JPH0898683A - 7-aminocephalosporanic acid esterase - Google Patents

Abstract

PURPOSE: To obtain the subject new enzyme catalyzing a reaction to hydrolyze a 7-aminocephalosporanic acid derivative into a deacetyl-7-aminocephalosporanic acid derivative and acetic acid and useful e.g. for the production of an intermediate for cephalosporin antibiotic substances. CONSTITUTION: A microbial strain capable of producing a 7- aminocephalosporanic acid (7-ACA) esterase [e.g. Rhodotorula glutinis 38B1 (FERM P-13039)] is cultured in a medium, the cultured cells are collected by centrifugal separation of the cultured product, the cells are disintegrated and extracted with a buffer solution and the extract is purified to obtain the new 7-ACA esterase catalyzing a reaction to hydrolyze a 7-aminocephalosporanic acid derivative of formula I (R is H or an acyl) into a deacetyl-7- aminocephalosporanic acid derivative of formula II and acetic acid, having a molecular weight of 141kDa (determined by gel-filtration), an optimum pH of 5.5, a stable pH range of 5.5-7.0 and an optimum temperature of 30-35 deg.C, containing >=20% mixed-type sugar chain and having an N-terminal amino acid sequence of formula III.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a 7-aminocephalosporanate esterase for use in producing a deacetylcephalosporanic acid derivative, which is a useful intermediate in the production of cephalosporin antibiotics, from a cephalosporanic acid derivative. Regarding

[0002]

2. Description of the Related Art As a method for producing a deacetylcephalosporanic acid derivative by removing the acetyl group of the acetoxymethyl group at the 3-position from a cephalosporanic acid derivative, an enzymatic method is more economically advantageous than a chemical method. It is known that there are various methods, and various methods are known (JP-A-49-1).
32294, JP-A-59-108790, JP-B-54-
43598, JP-B-56-46839, JP-B-61-1
1600, JP-A-61-67489, JP-B-1-471
58, etc.).

The enzyme used in the above-mentioned enzymatic method is called cephalosporin C deacetylase or 7-aminocephalosporanic acid esterase, and this enzyme itself is a cephalosporin C deacetylase derived from the genus Bacillus. WRRL-B-558 deacetylase; Appl. Microbiol. , 30 (3), 4
13 (1975) [Document A], ATCC6633 deacetylase; Biochim. Biophys. Act
a, 485, 367 (1977) [Literature B], SHS-
0133 Deacetylase; Ferment. Bi
oeng. , 77, 17 (1994) [Document C] as a cephalosporin C deacetylase derived from the genus Aureobasidium, 4466-I acetylesterase; JP-B-2-51594 [Document D] and 44.
66-II acetyl esterase; Japanese Patent Publication No. 2-515
Japanese Patent Laid-Open No. 94 [Document E] and the like have been reported.

A cephalosporin C deacetylase having an N-terminal amino acid sequence and a partial amino acid sequence has also been reported (JP-A-4-228079).

[0005]

[Problems to be Solved by the Invention] Conventional cephalosporin C deacetylase (Cec deacetylase) or 7
-Aminocephalosporanate esterase (7-ACA
Esterase), when used to convert a cephalosporanic acid derivative to a deacetylcephalosporanic acid derivative, it takes a long time for the enzymatic reaction and it is difficult to make the reaction yield 100% because of a large Km value. there were. Therefore, as CeC deacetylase, the specific activity (Vmax
Value) and the Km value is low.
It was required because it is advantageous for industrialization.

[0006]

Therefore, the present inventors have
As a result of continuing various studies to solve the above problems, the 7-ACA esterase produced by a microorganism belonging to the genus Rhodotorula isolated from soil by the present inventor was found to have the general formula (3):

[0007]

[Chemical 4] (In the formula, R ¹ represents a hydrogen atom, D-5-amino-5-carboxyvaleryl, chloroacetyl or benzyloxycarbonyl group.) A 7-aminocephalosporanic acid derivative [hereinafter, simply referred to as “7- ACA derivative (3) "
Is referred to as a general formula (4)

[0008]

[Chemical 5] (Wherein, R ¹ has the same meaning as described above) and conversion to a deacetyl-7-aminocephalosporanic acid derivative [hereinafter, simply referred to as "D-7-ACA derivative (4)"] They have found the present invention and completed the present invention.

That is, the object of the present invention is to provide a 7-ACA esterase (hereinafter sometimes simply referred to as "the present enzyme") having the following physicochemical properties. Enzyme action At least the general formula (1)

[Chemical 6] A 7-aminocephalosporanic acid derivative represented by the formula (wherein R represents a hydrogen atom or an acyl group) [hereinafter, simply referred to as "7-ACA derivative (1)"] is represented by the general formula (2).

[Chemical 7] (In the formula, R has the same meaning as described above) A deacetyl-7-aminocephalosporanic acid derivative [hereinafter,
Simply referred to as "D-7-ACA derivative (2)"] and catalyze the reaction of hydrolysis to acetic acid.

Substrate Specificity At least acts on acetic acid lower alkyl esters such as propyl acetate and butyl acetate, and 7-ACA derivative (3). In particular, cephalosporin C (CeC), 7-chloroacetylamino-cephalosporanic acid (7-CAAC
A), strongly acts on 7-aminocephalosporanic acid (7-ACA) and butyl acetate.

Molecular Weight The molecular weight is 141 kDa (gel filtration method). The molecular weight of the subunit is 82 kDa (SDS-PAG
E). Optimum pH and stable pH range The optimum pH is 5.5. pH 5.5-7.0 is stable p
It is the H region. Optimum temperature and stable temperature The optimum temperature is 30 to 35 ° C. It is stable up to 20 ° C when treated at pH 7.0 for 15 minutes.

Inhibition Inhibited by Cu ²⁺ , Hg ²⁺ , (p-amidinophenyl) methanesulfonyl fluoride, eserin sulfate and the like. Contains a mixed sugar chain with a sugar content of 20% or more.

N-terminal amino acid sequence Thr-Asn-Pro-Asn-Glu-Pro-P
ro-Pro-Val-Val-Asp-Leu-Gl
y-Tyr-Ala-

Partial Amino Acid Sequence At least the following four types of amino acid sequences are included. (A) Ala-Phe-Gly-Leu-X-Ala (wherein X represents an unidentified amino acid) (b) Gly-Phe-Ile-Phe-Thr-As
p-Ala

(C) Ala-Val-Asp-Ala-
Ala-Ala-Leu-Ala-Ala-Ala-G
ly-Val-Leu-Asn-Ser-Ala-Al
a-Phe (d) Thr-Ser-Ser-Leu-Thr-As
p-Phe-Gly-Thr-Ser-Gln-Ile
-Thr-Ile-Ile-Asp-Phe-Trp-
Arg-Gly-Gly-Ile

The 7-ACA esterase-producing bacterium of the present invention belongs to the genus Rhodotorula. For example, the present inventor isolated Rhodotorula glutinis (Rhodo) from soil in Kyoto Prefecture.
torula glutinis) 38B1 is an example of the strain most effectively used in the present invention, and this strain has been deposited with the Institute of Microbial Science and Technology as Microtechnology Research Institute No. 13039, and its mycological properties. Is JP-A-6-125
No. 785.

In the present invention, a 7-ACA esterase-producing bacterium belonging to the genus Rhodotorula is first cultivated in an appropriate medium. Examples of the 7-ACA esterase-producing bacterium include the above-mentioned Rhodotorula glutinis 38B1. However, since the mycological properties can be changed as a general property of the microorganism, it is natural or artificial. All strains belonging to the genus Rhodotorula and having the ability to produce the present enzyme can be used in the present invention, including naturally-occurring mutants as well as artificial mutants that can be mutated by dynamic mutation means.

The above culturing can be carried out according to a known culturing method generally used for culturing yeast. The medium to be used can be one known as a nutrient source for general yeast, for example, glucose, sucrose, maltose, ethanol, acetic acid, a carbon source such as ethyl oleate,
Nitrogen sources such as nitric acid, ammonium sulfate, ammonium chloride and ammonia, organic nutrient sources such as yeast extract, malt extract, peptone, meat extract and potato, amino acids such as L-valine and L-glutamic acid, phosphoric acid, magnesium, potassium and iron. Inorganic nutrient sources such as cobalt, cobalt and manganese can be appropriately combined and used.

Further, in order to induce the present enzyme activity, various fatty acid ester compounds such as methyl oleate may be added to the medium at 0.01 to 10%. The pH of the medium may be selected within the range of 3 to 10.

Culturing is usually carried out under aerobic conditions such as shaking or aeration-agitation culture, and deep aeration-agitation culture is industrially preferable. The culturing temperature can be appropriately changed within a range in which the present enzyme-producing bacterium grows and produces the present enzyme, but it is usually 15
C to 40C. Although the culturing time varies depending on the culturing conditions, it may be cultivated up to an appropriate time in consideration of the time when the present enzyme reaches the maximum titer, but it is usually in the range of 1 to 10 days.

Since the present enzyme thus obtained is mainly contained in the microbial cells, cells are collected from the obtained culture by means such as filtration or centrifugation, and the microbial cells are subjected to ultrasonic treatment, Dynomil. Treatment, French press treatment, freeze disruption treatment and other mechanical disruption treatment, and other known bacterial cell treatment means are appropriately combined and disrupted, and solids such as cell debris are removed by centrifugation to obtain crude 7-ACA esterase. A containing liquid is obtained.

From the crude enzyme-containing liquid, a further purified present enzyme can be obtained by using known means for isolating and purifying proteins, enzymes and the like. For example, the enzyme may be recovered as a precipitate by means such as ultracentrifugation fractionation, salting-out precipitation adding ammonium sulfate, sodium chloride, sodium sulfate, potassium phosphate, etc., organic solvent precipitation adding acetone, etc.

Further, the precipitate is purified by appropriately combining means such as dialysis using a semipermeable membrane, adsorption chromatography, ion exchange chromatography, hydrophobig chromatography and gel filtration chromatography. You can

The 7-ACA esterase thus obtained has the following physicochemical properties. 1) Enzyme action At least the 7-ACA derivative (1) is added to D-7-ACA.
It catalyzes the reaction of hydrolyzing the derivative (2) with acetic acid.

Here, the acyl group defined by the group R in the general formula (1) is an acyl group of a known cephalosporin compound, for example, 1 of an amino group, a carbonyl group or a carboxyl group. Straight or branched chain alkanoyl group having 2 to 7 carbon atoms which may be substituted by one to several; halogen atom, cyano group, lower alkoxy group, aryloxy group optionally having substituent (s) ,
An alkanoyl group having 2 to 4 carbon atoms substituted with an imino group, a lower alkylidene group or a heterocyclic group; 2 to 5 carbon atoms optionally substituted with an aryloxy group which may have a substituent A linear or branched alkoxycarbonyl group; and an aryl-lower alkanoyl group which may have a substituent.

2) Substrate specificity The substrate specificity was examined using this enzyme. The enzyme activity was measured according to the enzyme activity measuring method described below. When the 7-ACA derivative (1) was used as a substrate, the 7-ACA derivative (3) was selected from among them. Table 1 shows the results of measurement of the substrate specificity of the present enzyme for each substrate.

[Table 1]

From the above results, this enzyme acts on the 7-ACA derivative (3). In particular, CeC, 7-CAACA,
It acts strongly on 7-ACA. It also acts on acetic acid lower alkyl ester, and the longer the alcohol chain, the stronger the activity. On the other hand, by not hydrolyzing N-acetyl compounds such as N-acetylglucosamine and chitin, chitin deacetylase [Proc. Natl. Acad. Sci. U.
S. A. , 90, 2564 (1993)].

3) Molecular Weight Electrophoresis by SDS-PAGE by a known method,
By comparison with the standard protein, the molecular weight of the subunit of this enzyme is 82 kDa (SDS-PAGE).
In addition, in order to obtain the molecular weight of this enzyme under normal conditions, S
By elution with an upperdex200 prep grade column and comparison with a standard protein,
The molecular weight of this enzyme is 141 kDa (gel filtration method).

From the above results, the present enzyme is an enzyme in which two subunits of 82 kDa are associated with each other, and the present enzyme having such a molecular weight and a molecular weight of the subunit is different from the conventionally known CeC deacetylase.

4) Optimum pH and stable pH range The optimum pH of this enzyme is determined by the enzyme activity measuring method described below except for buffer solutions.
H was calculated. The buffer solution is succinic acid having a pH of 4.0 to 6.0.
A NaOH buffer solution, a sodium phosphate buffer solution having a pH of 5.5 to 7.8, and a Tris-hydrochloric acid buffer solution having a pH of 7.5 to 9.0 were used. The measurement result is as shown in FIG. 3, and the optimum pH is 5.5. On the other hand, pH 5.5 to 7.0 is the stable pH range.

5) Optimum temperature and stable temperature The optimum temperature of the present enzyme was determined by the enzyme activity measuring method described below except that the reaction temperature was changed. The measurement result is as shown in FIG. 2, and the optimum temperature is 30 to 35 ° C. On the other hand, pH
It is stable up to 20 ° C. in the treatment at 7.0 for 15 minutes.

6) Inhibition In the reaction system of the enzyme activity measuring method described below, the present enzyme activity in the presence of various metal ions or inhibitors was measured to determine the influence of various metal ions or inhibitors. Various additives are p
-All were added 1.0 mM except that 0.1 mM chloromercury benzoate was added. Table 2 shows the relative activity when the activity without addition is defined as 100%.

[0033]

[Table 2] From the above results, this enzyme is inhibited by Cu ²⁺ , Hg ²⁺ , (p-amidinophenyl) methanesulfonyl fluoride, eserin sulfate and the like.

7) Sugar content This enzyme was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and then treated with 25 mM sulfuric acid at 80 ° C. for 1 hour to remove sialic acid.
After washing, the enzymes on the membrane were various peroxidase-lectin reagents [Reference; Shinsei Chemistry Laboratory, 3. Sugar,
I, 31 (1990)]. Next, peroxidase staining was performed with 45 mM 4-aminoantipyrine 0.
3 ml, 60 mM phenol 0.3 ml, 30% hydrogen peroxide 2 μl, 15 mM sodium phosphate buffer (p
H6.8) 8.4 ml was used for the reaction mixture.

As a result, this enzyme was found to be concanavalin A (C
on A) and wheat germ lectin (Wheat germ)
Lactin), but Arachis hy
pogaea lectin (PNA), Ricinus
communis lectin (RCA ₁₂₀ ), Phas
eolus vulgaris lectin (PHA-E
₄ ) did not react. This indicates that this enzyme has a hybrid sugar chain attached to an asparagine residue.

Furthermore, in order to determine the content of this sugar,
When this enzyme was subjected to Endo H treatment and subjected to SDS-PAGE, three major bands and two minor bands were observed and the molecular weights were 74,000, 72000, 69000, 6
It became 7,000 and 65,000. Based on this, the present enzyme contains 20% or more of a hybrid sugar chain.

8) N-terminal amino acid sequence This enzyme was subjected to SDS-PAGE, transferred to a PVDF membrane, and then subjected to a protein sequencer by Edman method. The N-terminal amino acid sequence is as follows. Thr-Asn-Pro-Asn-Glu-Pro
-Pro-Pro-Val-Val-Asp-Leu-
Gly-Tyr-Ala-

9) Partial amino acid sequence In order to know the partial amino acid sequence of this enzyme, Staphy was used.
Peptide mapping by the Cleveland method using lococcus aureus V8 protease (protein structural analysis for gene cloning, Hisano Hirano, Tokyo Kagaku Dojin, pages 89-92) revealed that the enzyme had at least the following four amino acids: Contains an array.

(A) Ala-Phe-Gly-Leu-X-Ala (wherein X represents an unidentified amino acid) (b) Gly-Phe-Ile-Phe-Thr-As
p-Ala

(C) Ala-Val-Asp-Ala-
Ala-Ala-Leu-Ala-Ala-Ala-G
ly-Val-Leu-Asn-Ser-Ala-Al
a-Phe (d) Thr-Ser-Ser-Leu-Thr-As
p-Phe-Gly-Thr-Ser-Gln-Ile
-Thr-Ile-Ile-Asp-Phe-Trp-
Arg-Gly-Gly-Ile

10) Homogeneity of enzyme SDS-polyacrylamide gel electrophoresis [U. K.
Laemmli, Nature, 227, 680 (19)
70)], a uniform single protein band was observed, and the molecular weight of this protein was 82 kDa as compared with a known standard protein.

11) Method for measuring enzyme activity [Method 1 for measuring enzyme activity] 5 μmol of 7-ACA, succinic acid-sodium borate buffer solution (pH 5.5) 0.1 mmo
1 and an appropriate amount of enzyme were added and mixed so as to be 0.5 ml and reacted at 30 ° C. for 30 minutes, and then 2 ml of 50% dimethyl sulfoxide solution was added to stop the reaction. The produced deacetyl-7-aminocephalosporanic acid (D-7-ACA) was measured by high performance liquid chromatography (HPLC) under the following conditions.

Analytical conditions A; Column; Wacodil-II 5C 18 HG (250 × 4.6 m) equipped with a guard column (30 × 4.6 mm)
m, manufactured by Wako Pure Chemical Industries, Ltd.) (incubation at 35 ° C.) Mobile phase: 7.7% (V / V) acetonitrile, aqueous solution containing 1 mM tetrabutylammonium hydrogensulfate and 64 mM ammonium acetate Flow rate: 0.8 ml / Minute detection; absorbance at 254 nm One unit was an enzyme that produced 1 μmol of D-7-ACA in 1 minute.

In the measurement of the substrate specificity of the 7-ACA derivative (3), for substrates other than 7-ACA,
Using the same reaction conditions as above, but using 7-benzyloxycarbonylamino-cephalosporanic acid (7
-BZACA) and 7-CAACA with H
The PLC analysis conditions are as follows.

Analytical condition B: When a deacetylated product is produced from 7-BZACA Column: Cosmozil 5C18-AR (150 × 4.6)
mm, column (manufactured by Nacalai Tesque, Inc.) (incubated at 35 ° C.) Mobile phase: 27% (V / V) acetonitrile and 67 m
Aqueous solution containing M potassium phosphate buffer (pH 7.0) Flow rate: 1.6 ml / min Detection: Absorbance at 254 nm

Analytical condition C: When a deacetylated product is produced from 7-CAACA Column: Cosmozil 5C18-AR (150 × 4.6)
mm, manufactured by Nacalai Tesque, Inc. (incubated at 35 ° C.) Mobile phase; aqueous solution containing 7.7% (V / V) acetonitrile, 1 mM tetrabutylammonium hydrogensulfate and 64 mM ammonium acetate Flow rate; 1.6 ml / min Detection Absorbance at 254 nm

[Enzyme activity measurement method 2] Further, the enzyme activity was measured as follows when acetic acid lower alkyl ester was used as a substrate. That is, 4 μmol of substrate, succinic acid-sodium borate buffer (pH 5.5) 40 μmo
1 and an appropriate amount of enzyme were added and mixed to make 0.2 ml and reacted at 30 ° C. for 20 minutes, and then 50% dimethyl sulfoxide solution was added to stop the reaction. The generated acetic acid is a method of coupling three enzymes [H. U.
Bergmeyer, Methods Enzym. A
nal. , 1,172 (1974)].

12) Reaction rate (Km value) and specific activity (Vmax value) The Km value and Vmax value of this enzyme for the 7-ACA derivative (3) were determined to be 1.43 mM for 7-ACA, respectively. 422 μmol / min / mg, 8.39 mM for CeC, 1000 μmol / min / mg, respectively
Is.

Comparing the present enzyme having the above-mentioned physicochemical properties with the known CeC deacetylase of the prior art documents A to E, the present enzyme is clearly different from the known CeC deacetylase for the following reasons, and is therefore a novel enzyme. be able to.

(1) Molecular weight of 141 kDa and 82 kDa
The present enzyme having the subunit a is different from the known CeC deacetylase in the molecular weight and the molecular weight of the subunit. (2) The optimum pH of this enzyme is 5.5, while the optimum pH of known CeC deacetylase is the same as that of the literatures A to C.
It is 7.0-8.5 and that of the prior documents D-E is 4
This enzyme is different from the known CeC deacetylase because it is as follows.

(3) While the sugar content of the present enzyme is 20% or more, it is not known that a known CeC deacetylase contains a sugar. (4) The N-terminal amino acid sequence and partial amino acid sequence of this enzyme are different from the amino acid sequence of known CeC deacetylase. (5) This enzyme has a smaller Km value than the 7-ACA derivative (3) and a Vmax value far higher than that of the known CeC deacetylase.

[0052]

As described above, the 7-ACA esterase of the present invention converts the 7-ACA derivative (1) into the D-7-ACA derivative (2) by hydrolysis at room temperature and normal pressure, and Km value is small compared to the substrate, Vmax
Is much larger than the known CeC deacetylase, so that the reaction yield can be 100% with a small amount of enzyme, and thus an industrially very useful enzyme can be provided,
By using this enzyme, 7-ACA derivative (1)
To D-7-ACA derivative (2) can be industrially advantageously produced.

[0053]

EXAMPLES Next, the present invention will be described in more detail by way of examples. This does not limit the scope of the invention.

Example 1 Cultivation of 7-ACA Esterase-Containing Bacterial Cells Containing 2% malt extract and 0.5% peptone at pH 6.
Rhodotorula glutinis 38B, which had been cultivated in a test tube (7 ml) at 28 ° C. for 1 day, in 150 ml of a sterilized medium adjusted to 0 (using a 2 L Erlenmeyer flask) in advance, with a medium having the same composition as above
1 was transplanted and cultured at 28 ° C. for 1 day. This culture solution was transferred to a 15 L jar fermenter containing 10 L of sterilized medium containing 2% of malt extract, 0.5% of peptone and 0.5% of methyl oleate and adjusted to pH 6.0, and transferred to 5 L. The culture was carried out for 33 hours under an aeration condition of 1 / min and a stirring condition of 400 rpm. The obtained culture was centrifuged to collect the cells, and the cells were washed with 0.85% saline to obtain the cells. Looking at the hydrolysis activity of 7-ACA, 1.7 units /
ml (culture liquid).

[0054]

Example 2 Purification of 7-ACA Esterase The cells obtained in Example 1 were suspended in 50 mM sodium phosphate buffer (pH 7.0) containing 2 mM DTT, and the suspension was -20 ° C.
Frozen. The thawed microbial cell fluid was treated with glass beads for dyno mill (Willy A. Bachofen Ma).
The cells were disrupted by nufacturing Engineers). The precipitate was removed by centrifugation at 7,000 G for 60 minutes to obtain a cell-free extract. Streptomycin sulfate was added to this to a concentration of 1%, and the nucleic acid was removed as a precipitate by centrifugation. After adding ammonium sulfate to this so as to be 65% saturated, the supernatant was obtained by centrifugation. Ammonium sulfate was further added to this so that it would be 80% saturated, and the active fraction was collected as a precipitate by centrifugation.

This precipitate was dissolved in a succinic acid-NaOH buffer solution (pH 5.5) containing 2 mM DTT, and dialyzed twice with the same buffer solution. The dialyzed enzyme solution was concentrated by ultrafiltration using a YM30 membrane (manufactured by Amicon). CM-Toyopearl 650M pre-pa obtained by equilibrating the obtained enzyme solution with the same buffer solution as above.
A cked column (20 x 2.2 cm id) was charged. Fractions should be 5 ml and the same buffer should
Gradient elution (0-0.1 M, 30-55 min; 0.1) with the same buffer containing 0 to 0.2 M of salt concentration for 0 min.
-0.2M, 55-90 minutes). The active fractions were collected and then concentrated with a YM30 membrane filter and Centricon 30 concentrator.

The resulting concentrate was added with 2 mM DTT and 0.
Superdex 200 equilibrated with 50 mM sodium phosphate buffer (pH 7.0) containing 1 M NaCl.
prep grade column (60 x 1.6 cm
i. d. ) Was charged. Elution with the same buffer as above at a flow rate of 1 ml / min. Collect only the obtained active category,
Lyophilization gave the purified enzyme 7-ACA esterase.
The above purification process is shown in Table 3.

[Table 3]

[0057]

Example 3 Generation of D-7-ACA from 7-ACA Succinic acid-sodium borate buffer (pH 5.5) 1 m
Mol, 7-ACA 0.05 mmol, and 5 ml of a reaction solution containing 8 units of the purified enzyme obtained in Example 2 were reacted at 30 ° C. with stirring. 7-ACA, D-7-ACA and acetic acid were quantified. The results are shown in FIG. 1, and as the substrate 7-ACA decreases, D-7-ACA and acetic acid are produced in equimolar amounts, so that the purified enzyme is clearly
-Has been shown to be ACA esterase.

[0058]

Example 4 Km and Vmax Values for 7-ACA Derivative (3) Km Values and Vmax for 7-ACA Derivative (3)
In order to obtain the value, the purified 7-ACA esterase obtained in Example 2 was used to measure the activity by changing the substrate and its concentration in the above-mentioned enzyme activity measuring method, and the reciprocal plot (Lineweaver-Burk plots) was used.
Then, Km and Vmax were obtained.

As substrates, 7-ACA, CeC, 7-C
AACA and 7-BZACA were used. The Km values for these were 1.43, 8.39, 2.03 and 1.11 mM, respectively. Also, the Vmax values are 42 each.
2, 1000, 562 and 391 units / mg.

[Sequence list]

SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Asn Pro Asn Glu Pro Pro Pro Val Val Asp Leu Gly Tyr Ala 1 5 10 15

SEQ ID NO: 2 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 4 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Val Asp Ala Ala Ala Leu Ala Ala Ala Gly Val Leu Asn Ser Ala 1 5 10 15 Ala Phe

SEQ ID NO: 5 Sequence length: 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Ser Ser Leu Thr Asp Phe Gly Thr Ser Gln Ile Thr Ile Ile Asp 1 5 10 15 Phe Trp Arg Gly Gly Ile 20

[Brief description of drawings]

FIG. 1 is a schematic view of 7-aminocephalosporanic acid (7-aminocephalosporanic acid esterase using
It is a curve which shows the result of the production progress of deacetyl-7-amino cephalosporanic acid (D-7-ACA) from (ACA).

FIG. 2 is a curve showing the optimum temperature of 7-aminocephalosporanate esterase of the present invention.

FIG. 3 is a curve showing the optimum pH of 7-aminocephalosporanate esterase of the present invention.

Claims (1)

[Claims] 1. A 7-aminocephalosporanate esterase having the following physicochemical properties. Enzyme action At least the general formula (1) The 7-aminocephalosporanic acid derivative represented by the formula (wherein R represents a hydrogen atom or an acyl group) is represented by the general formula (2): (In the formula, R has the same meaning as described above) It catalyzes the reaction of hydrolyzing a deacetyl-7-aminocephalosporanic acid derivative with acetic acid. Substrate Specificity At least acetic acid lower alkyl ester such as propyl acetate and butyl acetate, and general formula (3) (In the formula, R ¹ represents a hydrogen atom, D-5-amino-5-carboxyvaleryl, chloroacetyl or benzyloxycarbonyl group) and acts on the 7-aminocephalosporanic acid derivative. Especially cephalosporin C,
7-chloroacetylamino-cephalosporanic acid, 7-
It acts strongly on aminocephalosporanic acid and butyl acetate. Molecular weight The molecular weight is 141 kDa (gel filtration method). The molecular weight of the subunit is 82 kDa (SDS-PAG
E). Optimum pH and stable pH range The optimum pH is 5.5. pH 5.5-7.0 is stable p
It is the H region. Optimum temperature and stable temperature The optimum temperature is 30 to 35 ° C. It is stable up to 20 ° C when treated at pH 7.0 for 15 minutes. Inhibition Inhibited by Cu ²⁺ , Hg ²⁺ , (p-amidinophenyl) methanesulfonyl fluoride, eserine sulfate and the like. Contains a mixed sugar chain with a sugar content of 20% or more. N-terminal amino acid sequence Thr-Asn-Pro-Asn-Glu-Pro-P
ro-Pro-Val-Val-Asp-Leu-Gl
y-Tyr-Ala- Partial amino acid sequence At least the following four types of amino acid sequences are included. (A) Ala-Phe-Gly-Leu-X-Ala (wherein X represents an unidentified amino acid) (b) Gly-Phe-Ile-Phe-Thr-As
p-Ala (c) Ala-Val-Asp-Ala-Ala-Al
a-Leu-Ala-Ala-Ala-Gly-Val
-Leu-Asn-Ser-Ala-Ala-Phe (d) Thr-Ser-Ser-Leu-Thr-As
p-Phe-Gly-Thr-Ser-Gln-Ile
-Thr-Ile-Ile-Asp-Phe-Trp-
Arg-Gly-Gly-Ile