U.S. patent application number 10/348455 was filed with the patent office on 2003-09-11 for d-aminoacylases, method for producing the same, and method for producing d-amino acids using the same.
This patent application is currently assigned to Daicel Chemical Industries, Ltd., a Japan corporation. Invention is credited to Matsuyama, Akinobu, Mitsuhashi, Kazuya, Tokuyama, Shinji, Yamamoto, Hiroaki.
Application Number | 20030170869 10/348455 |
Document ID | / |
Family ID | 16879455 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030170869 |
Kind Code |
A1 |
Mitsuhashi, Kazuya ; et
al. |
September 11, 2003 |
D-aminoacylases, method for producing the same, and method for
producing D-amino acids using the same
Abstract
D-aminoacylase derived from fungi is provided. The fungi capable
of producing D-aminoacylase include those belonging to the genus
Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis. The
fungal D-aminoacylase is useful for efficiently producing D-amino
acids from N-acetyl-D-amino acids.
Inventors: |
Mitsuhashi, Kazuya;
(Ibaraki, JP) ; Yamamoto, Hiroaki; (Ibaraki,
JP) ; Matsuyama, Akinobu; (Ibaraki, JP) ;
Tokuyama, Shinji; (Shizuoka, JP) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Daicel Chemical Industries, Ltd., a
Japan corporation
|
Family ID: |
16879455 |
Appl. No.: |
10/348455 |
Filed: |
January 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10348455 |
Jan 17, 2003 |
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09361901 |
Jul 27, 1999 |
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6514742 |
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Current U.S.
Class: |
435/228 ;
435/106; 435/254.11; 435/254.7; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/80 20130101; C12P
41/007 20130101; C12P 13/04 20130101 |
Class at
Publication: |
435/228 ;
435/69.1; 435/320.1; 435/254.7; 435/254.11; 536/23.2; 435/106 |
International
Class: |
C12N 009/78; C12N
009/80; C12P 013/04; C07H 021/04; C12P 021/02; C12N 001/16; C12N
015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 29, 1998 |
JP |
10/228636 |
Claims
What is claimed is:
1. A D-aminoacylase derived from a fungus.
2. The D-aminoacylase according to claim 1, wherein said fungus
belongs to the genus selected from the group consisting the genera
Hypomyces, Fusarium, Auricularia, Pythium, and Menisporopsis.
3. The D-aminoacylase according to claim 2, wherein said fungus
belongs to the species selected from the group consisting of
Hypomyces aurantius, Hypomyces broomeanus, Hypomyces chrysospermus,
Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces subiculosus,
Hypomyces mycophilus, Fusarium solani, Auricularia auriculajudae,
Pythium aphanidermaatum, and Menisporopsis novaezelandiae.
4. The D-aminoacylase according to claim 3, wherein said fungus
belongs to the strain selected from the group consisting of
Hypomyces aurantius IFO 6847, Hypomyces broomeanus IFO 9164,
Hypomyces rosellus IFO 6911, Hypomyces chrysospermus IFO 6817,
Hypomyces sepulcralis IFO 9102, Hypomyces subiculosus IFO 6892,
Hypomyces mycophilus ATCC 76474 or IFO 6785, Fusarium solani IFO
9974 or IFO 9975, Auricularia auriculajudae IFO 5949, Pythium
aphanidermaatum IFO 7030, and Menisporopsis novaezelandiae IFO
9179.
5. A D-aminoacylase, which has the physico-chemical properties (a)
through (f) below: (a) function: the enzyme acts on
N-acetyl-D-amino acids to produce corresponding D-amino acids; (b)
molecular weight: the molecular weight of the enzyme is estimated
to be about 64,000 daltons by SDS-polyacrylamide gel
electrophoresis, and about 56,000 daltons by gel filtration
chromatography on Superdex 200 Hi-Load 6/16 (Amersham Pharmacia
Biotech); (c) substrate specificity: the enzyme acts on
N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,
N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on
N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine,
N-acetyl-L-leucine, or N-acetyl-L-methionine; (d) thermostability:
when heated at pH 9.5 for 30 min, the enzyme is stable at
45.degree. C., but inactivated at higher than 60.degree. C.; (e)
optimal temperature for activity: for the reaction at pH 7.5, the
enzyme activity is optimal at about 45.degree. C.; and (f)
stabilizer: the enzyme activity is stabilized by reducing agents,
and further activated by ICH.sub.2CONH.sub.2.
6. The D-aminoacylase according to claim 5, which is derived from
the fungus belonging to the genus Hypomyces.
7. The D-aminoacylase according to claim 6, wherein said fungus
belongs to the species Hypomyces mycophilus.
8. The D-aminoacylase according to claim 7, wherein said fungus is
Hypomyces mycophilus ATCC 76474 or IFO 6785 strain.
9. The D-aminoacylase according to claim 5, comprising the amino
acid sequences described in SEQ ID NOs: 1 through 5.
10. A DNA encoding the D-aminoacylase according to claim 5.
11. A method for producing D-amino acids, wherein said method
comprises reacting a fungus capable of producing D-aminoacylase or
D-aminoacylase derived from a fungus with N-acyl-DL-amino acid or
its salt represented by the formula (I): 3wherein R.sub.1 and
R.sub.2 may be identical or different and each represents a
hydrogen atom or a substituted or unsubstituted hydrocarbon group,
provided that R.sub.2 does not represent a hydrogen atom; and X is
H, NH.sub.4, or a metal ion.
12. The method for producing D-amino acids according to claim 11,
wherein said fungus belongs to a genus selected from the group
consisting of the genera Hypomyces, Fusarium, Auricularia, Pythium
and Menisporopsis.
13. The method for producing D-amino acids according to claim 12,
wherein said fungus belongs to the species selected from the group
consisting of Hypomyces aurantius, Hypomyces broomeanus, Hypomyces
chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces
subiculosus, Hypomyces mycophilus, Fusarium solani, Auricularia
auriculajudae, Pythium aphanidermaatum, and Menisporopsis
novaezelandiae.
14. The method for producing D-amino acids according to claim 13,
wherein said fungus is a strain selected from the group consisting
of Hypomyces aurantius IFO 6847, Hypomyces broomeanus IFO 9164,
Hypomyces rosellus IFO 6911, Hypomyces chrysospermus IFO 6817,
Hypomyces sepulcralis IFO 9102, Hypomyces subiculosus IFO 6892,
Hypomyces mycophilus ATCC 76474 or IFO 6785, Fusarium solani IFO
9974 or IFO 9975, Auricularia auriculajudae IFO 5949, Pythium
aphanidermaatum IFO 7030, Menisporopsis novaezelandiae IFO
9179.
15. The method for producing D-amino acids according to claim 11,
wherein said D-aminoacylase has the physico-chemical properties (a)
through (f) below: (a) function: the enzyme acts on
N-acetyl-D-amino acids to produce the corresponding D-amino acids;
(b) molecular weight: apparent molecular weight of the enzyme is
estimated to be about 64,000 daltons by SDS-polyacrylamide gel
electrophoresis, and about 56,000 daltons by gel filtration
chromatography on Superdex 200 Hi-Load 6/16 (Amersham Pharmacia
Biotech); (c) substrate specificity: the enzyme acts on
N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,
N-acetyl-D-leucine and N-acetyl-D-methionine, but not on
N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine,
N-acetyl-L-leucine and N-acetyl-L-methionine; (d) thermostability:
when heated at pH 9.5 for 30 min, the enzyme is stable at
45.degree. C., but inactivated at higher than 60.degree. C.; (e)
optimal temperature for activity: for the reaction at pH 7.5, the
enzyme activity is optimal at about 45.degree. C.; and (f)
stabilizer: the enzyme activity is stabilized by reducing agents,
and further activated by ICH.sub.2CONH.sub.2.
16. The method for producing D-amino acids according to claim 15,
wherein said D-aminoacylase is derived from a fungus belonging to
the genus Hypomyces.
17. The method for preparing D-amino acids according to claim 16,
wherein said fungus belongs to the species Hypomyces
mycophilus.
18. The method for preparing D-amino acids according to claim 17,
wherein said fungus is Hypomyces mycophilus ATCC 76474 or IFO
6785.
19. The method for producing D-amino acids according to claim 15,
wherein said D-aminoacylase comprises the amino acid sequences
described in SEQ ID NOs: 1 through 5.
20. The method for producing D-amino acids according to claim 11,
wherein R1 and R2 in the above-described formula (I) may be
identical or different and each represents a substututed or
unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl
group.
21. The method for producing D-amino acids according to claim 20,
wherein R.sub.1 is an indolyl, benzyl, thiomethylethyl, isopropyl,
or 2-methyl-propyl group and R.sub.2 is a methyl, chloromethyl,
phenyl, or aminomethyl group.
22. A method for producing D-aminoacylase, wherein said method
comprises culturing a fungus.
23. The method for preparing D-aminoacylase according to claim 22,
wherein said fungus belongs to a genus selected from the group
consisting of the genera Hypomyces, Fusarium, Auricularia, Pythium,
and Menisporopsis.
24. The method for producing D-aminoacylase according to claim 23,
wherein said fungus belongs to the species selected from the group
consisting of Hypomyces aurantius, Hypomyces broomeanus, Hypomyces
chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis, Hypomyces
subiculosus, Hypomyces mycophilus, Fusarium solani, Auricularia
auriculajudae, Pythium aphanidermaatum, and Menisporopsis
novaezelandiae.
25. The method for producing D-aminoacylase according to claim 24,
wherein said fungus is a strain selected from the group consisting
of Hypomyces aurantius IFO 6847, Hypomyces broomeanus IFO 9164,
Hypomyces rosellus IFO 6911, Hypomyces chrysospermus IFO 6817,
Hypomyces sepulcralis IFO 9102, Hypomyces subiculosus IFO 6892,
Hypomyces mycophilus ATCC 76474 or IFO 6785, Fusarium solani IFO
9974 or IFO 9975, Auricularia auriculajudae IFO 5949, Pythium
aphanidermaatum IFO 7030, and Menisporopsis novaezelandiae IFO
9179.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a D-aminoacylase produced
by fungi, a method for producing said D-aminoacylase, and a method
for producing D-amino acids using said D-aminoacylase.
BACKGROUND OF THE INVENTION
[0002] Enzymes have excellent catalytic functions with substrate
specificity, reaction specificity, and stereospecificity.
Stereospecificity of enzymes, with some exceptions, are nearly
absolute.
[0003] Recent precise research has increased the importance of
optically active substances for use in drugs, pesticides, feeds,
and perfumes. Optical isomers sometimes have quite different
biological activities. D(R)-form thalidomide has no teratogenic
activity, but its L(S)-form shows strong teratogenicity. The
practical use of thalidomide racemate caused the drug injury
incidents. If one enantiomer shows an effective biological
activity, the other enantiomer may sometimes have no activity,
rather, it may competitively inhibit the activity of the effective
enantiomer. In some cases, the activity of the racemate is reduced
to half or less of the activity of the effective enantiomer.
Accordingly, it is industrially important to obtain (synthesize or
optically resolve) optically pure enantiomers. For this purpose,
techniques for effective resolution of synthetic racemate have been
widely used. Enzymatic optical resolution has drawn attention
because it does not produce by-products and a bulk of liquid
waste.
[0004] Generally, L-amino acids are widely and largely utilized in
seasonings, food and feed additives, and infusions and are thus
very highly demanded. L-Amino acids have been produced mainly by
direct fermentation using microorganisms. Optical resolution is
also a known method for producing L-amino acids by hydrolyzing
N-acyl-DL-amino acids using L-aminoacylases. It has been utilized
to industrially produce L-amino acids that are difficult to produce
by fermentation. L-aminoacylases are widely found in animals,
plants, and microorganisms. They have been purified from various
organisms, and their properties have been clarified. N-terminal
amino acids of many proteins are considered to be N-acetylated in
vivo. L-Aminoacylases presumably regenerate N-acetyl-amino acids
produced by decomposition of proteins to amino acids. Among
L-aminoacylases, acylase, which acts on N-acyl-L-glutamic acid, is
reportedly involved in arginine biosynthesis (Fruth, H., Leisinger,
T.: J. Gen. Microb. 125, pp1 (1981)).
[0005] In contrast, D-amino acids have not been a subject of
interest for a long time because they are nonprotein amino acids.
D-amino acids were known to occur in small cyclic peptides,
peptidoglycan of bacterial cell walls, and peptide antibiotics.
However, D-amino acids have been demonstrated to be constituents of
neuro peptides and to exist as binding forms in tooth enamel, the
lens, and cerebral proteins, resulting in investigation of
physiological significance and enzymatic synthesis of D-amino
acids.
[0006] At present, DL-amino acids have been optically resolved by
physicochemical, chemical, and enzymatic methods. The enzymatic
methods are the most convenient and industrially applicable for,
for example, continuously producing L-methionine from
N-acetyl-DL-methionine using a bioreactor of immobilized
L-aminoacylase. D-amino acids may also be produced using
hydantoinase. The method consists of two-step enzymatic reactions.
The first reaction uses D-specific hydantoinase to convert
D,L-5-substituted-hydantoin, which is synthesized at low cost from
aldehyde analogues, to a D-carbamyl derivative. The second reaction
uses D-amino acid carbamylase.
[0007] Another method comprises hydrolyzing N-acetyl-DL-amino acids
with D-aminoacylase to produce D-amino acids (Sugie, M. and Suzuki,
H.: Argric. Biol. Chem. 44, pp1089 (1980), Tsai, Y. C., Lin, C. S.,
Tseng, T. H., Lee, H. and Wang, Y. J.: J. Enzyme Microb. Technol.
14, pp384 (1992)).
[0008] D-aminoacylase was first reported to be found in Pseudomonas
sp. KT83 isolated from soil by Kameda et. al in 1952 (Kameda, Y.,
Toyoura, H., Kimura, Y. and Yasuda, Y.: Nature 170, pp888 (1952)).
This enzyme hydrolyzed N-benzoyl derivatives of D-phenylalanine,
D-tyrosine, and D-alanine. Thereafter, D-aminoacylases were derived
from microorganisms belonging to the genus Pseudomonas (Kubo, K.,
Ishikura, T., and Fukagawa, Y.: J. Antibiot. 43, pp550 (1980),
Kubo, K., Ishikura, T. and Fukagawa, Y.: J. Antibiot. 43, pp556
(1980), Kameda, Y., Hase, T., Kanatomo, S. and Kita, Y.: Chem.
Pharm. Bull. 26, pp2698 (1978), Kubo, K., Ishikura, T. and
Fukagawa, Y.: J. Antibiot. 43, pp543 (1980)), the genus
Streptomyces (Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 42,
pp107 (1978), Sugie, M. and Suzuki, H.: Argric. Biol. Chem. 44,
pp1089 (1980)), and the genus Alcaligenes (Tsai, Y. C., Tseng, C.
P., Hsiao, K. M. and Chen, L. Y.: Appl. Environ. Microbiol. 54,
pp984 (1988), Yang, Y. B., Hsiao, K. M., Li, H., Yano, Y., Tsugita,
A. and Tsai, Y. C.: Biosci. Biotech. Biochem. 56, pp1392 (1992),
Yang, Y. B., Lin, C. S., Tseng, C. P., Wang, Y. J. and Tsai, Y. C.:
Appl. Environ. Microbiol. 57, pp2767 (1991), Tsai, Y. C., Lin, C.
S., Tseng, T. H., Lee, H. and Wang: Microb. Technol. 14, pp384
(1992), Moriguchi, M. and Ideta, K.: Appl. Environ. Microbiol. 54,
pp2767 (1988), Sakai, K., Imamura, K., Sonoda, Y., Kido, H. and
Moriguchi, M.: FEBS, 289, pp44 (1991), Sakai, K., Obata, T., Ideta,
K. and Moriguchi, M.: J. Ferment. Bioeng. 71, pp79 (1991), Sakai,
K., Oshima, K. and Moriguchi, M.: Appl. Environ. Microbiol. 57,
pp2540 (1991), Moriguchi, M., Sakai, K., Katsuno, Y., Maki, T. and
Wakayama, M.: Biosci. Biotech. Biochem., 57, pp1145 (1993), Kayama,
M., Ashika, T., Miyamoto, Y., Yoshikawa, T., Sonoda, Y., Sakai, K.
and Moriguchi, M.: J. Biochem. 118, pp204 (1995)), Moriguchi, M.,
Sakai, K., Miyamoto, Y. and Wakayama, M.: Biosci. Biotech.
Biochem., 57, pp1149 (1993)).
[0009] Tsai et al. and Moriguchi et al. also clarified the
characteristics of D-aminoacylase derived from microorganisms
belonging to the genera Alcaligenes and Pseudomonas and the amino
acid and nucleotide sequences of the enzymes. Moriguchi et al.
found that microorganisms belonging to the genera Alcaligenes and
Pseudomonas produced three species of D-aminoacylase by using
different inducers (Wakayama, M., Katsumo, Y., Hayashi, S.,
Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci. Biotech.
Biochem. 59, pp2115 (1995)).
[0010] Furthermore, Moriguchi et al. determined the nucleotide
sequences of these D-aminoacylases derived from a microorganism
belonging to the genus Alcaligenes and compared it with
L-aminoacylases derived from Bacillus stereothermophilus, human,
and pig. The result demonstrated that these D-aminoacylases have a
low homology with L-aminoacylases (Wakayama, M., Katsuno, Y.,
Hayashi, S., Miyamoto, Y., Sakai, K. and Moriguchi, M.: Biosci.
Biotech. Biochem., 59, pp2115 (1995)).
[0011] Sugie et al. reported D-aminoacylase of a microorganism
belonging to the genus Streptomyces of actinomycetes (Sugie, M. and
Suzuki, H.: Argric. Biol. Chem. 42, pp107 (1978), Sugie, M. and
Suzuki, H.: Argric. Biol. Chem. 44, pp1089 (1980)). However, the
enzyme has not been purified yet, and its characteristics remain
unknown.
[0012] As described above, many D-aminoacylases have been isolated
from bacteria and have been used to produce D-amino acids. However,
the conventional methods for producing D-amino acids using bacteria
have the following problems.
[0013] Most of the conventional bacterial D-aminoacylases are
inducible enzymes, and N-acetyl-DL-amino acid is usually required
for their production. The culture medium of the bacterium used for
producing D-aminoacylase contains the unreacted N-acetyl-D-amino
acid as well as the degradation product of D-amino acid. In order
to react D-aminoacylase produced by these bacteria with a substrate
other than N-acetyl-D-amino acid used as the enzyme inducer, it was
necessary to isolate the cultured bacteria from the growth medium.
Even when the enzyme reacts with N-acetyl-D-amino acid as the
substrate, the bacteria must be removed to purify D-amino acid
produced. A continuous centrifuge, such as a Westfalia centrifuge,
and a Sharples centrifuge were usually used to remove the cultured
bacteria. One problem of the centrifugation is that it takes longer
to centrifuge a large volume of the culture medium, often causing
the inactivation of D-aminoacylase during centrifugation.
Furthermore, the bacteria and actinomycetes lyse as the reaction
proceeds and thus are difficult to separate centrifugation. As
described above, D-amino acids are not always efficiently produced
using the conventional bacterial D-aminoacylase.
SUMMARY OF THE INVENTION
[0014] An objective of the present invention is to provide an
efficient method for producing D-amino acids using microorganisms
other than the conventional bacteria and actinomycetes.
[0015] In order to solve the above-described problems, the present
inventors have attempted to produce D-amino acids using fungi which
are eukaryotic cells sharing a common cell structure and many
chemical properties and functions with animals and plants
(eukaryotic characteristics). Fungi are advantageous in that: (1)
Fungal cells are large and filamentous and thus can be readily
collected by filtration from the liquid culture; (2) fungi
efficiently utilize carbohydrates and are able to grow more rapidly
than usual at a high carbon/nitrogen ratio, reducing the cost of
fermentation materials per unit of enzyme; (3) fungi can be
economically cultured in a solid culture (i.e., without a liquid
culture) since they grow under natural aeration conditions; this
translates into less investment in equipment, high concentration
enzyme production, and less organic solvents to extract enzymes;
(4) fungi are able to grow well in the solid culture with a low
active water level, which can prevent contamination with bacteria
that grow poorly in a low active water level; and (5) most fungi
efficiently utilize starch, cellulose, and proteins, including
inexpensive nutrient sources such as cassava as a starch source,
wheat bran, rice, rice bran, barley, wheat, soybean, and cellulose
sources.
[0016] No fungi producing D-aminoacylase has been reported so far.
The present inventors first screened D-aminoacylase-producing fungi
and found that many fungi belonging to the genera Hypomyces,
Fusarium, Auricularia, Pythium, and Menisporopsis can produce
D-amino acid from N-acetyl-D-amino acid. These fungi thus have
D-aminoacylase activity.
[0017] The present inventors then succeeded in isolating and
purifying D-aminoacylases from fungi with D-aminoacylase activity
by salting-out with ammonium sulfate and various chromatographies.
Furthermore, the inventors studied various physico-chemical
properties of the purified D-aminoacylases such as their substrate
specificity and thermostability, confirming that D-amino acid can
be efficiently produced by reacting the fungal D-aminoacylases with
N-acetyl-D-amino acid under the appropriate conditions.
[0018] The present inventors are the first to discover
D-aminoacylase in eukaryotes, fungi. The enzyme had been found only
in prokaryote bacteria and actinomycetes. The fungal D-aminoacylase
can be used to produce D-amino acids.
[0019] Accordingly, the present invention relates to a
D-aminoacylase produced by fungi, a method for producing said
D-aminoacylase, and a method for producing D-amino acids using said
D-aminoacylase.
[0020] More specifically, the present invention provides a
D-aminoacylase derived from a fungus.
[0021] In another aspect, the invention provides a D-aminoacylase
having physico-chemical properties (a) through (f) below:
[0022] (a) function: the enzyme acts on N-acetyl-D-amino acids to
produce corresponding D-amino acids;
[0023] (b) molecular weight: the molecular weight of the enzyme is
about 64,000 daltons determined by SDS-polyacrylamide gel
electrophoresis, and about 56,000 daltons determined by gel
filtration chromatography on Superdex 200 Hi-Load 6/16 (Amersham
Pharmacia Biotech)
[0024] (c) substrate specificity: the enzyme acts on
N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,
N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on
N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine,
N-acetyl-L-leucine, or N-acetyl-L-methionine;
[0025] (d) thermostability: when heated at pH 9.5 for 30 min, the
enzyme is stable at 45.degree. C., but inactivated at 60.degree. C.
or higher;
[0026] (e) optimal temperature: for the reaction at pH 7.5, the
enzyme activity is optimal at about 45.degree. C.; and
[0027] (f) stabilizer: the enzyme activity is stabilized by
reducing agents and activated by ICH.sub.2CONH.sub.2.
[0028] The present invention also provides a DNA encoding
D-aminoacylase described above.
[0029] Furthermore, the invention provides a method for producing
D-amino acids, wherein said method comprises reacting a fungus
producing D-aminoacylase or D-aminoacylase derived from a fungus
with N-acyl-DL-amino acid or its salt represented by the formula
(I): 1
[0030] where R.sub.1 and R.sub.2 may be identical or different and
each represents a hydrogen atom or a substituted or unsubstituted
hydrocarbon group, provided that R.sub.2 does not represent a
hydrogen atom; and X is H, NH.sub.4, or a metal ion.
[0031] Moreover, the invention provides a method for producing
D-aminoacylase, wherein said method comprises culturing a
fungus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 shows the purification of the D-aminoacylase of the
present invention by DEAE-Sepharose FF 5.0/25 anion exchange
chromatography.
[0033] FIG. 2 shows the purification of the D-aminoacylase of the
present invention by Phenyl-Sepharose Hi-Load HP 2.6/10 hydrophobic
chromatography.
[0034] FIG. 3 shows the purification of the D-aminoacylase of the
present invention by Superdex 200 Hi-Load 1.6/60 gel filtration
chromatography.
[0035] FIG. 4 shows the molecular weight of the D-aminoacylase of
the present invention measured by Superdex 200 Hi-Load 1.6/60 gel
filtration chromatography.
[0036] FIG. 5 shows the electrophoretogram of the D-aminoacylase of
the present invention by SDS-PAGE.
[0037] FIG. 6 shows the optimal pH for the reactivity of the
D-aminoacylase of the present invention. Black diamond indicates
the enzyme activity in AcONa.AcOH; black square, the activity in
K.sub.2HPO.sub.4. KH.sub.2PO.sub.4; black triangle, the activity in
Tris-HCl; and black circle the activity in glycine.NaOH.
[0038] FIG. 7 shows the optimal temperature for the reactivity of
the D-aminoacylase of the present invention.
[0039] FIG. 8 shows pH stability of the D-aminoacylase of the
present invention.
[0040] FIG. 9 shows thermostability of the D-aminoacylase of the
present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0041] "D-aminoacylase" used herein means an enzyme reacting with
an N-acyl-D-amino acid to catalyze the production of an organic
acid and a D-amino acid.
[0042] D-Aminoacylase derived from fungi of the present invention
can be produced by culturing fungi to allow them produce it.
D-aminoacylase-producing fungi used in the present invention are
not particularly limited as long as they are capable of producing
D-aminoacylase. Fungi capable of producing D-aminoacylase include
those belonging to the genera Hypomyces, Fusarium, Auricularia,
Pythium, and Menisporopsis, but are not limited to them.
[0043] Examples of the fungi belonging to the genus Hypomyces
include the species Hypomyces aurantius, Hypomyces broomeanus,
Hypomyces chrysospermus, Hypomyces rosellus, Hypomyces sepulcralis,
Hypomyces subiculosus, and Hypomyces mycophilus. The fungi
belonging to the genus Fusarium include the species Fusarium
solani. The fungi belonging to the genus Auricular include the
species Auricular auriculajudae. The fungi belonging to the genus
Pythium include the species Pythium aphanidermaatum. The fungi
belonging to the genus Menisporopsis include the species
Menisporopsis novaezelandiae. However, fungi to be used are not
limited to these species.
[0044] More specifically, D-aminoacylase can be derived from
Hypomyces aurantius IFO 6847, Hypomyces broomeanus IFO 9164,
Hypomyces rosellus IFO 6911, Hypomyces chrysospermus IFO 6817,
Hypomyces sepulcralis IFO 9102, Hypomyces subiculosus IFO 6892,
Hypomyces mycophilus ATCC 76474 or IFO 6785, Fusarium solani IFO
9974 or IFO 9975, Auricularia auriculajudae IFO 5949, Pythium
aphanidermaatum IFO 7030, or Menisporopsis novaezelandiae IFO 9179.
However, fungi to be used are not limited to these strains.
[0045] The above microorganisms with IFO numbers are recited in the
List of Cultures 10th edition (1996) published by Institute of
Fermentation Research, Osaka (IFO) and are available from IFO. The
above microorganisms with ATCC numbers are recited in ATCC.TM.
Catalogue on CD 1995 edition provided by "American Type Culture
Collection (ATCC)" and are available from ATCC.
[0046] D-aminoacylase-producing fungi can be cultured using
conventional fermentation techniques. Either synthetic or natural
media can be used as long as they contain proper amounts of the
carbon source, nitrogen source, inorganic materials, and other
nutrients. The culture media may be either liquid orsolid. More
specifically, considering the planned utilization of the fungi,
examples of the carbon source include sugars such as glucose,
fructose, maltose, galactose, starch, starch hydrolyzate, molasses,
and blackstrap molasses; natural products such as wheat, barley,
and corn; alcohols such as glycerol, methanol, and ethanol;
aliphatic hydrocarbons such as acetic acid, gluconic acid, pyruvic
acid, and citric acid; hydrocarbons such as normal paraffin; and
amino acids such as glycine, glutamine, and asparagine. One or more
of the above carbon sources are used depending on assimilability of
the fungus used. Examples of the nitrogen sources include organic
nitrogen-containing compounds such as meat extract, peptone, yeast
extract, soybean hydrolyzate, milk casein, casamino acid, various
amino acids, corn steep liquor, and other hydrolyzates of animals,
plants and microorganisms; and inorganic nitrogen-containing
compounds such as ammonia, ammonium salts such as ammonium nitrate,
ammonium sulfate, ammonium chloride, nitrates such as sodium
nitrate, and urea. One or more of the above nitrogen sources are
used depending on assimilability of the fungus.
[0047] For fungi to efficiently produce D-aminoacylase,
N-acetyl-DL-amino acid can be used as the enzyme inducer depending
upon the fungus employed.
[0048] Furthermore, a minute amount of one or more inorganic salts
can be used. Examples thereof include phosphates; hydrochlorides;
nitrates; acetates; or similar salts of magnesium, manganese,
potassium, calcium, sodium, copper, or zinc. Antifoaming agents,
such as vegetable oil, surfactants, or silicon, may be added to the
culture medium.
[0049] Culturing can be performed in the liquid medium containing
the above-described ingredients using the usual culture methods,
such as shake culturing, aeration agitation culturing, continuous
culturing, or fed-batch culturing.
[0050] Culturing conditions may be properly selected depending upon
the fungal strain and culture method, and are not particularly
limited as long as the fungi used can proliferate to produce
D-aminoacylase. It is usually preferred to adjust the initial pH to
4 to 10, preferably 6 to 8, and to culture at a temperature of 15
to 50.degree. C., preferably 25 to 35.degree. C. The culturing time
is also not particularly limited as long as a sufficient amount of
fungal cells having the D-aminoacylase activity can be obtained.
The culturing is usually performed for 1 to 14 days, preferably for
1 to 3 days. D-Aminoacylase produced and accumulated in the culture
can be recovered and isolated by the following methods.
[0051] When D-aminoacylase is intracellularly produced, the fungal
cells are collected by filtration or centrifugation after the
culturing and washed with buffer, physiological saline, etc. The
enzyme can then be extracted by disrupting the fungal cells using
physical means such as freeze-thawing, ultrasonication,
compression, osmotic treatment, or trituration; using biochemical
means such as cell wall lysis with lysozyme; or using chemical
means such as surfactant treatment. One or more of these treatments
can be combined. The crude D-aminoacylase thus obtained can be
purified by a single or combined fractionation means including
salting out; fractional precipitation with organic solvents;
various chromatographies such as salting-out chromatography,
ion-exchange chromatography, gel filtration chromatography,
hydrophobic chromatography, dye chromatography, hydroxyl apatite
chromatography, or affinity chromatography; and electrophoresis
such as isoelectric focusing and native electrophoresis. The above
chromatographies can be performed using open columns or by means of
medium-pressure or high-performance liquid chromatography
(HPLC).
[0052] For example, the fungal cells collected by filtration are
freeze-thawed and triturated using a Dyno Mill to obtain a
D-aminoacylase extract. The extract is then successively subjected
to salting-out using ammonium sulfate, ion-exchange chromatography
on DEAE-Sepharose FF, hydrophobic chromatography on
Phenyl-Sepharose FF, Sephadex 200 gel filtration chromatography, or
MonoQ ion-exchange chromatography. The enzyme thus purified can be
detected as a single protein band on the SDS-polyacrylamide gel
electrophoresis.
[0053] The fungal D-aminoacylase of the present invention has the
physico-chemical properties (a) through (f) below:
[0054] (a) function: the enzyme acts on N-acetyl-D-amino acids to
produce corresponding D-amino acids;
[0055] (b) molecular weight: the molecular weight of the enzyme is
about 64,000 daltons determined by SDS-polyacrylamide gel
electrophoresis, and about 56,000 daltons determined by gel
filtration chromatography on Superdex 200 Hi-Load 6/16 (Amersham
Pharmacia Biotech);
[0056] (c) substrate specificity: the enzyme acts on
N-acetyl-D-tryptophan, N-acetyl-D-phenylalanine, N-acetyl-D-valine,
N-acetyl-D-leucine, and N-acetyl-D-methionine, but not on
N-acetyl-L-tryptophan, N-acetyl-L-phenylalanine, N-acetyl-L-valine,
N-acetyl-L-leucine, or N-acetyl-L-methionine;
[0057] (d) thermostability: when heated at pH 9.5 for 30 min, the
enzyme is stable at 45.degree. C., but inactivated at 60.degree. C.
or higher;
[0058] (e) optimal temperature: for the reaction at pH 7.5, the
enzyme activity is optimal at about 45.degree. C.; and
[0059] (f) stabilizer: the enzyme activity is stabilized by
reducing agents and activated by ICH.sub.2CONH.sub.2.
[0060] The D-aminoacylase of the present invention having the above
properties is preferably derived from fungi belonging to the genus
Hypomyces, more preferably the species Hypomyces mycophilus, and
still more preferably Hypomyces mycophilus ATCC 76474 or IFO 6785
strain. The enzyme may contain the amino acid sequences described
in SEQ ID Nos: 1 through 5.
[0061] D-aminoacylase of the present invention is capable of acting
on various N-acyl-D-amino acids to yield the corresponding D-amino
acids, enabling the industrially advantageous production of D-amino
acids using said enzyme. For example, D-amino acid can be
selectively produced by reacting D-aminoacylase of this invention
with N-acyl-DL-amino acid, a mixture of D- and L-enantiomers.
[0062] N-acyl-DL-amino acids used in the present invention are not
particularly limited and can be selected from a wide variety of
compounds. A typical N-acyl-DL-amino acid can be represented by the
formula (I): 2
[0063] where R.sub.1 and R.sub.2 may be identical or different and
each represents a hydrogen atom or a substituted or unsubstituted
hydrocarbon group, provided that R.sub.2 does not represent a
hydrogen atom; and X is H, NH.sub.4, or a metal ion.
[0064] The hydrocarbon group represented by R.sub.1 and R.sub.2 is
preferably alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or aralkyl,
which may be substituted. More specifically, the hydrocarbon group
includes linear or branched alkyl such as methyl, ethyl, n-propyl,
i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, i-pentyl,
n-hexyl, etc.; alkenyl such as ethenyl, 1-propenyl, 2-propenyl,
1-butenyl, 2-butenyl, 2-pentenyl, 4-pentenyl, 1-hexenyl, 3-hexenyl,
5-hexenyl, etc.; alkynyl such as ethynyl, 1-propynyl, 2-pentynyl,
etc.; aryl such as phenyl, naphthyl, etc.; cycloalkyl such as
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The
substituent of the hydrocarbon group for R.sub.1 and R.sub.2
includes halogen; alkyl as defined above; alkenyl as defined above;
alkynyl as defined above; aryl as defined above; heterocyclic such
as piridyl, indole, quinolyl, etc.; amino; hydroxyl; thio; etc.
R.sub.1 is preferably indolyl (N-acyl-DL-tryptophan), benzyl
(N-acyl-DL-phenylalanine), thiomethylethyl (N-acyl-DL-methionine),
isopropyl (N-acyl-DL-valine), or 2-methylpropyl
(N-acyl-DL-leucine). R.sub.2 is preferably methyl, chloromethyl,
phenyl, or aminomethyl, which may be substituted with the above
substituent(s). The metal ion represented by X includes sodium,
patassium, etc. Preferable examples of N-acyl-DL-amino acids are
N-acetyl-DL-amino acids such as N-acetyl-DL-methionine,
N-acetyl-DL-valine, N-acetyl-DL-tryptophan, N-acetyl-DL-asparagine,
N-acetyl-DL-phenylalanine, N-acetyl-DL-alanine, and
N-acetyl-DL-leucine.
[0065] N-acetyl-DL-amino acids can be used in the form of a salt
such as a sodium salt or a potassium salt.
[0066] D-Aminoacylase used for producing D-amino acid in the
present invention includes a partially purified enzyme as well as
the purified one. The enzyme also includes a recombinant protein
obtained by isolating a DNA encoding D-aminoacylase, introducing
said DNA into suitable host cells, and allowing the cells to
express the enzyme. A DNA encoding D-aminoacylase can be isolated,
for example, by obtaining the purified enzyme, determining its
partial amino acid sequence, designing suitable oligonucleotide
primers based on said determined amino acid sequence, and
performing the polymerase chain reaction using the primers and, as
a templete, the mRNA, cDNA, or genomic DNA prepared from the fungus
from which the purified enzyme is derived.
[0067] Besides these enzymes, the D-aminoacylase-producing fungal
cells themselves can also be used in the present invention. Namely,
D-amino acid can be produced by directly reacting the fungus
capable of producing D-aminoacylase with N-acetyl-DL-amino acid.
The fungus can be used in the form of the culture medium, cells
separated from the culture medium by centrifugation or the like, or
cells resuspended in buffer, water, or the like after they are
separated by centrifugation and washed. The separated cells can be
used in a state as they are recovered, as their disrupts, as
treated with acetone or toluene, or as lyophilizate.
[0068] D-aminoacylase or the fungus capable of producing said
enzyme is reacted with N-acyl-D-amino acid under conditions
suitable for the activity and stability of D-aminoacylase, and for
the reactivity of the fungus. Some D-aminoacylases are activated by
metal ions such as Co.sup.2+ and Ca.sup.2+. When such enzymes are
used, the divalent metal ions may be added to the reaction
solution. If the enzyme is inhibited by divalent metal ions, a
chelating reagent such as EDTA can be added. Though the
concentration of the substrate, N-acetyl-DL-amino acid, is not
particularly limited, it is usually employed at a concentration of
about 0.1 to 30 w/v %. Use of a large amount of D-aminoacylase
often accelerates the reaction rate, and the enzyme is usually used
in the amount of about 1 to 1,000 U/ml. One unit of the enzyme is
defined as the amount of the enzyme to produce 1 .mu.mol of
D-tryptophan at 30.degree. C. in 1 min when the enzyme is reacted
with N-acetyl-D-tryptophan as the substrate. It is preferred to
maintain the reaction temperature at which the D-aminoacylase
activity is exhibited, 30 to 50.degree. C. It is preferred to
maintain the reaction pH at which the D-aminoacylase is active, for
example, pH4 to 10. The reaction can be performed with or without
stirring.
[0069] The enzyme or the fungus can often be stabilized by
immobilization. It can be immobilized by a known method on a
suitable carrier such as polyacrylamide gel, sulfur-containing.
polysaccharide gel (carrageenan gel), alginic acid gel, or agar
gel.
[0070] The D-amino acids produced can be recovered from the
reaction mixture by a known method such as direct crystallization
by concentration or isoelectric precipitation, ion exchange resin
treatment, membrane filtration, or the like.
[0071] For example, D-tryptophan produced using
N-acetyl-DL-tryptophan as a substrate can be purified as follows.
After the enzymatic reaction, the reaction mixture is contacted
with strongly acidic cation exchange resin to adsorb D-tryptophan.
The resin was washed with water and eluted with 0.5 N aqueous
ammonia. After the eluate was concentrated, the thus-obtained crude
crystalline powder of D-tryptophan is dissolved in a small amount
of 50% hot ethanol, decolorized with activated charcoal, and cooled
to obtain purified crystals of D-tryptophan.
[0072] In the method of the present invention, D-valine can be
purified as follows. After the enzymatic reaction, the microbial
cells are removed by centrifugation or the like, and the resulting
supernatant is adjusted to pH 1 by adding 6N hydrochloric acid. The
precipitated N-acetyl-L-valine is removed by centrifugation. The
supernatant is treated with activated charcoal, adjusted to pH 7.0,
then added to an H.sup.+-type strongly acidic cation exchanger
(Amberlite IR-120B). Elution is performed with 5% aqueous ammonia,
and the resulting eluate is dried at 80.degree. C. under reduced
pressure, thereby producing D-valine.
[0073] The present invention will be described in more detail with
reference to the following examples but is not to be construed to
be limited thereto.
[0074] In the following examples, "%" for concentration denotes
weight per volume percent unless otherwise specified.
EXAMPLE 1
[0075] A platinum loopful of Hypomyces aurantius IFO 6847 strain,
Hypomyces broomeanus IFO 9164 strain, Hypomyces rosellus IFO 6911
strain, Hypomyces chrysospermus IFO 6817 strain, Hypomyces
sepuleralis IFO 9102 strain, Hypomyces subiculosus IFO 6892 strain,
Hypomyces mycophilus ATCC 76474 or IFO 6785 strain, or Fusarium
solani IFO 9974 or IFO 9975 strain was inoculated into 25 ml of YM
medium (containing 0.3% yeast extract, 0.3% malt extract, 0.5%
peptone, and 2.0% glucose, pH 6.0) in a 500-ml shouldered flask.
The fungi were shake-cultured at 24.degree. C. for 3 days. After
culturing, a 5-ml aliquot of the culture medium was centrifuged in
a refrigerated centrifuge to collect the fungal cells. The cells
were washed with physiological saline solution (5 ml) and collected
by centrifugation again. A reaction solution (containing 0.1%
N-acetyl-D-amino acid shown in Table 1 below in Tris-HCl buffer, pH
7.5) was then added to the cells thus collected. The mixture was
incubated in a 5-ml test tube with shaking at 30.degree. C. for 24
hours.
[0076] D-Amino acid thus produced was determined by
high-performance liquid chromatography (column, CROWNPAK CR (Daicel
Chemical); column temperature, 26.degree. C.; cooled in ice for
only valine; mobile phase, HClO.sub.4 aqueous solution, pH 2.0;
flow rate, 1 ml/min; and detection, 200 nm). The results are shown
in Table 1.
1 TABLE 1 D-Phenyl- alanine D-Valine D-Leucine D-Tryptophan
D-Methionine produced from produced produced produced from produced
from N-acetyl- from N- from N- N-acetyl-D- N-acetyl-D- D-phenyl-
acetyl-F- acetyl-D- tryptophan methionine alanine valine leucine
(mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) Hypomyces 0.008 0.02 0.005
0.14 0 aurantius IFO 6847 Hypomyces 0.002 0.01 0.004 0.18 0
broomeanus IFO 9164 Hypomyces 0.001 0.12 0.003 3.16 1.05
chrysospermu a IFO 6817 Hypomyces 0.08 0.14 0.06 2.73 0.3 rosellus
IFO 6911 Hypomyces 0.01 0.24 0.15 3.19 1.60 sepulcralis IFO 9102
Hypomyces 0.002 0.03 0.05 0.23 0.10 subiculosus IFO 6892 Hypomyces
0.27 0.10 0.003 0.11 0.11 mycophilus ATCC 76474 Hypomyces 0.40 0.71
0.65 0.98 0.55 mycophilus IFO 6785 Fusarium 0 0.04 0.001 0 0.14
solani IFO 9974 Fusarium 0.001 0.02 0.002 0.22 0.21 solani IFO
9975
[0077] From Table 1, it is clear that the D-aminoacylase producing
capability was found in all fungal strains tested.
EXAMPLE 2
[0078] A platinum loopful of Hypomyces rosellus IFO 6911 strain,
Hypomyces sepulcralis IFO 9102 strain, or Hypomyces mycophilus IFO
6785 strain was inoculated into 25 ml of YM medium (containing 0.3%
yeast extract, 0.3% malt extract, 0.5% peptone, and 2.0% glucose;
pH 6.0) in a 500-ml shouldered flask. The fungi were shake-cultured
at 24.degree. C. for 3 days. After culturing, a 5-ml aliquot of the
culture medium was centrifuged in a refrigerated centrifuge to
collect the fungal cells. The cells were washed with physiological
saline solution (5 ml), and collected by centrifugation again. A
reaction solution (containing 0.1% N-acetyl-DL-amino acid shown in
Table 2 below in Tris-HCl buffer, pH 7.5) was added to the cells
thus collected. The mixture was incubated in a 5-ml test tube with
shaking at 30.degree. C. for 24 hours. D-Amino acid thus produced
was assayed by high-performance liquid chromatography (column,
CROWNPAK CR (Daicel Chemical); column temperature, 26.degree. C.;
mobile phase, HClO.sub.4 aqueous solution pH 2.0; flow rate, 1
ml/min; and detection, 200 nm) and examined for the optical purity.
The results are shown in Table 2.
2 TABLE 2 D-Phenyl- D-Tryptophan alanine Optical produced from
Optical produced from purity of N-acetyl-DL- purity of N-acetyl-DL-
D-phenyl- tryptophan D-tryptophan phenylalanine alanine (mg/mi) (%
ee) (mg/ml) (% ee) Hypomyces 0.24 75 0.21 95 rosellus IFO 6911
Hypamyces 0.13 94 0.12 94 sepulcralis IFO 9102 Hypomyces 0.10 37
0.16 40 mycophilus IFO 6785
[0079] It was thus confirmed that all the strains tested produced
the D-enantiomer of a high optical purity from the corresponding
DL-racemate.
EXAMPLE 3
[0080] Auricularia auriculajudae IFO 5949, Pythium aphanidermaatum
IFO 7030, and Menisporopsis novaezelandiae IFO 9179 were each
inoculated into sterilized media (100 ml each) (Czapek Dox Broth
for IFO 5949, YM1 medium for IFO 7030, and POTATO DEXTROSE BROTH
for IFO 9179) in 500-ml Erlenmeyer flasks. The fungi were cultured
on a rotary shaker at 210 rpm at 24.degree. C. for 7 days and
collected by centrifugation using a HIMAC SCR 20B HITACHI
centrifuge with a RPR10-2 rotor at 8,000 rpm (12,500.times.g) for
20 min. The cells thus sedimented were washed with 50 mM phosphate
buffer (pH 7.0), dehydrated, and stored frozen.
[0081] In this experiment, POTATO DEXTROSE BROTH (DIFCO) was
prepared by dissolving 24 g of the dried medium powder (a 10:1
mixture of Potato infusion form and Bacto Dextrose) in 1 liter of
water, adding N-acetyl-DL-methionine (N-Ac-DL-Met) thereto to 0.1%,
and adjusting the pH to 5.1. Czapek Dox Broth consisted of 0.3%
NaNO.sub.3, 0.1% K.sub.2HPO.sub.4, 0.05% MgSO.sub.4.7H.sub.2O,
0.05% KCl, 0.001% FeSO.sub.4.7H.sub.2O, 3.0% sucrose, 0.2% yeast
extract, 0.2% polypeptone, and 0.1% N-Ac-DL-Met, and was adjusted
to pH 7.3. YM1 medium (Yeast extract-malt extract-peptone-glucose
medium) consisted of 0.5% polypeptone, 0.3% yeast extract, 0.3%
malt extract, 0.2% glucose, and 0.1% N-Ac-DL-Met, and was adjusted
to pH 5.5 to 6.0.
[0082] The frozen cells were disrupted by adding 50 mM phosphate
buffer (pH 7.0) to the cells to give a cell suspension of 1 g of
cells per 6 ml, putting the suspension (1.5 ml) and glass beads
(0.2 dia. to 0.5 mm) (1.5 g) in a 2.0-ml sample vial equipped with
a disrupter, and disrupting using a Mini-Bead Beater.TM. (BIOSPEC
PRODUCTS) at 2,500 rpm for 150 sec followed by 3.5-min intervals of
ice-cooling (a total of eight cycles). Disrupted cells thus
obtained were centrifuged using a HIMAC SCR20B HITACHI centrifuge
with a RPR20-2 rotor at 17,500 rpm (38,000.times.g) for 30 min at
4.degree. C. to obtain the supernatant as the crude enzyme. The
reaction was performed in a solution (containing 20 mM N-Ac-D-amino
acid shown in Table 3, 1 mM CoCl.sub.2, 50 mM phosphate buffer; pH
7.0) to assay the enzyme activity for each substrate. The results
are shown in Table 3.
3TABLE 3 Auricularia Pythiwn Menisporopsis auriculajudae
aphanidermaatum novaezelandiae Substrate IFO 5949 IFO 7030 IFO 9179
N-Acetyl-D- 2.45 0.90 0.89 methionine N-Acetyl-D-alanine 1.46 0.29
0.09 N-Acetyl-D-leucine 2.68 -- -- N-Acetyl-D- 4.34 -- 0.44
phenylalanine N-Acetyl-D- 2.44 -- 1.30 tryptophan N-Acetyl-D-valine
3.33 -- 0.16 N-Acetyl-D-aspartic 1.10 -- 0.66 acid
[0083] Figures in the table denote the amount (mM) of D-amino acids
produced from the corresponding N-acetyl-D-amino acids. The
D-aminoacylase activity was detected in all the strains tested,
producing D-amino acid from the corresponding N-acetyl-D-amino
acid.
EXAMPLE 4
[0084] (1) Fungal Strain and Flask Culture Method
[0085] YM medium (50 ml) (containing 0.3% yeast extract (Kyokuto
Seiyaku), 0.3% malt extract (Kyokuto Seiyaku), 0.5% polypeptone
(Nihon Seiyaku), 2.0% glucose (Wako Pure Chemical); pH 6.0) was
placed in a 500-ml baffled Erlenmeyer flask, autoclaved, and used
as the growth medium for producing D-aminoacylase by Hypomyces
mycophilus IFO 6785 strain. The fungal strain previously grown in
YM agar medium (containing 0.3% yeast extract, 0.3% malt extract,
0.5% polypeptone, 2.0% glucose (Wako Pure Chemical), and 1.5% agar
(Wako Pure Chemical); pH 6.0) on the plate was excised in about
5-cm square sections using a sterilized surgical knife, inoculated
into the above-described medium in the flask, and incubated on a
rotary shaker at 145 rpm and 25.degree. C. for 96 h. After
incubation, the cells were collected by centrifugation (with a TOMY
SEIKO MRX-150 centrifuge using a TMA-3 rotor) at 12,000 rpm
(12,000.times.g) and 4.degree. C. for 5 min. The cells thus
collected were washed with physiological saline solution and
centrifuged again using the same rotor at 12,000 rpm
(12,000.times.g) for 5 min to obtain the cells.
[0086] (2) Assay Method for D-Aminoacylase Activity
[0087] The cells obtained above were disrupted in 50 mM Tris-HCl
(pH 7.5) containing 0.01% 2-ME (.beta.-mercaptoethanol) and 1
mMPMSF (phenyl methyl sulfonyl fluoride) using a Mini Bead Beater 8
(BIOSPEC PRODUCTS) twice for 5 min each, then centrifuged at 15,000
rpm (18,000.times.g) for 5 min with an MRX-150 centrifuge using a
TMA-2 rotor (TOMY SEIKO) to obtain the supernatant, a crude
D-aminoacylase solution.
[0088] The enzyme reaction was performed in a reaction system
(total volume 1.0 ml) containing 20 mM N-acetyl-D-tryptophan
(Sigma), 50 mM Tris-HCl (pH 7.5), and an appropriate amount of the
enzyme at 30.degree. C. for 10 min. The reaction was then
terminated by adding a TCA reaction terminating solution (0.5 ml)
according to Tsai et al. (consisting of 110 mM trichloroacetic
acid, 220 mM sodium acetate, and 330 mM acetic acid).
[0089] The enzyme activity was assayed by determining the amount of
amino acid produced using the TNBS method and the HPLC method. In
the TMBS method, 100 mM Na.sub.2B.sub.4O.sub.7 (0.5 ml) was added
to a sample solution containing amino acid (0.5 ml), 20 .mu.l of
110 mM TNBS (trinitrobenzenesulfonic acid) was then added, and the
resulting mixture was quickly stirred. After 5 min, 100 mM
NaH.sub.2PO.sub.4 containing 1.5 mM Na.sub.2SO.sub.3 (2 ml) was
added to the reaction mixture to terminate the color reaction, and
the absorbance at 420 nm was measured. HPLC was performed by
subjecting a sample solution containing amino acid to
high-performance liquid chromatography using an ODS column (column,
Wakosil II 5C18 (4.6 dia..times.250 mm) (Wako Pure Chemical);
eluent, CH.sub.3CN/50 mM KH.sub.2PO.sub.4.H.sub.3PO.sub.4 (pH
2.5)=2:8; detection, A280 nm; and flow rate, 1.0 ml/min). The
retention times were 3.5 min for D-tryptophan and 9.8 min for
N-acetyl-D-tryptophan. Using D-tryptophan as the assay standard,
one unit (or U) of the enzyme was defined as the amount of the
enzyme required to produce 1 .mu.mol of D-tryptophan at 30.degree.
C. for 1 min.
[0090] It was found that Hypomyces mycophilus IFO 6785 has a
relatively high D-aminoacylase activity.
EXAMPLE 5
[0091] Purification of D-aminoacylase derived from Hypomyces
mycophilus IFO 6785 strain
[0092] 1. Conditions for D-Aminoacylase Production
[0093] This fungal strain was cultured in the YM liquid medium (50
ml) described in Example 4 supplemented with or without
N-acetyl-DL-valine, N-acetyl-DL-tryptophan, N-acetyl-DL-methionine,
DL-valine, DL-tryptophan, or DL-methionine (0.1% each) as the
enzyme inducer at 25.degree. C. for 96 h in a baffled Erlenmeyer
flask on a rotary shaker (145 rpm). After incubation, a crude
enzyme solution was prepared and assayed for enzyme activity by
HPLC in the same manner as in Example 4. The results are shown in
Table 4.
4 TABLE 4 DCW* Protein Activity Inducer pH g/l-broth mg/ml units/ml
units/mg None 5.02 5.188 0.248 0.00854 0.0345 5.12 1.925 0.771
0.0349 0.0453 DL-Val 5.08 3.100 0.854 0.0198 0.0232 4.88 5.025
1.520 0.0323 0.0213 DL-Trp 4.80 4.438 1.144 0.0213 0.0186 5.15
7.100 0.437 0.0118 0.0270 DL-Met 4.53 7.675 1.172 0.0283 0.0241
4.91 2.800 1.017 0.0241 0.0237 N-Ac-DL- 4.23 3.788 0.256 0.00468
0.0183 Val 4.27 1.938 0.475 0.0103 0.0216 N-Ac-DL- 4.44 7.688 0.497
0.00705 0.0142 Trp 4.51 3.013 1.101 0.0358 0.0325 N-Ac-DL- 4.31
1.975 0.520 0.0120 0.0231 Met 4.35 3.913 0.795 0.0221 0.0278 *DCW
stands for the dry cell weight.
[0094] The results indicate that D-aminoacylase is not an inducible
enzyme but a constitutive enzyme in this fungal strain.
[0095] 2. Culturing Method using Jar Fermentor
[0096] This fungal strain was cultured in 20 liters of a liquid
medium (containing 0.3% yeast extract (Kyokuto Seiyaku), 0.3% malt
extract (Kyokuto Seiyaku), 1.0% polypeptone (Wako Pure Chemical),
2.0% glucose (Wako Pure Chemical), and 0.01% silicon FS028; pH 6.0)
then placed in a 30-1 jar fermentor at 25.degree. C., 200 rpm, 1
v.v.m., without pressure for 44 h. The preculture used was obtained
by the flask culture method in Example 4.
[0097] After culturing, the culture was immediately cooled in
ice-water and filtered using No. 5A filter paper (Toyo Roshi) by
suction to collect the fungal cells. The cells thus collected were
washed with physiological saline solution, recovered again by the
suction filtration, and stored at -90.degree. C. till use.
[0098] 3. Purification of D-Aminoacylase
[0099] (1) Cell Disruption, Removal of Nucleic Acid, and Salting
Out with Ammonium Sulfate
[0100] Frozen cells (about 100 g) were wrapped in double-layered
plastic bags with zippers and pulverized in an aluminum vat using a
mallet. Pulverized cells were added to 50 mM Tris-HCl (pH 9.0)
containing 1 .mu.M leupeptin, 1 .mu.M pepstatin A, 1 mM PMSF, and
0.01% 2-ME to prepare a cell suspension. The suspension was then
subjected to continuous trituration with a Dyno mill type KDL
(Wiley A. Bachofen, Based, Switzerland) using 0.2 to 0.5 mm glass
beads.
[0101] The resulting cell triturate was centrifuged (with a Hitachi
Koki centrifuge 20PR-52D using a RPR-9 rotor) at 8,000 rpm
(6,000.times.g) at 4.degree. C. for 30 min to sediment unbroken
cells and cell debris. The supernatant thus obtained was assayed to
determine the protein concentration according to the method
described in Example 4. As a result, since the total protein
amounted to 61,500 mg, {fraction (1/10)}th weight of protamine
sulfate was added dropwise to the supernatant as a 3% solution (in
the same buffer used for the trituration) with stirring at low
temperature, and the mixture was stirred for an additional 2 h. The
resulting mixture was centrifuged (with a Hitachi Koki centrifuge
20PR-52D using an RPR-9 rotor) at 6,000 rpm (3,000.times.g) for
20min at 4.degree. C. to sediment microsomes and nucleic acids.
[0102] The supernatant was reversely dialyzed against 17 liters of
50 mM Tris-HCl (pH 9.0) containing 77% saturated ammonium sulfate,
0.1 mM PMSF, 0.1 .mu.M leupeptin, 0.1 .mu.M pepstatin A, and 0.01%
2-ME with stirring overnight. Since this did not completely salt
out D-aminoacylase, ammonium sulfate in excess to the dialyzate was
added directly with stirring at low temperature. The precipitate
was collected by centrifugation (with a Tomy Seiko RS-20BH
centrifuge using a BH-9 rotor) at 10,000 rpm (16,000.times.g) at
4.degree. C. for 20 min then suspended in a small amount of 10 mM
Tris-HCl (pH 9.0) containing 0.1 mM PMSF, 0.1 .mu.M leupeptin, 0.1
.mu.M pepstatin A, and 0.01% 2-ME. The suspension was dialyzed
against the same buffer (10 liter) for 4 h, and then against the
same freshly replaced buffer (10 liter) overnight.
[0103] (2) DEAE-Sepharose FF 5.0/25 Anion Exchange
Chromatography
[0104] The above-described enzyme thus prepared was further
purified by anion exchange chromatography. Namely, the crude enzyme
solution was adsorbed by an XK50 column (5.0 dia..times.25 cm, 500
ml) packed with DEAE-Sepharose FF (both from Amersham Pharmacia
Biotech) equilibrated with 10 mM Tris-HCl (pH 9.0) containing 0.01%
2-ME. After the column was washed with three volumes of the same
buffer, the enzyme was eluted with a linear gradient of NaCl from 0
M to 0.5 M in seven volumes of the same buffer. The amount of
protein in each fraction was estimated by measuring the absorbance
at 280 nm (FIG. 1).
[0105] The D-aminoacylase activity was assayed by performing the
enzyme reaction in 50 mM Tris-HCl (pH 7.5) containing 20 mM
N-acetyl-D-tryptophan (total volume of 100 .mu.l) at 30.degree. C.
for 30 min. An equal volume of 100 mM Na.sub.2B.sub.4O.sub.7-NaOH
solution containing 4 mM TNBS was added to the above reaction
mixture. The absorbance was measured at 450 nm to estimate the
amount of free D-tryptophan.
[0106] D-Aminoacylase was eluted with a buffer containing from 0.20
to 0.25 M NaCl. Fractions containing the D-aminoacylase activity
were combined, and concentrated fivefold using a UF membrane
(Amicon, YM-10 76 mm dia.). Ammonium sulfate was added to the
concentrate to 70% saturation, and the mixture was allowed to stand
overnight to complete the precipitation. The mixture was then
centrifuged (with a Hitachi Koki HIMAC CR26H centrifuge using a
RR18A rotor) at 12,000 rpm (18,000.times.g) and 4.degree. C. for 10
min to recover the precipitate. The recovered precipitate was
suspended in 10 ml of 200 mM KPB (pH 8.5) containing 0.01% 2-ME and
0.3 M Na.sub.2SO.sub.4, dialyzed against the same buffer (2 liters)
overnight. Dialysis was performed then again against the same but
freshly replaced buffer (2 liters) for 4 h.
[0107] (3) Phenyl-Sepharose HP 2.6/10 Hydrophobic
Chromatography
[0108] The enzyme obtained in (2) was further purified by
hydrophobic chromatography. Specifically, the enzyme was adsorbed
by a Phenyl-Sepharose Hi-Load HP2.6/10 column (Amersham Pharmacia
Biotech, 2.6 dia..times.10 cm, 50 ml) equilibrated with 200 mM KPB
(pH 8.5) containing 0.01% 2-ME and 0.3 M Na.sub.2SO.sub.4. After
the column was washed with four volumes of the same buffer, the
enzyme was eluted by linearly decreasing the concentration of
Na.sub.2SO.sub.4 in the above-described buffer (buffer A) from 0.3
M to 0 M, that is, linearly increasing the concentration of 10 mM
KPB (pH 8.5) containing 0.01% 2-ME from 0% to 100% in buffer A. The
amount of protein in each fraction was estimated by measuring the
absorbance at 280 nm. The D-aminoacylase activity was assayed in
the same manner as described in (2) (FIG. 2).
[0109] Ammonium sulfate was added to the fractions containing the
D-aminoacylase activity to 70% saturation, and the mixture was
gently stirred for 2 h to form precipitates, which were recovered
by centrifugation (with a Hitachi Koki HIMAC CR26H centrifuge using
a RR18A rotor) at 12,000 rpm (18,000.times.g) and 4.degree. C. for
10 min. The precipitate thus recovered was dissolved in about 3 ml
of 10 mM Tris-HCl (pH 9.5) containing 0.01% 2-ME and 0.3 M
NaCl.
[0110] (4) Superdex 200 Hi-Load 1.6/60 Gel Filtration
Chromatography
[0111] The enzyme solution obtained in (3) was further purified by
gel filtration chromatography. Specifically, the enzyme was applied
onto a Superdex 200 Hi-Load 1.6/60 column (Amersham Pharmacia
Biotech, 1.6 dia..times.60 cm, 120 ml) equilibrated with 10 mM
Tris-HCl (pH 9.5) containing 0.01% 2-ME and 0.3 M NaCl, and eluted
with the same buffer (240ml) at a flow rate of 1 ml/min. The amount
of protein in each fraction was estimated by measuring the
absorbance at 280 nm. The D-aminoacylase activity was assayed in
the same manner as described in (2) (FIG. 3).
[0112] The fractions containing the D-aminoacylase activity were
concentrated using a UF membrane (Amicon, YM-10, 43 mm dia.),
diluted with 10 mM Tris-HCl (pH 9.5) containing 0.01% 2-ME, and
concentrated again. The same procedure was repeated twice to desalt
the sample.
[0113] (5) MonoQ HR 5/5 Anion Exchange Chromatography
[0114] The above-purified enzyme was further purified by anion
exchange chromatography. Specifically, the enzyme was adsorbed by a
MonoQ HR 5/5 column (Amersham Pharmacia Biotech, 0.5 dia..times.5
cm, 1.0 ml) equilibrated with 10 mM Tris-HCl (pH 9.0) containing
0.01% 2-ME. After the column was washed with three volumes of the
same buffer, the enzyme was eluted with a linear gradient of NaCl
from 0 M to 0.6 M in 21-volumes of the same buffer. The amount of
protein in each fraction was estimated by measuring the absorbance
at 280 nm.
[0115] The D-aminoacylase activity was assayed in the same manner
as described in (2). A portion of the fraction having the
D-aminoacylase activity was subjected to SDS-polyacrylamide gel
electrophoresis (SDS-PAGE).
[0116] (6) SDS-Polyacrylamide Gel Electrophoresis
[0117] Electrophoresis was performed using a Phast-System (Amersham
Pharmacia Biotech) according to the method of Laemmli (Laemmli, U.
K.: Nature, 227, pp680). A Phast gel Homo 12.5 (Amersham Pharmacia
Biotech) was used as the gel. The sample for electrophoresis was
prepared by mixing the enzyme solution with an equal volume of the
sample treatment solution (125 mM Tris-HCl buffer (pH 6.8)
containing 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-ME,
and 0.005% Bromophenol Blue (BPB)); heating the mixture at
100.degree. C. for about 5 min in a block heater; then cooling it
to the room temperature. Aliquots (2 .mu.l) were subjected to
electrophoresis. The protein was detected by a staining method with
CBB-R. As a result, only a unique single band presumed to be the
desired D-aminoacylase was detected.
EXAMPLE 6
[0118] Property of D-aminoacylase derived from Hypomyces mycophilus
IFO 6785 strain
[0119] 1. Determination of Molecular Weight
[0120] Molecular weight was determined by (1) gel filtration and
(2) SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
[0121] (1) Gel Filtration
[0122] D-Aminoacylase derived from Hypomyces mycophilus IFO 6785
strain obtained in Example 2 was purified in the same manner as in
Example 5 and subjected to the gel filtration as described in
Example 5-3(4). MW-Marker proteins (HPLC) (Oriental Yeast)
consisting of glutamate dehydrogenase (290,000), lactate
dehydrogenase (142,000), enolase (67,000), myokinase (32,000), and
cytochrome C (12,400) were used as molecular weight markers. As a
result, the molecular weight of this enzyme was indicated to be
about 56,000 (FIG. 4).
[0123] (2) SDS-Polyacrylamide Gel Electrophoresis
[0124] D-Aminoacylase derived from Hypomyces mycophilus IFO 6785
strain in Example 2 was purified in the same manner as in Example 5
and subjected to electrophoresis as described in Example 5-3(6). An
Electrophoresis Caribration Kit (Amersham Pharmacia Biotech)
consisting of phosphorylase b (94,000), bovine serum albumin
(67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean
trypsin inhibitor (20,100), and .alpha.-lactoalbumin (14,400)
provided the molecular weight markers. As a result, the molecular
weight of this enzyme was indicated to be about 64,000 (FIG.
5).
[0125] 2. Substrate Specificity
[0126] Substrate specificity of the enzyme was expressed as the
percentage of the specific activity compared with that for
N-acetyl-D-methionine taken as 100%.
N-chloroacetyl-D-phenylalanine, N-acetyl-D-valine,
N-acetyl-D-phenylalanine, N-acetyl-D-tryptophan,
N-acetyl-D-leucine, N-acetyl-L-valine, N-acetyl-L-phenylalanine,
N-acetyl-L-tryptophan, and N-acetyl-L-leucine substrates were used
for the comparison. The enzyme reaction was performed in 50 mM
Tris-HCl (pH 7.5) containing 1 .mu.l of the enzyme solution and 20
mM of each substrate (total volume 1.0 ml) at 30.degree. C. for 20
min, and terminated by adding 0.5 ml of a TCA reaction terminating
solution according to Tsai et al. (consisting of 110 mM
trichloroacetic acid, 220 mM sodium acetate, and 330 mM acetic
acid). The enzyme activity was assayed by the TNBS method as
described in Example 4(2). The results are shown in Table 5.
5 TABLE 5 D-enantiomer L-enantiomer N-Acetyl-amino acid Phe 298 0
Trp 109 0 Met 100 0 Leu 77.5 0 Val 17.5 0 Ala 0 Asn 0 Ile 0 Pro 0
penicillin 0 N-Chloroacetyl-amino acid Phe 708 N-Benzoyl-amino acid
Phe 0
[0127] As indicated in the table, this enzyme was especially active
for N-acetyl-D-phenylalanine and N-chloroacetyl-D-phenylalanine,
very active for N-acetyl-D-tryptophan, N-acetyl-D-methionine and
N-acetyl-D-leucine, and somewhat active for N-acetyl-D-valine, but
inactive for N-acetyl-L-phenylalanine, N-acetyl-L-tryptophan,
N-acetyl-L-methionine; N-acetyl-L-valine, and
N-acetyl-L-leucine.
[0128] 3. Optimal pH
[0129] Enzyme assay was performed at 30.degree. C. for 5 min
according to the method described in Example 4(2) varying the pH of
the enzyme reaction system from pH 4.0 to pH 11.0. The amount of
enzyme was determined by HPLC as described in Example 4(2). The
buffers used were 50 mM AcONa.AcOH buffer for pH 4.0 to 6.0, 50 mM
K.sub.2HPO.sub.4.KH.sub.2PO- .sub.4 buffer for pH 5.5 to 8.0, 50 mM
Tris-HCl buffer for pH 7.0 to 9.0, and 50 mM glycine -NaOH buffer
for pH 8.0 to 11.0. The results are shown in FIG. 6. The results
indicated that the optimal pH for the reaction of this enzyme was
from 7.5 to 8.0 and that the enzyme exhibits not less than 80% of
its activity within the pH range from 6.5 to 9.0.
[0130] 4. Optimal Temperature
[0131] Enzyme assay was performed for 5 min according to the method
described in Example 4(2) while varying the temperature of the
enzyme reaction system from 20.degree. C. to 55.degree. C. The
enzyme was determined by HPLC as described in Example 4(2). In
order to prevent a temperature-dependent pH change, 50 mM
K.sub.2HPO.sub.4.KH.sub.2PO.sub.4 buffer (pH 7.5) was used. The
results are shown in FIG. 7. The results indicated that the optimal
temperature for reaction of this enzyme was about 45.degree. C.
[0132] 5. pH Stability
[0133] The enzyme solution was diluted 50-fold with each pH buffer,
kept at 30.degree. C. for 30 min, thoroughly cooled in ice, and
then assayed for the residual enzyme activity according to the
method described in Example 4(2). The enzyme was determined by HPLC
as described in Example 4(2). In order to minimize the influence of
buffer constituents, Britton and Robinson's buffer (Basal Methods
of Protein and Enzyme Experiments, 2nd Rev. Ed., Buichi Horio ed.,
1994) containing 0.01% 2-ME was used, changing pH from 5.0 to 11.0
at 0.5 intervals. The results are shown in FIG. 8 and revealed that
this enzyme was most stable at pH 9.5, expressing not less than 80%
of its activity within the pH range from pH 7.5 to 10.5.
[0134] 6. Thermostability
[0135] The enzyme solution was diluted with the buffer, kept at
each temperature from 15 to 60.degree. C. for 30 min, thoroughly
cooled in ice, and then assayed for the residual enzyme activity
according to the method described in Example 4(2). The enzyme was
determined by HPLC as described in Example 4(2). Britton and
Robinson's buffer (pH 9.5) containing 0.01% 2-ME was used as the
buffer. The diluted enzyme solution was either not treated (kept at
4.degree. C.) or treated at each temperature from 15.degree. C. to
65.degree. C. at 5.degree. C. intervals. The results shown in FIG.
9 indicate that this enzyme was activated by heating to 40.degree.
C. to 45.degree. C., but quickly inactivated when heated to
50.degree. C. or higher. The enzyme was also effectively activated
by heating at 40.degree. C. for 30 min. Both longer and shorter
heating periods were less effective, and once activated, enzyme
maintained its activity for about 3 days when stored at 4.degree.
C.
[0136] 7. Effects of Various Metal Salts and Reagents
[0137] The enzyme solution was diluted with Britton and Robinson's
buffer (pH 9.5) containing 1 mM DTT, activated by heating at
40.degree. C. for 30 min, diluted with the same buffer containing
various metal salts and reagents, and kept at 40.degree. C. for 30
min. After cooling sufficiently, the mixture was assayed for the
residual enzyme activity according to the method described in
Example 4(2). The amount of enzyme was determined by HPLC as
described in Example 4(2). The results are shown in Tables 6 and
7.
6 TABLE 6 Concentra- Relative Metal ion tion (mM) activity (%) KCl
1 102 MgCl.sub.2 1 98.0 CaCl.sub.2 1 104 ZnCl.sub.2 1 26.2
NiCl.sub.2 1 57.7 CoCl.sub.2 1 63.1 MnCl.sub.2 1 100 No addition
100
[0138]
7TABLE 7 Concen- Relative tration activity Compound Action (mM) (%)
Hydroxylamine Carbonyl reagent 1 96.2 lodoacetamide SH-enzyme 1 185
N-ethylmaleimide SH-enzyme 1 15.2 Sodium azide Respiratory chain 1
90.9 inhibitor P-Chloromercuri- SH-enzyflle 0.5 99.6 benzoic acid
HgCl.sub.2 SH-enzyme 0.01 13.4 CuSO.sub.4 SH-enzyme 1 20.4
ZnSO.sub.4 SH-enzyme 1 11.6 o-Phenanthroline metal enzyme 1 78.8
EDTA metal enzyme 1 96.0 EGTA metal ion 1 90.9 PMSF serine protease
or 1 94.2 choline esterase No addition 100
[0139] These data show that this enzyme was inhibited by metal ions
such as Co.sup.2+, Cu.sup.2+, Zn.sup.2+ and Hg.sup.2+ but also
exhibited previously unknown properties such as not being inhibited
by EDTA and being activated by ICH.sub.2CONH.sub.2.
[0140] 8. Determination of Internal Sequence
[0141] A solution containing the purified enzyme (1 nmol) was
concentrated by ultrafiltration to 50 .mu.l, 50 mM Tris-HCl (pH
9.0) containing 8 M urea (150 .mu.l) was added to the concentrated
solution, and the mixture was kept at 37.degree. C. for 1 h. After
200 .mu.l of 50 mM Tris-HCl (pH 9.0) was added, the mixture was
digested with lysyl endopeptidase (5 pmol) at 30.degree. C.
overnight. Digested products were fractionated by high-performance
liquid chromatography using an ODS column (column, TSK gel ODS-120T
(4.6dia..times.250 mm) (Tosoh); eluent, buffer A (0.1% TFA) and
buffer B (80% CH.sub.3CN containing 0.095% TFA); detection, 214 nm;
flow rate, 1.0 ml/min; eluted by a programmed gradient), and
fractions were recovered.
[0142] The resulting fractions were concentrated by a centrifugal
evaporator (UNISCIENCE, UNIVAP) and sequenced with a protein
sequencer (A477, Applied Biosystems). Partial amino acid sequences
thus determined are shown in SEQ ID Nos: 1 through 5.
Sequence CWU 1
1
5 1 25 PRT Hypomyces mycophilus 1 Gly Phe Ile Leu Ser Pro Gly Phe
Ile Asp Met His Ala His Ser Asp 1 5 10 15 Lys Tyr Leu Leu Ser His
Pro Thr His 20 25 2 20 PRT Hypomyces mycophilus 2 Val Leu Ala Asp
Glu Tyr Pro Gln Ala Phe Tyr Ala Pro His Ala Tyr 1 5 10 15 Ser Arg
Gly Phe 20 3 18 PRT Hypomyces mycophilus 3 Thr Ala Thr Asn Val Ala
Met Leu Val Pro Gln Gly Asn Leu Arg Leu 1 5 10 15 Leu Ala 4 18 PRT
Hypomyces mycophilus 4 Ile Gly Glu Pro Gly Ser Ile Ser His Asp Ser
Ala Arg Arg Val Asp 1 5 10 15 Ala Lys 5 20 PRT Hypomyces mycophilus
5 Ser Tyr Thr Gly Arg Phe Val Gly Glu Ile Ala Arg Glu Thr Asn Arg 1
5 10 15 Leu Pro Ile Glu 20
* * * * *