Note: Descriptions are shown in the official language in which they were submitted.
1 ~13~'~'~G
TITLE OF THE INVENTION
PROCESS FOR PRODUCING CIS-3-HYDROXY-L-PROLINE
Background of the Invention
The present invention relates to a process for
producing cis-3-hydroxy-L-proline. Cis-3-hydroxy-L-proline is
useful as a starting compound for medicines and an additive to
foods. The present invention also relates to a novel enzyme
capable of catalyzing the hydroxylation of L-proline at the 3-
position of L-proline. The novel enzyme is used in the above-
mentioned process.
Heretofore, chemosynthetic methods of producing cis-
3-hydroxy-L-proline are known [(J. Amer. Chem. Soc., 84, 3980
(1962); J. Amer. Chem. Soc., 85, 2824 (1963); Nature 289, 310
(1981); J. Org. Chem., 54, 1866 (1989); Acta Chemica
Scandinavica, 43, 290 (1989)].
The conventional chemosynthetic methods for
producing cis-3-hydroxy-L-proline are not satisfactory for
industrial production, because of (1) the expensive raw
materials, (2) too many the reaction steps, (3) the
complicated procedures for isolating and purifying the product
and/or (4) the lower productivity of cis-3-hydroxy-L-proline.
No chemosynthetic or biological method of producing
cis-3-hydroxy-L-proline by hydroxylating L-proline both
position-selectively and stereo-selectively, had been reported
yet.
The object of the present invention is to provide an
advantageous process for the production of cis-3-hydroxy-L-
proline, and the second object of the present invention is to
provide a novel enzyme which catalyzes the hydroxylation of L-
proline at the 3-position and which is useful in the above
process.
Summary of the Invention
The present invention provides a process for
2 213~.~r6
producing cis-3-hydroxy-L-proline, which comprises allowing L-
proline to coexit with 2-ketoglutaric acid, a divalent iron
ion and an enzyme source which catalyzes the hydroxylation of
L-proline at the 3-position of L-proline in an aqueous medium
to convert L-proline into cis-3-hydroxy-L-proline, and
recovering the cis-3-hydroxy-L-proline from the aqueous
medium. The present invention further provides a novel
hydroxylase having the following physicochemical properties
(hereinafter referred to as L-proline-3-hydroxylase):
(1) Action and Substrate Specificity:
The enzyme catalyzes the hydroxylation of L-proline
at the 3-position of L-proline in the presence of 2-
ketoglutaric acid and a divalent iron ion to produce cis-3-
hydroxy-L-proline.
(2) Optimum pH Range:
The enzyme has an optimum pH range of 6.5 to 7.5,
when it is allowed to stand at 30°C for 20 minutes.
(3) Stable pH Range:
The enzyme is stable at pH values of 6.5 to 8.0,
when it is allowed to stand at 4°C for 23 hours.
(4) Optimum Temperature Range:
The optimum temperature is 35 to 40°C when it is
allowed to stand at pH 7.0 for 15 minutes.
(5) Stable Temperature Range:
The enzyme is inactivated, when it is allowed to
stand at pH 7.5 and at 50°C for 30 minutes.
(6) Inhibitors:
The enzyme is inhibited by metal ions of Zn++, Cu++,
Co++ and Ba++ and ethylenediaminetetraacetic acid (EDTA).
(7) Activation:
The enzyme does not need any cofactor for its
activation. L-Ascorbic acid accelerates the activity of the
enzyme.
(8) Km Value:
Km value is 0.49 mM for L-proline and is 0.11 mM for
- 3 - ~ ~ 31 '~ '~
2-ketoglutaric acid, when determined in a 100 mM N-
tris(hydroxymethyl)methyl-2-aminoethansulfonic acid (TES)
buffer (pH 7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous
sulfate and a pre-determined amount of this enzyme.
(9) Isoelectric point:
The enzyme has an isoelectric point of 4.3 by Phast
system.
(10) Molecular Weight:
The enzyme has a molecular weight of 35,000 ~ 5,000
daltons by sodium dodecylsulfate-polyacrylamide gel
electrophoresis.
(11) N-terminal Amino Acid Sequence:
The enzyme has an N-terminal amino acid sequence
illustrated by Sequence Number 1.
Sequence Number 1:
(N-terminal) 1 MetCysSerHisIleLeuGlyArgIleGlu
11 LeuAspGlnGluArgLeuGlyArgAspLeu
21 GluTyrLeuAlaThrValProThrVa1
Detailed Description of the Invention
As the enzyme source to be used in the present
invention, any microorganism can be used so long as it has an
enzymatic activity of catalyzing the hydroxylation of L-
proline at the 3-position of L-proline in the presence of 2-
ketoglutaric acid and a divalent iron ion. As the
microorganism having such activity, mention may be made of
microorganisms belonging to the genus Streptomyces or
Bacillus. The preferred strain of such microorganism includes
Streptomyces canus ATCC 12647, Streptomyces canus ATCC 12646,
Streptomyces sp. TH1, and Bacillus sp. TH2 and TH3.
Specifically, a culture, cells or processed cells of these
strains can be used. Further, a crude enzyme preparation from
cells of the microorganism as mentioned above, a purified
product of such enzyme preparation, an immobilized enzyme
preparation, etc. can be used.
4 - z13~~~~
Streptomyces sp. TH1 was newly isolated by the
present inventors from the soil. The microbiological
properties of the strain TH1 is mentioned below.
1. Morphology:
The morphological properties when the strain was
cultivated on various media at 28°C for 14 days are shown in
Table 1 below.
Table 1 - Morpholoctical Properties
1) Hyphae Formation of aerial hyphae: Yes
Fragmentation and motility
of aerial hyphae: Not observed
Fragmentation and motility
of substrate hyphae: Not observed
2) Spores Sporulation and Positions to
which spores
adhere: Adhered to aerial hyphae
Formation of sporangia: Not observed
Number of spores on its
aerial mycelium: More than 10
Appearance of spore chain: spiral
Characteristics of spores
Surface structure: Spiny
Shape and size:
ellipsoidal, 0.5 ~ 0.7 x 1.0 ~ 1.2 pm
Motility and fragella: Not observed
3) Others Chlamydospores: Not observed
Synnemata: Not observed
Pseudosporangia: Not observed
Branching mode of hyphae: Simple branching
2. Cultural Characteristics in Various Media:
The strain TH1 grows normally or vigorously on usual
synthetic and natural media, while its substrate hyphae are
pale pink (rose), orange or greenish brown. On some media,
the strain often produces brown or blackish soluble pigments.
The cultural characteristics of the strain TH1, when
- 5 - 2131'~~~
the strain was cultivated on various media at 28°C for 14
days, are shown in Table 2. The designation of the colors has
been made, according to the classification of colors indicated
in Color Harmony Manual published by Container Corporation of
America.
Table 2 - Cultural Characteristics in Various Media
1) Sucrose-nitrate Growth: Moderate
agar Color of substrate hyphae:
Pearl pink (3 ca)
Adhesion of aerial hyphae: None
Soluble pigments: None
2) Glucose- Growth: Moderate
asparagine agar Color of substrate hyphae:
Pearl pink (3 ca)
Adhesion of aerial hyphae: None
Soluble pigments: None
3) Glycerol- Growth: Moderate
asparagine agar Color of substrate hyphae:
Pearl pink - bright orange
(3 ca ~ 4 na)
Adhesion and color of aerial hyphae:
Poor, white (a)
Soluble pigments:
Produced only a little
(Pale yellow)
4) Inorganic salts- Growth: Moderate
starch agar Color of substrate hyphae:
Pearl (2 ba)
Adhesion and color of aerial hyphae:
Moderate, white (a)
Soluble pigments: Negative
5) Tyrosine agar Growth: Moderate
Color of substrate hyphae:
Light olive (1 1/2 ie)
Adhesion and color of aerial hyphae:
Moderate, white - olive gray
Soluble pigments: Produced (black)
z~ ~~ ~~~
6) Nutrient agar Growth: Poor
Color substrate hyphae:
of
Light melon yellow (3 ea)
Adhesion of aerial hyphae: None
Soluble
pigments:
None
7) Yeast extract- Growth: Abundant
malt extract agar Color substrate hyphae:
of
Flesh pink (4 ca)
Adhesion and color of aerial hyphae:
Moderate, white (a)
Soluble pigments:
Produced only a little
(ocher)
8) Oatmeal agar Growth: Poor
Color substrate hyphae:
of
Yellow tint (1 ba)
Adhesion and color of aerial hyphae:
Moderate, white (a)
Soluble pigments: None
3. Physiological Properties:
The physiological properties of the strain TH1 is
shown in Table 3, in which the "temperature range for growth"
indicates the results of the strain after 7 day-cultivation by
shaking. The remaining items indicate the results after 2 to
3 week-cultivation at 28°C.
2.1~1~'~~
- 7 _
Table 3 - Physiological Properties
1) Temperature Range for Growth . 23 ~ 37C
2) Liquefaction of Gelatin . No
3) Hydrolysis of Starch . Yes
4) Coagulation of Skim Milk Powder . No
5) Peptonization of Skim Milk Powder : No
6) Formation of Melanoid Pigments
(1) Peptone-yeast extract-iron agar : Yes
(2) Tyrosine agar . Yes
7) Utilization of Carbon Sources(*)
L-Arabinose . +
D-Glucose : +
D-Xylose : +
Sucrose . +
Raffinose . +
D-Fructose . +
Rhamnose . -
Inositol ; +
D-Mannitol . +
(*) The medium of Pridham Gottlieb was used as basal
medium. + indicates that the strain utilized the carbon
source; and - indicates that the strain did not utilize the
carbon source.
4. Chemotaxonomic Properties:
The configuration of diaminopimelic acid in the
cells is LL type.
Accordingly, the strain TH1 is classified to the
genus Streptomyces of actinomycetes in view of its
morphological properties that it forms spiral spore chain
consisting of more than ten spores on its aerial mycelium, and
in view of its chemotaxonomic properties that the
8
diaminopimelic acid contained in its cell wall is L,L-
diaminopimelic acid (type I).
The strain TH1 was identified as Streptomyces sp.
TH1, and the strain has been deposited with National Institute
of Bioscience and Human-Technology, Agency of Industrial
Science and Technology in Japan as of September 1, 1993 under
FERM BP-4399 in terms of the Budapest Treaty.
Bacillus sp. TH2 and TH3 were newly isolated by the
present inventors from the soil. The microbiological
properties of the strains TH2 and TH3 are mentioned below.
Both the strains TH2 and TH3 have the following
properties except that the cell sizes of the strain TH2 and
TH3 are 1.5 to 1.8 x 3 to 4 pm and 1.2 to 1.5 x 3.5 to 4 pm,
respectively, and the base compositions of DNA (G + C mold) of
the strains TH2 and TH3 are 36.46 and 37.74, respectively.
1. Morphological Properties:
The strains had the morphological properties shown
in Table 4 below.
Table 4 - Morphological Properties
1) Cells Morphology : Rod
Polymorphism . Not observed
Motility : Not observed
(Position flagella)
of
2) Endo Sporulation . Observed
Spores Morphology . Ellipse
Positions . The end position
2. Cultural Characteristics in Various Media:
The characteristics of the strains TH2 and TH3 are
shown in Table 5 below.
_ g _
Table 5 - Cultural Characteristics in Various Media
Bouillon-Agar Growth : Abundant
Medium Shape of surface . Smooth
(Meat extract) Color : Pale yellow white
Gloss : None
Diffusible pigments : Negative
Bouillon-liquid Growth appearance . Grown only a
Medium little on surface
(Meat extract) of the medium
Turbidity . Positive
Bouillon- Liquefaction of
Gelatin-Medium gelatin . Positive
Litmus milk Reaction : Acid
Coagulation . Negative
Liquefaction . Positive
3. Physiological Properties:
The physiological properties of the strains TH2 and
TH3 are shown in Table 6.
Table
6 (1)
- Physiological
Pro
erties
(1) Gram staining . + (11)Pigment production
King A medium : -
(2) Reduction to nitrite : +
. King B medium . -
(3) Denitrification
(12)Urease . -
reaction ; +
(4) Methyl red test , - (13)Oxidase . -
(5) VP test - (14)Catalase : +
(15)Growth range:
(6) Indole production : -
, pH; 5 ~ 9.7
(7) Hydrogen sulfide Optimum pH; 7.1
production . - Temperature; 4 ~ 36C
(8) Hydrolysis of starch : +
Optimum temp;
around 33C
(9) Utilization of citric (16)Attitude towards oxygen
acid . Aerobic: +
Koser's method - . Anaerobic
Christensen's
(facultitive an
method W
aerobic): +
(10)Utilization of inorganic (17)OF test: oxidation
nitrogen:
Nitrates . -
Ammonium salts : +
+: Positive,
. Negative,
W:
Weak
- 10 - 2131 '~'~ 6
Table 6 (2)
Production of Acid and Gas from Carbohydrates
Acid Gas
production production
L-Arabinose - -
D-Xylose - -
D-Glucose + -
D-Mannose - -
D-Fructose + -
D-Galactose - -
Maltose + -
Sucrose + -
Lactose - -
Trehalose + -
D-Sorbitol - -
D-Mannitol - -
Inositol - -
Glycerol - -
Starch + -
+; Positive . Negative
4. The other Properties and the Chemotaxonomic Properties:
The other properties of the strains TH2 and TH3 are
shown in Table 7, and the Chemotaxonomic properties are shown
in Table 8 below.
Table 7 - Other Properties
Degradation of Esculin ; +
Degradation of Malonic acid . -
Degradation of Arginine . +
Decarboxylation of Lysine . -
Decarboxylation of Ornithine : -
Deamination of Phenylalanine : -
Resistance to NaCl (7.5~) . -
Phosphatase ; +
t: Positive . Negative
11
Table 8 - Chemotaxonomic Properties
1) Cellular lipids
~ Ubiquinone : Negative
~ Menaquinone . MK-7
2) Diamino acid composition of cell
wall peptidoglycan . meso-A2 pm
The strains having the microbiological properties
mentioned above were compared with the strains indicated in
Bergey's Manual of Systematic Bacteriology (Vol. 2, 1986).
Both the strains TH2 and TH3 are classified to the
genus Bacillus of bacteria. The strains TH2 and TH3 were
identified as Bacillus sp. TH2 and Bacillus sp. TH3,
respectively. These strains have been deposited with the
National Institute of Bioscience and Human-Technology, Agency
of Industrial Science and Technology in Japan as of September
1, 1993 under FERM B-4397 for the strain TH2 and under FERM
BP-4398 for the strain TH3, both in terms of the Budapest
Treaty.
The medium for cultivating these microorganisms may
be any of natural media and synthetic media, so long as it
contains carbon sources, nitrogen sources, inorganic salts,
etc. that may be assimilated by microorganisms having an
activity of catalyzing the hydroxylation of L-proline to
produce cis-3-hydroxy-L-proline.
As the carbon sources, carbohydrates such as
glucose, fructose, sucrose, molasses containing these
compounds, starch and starch hydrolysates, etc.; organic acids
such as acetic acid, propionic acid, etc.; alcohols such as
ethanol, propanol, etc. may be used.
As the nitrogen sources, ammonia; ammonium salts of
various inorganic and organic acids such as ammonium chloride,
ammonium sulfate, ammonium acetate, and ammonium phosphate,
etc.; other nitrogen-containing compounds; peptone, meat
- 12 - ~1~1~~~
extracts, yeast extracts, corn steep liquor, casein
hydrolysates, soy bean cakes, soy bean cake hydrolysates,
various cultured cells of microorganisms, their digested
products, etc. may be used.
The inorganic material includes, for example,
potassium dihydrogen phosphate, dipotassium hydrogen
phosphate, magnesium phosphate, magnesium sulfate, sodium
chloride, ferrous sulfate, manganese sulfate, copper sulfate,
and calcium carbonate, etc.
The cultivation of these microorganisms is carried
out under aerobic conditions, for example, by shaking culture
or by submerged-aerial stirring culture. The temperature for
the cultivation is preferably from 15 to 37°C, and the period
for the cultivation is generally from 16 to 96 hours. During
the cultivation, the pH of the medium is kept at 5.0 to 9.0
with inorganic or organic acids, alkaline solutions, urea,
calcium carbonate, ammonia, etc.
The amount of the enzyme source to be used in the
process for producing cis-3-hydroxy-L-proline depends on the
amount of the substrate to be used in the process. Usually,
it may be from 1.0 to 10,000,000 U, preferably from 1,000 to
2,000,000 U per one liter of the aqueous medium.
In the case of using microbial cells and products
obtained by processing microbial cells, the concentration of
wet cells to be used may be generally from 1 to 300 g/1.
The activity of the enzyme for producing one nmol of
cis-3-hydroxy-L-proline for one minute under the conditions
mentioned below is defined as one unit (U).
The enzyme preparation to be determined is added to
100 mM TES buffer (pH 7.0) containing 5 mM L-proline, 5 mM 2-
ketoglutaric acid, 1 mM ferrous sulfate and 5 mM L-ascorbic
acid to make 100 pl in total, and the mixture was allowed to
stand at 35°C for 10 minutes. The reaction mixture is heated
at 100°C for 2 minutes so as to stop the reaction, and the
amount of cis-3-hydroxy-L-proline produced in the reaction
- 13 - ~1~1'~~~
mixture is determined by HPLC.
For the determination, any method capable of
determining the amount of cis-3-hydroxy-L-proline may be
employed. For instance, generally usable are a post-column
derivatization method where HPLC is utilized, and a pre-column
derivatization method where the compound to be determined in
the reaction mixture is previously reacted with 7-chloro-4-
nitrobenz-2-oxa-1,3-diazole chloride (hereinafter referred to
as NBD) to form its NBD derivative, the derivative is
separated by reversed-phase chromatography using HPLC and the
thus-separated derivative is quantitatively determined by
spectrofluorometry (excitation wavelength: 503 nm, emission
wavelength: 541 nm). The pre-column derivatization method
may be conducted, according to the method of William J.
Lindblad & Robert F. Diegelmann, et al. (see Analytical
Biochemistry, Vol. 138, pp. 390 - 395, 1984).
The concentration of L-proline to be used in the
process for producing cis-3-hydroxy-L-proline may be from 1 mM
to 2M.
The process needs a divalent iron ion. The
concentration of the divalent iron ion may generally be from 1
to 100 mM. Any divalent iron ion may be used so long as it
does not interfere the reaction. For instance, sulfides such
as ferrous sulfate, chlorides such as ferrous chloride,
ferrous carbonate, the salts of organic acids such as
citrates, lactates, fumarates, etc. may be used.
The process also needs 2-ketoglutaric acid. 2-
Ketoglutaric acid itself may be added to the reaction system,
or alternatively, the compound that may be converted into 2-
ketoglutaric acid by the metabolic activity of the microbial
cells used in the reaction may be added thereto. The compound
includes, for example, saccharides such as glucose, glutamic
acid, succinic acid, etc. These compounds may be used singly
or in combination.
_ The aqueous medium to be used in the process for
i ~ ~, ~x
CA 02131776 2001-11-22
- 14 -
producing cis-3-hydroxy-L-proline includes, for example,
water, phosphates, carbonates, acetates, borates, citrates,
buffers such as tris-buffers, alcohols such as methanol and
ethanol, esters such as ethyl acetate, ketones such as
acetone, amides such as acetamide, etc.
The reaction may be carried out in the culture
medium where the above-mentioned microorganisms having an
activity of catalyzing the hydroxylation of L-proline to
produce cis-3-hydroxy-L-proline are being cultivated or have
been cultivated, or alternatively, the reaction may also be
carried out in an aqueous medium containing the cells of the
above-mentioned microorganisms separated from the culture,
processed cells, or a purified or crude enzyme derived from
the cells.
Processed cells of the microorganisms include, for
example, dried cells, lyophilized cells, surfactant-treated
cells, enzymatically-treated cells, ultrasonically-treated
cells, mechanically-ground cells, mechanically-compressed
cells, solvent-treated cells, cellular protein fractions,
immobilized cells, immobilized materials obtained by
processing their cells, etc.
The enzyme reaction is generally carried out at a
temperature of 15 to 50°C and at a pH of 6.0 to 9.0, for a
period of 1 to 96 hours. If desired, surfactants and/or
organic solvents may be added during the processing of the
cells or during the reaction.
As the surfactants, mention may be made of cationic
surfactants such as polyoxyethylene-stearylamine (e. g., Nymeen*
S-215. made by Nippon Oils and Fats Co.),
cetyltrimethylammonium bromide, Cation FB; Cation F2-40E;
etc.; anionic surfactants such as sodium oleylamidosulfate,
Newrex*TAB, Rapizole*80, etc.; ampholytic surfactants such as
polyoxyethylene-sorbitan monostearate (e. g., Nonion*ST221),
etc.; other tertiary amines PB, hexadecyldimethylamine, etc.
Any surfactant that may promote the reaction may be employed.
*Trademark
- 15 -
The concentration of the surfactant to be employed in the
reaction may be generally from 0.1 to 50 mg/ml, preferably
from 1 to 20 mg/ml.
As the organic solvent, mention may be made of
toluene, xylene, aliphatic alcohols, benzene, ethyl acetate,
etc. Generally, the concentration of the organic solvent in
the reaction system may be from 0.1 to 50 pl/ml, preferably
from 1 to 20 pl/ml.
To recover cis-3-hydroxy-L-proline from the aqueous
medium, ordinary separation methods such as column
chromatography using ion-exchange resins, crystallization,
etc. may be employed. The recovered cis-3-hydroxy-L-proline
may be identified by ordinary analytic means using, for
example, 13C-NMR spectrum 1H-NMR spectrum, mass spectrum,
specific rotatory power, etc.
Next, the novel enzyme, the L-proline-3-hydroxylase
of the present invention is described below.
The L-proline-3-hydroxylase may be obtained by
cultivating a microorganism having an ability to produce L-
proline-3-hydroxylase in a medium so as to produce and
accumulate the L-proline-3-hydroxylase in the culture medium,
and recovering the L-proline-3-hydroxylase from the cells.
As the microorganism having an ability to produce L-
proline-3-hydroxylase, mention may be made of a microorganism
belonging to the genus Streptomyces or Bacillus and having
such activity. Specific examples are Streptomyces canus
ATCC12647, Streptomyces canus ATCC12646, Streptomyces sp. TH1,
Bacillus sp. TH2 and Bacillus sp. TH3, a sub-cultivated strain
thereof, the mutant thereof, its derivative thereof, etc.
The medium for cultivating these microorganisms may
be any of natural media and synthetic media, so long as it
contains carbon sources, nitrogen sources, inorganic salts,
etc. that may be assimilated by the microorganism having an
activity to produce L-proline-3-hydroxylase.
The carbon source includes, for example,
-16- 21317'~~
carbohydrates such as glucose, fructose, sucrose, molasses
containing these compounds, starch and starch hydrolysates,
etc.; organic acids such as acetic acid, propionic acid, etc.;
alcohols such as ethanol, propanol, etc. which may be
assimilated by the microorganisms.
As the nitrogen source, ammonia; ammonium salts of
various inorganic and organic acids such as ammonium chloride,
ammonium sulfate, ammonium acetate, ammonium phosphate, etc.;
other nitrogen-containing compounds; peptone, meat extracts,
yeast extracts, corn steep liquor, casein hydrolysates, soy
bean cakes, soy bean cake hydrolysates, various microorganisms
for fermentation, their digested products, etc. may be used.
As the inorganic material, dipotassium hydrogen
phosphate, potassium dihydrogen phosphate, magnesium
phosphate, magnesium sulfate, sodium chloride, ferrous
sulfate, manganese sulfate, copper sulfate, calcium carbonate,
etc. can be used.
The cultivation of these microorganisms is carried
out under aerobic conditions, for example, by shaking culture
or by submerged-aeration stirring culture. The temperature of
the cultivation is preferably from 15 to 37°C, and the period
for the cultivation is generally from 16 to 96 hours.
During the cultivation, the pH of the medium is kept
at 5.0 to 9Ø The pH of the medium is adjusted with
inorganic or organic acids, alkaline solutions, urea, calcium
carbonate, ammonia, etc. During the cultivation, L-proline
may be added, if desired.
To isolate and purify the enzyme from the culture
medium containing the enzyme, any ordinary method for
isolating and purifying an enzyme may be employed. For
instance, the culture is subjected to centrifugation to
collect the cultivated cells therefrom, and the cells are
fully washed and then disrupted by an ultrasonic cell
disruptor, a French press, a Manton Gaulin homogenizer, a Dyno
mill, etc. to obtain a cell-free extract. The cell-free
i i
CA 02131776 2001-11-22
- 17 -
extract is again subjected to centrifugation, and the
resulting supernatant is then purified, for example, by
salting-out with ammonium sulfate or the like, by anion-
exchange chromatography with Diethylaminoethyl(DEAF)-Sepharose*
or the like, by hydrophobic chromatography with Butyl-
Sepharose; Phenyl-Sepharose*or the like, by dye affinity
chromatography with Red-Agarose or the like, by gel filtration
with molecular sieves or by electrophoresis such as
isoelectric point electrophoresis or the like. In this way, a
pure product of the enzyme is obtained. The activity of the
L-proline-3-hydroxylase thus isolated may be determined by the
same method as mentioned above.
The L-proline-3-hydroxylase thus obtained, has the
following physico-chemical properties (1) to (11):
(1) Action and Substrate Specificity:
The enzyme catalyzes the hydroxylation of L-proline
at the 3-position of L-proline in the presence of 2-
ketoglutaric acid and a divalent iron ion to produce cis-3-
hydroxy-L-proline.
(2) Optimum pH Range:
The optimum pH range is 6.5 to 7.5 when it is
allowed to stand at 30°C for 20 minutes.
(3) Stable pH Range:
The enzyme is stable at pH values ranging from 6.5
to 8.0, when it is allowed to stand at 4°C for 23 hours.
(4) Optimum Temperature Range:
The optimum temperature is in 35 to 40°C.
when it is allowed to stand at pH 7.0 for 15 minutes.
(5) Stable Temperature Range:
The enzyme is inactivated, when it is allowed to
stand at pH 7.5 and at 50°C for 30 minutes.
(6) Inhibitors:
The enzyme is inhibited by metal ions of Zn++, Cu++,
Co++ and Ba++ and ethylenediaminetetraacetic acid (EDTA).
(7) Activation:
* Trademark
- 18 - z~317~~
The enzyme does not need any cofactor for its
activation. L-Ascorbic acid accelerates the activity of the enzyme.
(8) Km Value:
The Km value is 0.49 mM for L-proline and is 0.11 mM
for 2-ketoglutaric acid, when determined in a 100 mM N-
tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)
buffer (pH7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous
sulfate and a pre-determined amount of this enzyme.
(9) Isoelectric point:
The enzyme has an isoelectric point of 4.3 by Phast
system.
(10) Molecular Weight:
The enzyme has a molecular weight of 35,000 ~ 5,000
daltons by sodium dodecylsulfate-polyacrylamide gel
electrophoresis.
(11) N-terminal Amino Acid Sequence:
The enzyme has an N-terminal amino acid sequence
illustrated by Sequence Number 1.
Sequence Number 1:
(N-terminal) 1 MetCysSerHisIleLeuGlyArgIleGlu
11 LeuAspGlnGluArgLeuGlyArgAspLeu
21 GluTyrLeuAlaThrValProThrVal
The present invention is described in more detail by
the following examples. However, the Examples are not
intended to restrict the scope of the present invention.
Example 1
Production of Cis-3-hydroxy-L-proline:
SR3 medium comprising 1.0~ glucose, 1.0~ soluble
starch, 0.5~ yeast extract, 0.5~ tryptone, 0.3~ meat extract
and 0.05 magnesium phosphate, and adjusted to pH 7.2 with 6N-
NaOH, was put in test tubes (diameter 25 mm x length 200 mm)
in an amount of 10 ml each and sterilized at 120°C for 20
minutes. One loopful of cells of Streptomyces sp. THl, that
-19 - z1 ~~ ~~ 6
had grown in HT-agar plate medium comprising 1~ soluble
starch, 0.2~ NZ amine, 0.1~ yeast extract, 0.1~ meat extract
and 1.5~ agar, adjusted to pH 7.2 with 6N-NaOH, and sterilized
at 120°C for 20 minutes, was inoculated into the SR3 medium in
each test tube and cultivated at 28°C for 2 days by shaking
culture. The resulting culture was used as a seed culture in
the subsequent steps.
SR3 medium was put in 2 liter-Erlenmeyer flasks in
an amount of 200 ml each and sterilized at 120°C for 20
minutes. The seed culture was inoculated in the SR3 medium in
each Erlenmeyer flask and cultivated at 28°C for 2 days by
shaking.
Df3 medium comprising 5~ soluble starch, 3~ corn
steep liquor, 0.05 potassium dihydrogen phosphate, 0.05
magnesium sulfate 7-hydrate and 0.5~ calcium carbonate, and
adjusted to pH 7.0 with 6N-NaOH, was put in 5 liter-jar
fermenters in an amount of 2 liters each and sterilized at
120°C for 20 minutes. The above-mentioned seed culture was
inoculated in the DF3 medium in each jar fermenter under germ-
free condition and cultivated under the condition of 700 rpm
and 1 vvm, at 28°C for 2 days. During the cultivation, the pH
of the medium was not adjusted. The thus-obtained culture
medium was subjected to centrifugation at 15,000 x g for 10
minutes at 4°C and 75g of the wet cells thus separated were
obtained per liter of the culture. The wet cells were washed
with a physiological saline at 4°C and then again subjected to
centrifugation and freezed at -80°C. The freezed cells were
stored at the temperature until just before use.
One gram of the thus-obtained wet cells was
suspended in 10 ml of a reaction mixture (a) [prepared by
adding 1.4~ (v/v) of Nymeen solution (prepared by dissolving
4g of Nymeen S-215 (made by Nippon Oils & Fats Co.) in 10 ml
of xylene) to 100 mM TES buffer (pH 7.5) containing 5 mM L-
proline, 5 mM 2-ketoglutaric acid, 5 mM L-ascorbic acid and 1
mM ferrous sulfate] and the mixture was allowed to stand at
30°C for 5 hours.
CA 02131776 2001-11-22
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After completion of the reaction, the cells were
removed from the reaction mixture by centrifugation, and the
amount of hydroxyproline produced in the supernatant was
determined.
The determination was carried out by HPLC under the
conditions mentioned below. To identify the intended product,
the resulting product was eluted from the column and reacted
with NBD in the column line to form its NBD-derivative and the
derivative was determined by spectrofluorometry.
Conditions for determination by HPLC
[1] Apparatus Used:
High Performance Liquid Chromatography Device (made
by Shimadzu Seisakusho K.K.)
Chromatopac CR6A
System Controller SCL-6B
Autoinjector SIL-6B
Liquid Chromatograph Pump LC-6A
Column Oven CTO-6A
Chemical Reaction Box CRB-6A
I Spectrofluorometric Detector RF-550A
[2] Column Used:
SUMCHIRAL*OA5000 (diameter 4.5 mm x length 250 mm,
made by Sumika Chemical Analysis Service Limited)
[3] Conditions for Analysis:
1) Mobile Phase: aqueous solution of 1 mM
copper sulfate
2) Flow Rate of Mobile Phase: 1.0 ml/min
3) Column Temperature: 3g~C
4) Buffer: 0.3M boric acid buffer (PH 9.6)
25 mM ethylenediaminetetraacetic
acid
5) Flow Rate of Buffer: 0.2 ml/min
6) NBD Chloride Solution: methanol solution of
0.5 g/liter
7) Flow Rate of NHD Chloride Solution: 0.5 ml/min
* Trademark
CA 02131776 2001-11-22
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8) Reaction Temperature: 60°C
9) Reaction Time: about 3 min
10) Wavelength for Detection:
excitation wavelength 503 nm
emission wavelength 541 nm
11) Sample: 10 pl
As a result of the determination, it was verified
that 910 pM (119 mg/liter) of cis-3-hydroxy-L-proline was
produced in the reaction mixture.
Example 2
Purification of Cis-3-hydroxy-L-proline:
1009 of the wet cells which were obtained in Example
1 was suspended in 1 liter of the reaction mixture (a) as
described in Example 1, that had been put in a 2 liter-beaker.
The suspension was allowed to stand at 30°C for 5 hours with
stirring.
After the reaction, the cells were removed from the
reaction mixture by centrifugation, and hydroxyprolines
produced in the supernatant were determined by the same method
as described in Example 1. As a result of the determination,
it was verified that 809 pM (106 mg/liter) of cis-3-hydroxy-L-
proline was produced in the reaction mixture.
The supernatant was separated from the reaction
mixture, adjusted to pH 4.5, and passed through a column
packed with 200 ml of an ion-exchange resin, Diaiori SK1B
(NHQ+ type, made by Mitsubishi Kasei Corp.). The fractions
containing cis-3-hydroxy-L-proline was concentrated under
reduced pressure and then passed through a column packed with
20 ml of an ion-exchange resin, Diaion*PA412 (OH-type, made by
Mitsubishi Kasei Corp.). The fraction containing cis-3-
hydroxy-L-proline was concentrated under reduced pressure, and
adjusted to pH 9.6, and 10 vol.% of OPA solution (0.0759 of o-
phthalaldehyde (OPA)/ml ethanol solution) and 2 vol.% of j3-
mercaptoethanol solution (10% v/v aqueous solution) were added
* Trademark
CA 02131776 2001-11-22
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thereto. The mixture was allowed to stand at 60°C for 5
minutes, whereby impurities of primary amino acids contained
therein were reated with OPA. The resulting mixture was
passed through a column packed with 10 ml of Sepabeads SP207
(made by Mitsubishi Kasei Corp.), to separate cis-3-hydroxy-L-
proline from the impurities of OPA-derivatized primary amino
acids. The fraction containing cis-3-hydroxy-L-proline was
concentrated under reduced pressure and again passed through a
column packed with 20 ml of an ion-exchange resin, Diaion*
PA412 (OH-type, made by Mitsubishi Kasei Corp.) to separate a
fraction containing cis-3-hydroxy-L-proline. The fraction was
concentrated and dried to obtain 68 mg of cis-3-hydroxy-L-
proline as white crystals (63$).
Physicochemical properties of the chemical compound
mentioned above were given below.
Specific rotation: [Q]D2i = -93.4 (c=0.503, H20)
FAB-mass spectrum: 132 (M+H)+
i3C-NMR spectrum (D20, 125 MHz) ppm:
33.9, 44.5, 68.3, 71.6. 171.3
1H-NMR spectrum (D20, 500 MHz) ppm:
2.18 (1H), 2.27 (1H), 3.52 (1H), 3.62 (1H), 4.18
(1H), 4.77 (1H)
The above-mentioned molecular weight data of its
mass spectrum and specific rotation, and the analytical
2:results of its 13C-NMR spectrum and 1H-NMR spectrum were found
to be identical as the data as described in the prior art [see
J. Hiol. Chem., 241, 1300 (1966), J. Antibiotics, 45, 824
(1992)] and the data of cis-3-hydroxy-L-proline which was
synthesized according to the methods of the prior art [see
Liebigs Ann. Chem., 1881 (1979), Tetrahedron, 42, 2421
(1986)].
Example 3
Production of cis-3-hydroxy-L-proline:
L-Proline was hydroxylated by use of Streptomyces
- 23 - 2~3~'~'~~
canus ATCC 12647, Streptomyces canus ATCC 12646, Bacillus sue.
TH2 and Bacillus sg. TH3.
SR3 medium comprising 1.0~ glucose, 1.0~ soluble
starch, 0.5~ yeast extract, 0.5~ tryptone, 0.3$ meat extract
and 0.05 magnesium phosphate, and adjusted to pH 7.2 with 6N-
NaOH was put in test tubes (diameter 25 mm x length 200 mm) in
an amount of 10 ml each and sterilized at 120°C for 20
minutes. One loopful of cells of each of the microorganisms
mentioned above that had grown in HT-agar plate medium was
inoculated in the medium in a test tube and cultivated at 28°C
for 2 days by shaking. The resulting culture was used as a
seed culture in the following steps.
Separately, Df4 medium comprising 2.5~ glycerol,
2.5~ glucose, 1.5~ soybean meal, 0.05$ dipotassium hydrogen
phosphate, 0.05 magnesium sulfate 7-hydrate and 0.5~ calcium
carbonate, and adjusted to pH 7.0 with 6N-NaOH, was put in
test tubes (diameter 25 mm x length 200 mm) in an amount of 10
ml each and sterilized at 120°C for 20 minutes. One ml of the
seed culture was inoculated in the medium in each test tube
under germ-free condition and cultivated at 28°C for 1 day by
shaking. The thus-obtained culture was subjected to
centrifugation at 15,000 x g for 10 minutes at 4°C. The cells
thus separated were washed with 80 mM TES buffer (pH 7.5) and
then again subjected to centrifugation. The thus-obtained wet
cells were suspended in 1 ml of the reaction mixture (a) and
allowed to stand at 30°C for 3 hours.
As a result, it was verified that 242 pM, 202 pM,
318 pM and 141 pM of cis-3-hydroxy-L-proline were produced in
the reaction mixture by use of the strains ATCC12647,
ATCC12646, TH2 and TH3 respectively.
Example 4
Production of Cis-3-hydroxy-L-proline:
In the same manner as in Example 3 except that Df2
medium comprising 5~ soluble starch, 1.5$ dry yeast, 0.05$
i i
CA 02131776 2001-11-22
- 24 -
potassium dihydrogen phosphate, 0.05% magnesium sulfate 7-
hydrate and 0.5% calcium carbonate and adjusted to pH 7.0 with
6N-NaOH, and further containing 0.1% L-proline was used in
place of Df4 medium, each of Bacillus sp. TH2 and TH3 were
cultivated.
As a result, it was verified that 745 pM (97.6 mg/1)
of cis-3-hydroxy-L-proline for the strain TH2 and 327 pM (42.8
mg/1) for strain TH3 were produced in the aqueous medium.
Example 5
Isolation and Purification of L-proline-3-hydroxylase:
(1) Preparation of Cell-free Extract:
Thirty grams of the freezed cells obtained in
Example 2 was thawed out and suspended in 200 ml of Buffer (A)
[20 mM piperazine buffer adjusted to 5.3 with 6N-HC1
containing 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% (v/v)
Tween*20 and 10% (v/v) glycerol] while cooling with ice. The
cells in the resulting suspension was disrupted by means of an
ultrasonic cell disruptor while cooling with ice. The thus-
disrupted cell suspension was subjected to centrifugation at
30,000 x g at 4°C for 30 minutes to separate a supernatant.
The subsequent operations were conducted under
cooling with ice or at a temperature of 4°C or lower.
(2) Isolation and Purification by Column Chromatography:
(2)-1 Acid Treatment
The supernatant obtained in the previous step was
adjusted to pH 4.5 with 6N-HC1, and centrifuged at.15,000 x g
for 30 minutes to obtain the precipitates formed by the above
treatment.
(2)-2 Resource Q Column Chromatography (I):
The supernatant obtained in the previous step was
passed through a RESOURCETM Q column (6 ml; made by Pharmacia
Co.) that had been equilibrated with Buffer (A). The column
was washed with Buffer (A), and the fraction containing the
enzyme was eluted with Buffer (A) having a linear
* Trademark
- 25 - 213 ~'~~ ~
concentration gradient of NaCl of 0 to 0.3M.
(2)-3 Resource Q Column Chromatography (II):
The active fraction obtained in the previous step
was diluted three times with Buffer (B) [20 mM TES buffer (pH
7.5) containing 1 mM DTT, 0.2 mM EDTA and 10~ (v/v) of
glycerol] and was passed through a RESOURCETM Q column (1 ml;
made by Pharmacia Co.) that had been equilibrated with Buffer
(B). The column was washed with Buffer (B), and the fraction
containing the enzyme was eluted with Buffer (B) having a
linear concentration gradient of NaCl of 0 to 0.3M.
(2)-4 Phenyl Sepharose Column Chromatography:
NaCl was added to the active fraction obtained in
the previous step so that the resulting solution had an NaCl
concentration to 2M. The mixture was applied to a Phenyl
Sepharose column (1 ml; Phenyl Sepharose HP HiLoad) that had
been equilibrated with Buffer (B) containing 2M NaCl. The
column was washed with Buffer (B) containing 2M NaCl, and the
fraction containing the enzyme was eluted with Buffer (B)
containing 0.1~ (v/v) of Tween 20.
(2)-5 Resource Q Column Chromatograph (III):
The active fraction obtained in the previous step
was de-salted using a PD-10 column (1 ml; made by Pharmacia
Co.), and the resulting mixture was passed through a
RESOURCETM Q column (1 ml) that had been equilibrated with
Buffer (A). After the column was washed with Buffer (A), the
fraction containing the enzyme was eluted with Buffer (A)
having a linear concentration gradient of NaCl of 0 to 0.3M.
(2)-6 Resource Q Column Chromatography:
The active fraction obtained in the previous step
was diluted three times with Buffer (B) containing 0.1~ (v/v)
Tween 20 and passed through a RESOURCETM Q column (1 ml; made
by Pharmacia Co.) that had been equilibrated with Buffer (B).
The fraction containing the enzyme was eluted with Buffer B
having a linear concentration gradient of NaCl of 0 to 0.3M.
The foregoing steps for the isolation and
CA 02131776 2001-11-22
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purification of the L-proline-3-hydroxylase are summarized in
Table 9.
Table 9
Isolation Purification
and of L-Proline-3-Hydroxylase
Total Total Specific
Fraction Protein Activity Activity yield ($)
(U/mg of
(m9) (U) protein)
Cell-free 542 3540 6.5 100
Extract
pH 4.5 treated 106 1800 17 56
supernatant
Resource Q (I) 7.5 1553 207 44
pH 5.3
Resource Q 3.3 10-36 306 29
(II) pH 7.5
Phenyl 0.43 188 437 5.3
Sepharose
Resource Q 0.16 90.5 566 2.6
(III) pH 5.3
Resource Q 0.035 60.3 1723 1.7
(IV) pH 7.5
Example 6
Properties of L-Proline-3-Hydroxylase:
(1) Analysis by Electrophoresis:
The purified enzyme preparation obtained in Example
5 was analyzed by sodium dodecylsulfate-polyacrylamide gel
electrophoresis, using polyacrylamide gel PAGEL*NPU-12.5L made
by Atto Co, and SDS-PAGE Molecular Weight Standard, Broad
Range made by Biorad Co. As a result, it was verified that
the enzyme was composed of almost uniform sub-unit having a
molecular weight of about 35,000 ~ 5.000 daltons.
(2) Properties of the Enzyme:
Using the reaction mixture mentioned below, the
enzyme was subjected to the substrate omission and addition
* Trademark
CA 02131776 2001-11-22
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tests, by which the compounds indispensable to the enzyme
reaction for hydroxylating L-proline at the 3-position of L-
proline, the promoters for the enzyme reaction and the
inhibitors against the enzyme reaction were investigated.
The reaction mixture was composed of 100 mM TES
buffer (pH 7.0) containing 5 mM L-proline, 5 mM 2-ketoglutaric
acid, 1 mM ferrous sulfate, 5 mM L-ascorbic acid and a pre-
determined amount of the pure enzyme, the total volume being
. 100 gl. The reaction was initiated by addition of the enzyme
and continued for 10 minutes at 35°C. The reaction was
stopped by heating the reaction mixture at 100°C for 2
minutes. The amount of cis-3-hydroxy-L-proline formed in the
reaction mixture was determined by the pre-column
derivatization method using HPLC. One hundred microliters of
0.3M boric acid buffer (pH 10.7), 4 pl of an aqueous solution
of 10% (v/v) mercaptoethanol and 16 pl of ethanol solution of
5% (w/v) OPA were added to 100 pl of the reaction mixture, and
the mixture was allowed to stand at 60°C for 30 seconds. In
addition, 50 pl of ethanol solution of 2% (w/v) NHD chloride
were added thereto and the mixture was allowed to stand at
60°C for 40 minutes. The reaction was stopped by adding 30 pl
of 1 N-hydrochloric acid to the reaction mixture, and the
precipitates formed were removed therefrom by centrifugation
and filtration. The cis-3-hydroxy-L-proline formed by the
reaction was quantitatively determined by HPLC analysis.
The HPLC was carried out for the determination under
the following conditions:
Mobile Phase: 10 mM Citric Acid (pH
4.0)/Methanol = 3/1 (v/v)
Flow Rate: 1 ml/min
Column: YMC Pack ODS AQ-312*
(made by
YMC Co., 6 x 150 mm)
Column Temperature: 50°C
Detection: Spectrofluorometry (excitation
wavelength: 503 nm, emission
wavelength: 541 nm)
* Trademark
- 2$ - 2131'~~~
The test results verified that L-proline, 2-
ketoglutaric acid and Fe++ ion are indispensable for the
enzyme reaction for hydroxylating L-proline at the 3-position
of L-proline, that L-ascorbic acid promotes the enzyme
reaction and that Zn++, Cu++, Co++, Ba++ and EDTA inhibit the
reaction.
The test results are shown in Table 10.
m~t,~ ~ i n
Components Influencing Enzyme Reaction of
L-Proline-3-Hydroxylase
Components in Added (+)2) Relative
Reaction Mixture Not Added (-) Activity3)
Basic Reaction 100
Mixtures)
- Pure Enzyme 0
- L-proline 0
- 2-Ketoglutaric Acid 0
_ Fe++ 0
- L-Ascorbic Acid 25
+ 2 mM EDTA 5
+ 1 mM Z n++ 0
+ 1 mM Cu++ 4
+ 1 mM Co++ 13
+ 1 mM Ba++ 42
1) The standard reaction mixture was composed of
100 mM TES buffer (pH 7.0) containing 5 mM L-proline, 5 mM 2-
ketoglutaric acid, 1 mM ferrous sulfate, 5 mM L-ascorbic acid
and a pre-determined amount of the pure enzyme, the total
volume being 100 pl. The reaction was carried out at 35°C for
10 minutes.
2) "(+)~~ means that the reaction mixture contained
the component shown in the table. "(-)" means that the
reaction mixture did not contain the component shown in the
29
table. The concentration shown in the table means the
concentration of the component in the reaction mixture.
3) The activity is indicated as the relative
activity, the activity in the standard reaction mixture being
defined as 100.
(3) Optimum pH Range:
In the determination of the enzymatic activity of
the L-proline-3-hydroxylase, the reaction was carried out
while the buffer component in the reaction mixture was changed
to MES buffer [2-(N-morpholino)ethanesulfonic acid] at pH of
5.5 to 6.5, it was changed to PIPES buffer [piperazine-N,N'-
bis(2-ethanesulfonic acid] at pH of 6.5 to 7.5, it was changed
to TES buffer at pH of 7.0 to 8.0, and it was changed to TAPS
buffer [N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic
acid] at pH of 8.0 to 9Ø As a result, the enzyme had an
activity of more than 80~ of the maximum activity thereof at
pH ranging from pH 6.5 to 7.5. The detailed test results are
shown in Table 11 below.
Table 11
Optimum pH Range for the Enzyme Reaction
pH (buffer) Relative Activityl)
5.5 (MES) 15.0
6.0 (MES) 70.9
6.5 (PIPES) 83.3
7.0 (PIPES) 92.1
7.0 (TES) 100
7.5 (PIPES) 80.4
7.5 (TES) 85.0
8.0 (TES) 72.2
8.0 (TAPS) 76.3
8.5 (TAPS) 52.9
9.0 (TAPS) 44.5
- 30 -
21317'~6
1) The activity is indicated as a relative activity,
defining the maximum activity as 100.
(4) Stable pH Range:
The enzyme solution in Buffer (B) was diluted three
times with 100 mM of a buffer (MES buffer at pH of 5.5 to 6.5,
PIPES buffer at pH of 6.1 to 7.5, TES buffer at pH of 7.0 to
8.0, TAPS buffer at pH of 8.0 to 9.0), kept at 4°C for 23
hours, and then its activity was measured. The enzyme kept at
pH ranging from 6.5 to 8.0 had an activity of 80~ or more of
the original activity of the enzyme before the test.
Accordingly, the enzyme was kept stable at pH ranging from 6.5
to 8Ø
(5) Optimum Temperature Range:
In the determination of the enzyme activity of the
L-proline-3-hydroxylase, the reaction was carried out at a
temperature ranging from 15 to 50°C for 15 minutes. As a
result, the enzyme had an activity of 80~ or more of the
maximum activity thereof at temperatures ranging from 35 to
40°C. The detailed test results are shown in Table 12 below.
m ..1.. i .. i ~
Temperature Ranqe for the Enzyme Reaction
Reaction Temperature Relative Activityl)
( C)
15 19
20 29
25 53
30 74
35 100
40 89
45 64
50 28
- 31 - 2~~1~~~
1) The activity is indicated as a relative
activity, the maximum activity being defined as 100.
(6) Stable Temperature Range:
The enzyme was kept in Buffer (B) at temperatures
ranging from 0 to 60°C for 30 minutes and thereafter the
activity of the enzyme was determined. As a result, the
enzyme was inactivated after it was kept at 50°C or higher for
30 minutes.
(7) Km Value:
The Km value is 0.49 mM for L-proline and is 0.11 mM
for 2-ketoglutaric acid, when determined in a TES buffer (pH
7.0) containing 5 mM L-ascorbic acid, 1 mM ferrous sulfate and
a pre-determined amount of this enzyme.
(8) Isoelectric point:
The enzyme was analyzed, using Phast system (made by
Pharmacia Co.), to determine the isoelectric point of the
enzyme. As a result, the isoelectric point of the enzyme was
4.3.
(9) N-terminal Amino Acid Sequence:
The enzyme was analyzed, using Protein Sequencer
Model PPSQ-10 (made by Shimadzu Seisakusho K.K.), to determine
the N-terminal amino acid sequence of the enzyme. The result
is as follows:
Sequence Number 1:
(N-terminal) 1 MetCysSerHisIleLeuGlyArgIleGlu
11 LeuAspGlnGluArgLeuGlyArgAspLeu
21 GluTyrLeuAlaThrValProThrVa1
Example 7
Production of Cis-3-hydroxy-L-proline:
The enzyme reaction by use of purified L-proline-3-
hydroxylase obtained in Example 5 was carried out. The
reaction mixture was composed of 200 mM TES buffer (pH 7.0),
20 mM L-proline, 20 mM 2-ketoglutarate, 5 mM L-ascorbic acid,
1 mM ferrous sulfate, and 106 U of purified enzyme
32
preparation, the total volume being 100 pl. The reaction was
carried out at 35°C for 30 minutes. As a result of the
reaction, 18 mM (2.4 g/1) of cis-3-hydroxy-L-proline was
produced in the reaction mixture.
- 33 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ozaki, Akio
Mori, Hideo
Shibasaki, Takeshi
Ando, Katsuhiko
Ochiai, Keiko
Chiba, Shigeru
Uosaki, Youichi
(ii) TITLE OF INVENTION: Process for Producing
Cis-3-Hydroxy-L-Proline
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ANTONELLI, TERRY, STOUT AND KRAUS
(B) STREET: 1300 NORTH SEVENTEENTH STREET
(C) CITY: ARLINGTON
(D) STATE: VIRGINIA
(E) COUNTRY: U.S.A.
(F) ZIP: 22209
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: JP 221941/93
(B) FILING DATE: 07-SEP-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Terry, David T.
(B) REGISTRATION NUMBER: 20178
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 703-312-6600
(B) TELEFAX: 703-312-6666
(C) TELEX: 248545
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Streptomyces sp.
(B) STRAIN: TH1
- 34 -
(~i) SEQUENCE DESCRIPTION: SEQ ID N0:1:
~~.~1'~'~G
Met Cys Ser His Ile Leu Gly Arg Ile Glu Leu Asp Gln Glu Arg Leu
1 5 10 15
Gly Arg Asp Leu Glu Tyr Leu Ala Thr Val Pro Thr Val
20 25
