoman Corporation. Invention is credited to Kozo Hirokawa, Naoki Kajiyama, Keiko Kurosawa.
Application Number | 20090011467 12/180780 |
Document ID | / |
Family ID | 27347435 |
Filed Date | 2009-01-08 |
United States Patent
Application |
20090011467 |
Kind Code |
A1 |
Kurosawa; Keiko ; et
al. |
January 8, 2009 |
NOVEL FRUCTOSYL PEPTIDE OXIDASE
Abstract
The present invention has an object of providing a novel
fructosyl peptide oxidase having superior physicochemical
properties such as stability that is useful as an enzyme for
clinical diagnosis, and an object of providing a method for
producing the fructosyl peptide oxidase. A novel fructosyl peptide
oxidase having physicochemical properties useful as an enzyme for
clinical diagnosis, and a method for producing a novel fructosyl
peptide oxidase are provided herein, the method comprising:
culturing a microorganism capable of producing the oxidase in a
medium; and collecting the oxidase from the culture. Furthermore, a
fructosyl peptide oxidase gene coding for a novel fructosyl peptide
oxidase, recombinant DNA wherein the gene is inserted into vector
DNA, and a method for producing a novel fructosyl peptide oxidase
are provided herein, the method comprising: culturing, in a medium,
a transformant or a transductant including the gene; and collecting
the novel fructosyl peptide oxidase from the culture.
Inventors: |
Kurosawa; Keiko; (Chiba,
JP) ; Hirokawa; Kozo; (Chiba, JP) ; Kajiyama;
Naoki; (Chiba, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Kikkoman Corporation
Noda-shi
JP
|
Family ID: |
27347435 |
Appl. No.: |
12/180780 |
Filed: |
July 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11073626 |
Mar 8, 2005 |
7419813 |
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12180780 |
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10232655 |
Sep 3, 2002 |
7018823 |
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11073626 |
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Current U.S.
Class: |
435/71.1 ;
435/191; 435/254.11; 435/320.1; 536/23.2; 536/24.5 |
Current CPC
Class: |
C12N 9/0022
20130101 |
Class at
Publication: |
435/71.1 ;
435/191; 536/23.2; 536/24.5; 435/320.1; 435/254.11 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 9/06 20060101 C12N009/06; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 1/15 20060101
C12N001/15 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2001 |
JP |
2001-266665 |
Dec 12, 2001 |
JP |
2001-378151 |
Aug 6, 2002 |
JP |
2002-228727 |
Claims
1. A fructosyl peptide oxidase which acts on fructosyl valyl
histidine in the presence of oxygen and catalyzes a reaction that
produces .alpha.-ketoaldehyde, valyl histidine and hydrogen
peroxide.
2. The fructosyl peptide oxidase of claim 1, wherein an activity of
80% or higher remains after a heat treatment at 45.degree. C. for
10 minutes.
3. The fructosyl peptide oxidase of claim 1 which has a molecular
weight of about 52,000 Da (SDS-PAGE).
4. The fructosyl peptide oxidase of claim 1 which: (a) acts on
fructosyl valyl histidine in the presence of oxygen and catalyzes a
reaction that produces a-ketoaldehyde, valyl histidine and hydrogen
peroxide; (b) exhibits optimal activity within the range of pH
6.0-8.0; (c) is active within the temperature range of
20-45.degree. C.; (d) retains at least 80% of its activity
following a heat treatment at 45.degree. C. for 10 minutes; (e) is
stable within the range of pH 6.0-9.0; and (f) has a molecular
weight of about 52,000 Da (SDS-PAGE).
5. A method for producing a fructosyl peptide oxidase comprising:
culturing a filamentous fungus that produces the fructosyl peptide
oxidase of claim 1 in a medium; and recovering the fructosyl
peptide oxidase.
6. The method of claim 5, wherein the filamentous fungus is
selected from a group consisting of Achaetomiella, Achaetomium,
Thielavia, Chaetomium, Gelasinospora, Microascus, Coniochaeta and
Eupenicillium.
7. The method of claim 6, wherein the filamentous fungus is
Achaetomiella virescens ATCC 32393 or Chaetomium is Chaetomium sp.
NISL 9335 (FERM BP-7799).
8. The fructosyl peptide oxidase of claim 1, which has less
activity on .epsilon.-fructosyl lysine than it does on fructosyl
valyl histidine or fructosyl glycine.
9. The fructosyl peptide oxidase of claim 8, which retains an
activity of 80% or higher after a heat treatment at 45.degree. C.
for 10 minutes.
10. The fructosyl peptide oxidase of claim 8 which: (a) exhibits an
optimal activity within the range of pH 6.0-8.0; (b) is active
within a temperature range of 20-40.degree. C.; (c) retains an
activity of 80% or higher following a heat treatment at 45.degree.
C. for 10 minutes; and (d) is stable within a range of pH
6.0-9.0.
11. A method for producing a fructosyl peptide oxidase comprising:
culturing a filamentous fungus that produces the fructosyl peptide
oxidase of claim 8 in a medium; and recovering the fructosyl
peptide oxidase.
12. The method of claim 11, wherein the filamentous fungus belongs
to Eupenicillium or Coniochaeta.
13. The method of claim 12, wherein the filamentous fungus is
selected from the group consisting of Eupenicillium terrenum ATCC
18547, Eupenicillium senticosum IFO 9158, Eupenicillium idahoense
IFO 9510, Eupenicillium euglaucum TO 31729, and Coniochaeta sp.
NISL 9330 (FERM BP-7798).
14. A protein having a fructosyl peptide oxidase activity selected
from the group consisting of: (a) a protein comprising an amino
acid sequence represented by SEQ ID NO: 1; (b) a protein comprising
an amino acid sequence having deletion, substitution and/or
addition of one to several amino acids relative to the amino acid
sequence represented by SEQ ID NO: 1, and having a fructosyl
peptide oxidase activity; and (c) a protein comprising an amino
acid sequence having at least 80% homology with the amino acid
sequence represented by SEQ ID NO: 1, and having a fructosyl
peptide oxidase activity.
15. A nucleic acid selected from the group consisting of: (a) a
protein comprising an amino acid sequence represented by SEQ ID NO:
1; (b) a protein comprising an amino acid sequence having deletion,
substitution and/or addition of one to several amino acids relative
to the amino acid sequence represented by SEQ ID NO: 1, and having
a fructosyl peptide oxidase activity; and (c) a protein comprising
an amino acid sequence having at least 80% homology with the amino
acid sequence represented by SEQ ID NO: 1, and having a fructosyl
peptide oxidase activity.
16. A nucleic acid selected from the group consisting of: (a) DNA
comprising a nucleotide sequence represented by SEQ ID NO: 2; (b)
DNA which hybridizes under stringent conditions with DNA comprising
a nucleotide sequence complementary to a full-length or 15 or more
consecutive bases of the DNA comprising the nucleotide sequence
represented by SEQ ID NO: 2; and (c) DNA which has at least 80%
homology with a full-length or 15 or more consecutive bases of the
DNA comprising the nucleotide sequence represented by SEQ ID NO:
2.
17. A DNA vector comprising the nucleic acid of claim 15.
18. A host cell transformed or transduced with the DNA vector of
claim 17.
19. A method for producing a fructosyl peptide oxidase, comprising:
culturing host cell of claim 18 in a medium; and recovering the
fructosyl peptide oxidase.
20. A protein having a fructosyl peptide oxidase activity selected
from the group consisting of: (a) a protein comprising an amino
acid sequence represented by SEQ ID NO: 3; (b) a protein comprising
an amino acid sequence having deletion, substitution and/or
addition of one to several amino acids relative to the amino acid
sequence represented by SEQ ID NO: 3, and having a fructosyl
peptide oxidase activity; and (c) a protein comprising an amino
acid sequence having at least 80% homology with the amino acid
sequence represented by SEQ ID NO: 3, and having a fructosyl
peptide oxidase activity.
21. A nucleic acid which encodes: (a) a protein comprising an amino
acid sequence represented by SEQ ID NO: 3; (b) a protein comprising
an amino acid sequence having deletion, substitution and/or
addition of one to several amino acids relative to the amino acid
sequence represented by SEQ ID NO: 3; or (c) a protein having at
least 80% homology with the amino acid sequence represented by SEQ
ID NO: 3.
22. A nucleic acid comprising: (a) DNA comprising a nucleotide
sequence represented by SEQ ID NO: 4; (b) DNA which hybridizes
under stringent conditions with DNA comprising a nucleotide
sequence complementary to a full-length or 15 or more consecutive
bases of the DNA comprising the nucleotide sequence represented by
SEQ ID NO: 4or (c) DNA which has at least 80% homology with a
full-length or 15 or more consecutive bases of the DNA comprising
the nucleotide sequence represented by SEQ ID NO: 4.
23. A DNA vector comprising the nucleic acid of claim 21.
24. A host cell that has been transformed or transduced with the
recombinant DNA vector of claim 23.
25. A method for producing a fructosyl peptide oxidase comprising:
culturing the host cell of claim 24 in a medium; and recovering a
fructosyl peptide oxidase encoded by said recombinant DNA
vector.
26. An isolated or purified polypeptide selected from the group
consisting of: (x) a polypeptide comprising the entire amino acid
sequence of SEQ ID NO: 1; (y) a polypeptide comprising SEQ ID NO:
1, but having the deletion, substitution and/or addition of one to
twenty amino acids relative to the amino acid sequence of SEQ ID
NO: 1; and (z) a polypeptide comprising an amino acid sequence
having at least 80% homology with the amino acid sequence of SEQ ID
NO: 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel fructosyl peptide
oxidase, to a fructosyl peptide oxidase gene encoding the same, and
to a method for producing the novel fructosyl peptide oxidase.
BACKGROUND OF THE INVENTION
[0002] Glycated protein is a non-enzymatically-glycated protein,
which is produced as a result of covalent bonding between an
aldehyde group of sugar, namely, aldose (i.e., a monosaccharide
potentially associated with an aldehyde group, and derivatives
thereof), and an amino group of a protein. Examples of the amino
group of the protein include an N-terminal .alpha.-amino group and
an internal lysine residue side chain .epsilon.-amino group. These
glycated proteins are also referred to as Amadori compounds for
being formed upon Amadori rearrangement of a Schiff base, a
reaction intermediate product.
[0003] The glycated proteins are contained in body fluid such as
blood in body or in a biological sample such as hair. The
concentration of the glycated protein in blood strongly depends on
the concentration of saccharide such as glucose dissolved in serum.
In diabetic conditions, production of the glycated protein is
accelerated. A concentration of glycated hemoglobin in erythrocytes
and a concentration of glycated albumin in serum indicate an
average blood sugar level for the past predetermined period.
Therefore, quantification of the glycated proteins is important for
diagnosis and control of the disease process of diabetes.
[0004] Examples of known conventional methods for quantifying a
glycated protein include, for example, a method employing
high-performance liquid chromatography (Chromatogr. Sci., 10, 659
(1979)), a method using a column loaded with a solid material bound
with boric acid (Clin. Chem., 28, 2088-2094 (1982)), a method
employing electrophoresis (Clin. Chem., 26, 1598-1602 (1980)), a
method using antigen-antibody reaction (JJCLA, 18, 620 (1993)), a
colorimetric method for determining reducibility using tetrazolium
salt (Clin. Chim. Acta, 127, 87-95 (1982)), a colorimetric method
using thiobarbituric acid following oxidation (Clin. Chim. Acta,
112, 197-204 (1981)), a method using an enzyme such as glycated
amino acid oxidase (Japanese Patent Examined Publication (kokoku)
No. 05-33997, Japanese Patent Application Laid-Open (kohyo) No.
11-127895, WO97-13872, Japanese Patent Examined Publication
(kokoku) No. 6-65300, and Japanese Patent Applications Laid-Open
(kohyo) Nos. 2-195900, 3-155780, 4-4874, 5-192193, 6-46846,
11-155596, 10-313893, 11-504808, 2000-333696, 2001-54398,
2001-204495 and 2001-204494). Furthermore, a new method for
quantifying a glycated protein was disclosed recently which is more
accurate than any of the above-mentioned methods (Japanese Patent
Application Laid-Open (kohyo) No. 2001-95598). According to this
quantification method, a sample containing a glycated protein is
treated with protease to release a fructosyl peptide from the
glycated protein, which is then exposed to oxidase. The resulting
product is quantified for quantification of the glycated protein.
This method has been recognized as an accurate quantification
method that requires short time and simple manipulation.
[0005] According to the conventional method using glycated amino
acid oxidase, a glycated protein is treated with protease and then
the produced glycated amino acid is quantified enzymatically.
Specifically, according to this method, a glycated protein is
treated with protease or the like to give fructosyl amino acid,
which is then exposed to fructosyl amino acid oxidase to quantify
the produced hydrogen peroxide. Examples of known fructosyl amino
acid oxidases that can be used in such quantification method
include oxidase produced by Corynebacterium (Japanese Patent
Examined Publications (kokoku) Nos. 5-33997 and 6-65300), oxidase
produced by Aspergillus (Japanese Patent Application Laid-Open
(kohyo) No. 3-155780), oxidase produced by Gibberella (Japanese
Patent Application Laid-Open (kohyo) No. 7-289253), oxidase
produced by Fusarium (Japanese Patent Applications Laid-Open
(kohyo) Nos. 7-289253 and 8-154672), oxidase produced by
Penicillium (Japanese Patent Application Laid-Open (kohyo) No.
8-336386), oxidase produced by Tricosporon (Japanese Patent
Application Laid-Open (kohyo) No. 2000-245454), and ketoamine
oxidase (Japanese Patent Application Laid-Open (kohyo) No.
5-192193). However, while these enzymes act well on fructosyl amino
acids, they do not act on fructosyl peptides. Oxidase produced by
E. coli DH5.alpha. (pFP1) (FERM BP-7297) described in Japanese
Patent Application Laid-Open (kohyo) No. 2001-95598 is known as an
enzyme that acts on fructosyl peptide. However, preparing a
quantification kit with this enzyme is difficult since its basic
physicochemical properties are unknown and it has poor
stability.
[0006] The objective of the present invention is to provide a novel
and stable fructosyl peptide oxidase, a fructosyl peptide oxidase
gene encoding the same, and a method for producing the novel
fructosyl peptide oxidase.
SUMMARY OF THE INVENTION
[0007] The present inventors have devoted themselves to solving the
above-described problems and found that various microorganisms
obtained by searching widely in nature produce novel fructosyl
peptide oxidases which have superior stability. The present
inventors have succeeded in acquiring these enzymes, found that
these fructosyl peptide oxidases have various novel physicochemical
properties that are advantageous for quantifying glycated proteins,
and succeeded in isolating fructosyl peptide oxidase genes, thereby
achieving the present invention.
[0008] Thus, the present invention relates to a fructosyl peptide
oxidase which acts on fructosyl valyl histidine in the presence of
oxygen and catalyzes a reaction that produces .alpha.-ketoaldehyde,
valyl histidine and hydrogen peroxide. The present invention also
relates to a fructosyl peptide oxidase which catalyzes the
above-mentioned reaction and whose remaining activity following a
heat treatment at 45.degree. C. for 10 minutes is 80% or higher,
and to a fructosyl peptide oxidase which catalyzes the
above-mentioned reaction and whose molecular weight is about 52,000
(SDS-PAGE). Moreover, the present invention relates to a fructosyl
peptide oxidase which catalyzes the above-mentioned reaction, whose
remaining activity following a heat treatment at 45.degree. C. for
10 minutes is 80% or higher, and whose molecular weight is about
52,000 (SDS-PAGE).
[0009] Furthermore, the present invention relates to a fructosyl
peptide oxidase having the following physicochemical
properties.
[0010] (a) Action and substrate specificity: Acts on fructosyl
valyl histidine in the presence of oxygen and catalyzes a reaction
that produces .alpha.-ketoaldehyde, valyl histidine and hydrogen
peroxide.
[0011] (b) Optimal pH: pH 6.0-8.0.
[0012] (c) Temperature range suitable for action: 20-45.degree.
C.
[0013] (d) Thermostability: a remaining activity of 80% or higher
following a heat treatment at 45.degree. C. for 10 minutes.
[0014] (e) Stable pH range: pH 6.0-9.0.
[0015] (f) Molecular weight: about 52,000 (SDS-PAGE).
[0016] The present invention also relates to a method for producing
the fructosyl peptide oxidase, the method comprising: culturing, in
a medium, a filamentous fungus capable of producing the
above-mentioned fructosyl peptide oxidase or a filamentous fungus
selected from the group consisting of Achaetomiella, Achaetomium,
Thielavia, Chaetomium, Gelasinospora, Microascus, Coniochaeta and
Eupenicillium which are capable of producing the above-mentioned
fructosyl peptide oxidases; and collecting the fructosyl peptide
oxidase from the culture.
[0017] The present invention further relates to a fructosyl peptide
oxidase which catalyzes the above-mentioned reaction and which acts
on fructosyl valyl histidine but has less action on
.epsilon.-fructosyl lysine, to the same fructosyl peptide oxidase
whose remaining activity following a heat treatment at 45.degree.
C. for 10 minutes is 80% or higher, and to the same fructosyl
peptide oxidase having the following physicochemical
properties:
[0018] (a) optimal pH: pH 6.0-8.0;
[0019] (b) temperature range suitable for action: 20-40.degree.
C.;
[0020] (c) thermostability: a remaining activity of 80% or higher
following a heat treatment at 45.degree. C. for 10 minutes; and
[0021] (d) stable pH range: pH 6.0-9.0.
[0022] The present invention also relates to a method for producing
the fructosyl peptide oxidase, the method comprising: culturing, in
a medium, a filamentous fungus capable of producing the
above-mentioned fructosyl peptide oxidase or a filamentous fungus
that belongs to Eupenicillium or Coniochaeta which is capable of
producing the above-mentioned fructosyl peptide oxidase; and
collecting the fructosyl peptide oxidase from the culture.
[0023] The present invention further relates to any of the
following proteins (a), (b) and (c) having a fructosyl peptide
oxidase activity:
[0024] (a) a protein comprising an amino acid sequence represented
by SEQ ID NO: 1;
[0025] (b) a protein comprising an amino acid sequence having
deletion, substitution and/or addition of one to several amino
acids relative to the amino acid sequence represented by SEQ ID NO:
1, and having a fructosyl peptide oxidase activity; and
[0026] (c) a protein having 80% or higher homology with the amino
acid sequence represented by SEQ ID NO: 1, and having a fructosyl
peptide oxidase activity.
[0027] The present invention further relates to a gene coding for
any of the following proteins (a), (b) and (c) having a fructosyl
peptide oxidase activity:
[0028] (a) a protein comprising an amino acid sequence represented
by SEQ ID NO: 1;
[0029] (b) a protein comprising an amino acid sequence having
deletion, substitution and/or addition of one to several amino
acids relative to the amino acid sequence represented by SEQ ID NO:
1, and having a fructosyl peptide oxidase activity; and
[0030] (c) a protein having 80% or higher homology with the amino
acid sequence represented by SEQ ID NO: 1, and having a fructosyl
peptide oxidase activity.
[0031] The present invention also relates to a gene comprising any
of the following DNAs (a), (b) and (c):
[0032] (a) DNA comprising a nucleotide sequence represented by SEQ
ID NO: 2;
[0033] (b) DNA which hybridizes under stringent conditions with DNA
comprising a nucleotide sequence complementary to a full-length or
15 or more consecutive bases of the DNA comprising the nucleotide
sequence represented by SEQ ID NO: 2, and which codes for a protein
having a fructosyl peptide oxidase activity; and
[0034] (c) DNA which has 80% or higher homology with a full-length
or 15 or more consecutive bases of the DNA comprising the
nucleotide sequence represented by SEQ ID NO: 2, and which codes
for a protein having a fructosyl peptide oxidase activity.
[0035] The present invention further relates to recombinant DNA
obtained by inserting the above-mentioned gene into vector DNA.
[0036] The present invention further relates to a transformant or a
transductant comprising the above-mentioned recombinant DNA.
[0037] The present invention also relates to a method for producing
a fructosyl peptide oxidase, comprising: culturing the
above-mentioned transformant or transductant in a medium; and
collecting the fructosyl peptide oxidase from the culture.
[0038] The present invention further relates to any of the
following proteins (a), (b) and (c) having a fructosyl peptide
oxidase activity:
[0039] (a) a protein comprising an amino acid sequence represented
by SEQ ID NO: 3;
[0040] (b) a protein comprising an amino acid sequence having
deletion, substitution and/or addition of one to several amino
acids relative to the amino acid sequence represented by SEQ ID NO:
3, and having a fructosyl peptide oxidase activity; and
[0041] (c) a protein having 80% or higher homology with the amino
acid sequence represented by SEQ ID NO: 3, and having a fructosyl
peptide oxidase activity.
[0042] The present invention further relates to a gene coding for
any of the following proteins (a), (b) and (c) having a fructosyl
peptide oxidase activity:
[0043] (a) a protein comprising an amino acid sequence represented
by SEQ ID NO: 3;
[0044] (b) a protein comprising an amino acid sequence having
deletion, substitution and/or addition of one to several amino
acids relative to the amino acid sequence represented by SEQ ID NO:
3, and having a fructosyl peptide oxidase activity; and
[0045] (c) a protein having 80% or higher homology with the amino
acid sequence represented by SEQ ID NO: 3, and having a fructosyl
peptide oxidase activity.
[0046] The present invention also relates to a gene comprising any
of the following DNAs (a), (b) and (c):
[0047] (a) DNA comprising a nucleotide sequence represented by SEQ
ID NO: 4;
[0048] (b) DNA which hybridizes under stringent conditions with DNA
comprising a nucleotide sequence complementary to a full-length or
15 or more consecutive bases of the DNA comprising the nucleotide
sequence represented by SEQ ID NO: 4, and which codes for a protein
having a fructosyl peptide oxidase activity; and
[0049] (c) DNA which has 80% or higher homology with a full-length
or 15 or more consecutive bases of the DNA comprising the
nucleotide sequence represented by SEQ ID NO: 4, and which codes
for a protein having a fructosyl peptide oxidase activity.
[0050] The present invention further relates to recombinant DNA
obtained by inserting the above-mentioned gene into vector DNA.
[0051] The present invention further relates to a transformant or a
transductant comprising the above-mentioned recombinant DNA.
[0052] The present invention also relates to a method for producing
a fructosyl peptide oxidase, comprising: culturing the
above-mentioned transformant or transductant in a medium; and
collecting the fructosyl peptide oxidase from the culture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1 shows the optimum pH for the inventive oxidase
produced by a filamentous fungus belonging to Achaetomiella.
[0054] FIG. 2 shows the stable pH range for the inventive oxidase
produced by a filamentous fungus belonging to Achaetomiella.
[0055] FIG. 3 shows the optimum temperature range for the inventive
oxidase produced by a filamentous fungus belonging to
Achaetomiella.
[0056] FIG. 4 shows the thermostability of the inventive oxidase
produced by a filamentous fungus belonging to Achaetomiella.
[0057] FIG. 5 shows the optimum pH for the inventive oxidase
produced by a filamentous fungus belonging to Chaetomiumn.
[0058] FIG. 6 shows the stable pH range for the inventive oxidase
produced by a filamentous fungus belonging to Chaetomium.
[0059] FIG. 7 shows the optimum temperature range for the inventive
oxidase produced by a filamentous fungus belonging to
Chaetomium.
[0060] FIG. 8 shows the thermostability of the inventive oxidase
produced by a filamentous fungus belonging to Chaetomium.
[0061] FIG. 9 shows the optimum pH for the inventive oxidase
produced by a filamentous fungus belonging to Coniochaeta which has
less action on .epsilon.-fructosyl lysine.
[0062] FIG. 10 shows the stable pH range for the inventive oxidase
produced by a filamentous fungus belonging to Coniochaeta which has
less action on .epsilon.-fructosyl lysine.
[0063] FIG. 11 shows the optimum temperature range for the
inventive oxidase produced by a filamentous fungus belonging to
Coniochaeta which has less action on .epsilon.-fructosyl
lysine.
[0064] FIG. 12 shows the thermostability of the inventive oxidase
produced by a filamentous fungus belonging to Coniochaeta which has
less action on .epsilon.-fructosyl lysine.
[0065] FIG. 13 shows the optimum pH for the inventive oxidase
produced by a filamentous fungus belonging to Eupenicillium which
has less action on .epsilon.-fructosyl lysine.
[0066] FIG. 14 shows the stable pH range for the inventive oxidase
produced by a filamentous fungus belonging to Eupenicillium which
has less action on .epsilon.-fructosyl lysine.
[0067] FIG. 15 shows the optimum temperature range for the
inventive oxidase produced by a filamentous fungus belonging to
Eupenicillium which has less action on .epsilon.-fructosyl
lysine.
[0068] FIG. 16 shows the thermostability of the inventive oxidase
produced by a filamentous fungus belonging to Eupenicillium which
has less action on .epsilon.-fructosyl lysine.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0069] Hereinafter, the present invention will be described in more
detail. The fructosyl peptide oxidases according to the present
invention (hereinafter, referred to as "the oxidases of the
invention") are oxidases which act on fructosyl valyl histidine in
the presence of oxygen and catalyze the following reaction formula
that results in .alpha.-ketoaldehyde, valyl histidine and hydrogen
peroxide. Any oxidase with this action is considered as the oxidase
of the invention.
Fructosyl valyl
histidine+H.sub.2O+O.sub.2.fwdarw..alpha.-ketoaldehyde+valyl
histidine+H.sub.2O.sub.2
[0070] Any oxidase that catalyzes the above-mentioned reaction is
contemplated as the present invention (and referred to as "the
oxidase of the invention"), including, for example, oxidases that
act on other fructosyl amino acids such as
N.sup..epsilon.-fructosyl lysine (.epsilon.-fructosyl lysine) and
N-fructosyl glycine (fructosyl glycine), and oxidases that act on
various fructosyl peptides. On the other hand, fructosyl peptide
oxidases that act on fructosyl valyl histidine but have less action
on .epsilon.-fructosyl lysine (referred to as "the oxidases of the
invention that have less action on .epsilon.-fructosyl lysine")
refer to the oxidases of the invention which catalyze the
above-mentioned reaction formula and which have a lower activity
upon use of .epsilon.-fructosyl lysine substrate as compared to the
activity upon use of fructosyl valyl histidine substrate, and may
be any oxidase which can accurately quantify fructosyl valyl
histidine in a sample containing .epsilon.-fructosyl lysine. The
oxidases of the invention which do not act on .epsilon.-fructosyl
lysine are also contemplated as the present invention. The oxidases
of the invention that have less action on .epsilon.-fructosyl
lysine are particularly preferable for quantifying fructosyl valyl
histidine in a biological sample containing .epsilon.-fructosyl
lysine.
[0071] In addition, some embodiments of the oxidases of the
invention may comprise: a fructosyl peptide oxidase with a
molecular weight of about 52,000 (SDS-PAGE) which catalyzes the
above-mentioned reaction; a fructosyl peptide oxidase which
catalyzes the above-mentioned reaction and whose remaining activity
following a heat treatment at 45.degree. C. for 10 minutes is 80%
or higher; and a fructosyl peptide oxidase with a molecular weight
of about 52,000 (SDS-PAGE) which catalyzes the above-mentioned
reaction and whose remaining activity following a heat treatment at
45.degree. C. for 10 minutes is 80% or higher. Furthermore, the
oxidase of the invention may also comprise a fructosyl peptide
oxidase which catalyzes the above-mentioned reaction and at the
same time has any or a combination of the following physicochemical
properties (a) to (e).
[0072] (a) Optimal pH: pH 6.0-8.0
[0073] For example, 200 mM acetic acid buffer (pH 3.0-6.0), 200 mM
MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH 6.8-9.0), 200 mM
potassium phosphate buffer (pH 6.5-8.0) and 200 mM glycine buffer
(pH 8.0-12.0) are used as buffers to perform enzyme reactions at
the indicated pH at 30.degree. C., thereby determining optimal pH.
For example, the oxidases of the invention may have an optimal pH
at 5.0-9.0, preferably pH 6.0-8.0.
[0074] (b) Temperature range suitable for action: 20-45.degree.
C.
[0075] For example, a reaction solution having the same composition
as that of a reaction solution used for the activity assay
described later is used to determine activities of the enzyme at
various temperatures, thereby determining a suitable temperature
range for action. The oxidases of the invention may, for example,
have a suitable temperature range of 20-50.degree. C., preferably
25-45.degree. C.
[0076] (c) Thermostability: a remaining activity of 80% or higher
following a heat treatment at 45.degree. C. for 10 minutes.
[0077] For example, 200 mM potassium phosphate buffer (pH 8.0) is
used for a treatment at 45.degree. C. for 10 minutes to determine
the remaining activities of the oxidases of the invention. The
oxidases of the invention having high stability at a high
temperature range are particularly preferable for industrial use.
The oxidases of the invention may, for example, have a remaining
activity of 50% or higher, preferably 70% or higher, and more
preferably 80% or higher under the above-described conditions.
[0078] (d) Stable pH range: pH 6.0-9.0
[0079] For example, 200 mM acetic acid buffer (pH 3.0-6.0), 200 mM
MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH 6.8-9.0), 200 mM
potassium phosphate buffer (pH 6.5-8.0) and 200 mM glycine buffer
(pH 8.0-12.0) are used as buffers to perform enzyme reactions at
the indicated pH at 30.degree. C. for 10 minutes, thereby
determining the remaining activities of the oxidases of the
invention. For example, the oxidases of the invention may have a
stable pH at 5.0-10.0, preferably at 6.0-9.0.
[0080] (e) Molecular weight: about 52,000 (SDS-PAGE)
[0081] For example, the molecular weight is determined by SDS-PAGE
technique using Multigel 10/20 (Daiichi Pure Chemicals Co., Ltd.).
The oxidases of the invention may have a molecular weight of
45,000-60,000, preferably 47,000-57,000 (SDS-PAGE). Presently,
SDS-PAGE technique is a general technique frequently used for
determining the molecular weight of a protein. Considering the
possible error of the molecular weight determined by this
determination method, the molecular weight of about 52,000
(SDS-PAGE) is considered to cover molecular weights within a range
of 47,000 to 57,000.
[0082] On the other hand, the oxidases of the invention that have
less action on .epsilon.-fructosyl lysine refer to the oxidases of
the invention which have a lower activity upon use of
.epsilon.-fructosyl lysine substrate as compared to the activity
upon use of fructosyl valyl histidine substrate, and may be any
oxidase which can accurately quantify fructosyl valyl histidine in
a sample containing .epsilon.-fructosyl lysine. Specifically, if an
activity of the oxidases of the invention that have less action on
.epsilon.-fructosyl lysine is defined as 100 upon use of fructosyl
valyl histidine substrate, an activity upon use of
.epsilon.-fructosyl lysine substrate is 70 or less, preferably 50
or less, more preferably 20 or less.
[0083] Embodiments of the oxidases of the invention that have less
action on .epsilon.-fructosyl lysine may comprise: a fructosyl
peptide oxidase which catalyzes the above-mentioned reaction and
which acts on fructosyl valyl histidine but acts less on
.epsilon.-fructosyl lysine, and whose remaining activity following
a heat treatment at 45.degree. C. for 10 minutes is 80% or higher;
and a fructosyl peptide oxidase which has the above action and
properties as well as any or a combination of the following
physicochemical properties (a) to (d).
[0084] (a) Optimal pH: pH 6.0-8.0
[0085] For example, 200 mM acetic acid buffer (pH 3.0-6.0), 200 mM
MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH 6.8-9.0), 200 mM
potassium phosphate buffer (pH 6.5-8.0) and 200 mM glycine buffer
(pH 8.0-12.0) are used as buffers to perform enzyme reactions at
the indicated pH at 30.degree. C., thereby determining optimal pH.
For example, the oxidases of the invention that have less action on
.epsilon.-fructosyl lysine may have an optical pH at 5.0-9.0,
preferably at 6.0-8.0.
[0086] (b) Temperature range suitable for action: 20-40.degree.
C.
[0087] For example, a reaction solution having the same composition
as that of a reaction solution for the activity assay described
later is used to determine activities of the enzyme at various
temperatures, thereby determining a suitable temperature range. The
oxidases of the invention that have less action on
.epsilon.-fructosyl lysine may, for example, have a suitable
temperature range of 20-50.degree. C., preferably 25-40.degree.
C.
[0088] (c) Thermostability: a remaining activity of 80% or higher
following a heat treatment at 45.degree. C. for 10 minutes
[0089] For example, 200 mM potassium phosphate buffer (pH 8.0) is
used for a treatment at 45.degree. C. for 10 minutes. Then, the
remaining activities of the enzymes of the invention are
determined. The oxidases of the invention having high stability at
a high temperature range are particularly preferable for industrial
use. The oxidases of the invention that have less action on
.epsilon.-fructosyl lysine may, for example, have a remaining
activity of 50% or higher, preferably 70% or higher, more
preferably 80% or higher under the above-described conditions.
[0090] (d) Stable pH range: pH 6.0-9.0
[0091] For example, 200 mM acetic acid buffer (pH 3.0-6.0), 200 mM
MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH 6.8-9.0), 200 mM
potassium phosphate buffer (pH 6.5-8.0) and 200 mM glycine buffer
(pH 8.0-12.0) are used as buffers to perform enzyme reactions at
the indicated pH at 30.degree. C. for 10 minutes, thereby
determining the remaining activities of the oxidases of the
invention. For example, the oxidases of the invention that have
less action on .epsilon.-fructosyl lysine may have a stable pH
range at 5.0-10.0, preferably at 6.0-9.0.
[0092] Enzyme activities of the oxidase of the invention and the
oxidase of the invention that have less action on
.epsilon.-fructosyl lysine (hereinafter, the term "the oxidases of
the invention" may comprise both the oxidase of the invention and
the oxidase of the invention that have less action on
.epsilon.-fructosyl lysine) may principally be determined by
determining the amount of .alpha.-ketoaldehyde, peptide, hydrogen
peroxide or the like which are produced through enzyme reaction or
by determining the amount of oxygen consumed by the enzyme
reaction. Hereinafter, a method for determining the amount of
hydrogen peroxide will be described as one example. Unless
otherwise noted, fructosyl valyl histidine is used as a substrate
for determining the activities of the oxidases of the invention.
Titer of the enzyme is defined such that an amount of enzyme for
producing 1 .mu.mol hydrogen peroxide per minute is 1 U as
determined using a fructosyl valyl histidine substrate.
[0093] A. Preparation of Reagents
[0094] (1) Reagent 1: POD-4-AA solution
[0095] 1.0 kU of peroxidase (Toyobo Co., Ltd, TYPE III) and 100 mg
of 4-aminoantipyrine (Tokyo Kasei Kogyo Co., Ltd.) are dissolved in
0.1 M potassium phosphate buffer (pH 8.0) to a constant volume of 1
L.
[0096] (2) Reagent 2: 2,4-dichlorophenol sulfate solution
[0097] 25 ml of a commercially available 2% solution (available
from Nacalai Tesque, Inc.) is dissolved in ion exchange water to a
constant volume of 100 ml.
[0098] (3) Reagent 3: substrate solution (150 mM; final
concentration 5 mM)
[0099] 624 mg of fructosyl valyl histidine is dissolved in ion
exchange water to a constant volume of 10 ml. Alternatively,
fructosyl glycine or .epsilon.-fructosyl lysine of 357 mg or 462
mg, respectively, may be dissolved in ion exchange water to a
constant volume of 10 ml to be used as a substrate. Fructosyl
valine histidine was prepared as described in Japanese Patent
Application Laid-Open (kohyo) No. 2001-95598. Fructosyl glycine and
.epsilon.-fructosyl lysine were prepared according to a method of
Horiuchi et al. (Agric. Biol. Chem., 53, 103-110, 1989; Agric.
Biol. Chem., 55, 333-338, 1991).
[0100] B. Determination Assay
[0101] 2.7 ml of Reagent 1, 100 .mu.l of Reagent 2 and 100 .mu.l of
enzyme solution are mixed and pre-heated at 30.degree. C. for 5
minutes. Then, 100 .mu.l of Reagent 3 is added and thoroughly mixed
to determine absorbance at 510 nm using a spectrophotometer
(U-2000A, Hitachi, Ltd.). Measurement values indicate changes in
the absorbance per minute at 510 nm after 1 to 3 minutes. A control
solution was prepared in the same manner as described above except
100 .mu.l ion exchange water was added instead of 100 .mu.l Reagent
3. A standard solution of hydrogen peroxide and ion exchange water
were used instead of Reagent 3 and the enzyme solution,
respectively, to prepare a graph showing relationship with respect
to the level of color. Referring to this graph, micromole of
hydrogen peroxide produced per minute at 30.degree. C. is
calculated and used as the unit for expressing the activity of the
enzyme solution. The presence of action on .epsilon.-fructosyl
lysine can be determined in the same manner by using Reagent 3
(substrate solution) containing .epsilon.-fructosyl lysine
substrate instead of fructosyl valyl histidine substrate.
[0102] Thus, the oxidases of the invention comprise oxidase that
acts on fructosyl valyl histidine in the presence of oxygen and
catalyzes reaction represented by the above-described formula which
results in .alpha.-ketoaldehyde, valyl histidine and hydrogen
peroxide. Such oxidases of the invention may, for example, have the
above-described behavior and any or a combination of the
above-described physicochemical properties. Such oxidases of the
invention act efficiently upon free fructosyl valyl histidine
resulting from protease treatment of a sample containing a glycated
protein such as glycated hemoglobin, and thus can be used as an
effective enzyme for quantifying the glycated protein. The oxidases
of the invention having high thermostability are particularly
preferable for use as a clinical diagnostic enzyme.
[0103] When a sample containing a glycated protein is treated with
protease, .epsilon.-fructosyl lysine may also be released together
with fructosyl valyl histidine depending on the sample used. In
this case, when the amount of fructosyl valyl histidine is
determined with the oxidase of the invention which acts well on
.epsilon.-fructosyl lysine, accurate determination may be
difficult. In such case, the oxidase of the invention that has less
action on .epsilon.-fructosyl lysine is more preferable.
[0104] The oxidases of the invention can be acquired from nature by
conducting searches of enzymes derived from microorganisms, animals
and plants. For example, in order to search for microorganisms
capable of producing the oxidases of the invention, microorganisms
are cultured in media supplied with an enzyme-producing inducer
such as fructosyl valyl histidine. The obtained microorganism cells
are disrupted to test for fructosyl peptide oxidase activities
using fructosyl valyl histidine as a substrate. Thus,
microorganisms capable of producing the oxidases of the invention
can be obtained. The microorganisms used here may be newly isolated
from soil, or obtained from a microorganism collection organization
or the like. Furthermore, in order to obtain the oxidases of the
invention with superior storage stability, solutions containing the
disrupted microorganism cells obtained as described above may be
subjected to a heat treatment, for example, at 45.degree. C. for 10
minutes, after which activities are determined and those with
higher remaining activities are selected. As organisms capable of
producing the oxidases of the invention, in terms of easy handling,
productivity and the like, for example, microorganisms are
preferable, particularly filamentous fungi that belong to genus
such as Achaetomiella, Achaetomium, Thielavia, Chaetomium,
Gelasinospora, Microascus, Coniochaeta and Eupenicillium.
Preferable microorganisms may be filamentous fungi belonging to
Achaetomiella, Chaetomium, Coniochaeta and Eupenicillium,
particularly microorganisms such as Achaetomiella virescens ATCC
32393, Chaetomium sp. NISL 9335 (FERM BP-7799), Coniochaeta sp.
NISL 9330 (FERM BP-7798) and Eupenicillium terrenum ATCC 18547.
[0105] In order to search for microorganisms capable of producing
the oxidases of the invention that act on fructosyl valyl histidine
but have less action on .epsilon.-fructosyl lysine, microorganisms
capable of producing the oxidases of the invention which have lower
activity upon use of .epsilon.-fructosyl lysine substrate as
compared to the activity upon use of fructosyl valyl histidine
substrate are selected from the microorganisms capable of producing
the oxidases of the invention obtained by the above-described
search. As microorganisms capable of producing the oxidases of the
invention that have less action on .epsilon.-fructosyl lysine,
filamentous fungi are preferable, such as those that belong to
Eupenicillium and Coniochaeta. Preferable filamentous fungi are,
for example, Eupenicillium terrenum ATCC 18547, Eupenicillium
senticosum IFO 9158, Eupenicillium idahoense IFO 9510,
Eupenicillium euglaucum IFO 31729 and Coniochaeta sp. NISL 9330
(FERM BP-7798).
[0106] The oxidases of the invention are obtained not only by
modifying native oxidases of the invention by techniques such as
gene engineering or mutation process but also by modifying
conventionally known enzyme genes such as fructosyl amino acid
oxidase genes.
[0107] Examples of such modification techniques include, for
example, irradiating organisms capable of producing the
above-described oxidases with UV ray, X-ray, radiation or the like,
or bringing organisms capable of producing the above-described
oxidases into contact with a mutagen such as ethylmethanesulfonate,
N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid, thereby
obtaining a microorganism that produces modified oxidase of the
invention. Then, from the obtained microorganism, the oxidase of
the invention can be obtained. In general, gene engineering can be
employed to modify genes encoding oxidases with different
properties so as to obtain the oxidases of the invention.
[0108] Hereinafter, a method for producing the oxidases of the
invention will be described. According to the present invention,
the oxidases of the invention are collected and produced from
organisms having activities of the oxidases of the invention,
according to a variety of generally employed methods for isolating
proteins. For example, a preferable method for producing the
present oxidase comprises culturing a microorganism capable of
producing the above-described oxidase of the invention in a medium,
and collecting from the culture a protein having a fructosyl
peptide oxidase activity or a protein having a fructosyl peptide
oxidase activity but having less action on .epsilon.-fructosyl
lysine. Specifically, as an example, the following method may be
employed which uses a microorganism capable of producing the
oxidase of the invention.
[0109] First, searched microorganisms that are found to produce the
oxidases of the invention (collectively referred to as "the
microorganisms") are cultured. The microorganisms may be cultured
by a solid culture method but more preferably by a liquid culture
method. As the media for culturing the above-mentioned
microorganisms, one or more types of inorganic salts such as
potassium dihydrogenphosphate, dipotassium hydrogenphosphate,
magnesium sulfate, ferric chloride, ferric sulfate and manganese
sulfate, and, if necessary, sugars, vitamins or the like, are added
to one or more types of nitrogen sources such as infusions of yeast
extract, peptone, meat extract, corn steep liquor, soybean or wheat
malt. As an enzyme inducible substrate, an enzyme substrate such as
fructosyl glycine, fructosyl valyl histidine, .epsilon.-fructosyl
lysine or the like may appropriately be added to increase the
yield.
[0110] Initial pH of the medium may suitably be adjusted to 7 to 9.
Preferably, culture is performed at 25-42.degree. C., preferably at
about 30.degree. C., for 1 to 5 days by submerged aeration culture,
shake culture, standing culture or the like. Following culture, the
oxidases of the invention may be collected from the cultures with
generally employed enzyme collecting means.
[0111] Specifically, cells are separated from the culture
solutions, for example, by filtration, centrifugation or the like
and then washed. The oxidases of the invention are preferably
collected from these cells. These cells can be used directly.
However, it is more preferable to disrupt the cells with disrupting
means such as an ultrasonic homogenizer, a French Press, a Dynomill
and the like, disrupt the cells by lysing cell walls of the cells
with a cell wall lytic enzyme such as lysozyme, or extract the
enzymes from the cells by using a surfactant such as Triton X-100
so as to collect the oxdases of the invention from these cells.
[0112] A general enzyme purification method may be employed to
purify and isolate the oxidases of the invention from the obtained
crude enzyme solutions. Preferably, a combination of, for example,
ammonium sulfate salting out method, organic solvent precipitation,
ion exchange chromatography, gel filtration chromatography,
hydrophobic chromatography, absorption chromatography,
electrophoresis and the like may be performed. Thus, the oxidases
of the invention may be isolated until a generally single band
appears on SDS-PAGE. The above-mentioned purification methods can
suitably be combined to prepare enzyme preparations with different
degrees of purification according to use. The oxidases of the
invention that have less action on .epsilon.-fructosyl lysine may
also be produced according to a method similar to the
above-described method for producing the oxidases of the
invention.
[0113] The produced oxidases of the invention may effectively be
used for quantifying a glycated protein as described below. First,
a glycated protein such as HbA.sub.1c is digested by protease such
as Molsin, AO-protease, peptidase (available from Kikkoman Corp.),
carboxypeptidase Y or Protin P (available from Daiwa Fine Chemicals
Co., Ltd.) to release fructosyl peptide. Then, the released
fructosyl valyl histidine is quantified with the oxidase of the
invention. When .epsilon.-fructosyl lysine which is also released
at the same time causes a problem, an enzyme that acts on
.epsilon.-fructosyl lysine, for example, fructosyl amino acid
oxidase derived from fungus (Japanese Patent Applications Laid-Open
(kohyo) Nos. 7-289253, 8-154672, 8-336386, etc.) or fructosyl amine
oxidase (Japanese Patent Application Laid-Open (kohyo) No.
03-155780) may be used to digest the .epsilon.-fructosyl lysine.
Subsequently, fructosyl valyl histidine can be quantified with the
oxidase of the invention. Alternatively, an oxidase of the
invention that has less action on .epsilon.-fructosyl lysine may be
used so that fructosyl valyl histidine can accurately be quantified
without digesting the released .epsilon.-fructosyl lysine.
[0114] The oxidases of the invention may comprise the following
oxidase (a), (b), (c), (d), (e) or (f):
[0115] (a) a fructosyl peptide oxidase comprising an amino acid
sequence represented by SEQ ID NO: 1;
[0116] (b) a fructosyl peptide oxidase comprising an amino acid
sequence having deletion, substitution and/or addition of one to
several amino acids relative to the amino acid sequence represented
by SEQ ID NO: 1;
[0117] (c) a fructosyl peptide oxidase comprising an amino acid
sequence having 80% or higher homology with the amino acid sequence
represented by SEQ ID NO: 1;
[0118] (d) a fructosyl peptide oxidase comprising an amino acid
sequence represented by SEQ ID NO: 3;
[0119] (e) a fructosyl peptide oxidase comprising an amino acid
sequence having deletion, substitution and/or addition of one to
several amino acids relative to the amino acid sequence represented
by SEQ ID NO: 3; and
[0120] (f) a fructosyl peptide oxidase comprising an amino acid
sequence having 80% or higher homology with the amino acid sequence
represented by SEQ ID NO: 3.
[0121] Herein, "deletion, substitution and/or addition of one to
several amino acids" means deletion, substitution and/or addition
of, for example, 1 to 20, preferably 1 to 10, and more preferably 1
to 5 amino acids.
[0122] Herein, "having 80% or higher homology" has no limitation as
long as homology with the amino acid sequence represented by SEQ ID
NO: 1 or 3 is 80% or higher, for example, 80% or higher, preferably
90% or higher, and most preferably 95% or higher.
[0123] The above-described oxidases of the invention may be
obtained by cloning and expressing a native fructosyl peptide
oxidase gene derived from chromosomal DNA or cDNA of Coniochaeta
sp. NISL 9330 (FERM BP-7798) or a native fructosyl peptide oxidase
gene derived from Eupenicillium terrenum ATCC 18547 in a suitable
vector host system. The oxidases of the invention may also be
obtained from fructosyl peptide oxidase derived from various
sources acquired from nature.
[0124] The fructosyl peptide oxidase genes (hereinafter, referred
to as "the genes of the invention") coding for the fructosyl
peptide oxidases of the invention may comprise genes coding for the
oxidases of the invention (a) to (f) above, and genes coding for
the oxidases of the invention comprising the following DNA (g),
(h), (i), (j), (k) or (l):
[0125] (g) DNA comprising a nucleotide sequence represented by SEQ
ID NO: 2;
[0126] (h) DNA which hybridizes with DNA comprising a nucleotide
sequence complementary to a full-length or 15 or more consecutive
bases of the DNA comprising the nucleotide sequence represented by
SEQ ID NO: 2 under stringent conditions, and which codes for a
protein having a fructosyl peptide oxidase activity;
[0127] (i) DNA which indicates 80% or higher homology with a
full-length or 15 or more consecutive bases of the DNA comprising
the nucleotide sequence represented by SEQ ID NO: 2, and which
codes for a protein having a fructosyl peptide oxidase
activity;
[0128] (j) DNA comprising a nucleotide sequence represented by SEQ
ID NO: 4;
[0129] (k) DNA which hybridizes with DNA comprising a nucleotide
sequence complementary to a full-length or 15 or more consecutive
bases of the DNA comprising the nucleotide sequence represented by
SEQ ID NO: 4 under stringent conditions, and which codes for a
protein having a fructosyl peptide oxidase activity; and
[0130] (l) DNA which indicates 80% or higher homology with a
full-length or 15 or more consecutive bases of the DNA comprising
the nucleotide sequence represented by SEQ ID NO: 4, and which
codes for a protein having a fructosyl peptide oxidase
activity.
[0131] Herein, "stringent conditions" refer to conditions under
which a signal from a specific hybrid is clearly distinguished from
a signal from a non-specific hybrid upon colony hybridization,
plaque hybridization, Southern blot hybridization or the like
(Current Protocols in Molecular Biology (WILEY, Interscience,
1989)). These conditions differ depending on the hybridization
system used as well as the type, sequence and length of the probe.
These conditions can be determined by changing the hybridization
temperature, washing temperature and salt concentration. For
example, when a signal from a non-specific hybrid is positively
detected, the hybridization and washing temperatures can be raised
while the salt concentration is decreased, thereby enhancing
specificity. When a signal from a specific hybrid is not detected,
the hybridization and washing temperatures can be decreased while
the salt concentration is increased, thereby stabilizing the
hybrid. More specifically, DNA that is subjected to hybridization
under stringent conditions may have a certain homology with a
nucleotide sequence of probe DNA. Homology as used in "DNA
comprising a nucleotide sequence indicating 80% or higher homology
with a full-length or 15 or more consecutive bases of a nucleotide
sequence" refers to a homology of, for example, 80% or higher,
preferably 90% or higher, and most preferably 95% or higher.
[0132] Genes coding for the above-mentioned oxidases of the
invention may be, for example, a gene coding for an oxidase
obtained by cloning and expressing a native fructosyl peptide
oxidase gene derived from chromosomal DNA or cDNA of Coniochaeta
sp. NISL 9330 (FERM BP-7798) in a suitable vector host system, or a
gene coding for an oxidase obtained by cloning and expressing a
native fructosyl peptide oxidase gene derived from chromosomal DNA
or cDNA of Eupenicillium terrenum ATCC 18547 in a suitable vector
host system.
[0133] The above-mentioned DNA may be, for example, a native
fructosyl peptide oxidase gene derived from chromosomal DNA or cDNA
of Coniochaeta sp. NISL 9330 (FERM BP-7798), or a native fructosyl
peptide oxidase gene derived from chromosomal DNA or cDNA of
Eupenicillium terrenum ATCC 18547. The genes coding for the
oxidases of the invention or the DNA thereof may also be obtained
from fructosyl peptide oxidases derived from various sources
acquired from nature.
[0134] In addition, the oxidases of the invention, genes coding for
the oxidases of the invention or DNA thereof may also be obtained
from various mutant fructosyl peptide oxidases obtained from native
fructosyl peptide oxidases.
[0135] Hereinafter, a method for obtaining the genes of the
invention will be described.
[0136] In order to obtain the genes of the invention, a generally
employed gene cloning method is used. For example, chromosomal DNA
or mRNA is extracted from cells capable of producing the
above-described oxidases of the invention according to a routine
method (e.g., a method described in Current Protocols in Molecular
Biology (WILEY, Interscience, 1989)). Moreover, cDNA can be
synthesized using mRNA as a template. Thus, chromosomal DNA or a
cDNA library can be obtained. Next, suitable probe DNA is
synthesized based on the amino acid sequence of the oxidase of the
invention, which is used to screen for DNA from the chromosomal DNA
or cDNA library. Alternatively, DNA containing a gene fragment of
interest is amplified by polymerase chain reaction (PCR method)
such as the 5'-RACE or 3'-RACE method by producing suitable primer
DNA based on the above-mentioned amino acid sequence. Then, the
obtained DNA fragments are linked to obtain DNA containing a
full-length gene of the invention. Furthermore, the genes of the
invention coding for the oxidases of the invention can be obtained
from various organisms through hybridization with the
above-mentioned probe DNA.
[0137] For example, the genes of the invention from Coniochaeta sp.
NISL 9330 (FERM BP-7798) or Eupenicillium terrenum ATCC 18547 can
be obtained as follows.
[0138] First, the above-described microorganism is cultured. The
obtained cells are frozen in liquid nitrogen, followed by physical
disruption using, for example, a mortar. From the obtained fine
powdery cell debris, chromosomal DNA is extracted according to a
general method using a commercially available DNA extraction kit or
the like. Then, a total RNA fraction is extracted from the cell
debris according to a general method by using a commercially
available RNA extraction kit or the like. Then, RNA is collected
from this extract through ethanol precipitation. If necessary, a
commercially available Oligo dT column is used to fractionate RNA
having a poly-A chain according to a general method.
[0139] Next, the oxidase of the invention produced from the
above-mentioned microorganism is purified, isolated and sequenced
to determine the N-terminal amino acid sequence thereof. The
obtained peptide fragment is digested with trypsin or lysyl-end
peptidase to give a peptide fragment, and the amino acid sequence
thereof (i.e., the internal amino acid sequence) is determined.
Then, considering the information of the obtained partial amino
acid sequence and the codon usage frequency of the above-mentioned
microorganism, primers are synthesized for PCR. These primers as
well as the obtained chromosomal DNA or RNA as a template are used
to perform PCR or RT-PCR, thereby obtaining a DNA fragment coding
for a part of the oxidase of the invention. Furthermore, primers
are appropriately synthesized based on the nucleotide sequence of
the obtained DNA fragment.
[0140] Next, using the above-mentioned primers and RNA, cDNA
containing the fragment of the present gene is amplified by a
suitable RT-PCR method such as the 5'-RACE or 3'-RACE method. The
amplified products are linked to obtain cDNA containing a
full-length gene of the invention. RT-PCR using the RNA as a
template and synthesized primers complementary to the 5'- and
3'-terminal sequences can amplify cDNA containing the present
gene.
[0141] The amplified DNA can be cloned following a general method.
The amplified DNA is inserted into a suitable vector to obtain
recombinant DNA. For cloning, a commercially available kit such as
TA Cloning Kit (available from Invitrogen), plasmid vector DNA such
as pUC119 (available from Takara Bio Inc.), pBR322 (available from
Takara Bio Inc.), pMAL-C2 (available from New England Labs),
pBluescript II SK.sup.+ (available from Stratagene) and pKK223-3
(available from Amersham Bioscience K.K.), bacteriophage vector DNA
such as .lamda.ENBL3 (available from Stratagene) and .lamda.DASH II
(available from Funakoshi Co., Ltd.), and the like can be used.
[0142] The thus-obtained recombinant DNA is used to transform or
transduce, for example, E. coli K12, preferably E. coli JM109
(available from Toyobo Co., Ltd.), DH5.alpha. (available from
Toyobo Co., Ltd.), XL1-Blue (available from Funakoshi Co., Ltd.)
and the like, thereby obtaining a transformant or a transductant
containing the recombinant DNA. Transformation can be performed,
for example, according to the method by D. M. Morrison (Methods in
Enzymology, 68, 326-331, 1979). Transduction can be performed, for
example, according to the method by B. Hohn (Methods in Enzymology,
68, 299-309, 1979). As a host cell, microorganisms other than E.
coli, for example, other bacteria, yeasts, filamentous fungi or
actinomycetes, or animal cells may be used.
[0143] The total nucleotide sequence of the above-described
amplified DNA may be analyzed by using, for example, LI-COR MODEL
4200L sequencer (LI-COR, Inc.) or 370A DNA sequence system (Perkin
Elmer). The nucleotide sequence is compared with information of the
partial amino acid sequences so as to confirm whether or not the
present gene is obtained. By analyzing the obtained gene of the
invention, the translated polypeptide, namely, the amino acid
sequence of the oxidase of the invention, is determined.
[0144] Examples of the genes of the invention include genes
containing DNA comprising a nucleotide sequence represented by SEQ
ID NO: 2 or 4. Plasmid pKK223-3-CFP containing DNA comprising a
nucleotide sequence represented by SEQ ID NO: 2 was deposited at
the International Patent Organism Depositary, the National
Institute of Advanced Industrial Science and Technology, an
Independent Administrative Institution under the Ministry of
Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1,
Tsukuba, Ibaraki, Japan as FERM BP-8132, while plasmid puc-EFP
containing DNA comprising a nucleotide sequence represented by SEQ
ID NO: 4 was deposited at the AIST as FERM BP-8131.
[0145] The above-mentioned genes of the invention may be modified
to give various modified oxidases of the invention.
[0146] In order to modify the above-mentioned genes, any known
method may be used, for example, a method in which the
above-mentioned recombinant DNA is brought into contact with a
chemical mutation agent such as hydroxylamine or nitrous acid, a
point mutation method for random modification using a PCR method, a
well-known site-directed mutagenesis for site-specific substitution
or deletion using a commercially available kit, a method in which
the recombinant DNA is selectively cleaved, a selected
oligonucleotide is then removed from or added thereto, followed by
linking (i.e., an oligonucleotide mutagenesis method). Then, the
treated recombinant DNA is purified using desalting column, QIAGEN
(available from Qiagen) or the like to obtain various recombinant
DNAs.
[0147] Through these modifications, for example, the following
genes can be obtained: the genes of the invention containing DNA
which hybridizes under stringent conditions with DNA comprising a
nucleotide sequence complementary to a full-length or 15 or more
consecutive bases of the DNA comprising the nucleotide sequence
represented by SEQ ID NO: 2 or 4 and which codes for a protein
having a fructosyl peptide oxidase activity; or the genes of the
invention containing DNA which indicates 80% or higher homology
with a full-length or 15 or more consecutive bases of the DNA
comprising the nucleotide sequence represented by SEQ ID NO: 2 or
4, and which codes for a protein having a fructosyl peptide oxidase
activity.
[0148] Furthermore, by modifying the genes of the invention, for
example, the following oxidases of the invention can be obtained: a
protein comprising an amino acid sequence having deletion,
substitution and/or addition of one to several amino acids relative
to the amino acid sequence represented by SEQ ID NO: 1 or 3, and
having a fructosyl peptide oxidase activity; or a protein having
80% or higher homology with the amino acid sequence represented by
SEQ ID NO: 1 or 3, and having a fructosyl peptide oxidase
activity.
[0149] Next, the transformant or transductant including the
above-described recombinant DNA is cultured in a medium, and
fructosyl peptide oxidase is collected from the culture. In
general, the transformant or transductant is cultured by using a
medium suitable for growing a host used. For example, when E. coli
is used as a host, the transformant is seeded in 10 L LB medium,
and cultured using a jar fermenter at 30.degree. C. for 24 hours
with an air flow of 1 L/min and an agitation rate of 600 rpm. The
obtained 10 L culture is centrifuged at 7,000 rpm for 10 minutes,
thereby collecting and obtaining cells. From the obtained cells,
the enzyme of the invention can be obtained according to the
above-described methods.
EXAMPLES
[0150] Hereinafter, the present invention will be described in more
detail with reference to the Experimental Example and Examples
described below, although the technical scope of the present
invention is not limited to these Examples.
Experimental Example 1
Search for Microorganism Capable of Producing the Inventive
Oxidase
[0151] Soil-derived microorganisms and deposited microorganisms
provided by microorganism collection organizations were used for
the search. Soil-derived microorganisms were isolated from about
100 soil samples obtained at different places in and around
Noda-City (Chiba) and Tsukuba-City (Ibaraki). Particularly, one
small spatula of each soil sample was grown by shake-culture on a
enrichment medium (0.5% yeast extract, 0.2% potassium
dihydrogenphosphate, 0.05% magnesium sulfate, 0.1% fructosyl valyl
histidine, pH 6.5) at 30.degree. C. for one day, and microorganisms
were isolated on a plate medium (enrichment medium+1.2% agar).
About 5,000 strains were isolated, most of which were bacteria and
yeast. Deposited strains used included 380 strains of yeast, 480
strains of filamentous fungi and 700 strains of actinomycetes.
[0152] Soil-derived bacteria and yeast were inoculated on 3 ml of
the above-described enrichment medium, filamentous fungi were
inoculated on 3 ml of enzyme inducible medium 1 (0.1% yeast
extract, 0.1% malt extract, 0.1% potassium dihydrogenphosphate,
0.05% magnesium sulfate, 0.1% fructosyl valyl histidine, pH 7.3)
and actinomycetes were inoculated on 3 ml of enzyme-induction media
2 (0.2% dry yeast, 1.25% soybean powder, 2% fructosyl valyl
histidine). Soil-derived microorganisms were grown by shake-culture
for 24 hours while yeast, filamentous fungi and actinomycetes were
grown by shake-culture for 3-5 days at 30.degree. C. Each culture
solution was centrifuged at 3,000 rpm for 10 minutes to obtain
cells. Next, cells were suspended in a lysis buffer (100 mM
phosphate buffer (pH 8), 1 mM EDTA, 1 mg/ml lysozyme, 0.5 mM PMSF),
disrupted by, for example, ultrasonication (1-3 minutes) or using a
Physcotron (50 power, 30 seconds, twice, available from Microtec
Nition Co., Ltd.), added with Triton X-100 to a final concentration
of 0.5%, and centrifuged at 15,000 rpm, 4.degree. C. for 10 minutes
to collect supernatant as crude enzyme solution. Each of the crude
enzyme solutions obtained was examined for the presence or absence
of fructosyl peptide oxidase activity by the above-described
activity assay to select 19 strains with the same activity, all of
which were filamentous fungi. Particularly, these filamentous fungi
included Achaetomiella (1 strain), Achaetomium (8 strains),
Thielavia (1 strain), Chaetomium (2 strains), Gelasinospora (1
strain), Microascus (1 strain), Coniochaeta (1 strain), and
Eupenicillium (4 strains).
Example 1
Preparing the Inventive Oxidase Produced by the Filamentous Fungus
Belonging to the Genus Achaetomiella
[0153] Achaetomiella virescens ATCC 32393 was inoculated on 0.05 L
of a medium (0.4% yeast extract, 1% malt extract, 2% glucose, 0.1%
tryptone, 0.1% potassium dihydrogenphosphate, 0.05% magnesium
sulfate, pH 7) contained in a 0.15 L Erlenmeyer flask, and grown by
rotary shaking culture at 120 rpm, 30.degree. C. for 3 days. Next,
the culture solution (seed) was dispensed (10 mL/flask) into 5 L
Erlenmeyer flasks (each containing 1 L of the above-described
medium) and grown by rotary shaking culture at 90 rpm, 30.degree.
C. for 4 days. Cells were collected from the culture solution using
a Buchner funnel with a filter. The cells obtained were frozen for
storage at -80.degree. C.
[0154] Frozen cells (collected from 6 L of culture solution) were
suspended in 1 L of buffer A (0.4M sodium chloride, 20 mM phosphate
buffer, 1 mM EDTA, 5% glycerol, 0.5 mM PMSF, pH 8), and disrupted
by a French press. Solution containing disrupted cells was
centrifuged at 9,000 rpm for 15 minutes, and supernatant was loaded
on a DEAE Sepharose FF (available from Amersham Biotech) column (5
cm.times.18 cm) pre-equilibrated with buffer A. Additionally, 500
ml of buffer A was added, and the whole elution solution was
collected. The elution solution was added slowly with ammonium
sulfate to 40% saturation to precipitate an excess amount of
protein. The solution was left to stand at 4.degree. C. overnight,
and then centrifuged at 9,000 rpm, 4.degree. C. for 15 minutes.
Next, ammonium sulfate was added slowly to supernatant to 65%
saturation to precipitate the protein of interest. The supernatant
solution was left to stand at 4.degree. C. overnight, and
centrifuged at 9,000 rpm, 4.degree. C. for 15 minutes to collect
precipitant.
[0155] The precipitant was dissolved in 30 ml of buffer B (10 mM
Tris-HCl, 0.2 mM EDTA, 1% glycerol, pH 8.6), desalted with PD-10
(available from Amersham Biotech), and then applied to a Q
Sepharose FF (available from Amersham Biotech) column (2.5
cm.times.15 cm) pre-equilibrated with buffer B. The gel was washed
with 150 ml of buffer B and eluted with a linear gradient from
buffer B to buffer C (150 mM sodium chloride, 50 mM Tris-HCl, 1 mM
EDTA, 5% glycerol, pH 8.6). Active fraction was eluted at about
0.08M sodium chloride.
[0156] The eluted active fraction was concentrated in CentriPrep 10
(available from Amicon), dialyzed, and loaded on TSK gel super Q
(available from Tosoh Co., Ltd.). Elution was performed using a
linear gradient from buffer B to buffer C. Activity was monitored
at 280 nm using a flow rate of 1 ml/min. Active fraction was eluted
at about 0.08M sodium chloride.
[0157] The active fraction obtained was concentrated in Microcon 10
(available from Amicon), and loaded on a POROS PE (available from
Perceptive Biosystems). Elution was performed using a linear
gradient from buffer D (2M ammonium sulfate, 20 mM phosphate
buffer, 1 mM EDTA, 5% glycerol, pH 7) to buffer E (20 mM phosphate
buffer, 1 mM EDTA, 5% glycerol, pH 8). Activity was monitored at
280 nm using a flow rate of 2 ml/min. Active fraction was eluted at
about 1M ammonium sulfate. The active fraction obtained was
analyzed by SDS-PAGE to obtain a single band (molecular
weight=about 52,000). The active fraction obtained was used to
determine the following physiochemical properties.
Example 2
Physiochemical Properties of the Inventive Oxidase Produced by
Filamentous Fungus Belonging to the Genus Achaetomiella
[0158] The physiochemical properties of the inventive oxidase
obtained in Example 1 will be described below.
(a) Activity and Substrate Specificity
[0159] The activity of the inventive oxidase was assayed by the
above-described enzyme activity assay using, as substrate,
fructosyl valyl histidine, fructosyl glycine or .epsilon.-fructosyl
lysine. The inventive oxidase had 42% relative activity for
fructosyl valyl histidine and 18% for fructosyl glycine when
compared to 100% activity for .epsilon.-fructosyl lysine.
(b) Optimum pH
[0160] Enzyme reaction was monitored in 200 mM acetic acid buffer
(pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH
6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0) or 200 mM
glycine buffer (pH 8.0-12.0) at the indicated pH values at
30.degree. C. The results are shown in FIG. 1. The inventive
oxidase exhibited its maximum activity (100%) at pH 8.0 and
relative activities of 70% or higher at pH 7.0-8.0. From these
results, the optimum pH for the inventive oxidase was determined to
be pH 7.0-8.0, and most preferably pH 8.0.
(c) Km Value for Fructosyl Valyl Histidine
[0161] In the above-described activity assay, the activity of the
oxidase was monitored using different concentrations of fructosyl
valyl histidine (substrate), and Michaelis constant (Km) was
determined from a Lineweaver-Burk plot. The Km value for fructosyl
valyl histidine was found to be 2.3 mM.
(d) Optimum Temperature Range
[0162] The activity of the inventive oxidase was assayed at
different temperatures using reactions which consisted of the same
compositions as those used in the above-described activity assay.
The results are shown in FIG. 3. The enzyme exhibited its maximum
activity (100%) at around 40.degree. C. and relative activities of
50% or higher at from 30 to 45.degree. C.
[0163] From these results, the optimum temperature range of the
inventive oxidase was determined to be 30-45.degree. C.
(e) Thermostability
[0164] The thermostability of the inventive oxidase following a
treatment with 200 mM potassium phosphate buffer (pH 8.0) at
different temperatures for 10 minutes is shown in FIG. 4 which
illustrates that the inventive oxidase remained stable up to about
50.degree. C.
(f) Stable pH Range
[0165] The inventive oxidase was treated with 200 mM acetic acid
buffer (pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris
buffer (pH 6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0)
or 200 mM glycine buffer (pH 8.0-12.0) at the indicated pH values
at 30.degree. C. for 10 minutes, and its remaining activity was
determined. The results are shown in FIG. 2. The inventive oxidase
exhibited its maximum activity (100%) at around pH 7.0 and
remaining activities of 70% or higher at pH 6.0-9.0.
[0166] From these results, the stable pH range for the inventive
oxidase was determined to be pH 6.0-9.0.
(g) Molecular Weight
[0167] Molecular weight was determined by SDS-PAGE on Multigel
10/20 (available from DAIICHI PURE CHEMICALS CO., LTD.). The
molecular weight of the inventive oxidase was determined to be
about 52,000.
(h) Identifying Reaction Product
[0168] Reaction solution was assayed by HPLC to identify reaction
products. First, 50 .mu.l of reaction solution (2 mM fructosyl
valyl histidine, 5 mM phosphate buffer (pH 8.0), 0.003 U of the
inventive oxidase) was incubated at 37.degree. C. for 2 hours,
diluted (10.times.) and then assayed for the reaction products in a
TSK gel Amide-80 column (available from Tosoh Co., Ltd.). As a
control, the same procedure was repeated except for using a buffer
instead of the enzyme. As a result, a peak was detected only for
fructosyl valyl histidine in the control case, while in the enzyme
case a peak was detected only for valyl histidine and no peak was
detected for fructosyl valyl histidine. From these results, it was
confirmed that the inventive oxidase catalyzes the decomposition of
fructosyl valyl histidine to produce valyl histidine. Further, it
was suggested that this reaction cleaved an .alpha.-ketoamine bond,
as in the glycated amino acid oxidase reaction.
Example 3
Comparison of Thermostability
[0169] The thermostability of the inventive oxidase produced by the
filamentous fungus belonging to the genus Achaetomiella was
compared to that of the oxidase disclosed in Japanese Patent
Application Laid-Open (kohyo) No. 2001-95598. E. coli strain
DH5.alpha. (pFP1) (FERM BP-7297) which produces the oxidase
disclosed in Japanese Patent Application Laid-Open (kohyo) No.
2001-95598 was inoculated on 10 ml of LB-amp medium (1%
bactotryptone, 0.5% bactoyeast extract, 0.5% sodium chloride, 50
.mu.g/mL ampicillin, pH 7), and grown by reciprocal shaking culture
at 120 rpm, 30.degree. C. for 20 hours. The resultant culture
solution was centrifuged at 12,000 rpm for 10 minutes to collect
cells which were then suspended in 10 ml of lysis buffer (50 mM
phosphate buffer, 1 mM EDTA, 5% glycerol, 0.5 mM PMSF, pH 8), and
then disrupted by ultrasonication. The suspension containing
disrupted cells was centrifuged at 12,000 rpm for 10 minutes and
supernatant obtained was used as crude enzyme solution. The crude
enzyme solution was treated with 200 mM potassium phosphate buffer
(pH 8.0) at 45.degree. C. for 10 minutes, and then assayed for its
activity using fructosyl valyl histidine as the substrate. No
activity was detected. These results show that the oxidase
disclosed in Japanese Patent Application Laid-Open (kohyo) No.
2001-95598 exhibited a low thermostability, which is
disadvantageous in that it may not be stable during storage when it
is formulated into a reagent (an enzyme) contained in a kit for
clinical diagnosis. On the other hand, the inventive oxidase
exhibited, as described above, an extremely high thermostability
with 80% or higher activity following a heat treatment at
45.degree. C. for 10 minutes.
Example 4
The Inventive Oxidases Produced by Filamentous Fungi
[0170] Among the filamentous fungi obtained in the search described
in Experimental Example 1 above, the above-listed filamentous
fungi, Achaetomiella (1 strain), Achaetomium (8 strains), Thielavia
(1 strain), Chaetomium (2 strains), Gelasinospora (1 strain),
Microascus (1 strain), Coniochaeta (1 strain) and Eupenicillium (1
strain) were used to produce the inventive oxidases. Next, the
physiochemical properties of these oxidases were determined. The
results are shown in Tables 1 and 2. Each of these 16 strains was
cultured on 3 ml of the above-described enzyme inducible medium 1
at 30.degree. C. for 4 days and cells were then collected. The
cells collected were suspended in 0.9 ml of lysis buffer, disrupted
by using a Physcotron and by ultrasonication, added with Triton
X-100 to a final concentration of 0.5%, and then centrifuged at
15,000 rpm, 4.degree. C. for 10 minutes to collect supernatant
which was then used as crude enzyme sample. Each of the crude
enzyme samples obtained was examined for its activity for fructosyl
valyl histidine (FVH), fructosyl glycine (FG) or
.epsilon.-fructosyl lysine (.epsilon.FL). For comparison,
activities of each enzyme are shown as % relative activities when
compared to its activity for .epsilon.-fructosyl lysine (100%).
Each crude enzyme sample was heat-treated at 45.degree. C. for 10
minutes, and then its activity for fructosyl valyl histidine was
determined and compared to the activity before treatment. As shown
in Tables 1 and 2, all of these sample strains showed activities on
fructosyl valyl histidine though with different levels of activity
per medium. Although these samples obtained from different strains
exhibited slightly different substrate specificities, the inventive
oxidases produced by Eupenicillium terrenum ATCC 18547 and
Coniochaeta sp. NISL 9330 (FERM BP-7798) exhibited particularly
preferable properties in that they acted well on fructosyl valyl
histidine but less on .epsilon.-fructosyl lysine. It was found that
the inventive oxidases produced by Achaetomiella virescens ATCC
32393 and Chaetomium sp. NISL 9335 (FERM BP-7799) exhibit a
relatively strong action on fructosyl valyl histidine. It was also
shown that under the above-described heat-treatment conditions, the
inventive oxidase samples produced by 12 out of 16 strains
exhibited remaining activities of 100% or higher while those
produced by the remaining 4 strains exhibited 80-100% remaining
activities, which indicated that all of the oxidase samples
produced by those 16 strains had extremely high stability.
TABLE-US-00001 TABLE 1 Activity per Substrate specificity Thermo-
medium (% relative activity) stability Strain (U/L) FVH FG
.epsilon.FL (%) Thielavia novoquineensis 0.9 17 10 100 100 NISL
9334 Chaetomium quatrangulatum 1.0 8 15 100 107 NISL 9329
Achaetomium luteum 1.6 39 22 100 100 ATCC 18524 Achaetomium
strumarium 1.1 26 21 100 100 NISL 9324 Achaetomium globosum 4.1 18
10 100 104 NISL 9321 Achaetomium luteum 7.2 38 18 100 108 NISL 9323
Gelasinospora 1.2 17 14 100 102 pseudoreticulata NISL 9332
TABLE-US-00002 TABLE 2 Activity per Substrate specificity Thermo-
medium (% relative activity) stability Stain (U/L) FVH FG
.epsilon.FL (%) Achaetomiella virescens 11.6 71 25 100 88 ATCC
32393 Achaetomium strumarium 20.9 42 24 100 107 NISL 9325
Achaetomium strumarium 6.0 15 6 100 102 NISL 9326 Achaetomium sp.
3.2 26 21 100 104 NISL 9327 Chaetomium sp. 13.2 69 88 100 100 NISL
9335 Eupenicillium terrenum 2.8 1023 1862 100 100 ATCC 18547
Microascus sp. 19.1 27 37 100 94 NISL 9333 Achaetomium sp. 19.4 38
21 100 80 NISL 9328 Coniochaeta sp. 70.1 165 65 100 80 NISL
9330
Example 5
Preparing the Inventive Oxidases Produced by Linear Fungi Belonging
to the Genera Chaetomium and Coniochaeta
[0171] Chaetomium sp. NISL 9335 (FERM BP-7799) and Coniochaeta sp.
NISL 9330 (FERM BP-7798) cells were cultured, and the inventive
oxidases were purified therefrom. Both oxidases were obtained using
the same purification procedure.
[0172] Each of the oxidases obtained was inoculated on 3 ml of a
medium (0.4% yeast extract, 1% malt extract, 2% glucose, 0.1%
tryptone, 0.1% potassium dihydrogenphosphate, 0.05% magnesium
sulfate, pH 7) contained in a glass tube (1.6 cm
(diameter).times.12.5 cm), and grown by reciprocal shaking culture
at 120 rpm, 30.degree. C. for 1 day. Then, 3 ml of the culture
(seed) was dispensed (3 ml/flask) into 1 L Erlenmeyer flasks each
containing 0.4 L of the above-described medium, and grown by rotary
shaking culture at 130 rpm, 30.degree. C. for 4 days. Cells were
collected from the culture solution by filtration using a Buchner
funnel with a filter or by centrifugation at 12,000 rpm for 10
minutes. The cells obtained were frozen for storage at -80.degree.
C.
[0173] Frozen cells (collected from 0.4 L of culture solution) were
suspended in 0.025 L of buffer A, and disrupted by a French press.
The suspension containing disrupted cells was centrifuged at 12,000
rpm for 10 minutes, and ammonium sulfate was added slowly to the
supernatant to 65% saturation to precipitate the protein of
interest. The supernatant solution was left to stand at 4.degree.
C. overnight, and centrifuged at 12,000 rpm for 10 minutes to
collect precipitant.
[0174] The precipitant was then dissolved in 5 ml of buffer D and
centrifuged at 15,000 rpm for 10 minutes to give a supernatant
which was then subjected to POROS PE. Elution was performed using a
linear gradient from buffer D to buffer E. Activity was monitored
at 280 nm using a flow rate of 2 ml/min. Active fractions were
eluted at about 0.25 M ammonium sulfate for both of the enzymes.
Both of the active fractions obtained were concentrated and
demineralized in Microcon 10 (available from Amicon) and used to
determine their physiochemical properties as described below.
Example 6
The Physiochemical Properties of the Inventive Oxidase Produced by
Chaetomium sp
[0175] The physiochemical properties of the inventive oxidase
produced by Chaetomium sp. NISL 9335 (FERM BP-7799) obtained in
Example 5 will be described below.
(a) Activity and Substrate Specificity
[0176] The activity of the inventive oxidase was assayed by the
above-described enzyme activity assay using, as substrate,
fructosyl valyl histidine, fructosyl glycine or .epsilon.-fructosyl
lysine. The inventive oxidase exhibited 40% relative activity for
fructosyl valyl histidine and 28% for fructosyl glycine when
compared to 100% activity for .epsilon.-fructosyl lysine.
(b) Optimum pH
[0177] Enzyme reaction was monitored in 200 mM acetic acid buffer
(pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH
6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0) or 200 mM
glycine buffer (pH 8.0-12.0) at the indicated pH values at
30.degree. C. The results are shown in FIG. 5. The inventive
oxidase exhibited its maximum activity at pH 8.0. It still
exhibited relative activities of 70% or higher at pH 6.0-8.0 when
compared to the maximum activity (100%) at around pH 8.0. From
these results, the optimum pH for the inventive oxidase was
determined to be pH 6.0-8.0, and most preferably pH 8.0.
(c) Optimum Temperature Range
[0178] The activity of the inventive oxidase was assayed at
different temperatures using reaction solutions which consisted of
the same compositions as those used in the above-described activity
assay. The results are shown in FIG. 7. The enzyme exhibited its
maximum activity (100%) at around 37.degree. C. and relative
activities of 60% or higher at from 20 to 45.degree. C.
[0179] From these results, the optimum temperature range of the
inventive oxidase was determined to be from 20 to 45.degree. C.
(d) Thermostability
[0180] The inventive oxidase was treated with 200 mM potassium
phosphate buffer (pH 8.0) at different temperatures for 10 minutes
and the thermostability thereof was determined. The results are
shown in FIG. 8 which illustrates that the inventive oxidase
remained stable up to about 55.degree. C.
(e) Stable pH Range
[0181] The inventive oxidase was treated with 200 mM acetic acid
buffer (pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris
buffer (pH 6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0)
or 200 mM glycine buffer (pH 8.0-12.0) at the indicated pH values
at 30.degree. C. for 10 minutes, and the remaining activity thereof
was determined. The results are shown in FIG. 6. The inventive
oxidase exhibited its maximum activity at around pH 7.0 and
remaining activities of 70% or higher at pH 5.0-9.0.
[0182] From these results, the stable pH range for the inventive
oxidase was determined to be pH 5.0-9.0.
Example 7
The Physiochemical Properties of the Inventive Oxidase Produced by
Coniochaeta sp. which has Less Action on .epsilon.-Fructosyl
Lysine
[0183] The physiochemical properties of the inventive oxidase
produced by Coniochaeta sp. NISL 9330 (FERM BP-7798) obtained in
Example 5 will be described below.
(a) Activity and Substrate Specificity
[0184] The activity of the inventive oxidase was assayed by the
above-described enzyme activity assay using, as substrate,
fructosyl valyl histidine, fructosyl glycine or .epsilon.-fructosyl
lysine. The inventive oxidase exhibited 61% relative activity for
.epsilon.-fructosyl lysine and 39% for fructosyl glycine when
compared to 100% activity for fructosyl valyl histidine, which
shows that the enzyme had less action on .epsilon.-fructosyl
lysine.
(b) Optimum pH
[0185] Enzyme reaction was monitored in 200 mM acetic acid buffer
(pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH
6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0) or 200 mM
glycine buffer (pH 8.0-12.0) at the indicated pH values at
30.degree. C. The results are shown in FIG. 9. The inventive
oxidase exhibited its maximum activity at pH 7.0. It still
exhibited relative activities of 70% or higher at pH 6.0-8.0 when
compared to the maximum activity (100%) obtained at around pH 7.0.
From these results, the optimum pH for the inventive oxidase was
determined to be pH 6.0-8.0, and most preferably pH 7.0.
(c) Optimum Temperature Range
[0186] The activity of the inventive oxidase was assayed at
different temperatures using reaction solutions which consisted of
the same compositions as those used in the above-described activity
assay. The results are shown in FIG. 11. The enzyme exhibited its
maximum activity (100%) at around 40.degree. C. and relative
activities of 60% or higher at 20-40.degree. C.
[0187] From these results, the optimum temperature range of the
inventive oxidase was determined to be from 20 to 40.degree. C.
(d) Thermostability
[0188] The inventive oxidase was treated with 200 mM potassium
phosphate buffer (pH 8.0) at different temperatures for 10 minutes
and the thermostability thereof was determined. The results are
shown in FIG. 12 which illustrates that the inventive oxidase
remained stable up to about 50.degree. C.
(e) Stable pH Range
[0189] The inventive oxidase was treated with 200 mM acetic acid
buffer (pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris
buffer (pH 6.8-9.0), 200 mM potassium phosphate buffer (pH 6.5-8.0)
or 200 mM glycine buffer (pH 8.0-12.0) at the indicated pH values
at 30.degree. C. for 10 minutes, and the remaining activity thereof
was determined. The results are shown in FIG. 10. The inventive
oxidase exhibited its maximum remaining activity at around pH 7.0
and remaining activities of 70% or higher at pH 5.0-9.0.
[0190] From these results, the stable pH range for the inventive
oxidase was determined to be pH 5.0-9.0.
Example 8
Preparing the Inventive Oxidase Produced by Eupenicillium terrenum
which has Less Action on .epsilon.-Fructosyl Lysine
[0191] Eupenicillium terrenum ATCC 18547 cells were inoculated on
0.05 L of a medium (0.1% yeast extract, 0.1% malt extract, 0.1%
potassium dihydrogenphosphate, 0.05% magnesium sulfate, pH 7.3)
contained in a 0.15 L Erlenmeyer flask, and grown by rotary shaking
culture at 120 rpm, 25.degree. C. for 3 days. Next, the culture
(seed) was dispensed (10 mL/flask) into 5 L Erlenmeyer flasks (each
containing 1 L of the above-described medium) and grown by rotary
shaking culture at 100 rpm, 25.degree. C. for 4 days. Cells were
collected from the culture solution by filtration using a Buchner
funnel with a filter. The cells obtained were then frozen for
storage at -80.degree. C.
[0192] Frozen cells (collected from 6 L of culture solution) were
suspended in 500 mL of buffer F (10 mM phosphate buffer, 1 mM EDTA,
5% glycerol, 0.5 mM PMSF, pH 8), and disrupted by a French press.
The suspension containing disrupted cells was centrifuged at 9,000
rpm for 15 minutes, and buffer F was added to the supernatant to
give a crude enzyme solution (500 mL). Ammonium sulfate was then
added slowly to the crude enzyme solution to 40% saturation to
precipitate an excess amount of protein. The solution was then left
to stand at 4.degree. C. overnight and then centrifuged at 9,000
rpm, 4.degree. C. for 15 minutes to collect supernatant. Further,
ammonium sulfate was added slowly to the supernatant to 60%
saturation to precipitate protein. The supernatant solution was
left to stand at 4.degree. C. overnight, followed by centrifugation
(9,000 rpm, 4.degree. C., 15 minutes) to collect precipitant.
[0193] The precipitant was dissolved in 10 ml of buffer G (10 mM
phosphate buffer, 1 mM EDTA, 5% glycerol, 0.2M NaCl, pH 8) followed
by buffer replacement with PD-10 (available from Amersham Biotech),
and then applied to an UltrogelAcA34 (available from IBF
Biotechnics) column (2.8 cm.times.85 cm) pre-equilibrated with
buffer G. The column was eluted with 1 L of buffer G to collect
active fraction.
[0194] The active fraction obtained was concentrated in CentriPrep
10 (available from Amicon) followed by buffer replacement with
buffer F, and then applied to a Q Sepharose FF (available from
Amersham Biotech) column (1.0 cm.times.8 cm).
[0195] Elution was performed using a linear gradient from buffer H
(10 mM phosphate buffer, 1 mM EDTA, 5% glycerol, pH 8) to buffer I
(10 mM phosphate buffer, 1 mM EDTA, 5% glycerol, 0.5 M NaCl, pH
8).
[0196] The active fraction obtained was again concentrated in
CentriPrep 10 (available from Amicon) and then applied to POROS PE
(available from Perceptive Biosystems). Elution was performed using
a linear gradient from buffer D to buffer E. Protein amount was
monitored at O.D. 280 nm using a flow rate of 2 ml/min. Active
fraction was eluted at about 0.01 M ammonium sulfate. Obtained
active fraction was analyzed by SDS-PAGE to confirm a single band
(molecular weight=about 53 kDa). The active fraction obtained was
used to determine its physiochemical properties as described
below.
Example 9
The Physiochemical Properties of the Inventive Oxidase Produced by
Eupenicillium terrenum which has Less Action on .epsilon.-Fructosyl
Lysine
[0197] The physiochemical properties of the inventive oxidase
produced by Eupenicillium terrenum ATCC 18547 which has less action
on .epsilon.-fructosyl lysine were as described below.
(a) Activity and Substrate Specificity
[0198] The activity of the inventive oxidase was assayed by the
above-described enzyme activity assay using, as substrate,
fructosyl valyl histidine, fructosyl glycine or .epsilon.-fructosyl
lysine. The inventive oxidase with less action on
.epsilon.-fructosyl lysine exhibited 182% relative activity for
fructosyl glycine and 9.78% for .epsilon.-fructosyl lysine when
compared to 100% activity for fructosyl valyl histidine, indicating
that the inventive oxidase had high specificity for fructosyl valyl
histidine and fructosyl glycine.
(b) Optimum pH
[0199] Enzyme reaction was monitored in 200 mM acetic acid buffer
(pH 4.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris buffer (pH
7.0-8.5), 200 mM potassium phosphate buffer (pH 6.0-8.0) or 200 mM
glycine buffer (pH 8.0-9.0) at the indicated pH values at
30.degree. C. The results are shown in FIG. 13. The inventive
oxidase with less action on .epsilon.-fructosyl lysine exhibited
its maximum activity in potassium phosphate buffer at pH 7.0. It
still exhibited relative activities of 70% or higher in the same
buffer at pH 6.0-8.0 when compared to the maximum activity (100%)
at around pH 7.0. From these results, the optimum pH for the
inventive oxidase with less action on .epsilon.-fructosyl lysine
was determined to be pH 6.0-8.0, and most preferably pH 7.0.
(c) Km Value for Fructosyl Valyl Histidine
[0200] In the above-described activity assay, activity was
monitored using different concentrations of fructosyl valyl
histidine (substrate), and Michaelis constant (Km) was determined
from a Lineweaver-Burk plot. The Km value of the enzyme for
fructosyl valyl histidine was found to be 4.25 mM.
(d) Optimum Temperature Range
[0201] The activity of the inventive oxidase was assayed at
different temperatures using reaction solutions which consisted of
the same compositions as those used in the above-described activity
assay. The results are shown in FIG. 15. The enzyme exhibited its
maximum activity (100%) at around 35.degree. C. and relative
activities of 60% or higher at from 25 to 40.degree. C.
[0202] From these results, the optimum temperature range of the
inventive oxidase with less action on .epsilon.-fructosyl lysine
was determined to be 25-40.degree. C.
(e) Thermostability
[0203] The inventive oxidase was treated with 200 mM potassium
phosphate buffer (pH 8.0) at different temperatures for 10 minutes
and the remaining activity thereof was monitored. The results are
shown in FIG. 16 which illustrates that the inventive oxidase with
less action on .epsilon.-fructosyl lysine exhibited high stability,
with approximately 100% remaining activity up to about 45.degree.
C.
(f) Stable pH Range
[0204] The inventive oxidase was treated with 200 mM acetic acid
buffer (pH 3.0-6.0), 200 mM MES-NaOH (pH 6.0-7.0), 200 mM Tris
buffer (pH 7.0-8.5), 200 mM potassium phosphate buffer (pH 6.0-8.0)
or 200 mM glycine buffer (pH 8.0-12.0) at the indicated pH values
at 30.degree. C. for 10 minutes, and the remaining activity thereof
was monitored. The results are shown in FIG. 14. The inventive
oxidase exhibited its maximum activity in potassium phosphate
buffer at pH 8.0 and remaining activities of 60% or higher at pH
6.0-9.0.
[0205] From these results, the stable pH range for the inventive
oxidase with less action on .epsilon.-fructosyl lysine was
determined to be pH 6.0-9.0.
(g) Molecular Weight
[0206] Molecular weight was determined by SDS-PAGE using Multigel
10/20 (available from DAIICHI PURE CHEMICALS CO., LTD.). The
molecular weight of inventive oxidase with less action on
.epsilon.-fructosyl lysine was about 53,000.
(h) Identifying Reaction Product
[0207] Reaction solution was assayed by HPLC to identify reaction
products. First, 50 .mu.l of reaction solution (2 mM fructosyl
valyl histidine, 5 mM phosphate buffer (pH 8.0), 0.003 U of the
inventive oxidase) was incubated at 37.degree. C. for 2 hours,
diluted (10.times.) and then assayed for the reaction products in a
TSK gel Amide-80 column (available from Tosoh Co., Ltd.).
Similarly, a control sample was obtained by using a buffer instead
of the enzyme and assayed. As a result, a peak was detected only
for fructosyl valyl histidine in the control sample, while in the
enzyme sample a peak was detected only for valyl histidine with no
peak detected for fructosyl valyl histidine. From these results, it
was confirmed that the inventive oxidase catalyzes the
decomposition of fructosyl valyl histidine to produce valyl
histidine. Further, it was suggested that this reaction cleaved an
.alpha.-ketoamine bond, as in the glycated amino acid oxidase
reaction.
Example 10
The Inventive Oxidases Produced by Filamentous Fungi Belonging to
the Genus Eupenicillium which have Less Action on
.epsilon.-Fructosyl Lysine
[0208] Among the inventive filamentous fungi selected in
Experimental Example 1, in addition to Eupenicillium terrenum ATCC
18547, three strains of filamentous fungi belonging to the genus
Eupenicillium were obtained which produce the inventive oxidases
with less action on .epsilon.-fructosyl lysine. These filamentous
fungi belonging to the genus Eupenicillium (i.e., Eupenicillium
terrenum ATCC 18547, Eupenicillium senticosum IFO 9158,
Eupenicillium idahoense IFO 9510, and Eupenicillium euglaucum IFO
31729) were used to prepare the inventive oxidases with less action
on .epsilon.-fructosyl lysine. The physiochemical properties of the
oxidases obtained were then assayed. The results are shown in Table
3.
[0209] Each of the above-described 4 strains was cultured on the
above-described enzyme induction media 1 (3 ml) at 30.degree. C.
for 4 days and then cells were collected. The cells obtained were
suspended in 0.9 ml of a lysis buffer, disrupted by using a
Physcotron and by ultrasonication, added with Triton X-100 to a
final concentration of 0.5%, and centrifuged at 15,000 rpm,
4.degree. C. for 10 minutes to collect supernatant (crude enzyme
sample). Each of the crude enzyme samples obtained was assayed for
its activity for fructosyl valyl histidine (FVH), fructosyl glycine
(FG) or .epsilon.-fructosyl lysine (.epsilon.FL). The activities
were indicated as % relative activities when compared to the
activity for fructosyl valyl histidine (100%). Moreover, each crude
enzyme sample was heat-treated at 45.degree. C. for 10 minutes and
its activity for fructosyl valyl histidine was assayed. The results
were compared to the activity obtained before heat-treatment. As
shown in Table 3, all the strain samples tested acted on fructosyl
valyl histidine and fructosyl glycine, although the level of
activity per medium was different depending on the strains.
Particularly, two out of the four stains exhibited 2-fold or higher
activities, one exhibited 3-fold or higher activity, and the
remaining one exhibited even 10-fold or higher activity for
fructosyl valyl histidine when compared to their activities for
.epsilon.-fructosyl lysine. Under the above-described
heat-treatment conditions, all of the inventive oxidase samples
obtained from the 4 strains exhibited remaining activities of 90%
or higher, indicating that they are very stable.
TABLE-US-00003 TABLE 3 Activity per Substrate specificity Thermo-
medium (% Relative activity) stability Strain (U/L) FVH FG
.epsilon.FL (%) Eupenicillium terrenum 2.8 100 182 9.78 100 ATCC
18547 Eupenicillium senticosum 0.52 100 83.1 30.8 92.9 IFO 9158
Eupenicillium idahoense 0.25 100 86.1 41.0 90.0 IFO 9510
Eupenicillium euglaucum 0.095 100 83.3 38.9 116 IFO 31729
Example 11
Reaction of the Inventive Oxidase with Products Obtained by
Treatment of Glycated Peptide with Protease
[0210] Glycated hemoglobin (HbA.sub.1c) may be treated with
endoproteinase Glu-C to liberate .alpha.-glycated hexapeptide
(fructosyl Val-His-Leu-Thr-Pro-Glu) from the glycated hemoglobin
.beta.-chain (Clin. Chem., 43; 10 1944-1951, 1997). Fructosyl
Val-His-Leu-Thr-Pro-Glu (available from PEPTIDE INSTITUTE, INC),
which is the same substance as the .alpha.-glycated hexapeptide,
was used in the following experiment.
(1) Preparation of Reagents
[0211] (A) 20 mM .alpha.-glycated hexapeptide
[0212] .alpha.-glycated hexapeptide (fructosyl
Val-His-Leu-Thr-Pro-Glu, MW=856, available from PEPTIDE INSTITUTE,
INC) (3.434 mg) was dissolved in 0.2 ml of water.
(B) Protease
[0213] Molsin (15 mg, available from Kikkoman Corp.) was dissolved
in 0.2 ml of buffer (10 mM acetate buffer, pH 3.0)
(C) 0.2 M acetate buffer, pH 3.0 (D) Reaction substrate (a)
Products obtained by digesting .alpha.-glycated hexapeptide with
protease
[0214] A (100 .mu.l), B (5 .mu.l) and C (5 .mu.l) were mixed in a
microtube and incubated at 37.degree. C. for 17 hours. The reaction
solution was then filtered using Microcon 10 (available from
Amicon) to remove protease.
(b) 20 mM fructosyl glycine (control)
[0215] Fructosyl glycine (4.5 mg) was dissolved in 1 ml of
water.
(E) Developer solution
[0216] Developer solution consisted of the following materials:
[0217] 5 .mu.l of 10 mg/ml 4-aminoantipyrine;
[0218] 7.5 .mu.l of 2% 2,4-dichlorophenol sulfate;
[0219] 1 .mu.l of 3300 U/ml peroxidase;
[0220] 100 .mu.l of 1M phosphate buffer (pH 8); and
[0221] 286.5 .mu.l of water.
(F) The inventive oxidases
[0222] (a) Enzyme purified from Achaetomiella virescens ATCC
32393
[0223] (b) Enzyme purified from Eupenicillium terrenum ATCC
18547
[0224] (c) Enzyme purified from Coniochaeta sp. NISL 9330 (FERM
BP-7798)
[0225] (d) 20 mM phosphate buffer, pH 8.0
(2) Reaction Assay
[0226] Reaction substrate (D: a, b) (20 .mu.l), developer solution
(E) (20 .mu.l) and the inventive oxidase (F: a-d) (10 .mu.l) were
mixed in 96-well assay plates for reaction. Each plate was
monitored at 510 nm using a plate reader (Immuno Mini NJ-2300,
available from Nalge Nunc International K. K.) for 70 minutes. The
results are shown as % relative activity when compared to 100%
activity for fructosyl glycine (Table 4). N. D. indicates that
color development was undetectable. It was confirmed that all of
the inventive oxidases were reactive with the protease-digested
products of .alpha.-glycated hexapeptide.
TABLE-US-00004 TABLE 4 Achaetomiella Eupenicillium Coniochaeta
virescens terrenum sp. Phosphate ATCC 32393 ATCC 18547 NISL 9330
buffer .alpha.-glycated 9.2% 9.5% 10.0% N.D. hexapeptide fructosyl
100% 100% 100% N.D. glycine
Example 12
Cloning and Expression of the Inventive Gene (Derived from
Coniochaeta sp.) by Using Transformant
[0227] (1) Preparation of Coniochaeta sp. mRNA
[0228] Coniochaeta sp. was inoculated on 0.05 L of a medium (0.4%
yeast extract, 1% malt extract, 2% glucose, 0.1% tryptone, 0.1%
potassium dihydrogenphosphate, 0.05% magnesium sulfate, pH 7)
contained in a 0.15 L Erlenmeyer flask, and grown by rotary shaking
culture at 120 rpm, 30.degree. C. for 1 day. Next, the culture
(seed) was dispensed (1 ml/flask) into 1 L Erlenmeyer flasks (each
containing 0.5 L of the above-described medium) and grown by rotary
shaking culture at 120 rpm, 30.degree. C. for 2 days. The culture
solution was centrifuged at 12,000 rpm for 10 minutes to collect
cells. The cells obtained were disrupted in a mortar with a pestle
in the presence of liquid nitrogen, suspended in 10 ml of RNA
extraction reagent ISOGEN (available from Wako Pure Chemical
Industries, Ltd.), and centrifuged at 2,700 rpm for 5 minutes to
obtain RNA fraction, from which mRNA (0.51 mg) was obtained
according to the method described in Current Protocols in Molecular
Biology (WILEY, Interscience, 1989).
(2) Synthesis of Primers
[0229] Fructosyl peptide oxidase (about 10 .mu.g) purified
according to the method described in Example 5 was digested with
trypsin and subjected to preparative HPLC to obtain 7 peptides. The
7 peptides obtained were read on an ABI 470A protein sequencer
(available from Perkin-Elmer Corp.) to determine the internal amino
acid sequences. The sequences determined were used to synthesize
primers shown in SEQ ID NOS: 5 and 6 utilizing Amersham Biotech
Custom Synthesis Service.
(3) RT-PCR
[0230] A first reaction solution was prepared using the following
materials:
TABLE-US-00005 Mg Cl.sub.2 5 mM; *10x RNA PCR buffer 2 .mu.l;
H.sub.2O 8.5 .mu.l; dNTPs 1 mM each; RNase inhibitor 1 U/.mu.l;
*AMV reverse transcriptase XL 0.25 U/.mu.l; *oligo dT adapter
primer 0.125 .mu.M; and mRNA 1 .mu.g. Note) *available from Takara
Bio Inc.
[0231] The first reaction solution was left to stand at 42.degree.
C. for 30 minutes for reverse transcription reaction, denatured at
99.degree. C. for 5 minutes and then stored at 5.degree. C.
[0232] Next, a second reaction solution was prepared using the
following materials:
TABLE-US-00006 Primer (SEQ ID NO: 5) 0.2 .mu.M; Primer (SEQ ID NO:
6) 0.2 .mu.M; *10x RNA PCR buffer 8 .mu.l; Magnesium chloride 2.5
mM; *Taq polymerase 2.5 U; and H.sub.2O (to a final total volume of
80 .mu.l). Note) *available from Takara Bio Inc.
[0233] Then, 80 .mu.l of the second reaction solution was added to
the tube containing reverse-transcription, and 30 cycles of PCR
amplification were performed. The amplification cycle consisted of
denaturing at 94.degree. C. for 30 seconds, annealing at 62.degree.
C. for 30 seconds and extension at 72.degree. C. for 1.5
minutes.
[0234] After completion of PCR reaction, the reaction solution was
subjected to electrophoresis on agarose gel to confirm a band at
approximately 0.77 kb, which seemed to be the fragment of interest.
The region of the gel containing the band was excised and DNA
fragment was purified in GENECLEAN II (available from BIO 101
Inc.
(4) Analysis of Purified DNA Fragment
[0235] Purified DNA fragment was sequenced and analyzed using a
370A DNA sequencing system (available from Perkin-Elmer Corp.) to
confirm the presence of the above-described amino acid sequence
(Leu-Ser-Lys-Met-Pro-Leu-Leu-Gln-Arg) in the putative amino acid
sequence deduced from the nucleotide sequence determined. From
these results, it was confirmed that the DNA fragment obtained by
the above-described RT-PCR amplification contained a portion of the
genetic sequence encoding the fructosyl peptide oxidase derived
from Coniochaeta sp.
(5) Analysis of the Downstream Region by 3'-RACE
[0236] First, a primer (SEQ ID NO: 7) was designed using the DNA
sequence data obtained as described above and synthesized utilizing
Amersham Biotech Custom Synthesis Service. This primer, the
above-described mRNA and 3'-Full RACE Core Set (available from
Takara Bio Inc.) were used to perform RT-PCR to amplify the
3'-unknown region. The reaction solution was subjected to
electrophoresis on agarose gel followed by purification and
extraction of an about 500 bp DNA fragment using Reco Chip
(available from Takara Bio Inc.). The DNA fragment purified was
then sequenced and analyzed in a DNA sequencer to confirm the
presence of the same sequence as the 3' sequence of a portion of
the genetic sequence encoding the fructosyl peptide oxidase derived
from Coniochaeta sp. in the 5'-region of the nucleotide sequence
determined. Further, the presence of the above-described amino acid
sequence (Phe-Gln-Asp-Lys-Glu-Leu-Phe-Asn-Arg) was confirmed in the
putative amino acid sequence deduced from the nucleotide sequence
determined.
(6) Analysis of the Upstream Region by 5'-RACE
[0237] First, primers (SEQ ID NOS: 8-12) were designed using the
DNA sequence data obtained as described above and synthesized
utilizing Amersham Biotech Custom Synthesis Service. These primers,
the above-described mRNA and 5'-Full RACE Core Set (available from
Takara Bio Inc.) were used to perform RT-PCR to amplify the
5'-unknown region. The reaction solution was subjected to
electrophoresis on agarose gel followed by purification and
extraction of an about 450 bp DNA fragment using Reco Chip
(available from Takara Bio Inc.). The DNA fragment purified was
then sequenced and analyzed in a DNA sequencer to confirm the
presence of the same sequence as the 5' sequence of a portion of
the genetic sequence encoding the fructosyl peptide oxidase derived
from Coniochaeta sp. in the 3'-region of the nucleotide sequence
determined. Further, the presence of the above-described amino acid
sequence (Ser-Gly-Tyr-Ala-Pro-Ala-Asn-Ile-Thr) was confirmed in the
putative amino acid sequence deduced from the nucleotide sequence
determined.
(7) Obtaining Genetic Fragments by RT-PCR
[0238] Translation start and stop codons were deduced from the
above-described 3 nucleotide sequences, and primer DNAs (SEQ ID
NOS: 13 and 14) for the N- and C-terminal regions were synthesized
utilizing Amersham Biotech Custom Synthesis Service. These primers
and the above-described mRNA were used to perform RT-PCR. The
reaction solution was subjected to electrophoresis on agarose gel
to confirm the presence of an approximately 1.3 kb band. Then, DNA
fragment contained in the band was purified and extracted using
Reco Chip (available from Takara Bio Inc.). Further, plasmid
pKK223-3 (available from Amersham) was digested with restriction
enzyme EcoRI, blunt-ended using BluntingKit (available from Takara
Bio Inc.) and ligated with the DNA fragment purified and extracted
as described above. The ligated DNA was then used to transform E.
coli JM 109. The plasmid pKK223-3-CFP obtained as described above
was deposited at the International Patent Organism Depositary, the
National Institute of Advanced Industrial Science and Technology,
an Independent Administrative Institution under the Ministry of
Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1,
Tsukuba, Ibaraki, Japan as FERM BP-8132.
(8) Confirmation of Activity
[0239] E. coli JM 109 (pKK223-3-CFP) cells were grown in 10 ml of
TY-medium containing 50 .mu.g/ml ampicillin (1% bactotryptone, 0.5%
bactoyeast extract, 0.5% NaCl, pH 7.0) by shake-culture at
32.degree. C. to Klett 100, added with IPTG to a final
concentration of 1 mM, and cultured for an additional 3 hours. The
culture solution was treated in an Ultrasonicgenerator (available
from Nissei) for 20 sec..times.4 times while cooling on ice. The
culture solution was loaded in a micro tube, and centrifuged in a
microcentrifuge at 12,000 rpm for 10 minutes to give supernatant
and precipitate fractions. The supernatant fraction obtained was
transferred to another micro tube, and fructosyl peptide oxidase
activity was determined according to the above-described enzyme
activity assay to find that JM109 (pKK 223-3-CFP) had 4.74 U/ml
fructosyl peptide oxidase activity.
(9) Analysis of Gene Encoding Fructosyl Peptide Oxidase
[0240] Since it was confirmed that E. coli JM109 (pKK223-3-CFP) had
fructosyl peptide oxidase activity, it became clear that the insert
fragment incorporated in pKK223-3-CFP contained the gene encoding
fructosyl peptide oxidase. Therefore, the nucleotide sequence of
the plasmid DNA was determined using a 370A DNA sequencing system
(available from Perkin-Elmer Corp.). The nucleotide sequence of the
plasmid DNA determined and the putative amino acid sequence of the
polypeptide which may be translated therefrom are shown in SEQ ID
NOS: 2 and 1, respectively. The gene encoding fructosyl peptide
oxidase had a 1314 bp coding region which encoded 437 amino
acids.
Example 13
Cloning and Expression of the Inventive Gene (Derived from
Eupenicillium terrenum) by Transformant
[0241] (1) Preparation of Eupenicillium terrenum mRNA
[0242] Eupenicillium terrenum ATCC 18547 was inoculated on 0.05 L
of a medium (0.1% yeast extract, 0.1% malt extract, 0.1% potassium
dihydrogenphosphate, 0.05% magnesium sulfate, pH 7.3) contained in
a 0.15 L Erlenmeyer flask, and grown by rotary shaking culture at
120 rpm, 25.degree. C. for 3 days. Next, the culture (seed) was
dispensed (1 ml/flask) into 1 L Erlenmeyer flasks (each containing
0.5 L of the above-described medium) and grown by rotary shaking
culture at 120 rpm, 25.degree. C. for 4 days. The culture solution
was centrifuged at 12,000 rpm for 10 minutes to collect cells. The
cells obtained were disrupted in a mortar with a pestle in the
presence of liquid nitrogen, suspended in 10 ml of RNA extraction
reagent ISOGEN (available from Wako Pure Chemical Industries,
Ltd.), and centrifuged at 2,700 rpm for 5 minutes to give RNA
fraction, from which mRNA was obtained according to the method
described in Current Protocols in Molecular Biology (WILEY,
Interscience, 1989).
(2) Synthesis of Primers
[0243] Fructosyl peptide oxidase (about 10 .mu.g) purified
according to the method described in Example 8 was digested with
trypsin and subjected to preparative HPLC to obtain 6 peptides. The
6 peptides obtained were read on an ABI 470A protein sequencer
(available from Perkin-Elmer Corp.) to determine the internal amino
acid sequences (Thr-Asn-Val-Trp-Leu-Glu-Ser-Glu,
Asp-Leu-Ala-Glu-Met-Pro-Gly, Asn-Phe-Ile-Leu-Ala,
Leu-Pro-Asn-Ile-Gly, x-Pro-Thr-Asp-x-Tyr-Pro,
Leu-His-Gln-Pro-Tyr-Gly-Ala-x-x-Pro). The sequences determined were
used to synthesize primers shown in SEQ ID NOS: 15 and 16 utilizing
Amersham Biotech Custom Synthesis Service.
(3) RT-PCR
[0244] A first reaction solution was prepared using the following
materials:
TABLE-US-00007 Mg Cl.sub.2 5 mM; *10x RNA PCR buffer 2 .mu.l;
H.sub.2O 8.5 .mu.l; dNTPs 1 mM each; RNase inhibitor 1 U/.mu.l;
*AMV reverse transcriptase XL 0.25 U/.mu.l; *oligo dT adapter
primer 0.125 .mu.M; and mRNA 1 .mu.g. Note) *available from Takara
Bio Inc.
[0245] The first reaction solution was left to stand at 42.degree.
C. for 30 minutes for reverse transcription reaction, denatured at
99.degree. C. for 5 minutes and then stored at 5.degree. C.
[0246] Next, a second reaction solution was prepared using the
following materials:
TABLE-US-00008 Primer (SEQ ID NO: 15) 0.2 .mu.M; Primer (SEQ ID NO:
16) 0.2 .mu.M; *10x RNA PCR buffer 8 .mu.l; Magnesium chloride 2.5
mM; *Taq polymerase 2.5 U; and H.sub.2O (to a final total volume of
80 .mu.l). Note) *available from Takara Bio Inc.
[0247] Then, 80 .mu.l of the second reaction solution was added to
the tube containing reverse-transcription, and 30 cycles of PCR
amplification were performed. The amplification cycle consisted of
denaturing at 94.degree. C. for 30 seconds, annealing at 62.degree.
C. for 30 seconds and extension at 72.degree. C. for 1.5
minutes.
[0248] After completion of PCR reaction, the reaction solution was
subjected to electrophoresis on agarose gel to confirm a band at
approximately 0.9 kb, which seemed to be the fragment of interest.
The region of the gel containing the band was excised and DNA
fragment was purified in GENECLEAN II (available from BIO 101,
Inc.)
(4) Analysis of Purified DNA Fragment
[0249] Purified DNA fragment was sequenced and analyzed on a 370A
DNA sequencing system (available from Perkin-Elmer Corp.) to
confirm the presence of the above-described amino acid sequences
(Asn-Phe-Ile-Leu-Ala, Leu-Pro-Asn-Ile-Gly, x-Pro-Thr-Asp-x-Tyr-Pro,
Leu-His-Gln-Pro-Tyr-Gly-Ala-x-x-Pro) in the putative amino acid
sequence deduced from the nucleotide sequence determined. From
these results, it was confirmed that the DNA fragment obtained by
the above-described RT-PCR amplification contained a portion of the
genetic sequence encoding the fructosyl peptide oxidase derived
from Eupenicillium terrenum.
(5) Analysis of the Downstream Region by 3'-RACE
[0250] First, a primer (SEQ ID NO: 17) was designed using the DNA
sequence data obtained as described above and synthesized utilizing
Amersham Biotech Custom Synthesis Service. This primer, the
above-described mRNA and 3'-Full RACE Core Set (available from
Takara Bio Inc.) were used to perform RT-PCR to amplify the
3'-unknown region. The reaction solution was subjected to
electrophoresis on agarose gel followed by purification and
extraction of an about 400 bp DNA fragment using Reco Chip
(available from Takara Bio Inc.). The DNA fragment purified was
then sequenced and analyzed in a DNA sequencer to confirm the
presence of the same sequence as the 3' sequence of a portion of
the genetic sequence encoding the fructosyl peptide oxidase derived
from Eupenicillium terrenum in the 5'-region of the nucleotide
sequence determined.
(6) Analysis of the Upstream Region by 5'-RACE
[0251] First, primers (SEQ ID NOS: 18-22) were designed using the
DNA sequence data obtained as described above and synthesized
utilizing Amersham Biotech Custom Synthesis Service. These primers,
the above-described mRNA and 5'-Full RACE Core Set (available from
Takara Bio Inc.) were used to perform RT-PCR to amplify the
5'-unknown region. The reaction solution was subjected to
electrophoresis on agarose gel followed by purification and
extraction of an about 600 bp DNA fragment using Reco Chip
(available from Takara Bio Inc.). The DNA fragment purified was
then sequenced and analyzed in a DNA sequencer to confirm the
presence of the same sequence as the 5' sequence of a portion of
the genetic sequence encoding the fructosyl peptide oxidase derived
from Eupenicillium terrenum in the 3'-region of the nucleotide
sequence determined.
(7) Obtaining Genetic Fragments by RT-PCR
[0252] Translation start and stop codons were deduced from the
above-described 3 nucleotide sequences, and primer DNAs (SEQ ID
NOS: 23 and 24) for the N- and C-terminal regions were synthesized
utilizing Amersham Biotech Custom Synthesis Service. These primers
and the above-described mRNA were used to perform RT-PCR. The
reaction solution was then subjected to electrophoresis on agarose
gel to confirm the presence of an approximately 1.3 kb band. Then,
DNA fragment contained in the band was purified and extracted using
Reco Chip (available from Takara Bio Inc.). Further, plasmid pUC19
(available from Takara Bio Inc) was digested with restriction
enzyme SmaI, and ligated with the DNA fragment purified and
extracted as described above. The ligated DNA was then used to
transform E. coli JM 109. The obtained plasmid puc-EFP was
deposited at the International Patent Organism Depositary, the
National Institute of Advanced Industrial Science and Technology,
an Independent Administrative Institution under the Ministry of
Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1,
Tsukuba, Ibaraki, Japan as FERM BP-8131.
(8) Confirmation of Activity
[0253] E. coli JM 109 (puc-EFP) cells were grown in 10 ml of
TY-medium containing 50 .mu.g/ml ampicillin (1% bactotryptone, 0.5%
bactoyeast extract, 0.5% NaCl, pH 7.0) by shake-culture at
30.degree. C. to Klett 100, added with IPTG to a final
concentration of 1 mM, and cultured for an additional 3 hours. The
culture solution was treated in an Ultrasonicgenerator (available
from Nissei) for 20 sec..times.4 times while cooling on ice. The
culture solution was loaded in a microtube, and centrifuged in a
microcentrifuge at 12,000 rpm for 10 minutes to obtain supernatant
and precipitate fractions. The supernatant fraction obtained was
transferred to another microtube and determined for fructosyl
peptide oxidase activity according to the above-described enzyme
activity assay to find that JM109 (puc-EFP) had a fructosyl peptide
oxidase activity of 0.01 U/ml.
(9) Analysis of Gene Encoding Fructosyl Peptide Oxidase
[0254] Since it was confirmed that E. coli JM109 (puc-EFP) had
fructosyl peptide oxidase activity, it became clear that the insert
fragment incorporated in puc-EFP contained the gene encoding
fructosyl peptide oxidase. Therefore, the nucleotide sequence of
the plasmid DNA was determined using a 370A DNA sequencing system
(available from Perkin-Elmer Corp.). The nucleotide sequence of the
plasmid DNA determined and the putative amino acid sequence of the
polypeptide which may be translated from the DNA are shown in SEQ
ID NOS: 4 and 3, respectively. The gene encoding fructosyl peptide
oxidase had a 1314 bp coding region which coded for 437 amino
acids.
[0255] According to the present invention, novel fructosyl peptide
oxidases with a variety of physicochemical properties and a method
for producing them are provided. The inventive fructosyl peptide
oxidases are advantageous in that they can be used easily as
diagnostic enzymes in an assay kit and that they can be
mass-produced at low cost, and are therefore useful in the
industry. Further, the present invention also provides oxidases
which have less action on fructosyl lysine. Those oxidases have
improved stability and are particularly useful as enzymes for
diagnosing diabetes. The present invention also provides a method
for producing those oxidases. In this way, the present invention
allows for the development of a kit for clinical diagnosis with
improved storage stability. Those oxidases with less action on
fructosyl lysine are more useful in the industry.
[0256] This application includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application Nos. 2001-266665, 2001-378151 and 2002-228727, which
are priority documents of the present application. All
publications, patents and patent applications cited herein are
incorporated in the present specification by reference in their
entirety.
Sequence CWU 1
1
241437PRTConiochaeta sp. 1Met Thr Ser Asn Arg Ala Asp Thr Arg Val
Ile Val Val Gly Gly Gly1 5 10 15Gly Thr Ile Gly Ser Ser Thr Ala Leu
His Leu Val Arg Ser Gly Tyr 20 25 30Ala Pro Ala Asn Ile Thr Val Leu
Asp Thr Phe Glu Ile Pro Ser Ala 35 40 45Gln Ser Ala Gly His Asp Leu
Asn Lys Ile Met Gly Ile Arg Leu Arg 50 55 60Asn Lys Val Asp Leu Gln
Met Ser Leu Glu Ala Arg Gln Met Trp Lys65 70 75 80Glu Asp Glu Leu
Phe Gln Pro Phe Phe His Asn Thr Gly Arg Met Asp 85 90 95Cys Glu His
Thr Pro Glu Gly Ile Glu Asp Leu Lys Lys Gln Tyr Gln 100 105 110Ala
Leu His Asp Ala Gly Ala Gly Leu Glu Lys Thr His Ala Trp Leu 115 120
125Asp Asn Glu Asp Glu Ile Leu Ser Lys Met Pro Leu Leu Gln Arg Asp
130 135 140Gln Ile Gln Gly Trp Lys Ala Ile Trp Ser Gln Asp Gly Gly
Trp Leu145 150 155 160Ala Ala Ala Lys Ala Ile Asn Ala Ile Gly Gln
Phe Leu Lys Glu Arg 165 170 175Gly Val Lys Phe Gly Phe Gly Gly Ala
Gly Ser Phe Lys Gln Pro Leu 180 185 190Phe Asp Asp Glu Gly Thr Thr
Cys Ile Gly Val Glu Thr Ala Asp Gly 195 200 205Thr Lys Tyr Tyr Ala
Asp Lys Val Val Leu Ala Ala Gly Ala Trp Ser 210 215 220Pro Thr Leu
Val Asp Leu Glu Asp Gln Cys Cys Ser Lys Ala Trp Val225 230 235
240Tyr Ala His Ile Gln Leu Thr Pro Glu Glu Ala Ala Glu Tyr Lys Gly
245 250 255Val Pro Val Val Tyr Asn Gly Glu Phe Gly Phe Phe Phe Glu
Pro Asn 260 265 270Glu Phe Gly Val Ile Lys Val Cys Asp Glu Phe Pro
Gly Phe Ser Arg 275 280 285Phe Lys Glu His Gln Pro Tyr Gly Ala Pro
Ser Pro Lys His Ile Ser 290 295 300Val Pro Arg Ser His Ala Lys His
Pro Thr Asp Thr Tyr Pro Asp Ala305 310 315 320Ser Glu Val Ser Ile
Lys Lys Ala Ile Ala Thr Phe Leu Pro Arg Phe 325 330 335Gln Asp Lys
Glu Leu Phe Asn Arg Ala Leu Cys Trp Cys Thr Asp Thr 340 345 350Ala
Asp Ala Ala Leu Leu Met Cys Glu His Pro Lys Trp Lys Asn Phe 355 360
365Ile Leu Ala Thr Gly Asp Ser Gly His Ser Phe Lys Ile Leu Pro Asn
370 375 380Val Gly Lys His Val Val Glu Leu Ile Glu Gly Arg Leu Pro
Glu Glu385 390 395 400Met Ala Tyr Gln Trp Arg Trp Arg Pro Gly Gly
Asp Ala Leu Lys Ser 405 410 415Arg Arg Ala Ala Pro Pro Lys Asp Leu
Ala Asp Met Pro Gly Trp Lys 420 425 430His Asp Pro Lys Leu
43521314DNAConiochaeta sp. 2atgacgtcga atcgtgcaga tacaagggtg
attgtcgtcg gtggcggagg aacgattggt 60tcctcgacag cgctgcatct tgtgaggagt
ggttatgctc ccgcaaatat cacggtcttg 120gacacatttg agattccatc
ggctcaatca gccggccatg atctcaacaa gatcatggga 180atacgactgc
gcaacaaggt ggacctgcaa atgagtctag aggctagaca gatgtggaag
240gaggatgagt tattccagcc cttctttcac aataccggca gaatggactg
cgaacacacg 300cctgagggta tcgaggacct gaaaaagcag taccaggcac
tgcacgatgc cggtgcgggt 360ctggagaaga ctcatgcctg gttggacaac
gaggatgaga tcttatccaa gatgccgttg 420cttcaacgtg accaaataca
aggatggaaa gcaatatgga gtcaagatgg cggctggtta 480gctgcggcaa
aggccatcaa tgcgatcgga cagttcttga aagaacgtgg tgtaaagttc
540ggattcggcg gcgctggatc cttcaagcaa ccccttttcg acgatgaagg
cacaacttgc 600attggcgttg agacggcaga tggtaccaaa tattacgctg
acaaggtggt cttagcagct 660ggcgcatgga gcccaaccct ggtggacctg
gaagatcaat gttgctcgaa ggcttgggtg 720tatgctcata ttcagttgac
gcctgaagag gccgctgagt ataagggtgt cccagttgtg 780tataatggcg
aatttggctt cttctttgag cctaatgagt ttggtgtaat aaaggtgtgc
840gacgaattcc caggattctc gcgcttcaag gaacatcaac cctatggcgc
cccatctccg 900aaacacatat cagtaccacg atcgcacgcc aagcatccca
cagacactta tccagacgca 960tccgaagtca gcatcaaaaa agcaatcgcg
acgtttctcc ctcgatttca ggacaaggag 1020ctcttcaatc gcgccttgtg
ctggtgtaca gacactgcgg acgctgctct cttgatgtgt 1080gaacacccca
aatggaagaa tttcattcta gcgaccggcg acagcggaca ctcattcaaa
1140atcttgccta acgtcggaaa acatgtagtc gagttgatag agggccgcct
gccggaggaa 1200atggcttatc aatggaggtg gcggccagga ggcgatgcac
tcaagtctag acgtgcggca 1260ccgccaaaag atcttgcaga catgccagga
tggaaacatg atccgaaatt gtaa 13143437PRTEupenicillium terrenum 3Met
Ala His Ser Arg Ala Ser Thr Lys Val Val Val Val Gly Gly Gly1 5 10
15Gly Thr Ile Gly Ser Ser Thr Ala Leu His Leu Ile Arg Ser Gly Tyr
20 25 30Thr Pro Ser Asn Ile Thr Val Leu Asp Val Tyr Lys Thr Pro Ser
Leu 35 40 45Gln Ser Ala Gly His Asp Leu Asn Lys Ile Met Gly Ile Arg
Leu Arg 50 55 60Asn Gly Pro Asp Leu Gln Leu Ser Leu Glu Ser Leu Asp
Met Trp Gln65 70 75 80Asn Asp Glu Leu Phe Lys Pro Phe Phe His Gln
Val Gly Met Ile Asp 85 90 95Cys Ser Ser Ser Lys Glu Gly Ile Glu Asn
Leu Arg Arg Lys Tyr Gln 100 105 110Thr Leu Leu Asp Ala Gly Ile Gly
Leu Glu Lys Thr Asn Val Trp Leu 115 120 125Glu Ser Glu Asp Glu Ile
Leu Ala Lys Ala Pro Asn Phe Thr Arg Glu 130 135 140Gln Val Lys Gly
Trp Lys Gly Leu Phe Cys Thr Asp Gly Gly Trp Leu145 150 155 160Ala
Ala Ala Lys Ala Ile Asn Ala Ile Gly Ile Phe Leu Gln Asp Lys 165 170
175Gly Val Lys Phe Gly Phe Gly Gly Ala Gly Thr Phe Gln Gln Pro Leu
180 185 190Phe Ala Ala Asp Gly Lys Thr Cys Ile Gly Leu Glu Thr Thr
Asp Gly 195 200 205Thr Lys Tyr Phe Ala Asp Lys Val Val Leu Ala Ala
Gly Ala Trp Ser 210 215 220Pro Thr Leu Val Asp Leu Glu Asp Gln Cys
Val Ser Lys Ala Trp Val225 230 235 240Phe Ala His Ile Gln Leu Thr
Pro Lys Glu Ala Asp Ala Tyr Lys Asn 245 250 255Val Pro Val Val Tyr
Asp Gly Glu Tyr Gly Phe Phe Phe Glu Pro Asn 260 265 270Glu Tyr Gly
Val Ile Lys Val Cys Asp Glu Phe Pro Gly Phe Ser Arg 275 280 285Phe
Lys Leu His Gln Pro Tyr Gly Ala Ala Ser Pro Lys Met Ile Ser 290 295
300Val Pro Arg Ser His Ala Lys His Pro Thr Asp Thr Tyr Pro Asp
Ala305 310 315 320Ser Glu Val Thr Ile Arg Lys Ala Ile Ala Arg Phe
Leu Pro Glu Phe 325 330 335Lys Asp Lys Glu Leu Phe Asn Arg Thr Met
Cys Trp Cys Thr Asp Thr 340 345 350Ala Asp Ala Asn Leu Leu Ile Cys
Glu His Pro Lys Trp Lys Asn Phe 355 360 365Ile Leu Ala Thr Gly Asp
Ser Gly His Ser Phe Lys Leu Leu Pro Asn 370 375 380Ile Gly Lys His
Val Val Glu Leu Leu Glu Gly Ser Leu Ser Gln Glu385 390 395 400Met
Ala Gly Ala Trp Arg Trp Arg Pro Gly Gly Asp Ala Leu Arg Ser 405 410
415Arg Arg Gly Ala Pro Ala Lys Asp Leu Ala Glu Met Pro Gly Trp Lys
420 425 430His Asp Ala His Leu 43541314DNAEupenicillium terrenum
4atggctcatt cgcgtgcaag caccaaagtc gtcgtggttg ggggaggtgg tacgatcggg
60tcttcgacgg ctctgcactt aatccgctct ggatataccc cctcaaatat caccgtgctt
120gacgtataca agaccccttc attgcaatct gcaggacatg atttgaacaa
gatcatgggc 180attcgattgc gcaacgggcc tgacttgcag ctttcgctgg
aatcactcga catgtggcaa 240aacgatgagt tgttcaagcc attctttcac
caagtgggca tgattgattg ttcgtcatcc 300aaagagggta ttgaaaatct
tcgacgaaaa taccagaccc tcctcgatgc gggcattggg 360ctggagaaga
cgaacgtttg gctggaatct gaagatgaga tcctcgccaa agcgccgaat
420ttcacgcgtg aacaagtcaa ggggtggaaa ggcttatttt gcactgatgg
aggctggctt 480gctgcagcca aggctatcaa tgcgatcgga attttcctcc
aggacaaagg tgtcaagttt 540ggctttggag gtgctggaac atttcagcaa
cctctgttcg ccgctgatgg aaaaacttgc 600atcggacttg aaactacaga
cggaaccaag tactttgctg acaaggttgt cttggctgct 660ggtgcgtgga
gtcccacctt ggtggatcta gaagatcagt gtgtttcaaa ggcctgggtt
720ttcgctcata ttcaactcac acccaaagaa gcggacgcgt acaagaatgt
gcctgtggtc 780tatgatggtg aatatgggtt cttttttgag cccaacgagt
atggggtgat caaagtctgt 840gacgagttcc ctggtttctc tcgcttcaaa
ctgcatcaac cgtacggggc tgcatctccc 900aagatgatat ccgtaccgcg
atcacacgcc aagcatccca cagataccta ccctgatgcc 960tccgaagtca
ccatacgcaa agcgatcgca aggttcctgc cagaatttaa agacaaggag
1020ctcttcaacc gtaccatgtg ctggtgtaca gatacggccg atgctaactt
attgatttgc 1080gaacacccga agtggaagaa tttcattctg gccactggag
atagcggaca ttccttcaag 1140ctgttgccaa acatcgggaa acacgttgtt
gagcttttag agggatctct atcgcaggaa 1200atggctggtg cctggagatg
gagacccgga ggtgatgctc ttagatctag acgcggtgct 1260ccggcaaagg
atcttgctga gatgccggga tggaagcatg atgcacattt gtga
1314523DNAARTIFICIAL SEQUENCESYNTHETIC DNA 5tggytngaya aygargayga
rat 23626DNAARTIFICIAL SEQUENCESYNTHETIC DNA 6ttraarttrt gnccraartc
nccngt 26720DNAARTIFICIAL SEQUENCESYNTHETIC DNA 7cccacagaca
cttatccaga 20815DNAARTIFICIAL SEQUENCESYNTHETIC DNA 8actcagcggc
ctctt 15928DNAARTIFICIAL SEQUENCESYNTHETIC DNA 9agatggtacc
aaatattacg ctgacaag 281027DNAARTIFICIAL SEQUENCESYNTHETIC DNA
10tttacaccac gttctttcaa gaactgt 271126DNAARTIFICIAL
SEQUENCESYNTHETIC DNA 11aaggcttggg tgtatgctca tattca
261227DNAARTIFICIAL SEQUENCESYNTHETIC DNA 12tccttgtatt tggtcacgtt
gaagcaa 271323DNAARTIFICIAL SEQUENCESYNTHETIC DNA 13atgacgtcga
atcgtgcaga tac 231425DNAARTIFICIAL SEQUENCESYNTHETIC DNA
14ttacaatttc ggatcatgtt tccat 251522DNAARTIFICIAL SEQUENCESYNTHETIC
DNA 15acnaaygtnt ggctngarws ng 221619DNAARTIFICIAL
SEQUENCESYNTHETIC DNA 16kccanccngg catytcngc 191721DNAARTIFICIAL
SEQUENCESYNTHETIC DNA 17catcccacag atacctaccc t 211820DNAARTIFICIAL
SEQUENCESYNTHETIC DNA 18tcccgatgtt tggcaacagc 201922DNAARTIFICIAL
SEQUENCESYNTHETIC DNA 19gtgcctgtgg tctatgatgg tg
222020DNAARTIFICIAL SEQUENCESYNTHETIC DNA 20atcaaccgta cggggctgca
202121DNAARTIFICIAL SEQUENCESYNTHETIC DNA 21gatcgcattg atagccttgg c
212230DNAARTIFICIAL SEQUENCESYNTHETIC DNA 22ctccacgcac cagcagccaa
gacaaccttg 302324DNAARTIFICIAL SEQUENCESYNTHETIC DNA 23gacatggctc
attcgcgtgc aagc 242425DNAARTIFICIAL SEQUENCESYNTHETIC DNA
24caagaatcac aaatgtgcat catgc 25
* * * * *