JPS5858080B2 - Method for producing 7-aminocephem compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the microbiologically derived homolog of cephalosporin C (referred to as Ce-C under IJ), which has the following general formula (wherein X is a hydrogen atom, a hydroxyl group, an acetoxy group, or Indicates nuclear residues.

) The present invention relates to an improvement in a method for producing a 7-aminocephem compound represented by

Cephalosporin antibiotics are excellent antibacterial agents with low toxicity and a wide range of efficacy compared to penicillins. Cephalosporanic acid (hereinafter simply 7
Since it is extremely difficult to microbiologically produce 7-ACA (referred to as Ce-C
has been supplied by chemical transformation of

However, in the laboratory to which one of the present inventors belongs, the general formula C containing 7-ACA as a member
We have developed and filed an application for a microbiological production method for the 7-aminocephem compound represented by I) (referred to as iu-ago gold compound CD) (Japanese Unexamined Patent Publication No. 101584/1984).

The method is based on the following general formula (II) which is a homolog obtained by converting the side chain of Ce-C] (R represents a -C00I( group or a -COCOOI-(group) or the same as that of the formula [1] ) is a method of decomposing the amide bond of a compound (hereinafter referred to as ID) using a microorganism to obtain the corresponding 7-aminocephem compound.
anas oval is ) ATCC950, or a strain belonging to the genus Comamonas 5Y-77-
One strain (Feikoken Bacillus No. 2410) was discovered.

Although the invention disclosed in the above-mentioned publication also discloses the usage form of the amide bond degrading enzyme produced by the above microorganism, the purpose of the present invention is to provide a more significantly improved usage form of the amide bond degrading enzyme. Our goal is to provide the following.

It has been found that this object can be achieved by a method characterized in that an immobilized enzyme obtained by adsorbing the above-mentioned amide bond-degrading enzyme on a porous copolymer to be specified later is allowed to act on compound (II). It was done.

The compound (ID) which is the starting material of the present invention is a known compound, and is produced, for example, by the following known method.

That is, a compound represented by the following general formula (IV) (X is the same as [formula 13]) or a salt thereof is treated with Aspergillus, Penicillium, Neurospora, Aerobacter, or a salt thereof under aerobic conditions. Method for using D-amino acid oxidase obtained from Trigo/Psis parabilis (
British Patent No. 1,272,769), method for causing D-amino acid oxidase derived from Fusarium and Cephalosporium to act under aerobic conditions (Japanese Unexamined Patent Publication No. 51-44695), glyoxyl A method of acting with an acid or its homolog (Japanese Patent Application Laid-Open No. 51-86490, Japanese Patent Application No. 50-83574, Japanese Patent Application No. 50-83575,
and 50-153.241).

From the compound [■] used in the present invention to the general formula CI
) The amide bond degrading enzyme for obtaining the 7-aminocephem compound (compound CI) may be of any origin, and may be of any origin.
Bacterial strains belonging to C950 and Microtechnical Research Institute No. 2410 are:
Of course it can be used.

When using these microorganisms, please refer to JP-A-50-101.
The microorganism is first cultured according to the method described in Publication No. 58, and a cell-free extract obtained by applying physical or chemical means from the bacterial cells collected from the culture, such as polishing treatment, ultrasonic treatment, etc. A known method such as salting out, fractional precipitation, dialysis, adsorption chromatography, gel filtration, etc. Partially purified or completely purified amide bond-degrading enzymes obtained by applying the enzyme separation and purification method described above can be used.

The present invention relates to a method for producing compound [■] from compound [■] using an immobilized enzyme in which the above-mentioned amide bond-degrading enzyme is supported on a specific carrier. This has a remarkable effect.

This remarkable effect largely depends on the properties of the immobilized enzyme used, and the properties of the immobilized enzyme are extremely dependent on the properties of the carrier supporting it.

The carrier will be described in detail below.

The most suitable carrier for this was discovered in 1975.
This is a porous copolymer disclosed in an invention entitled Protein Adsorbent (hereinafter referred to as the prior invention) filed by Asahi Kasei Kogyo Co., Ltd. on May 28th.

This porous copolymer consists of a cyano-containing polymer represented by the general formula (I) (wherein R represents hydrogen, an alkyl group, etc.) having a weight φ of 20 to 98 weight φ and a cyano-containing polymer having a weight φ of 20 to 98 weight φ. A copolymer obtained by copolymerizing a monomer mixture containing a crosslinking polymerizable monomer, the average pore size (a) of which is 40 to 90
It is a porous copolymer with a thickness of 0.00.

Specific examples of the cyano-containing monomer represented by the general formula CI), which is a constituent unit of this porous copolymer, include acrylonitrile, methacrylonitrile, α-chloroacrylonitrile, cinnamitrile, and the like.

The content of these cyano-containing monomers ranges from 20 to 98% by weight, preferably from 30 to 85% by weight.

When the content of the shea-containing monomer is less than 20% by weight, the hydrophilicity of the copolymer decreases, and the adsorption ability of amino bond-degrading enzymes decreases.

This copolymer can contain other monomers copolymerizable with the cyano-containing monomer.

These monomers include styrene, methylstyrene,
Hydrocarbon compounds such as ethylstyrene, vinylnaphthalene, butadiene, isoprene, and hyperylene; Styrene derivatives such as chlorstyrene, bromustyrene, N,N-dimethylaminostyrene, nitrostyrene, and chloromethylaminostyrene; methylvinyl sulfide, phenylvinyl sulfide Vinyl sulfide derivatives such as Niacrylic acid: Methyl meccrylate, Chlormethyl acrylate, etc. Acrylic acid esters: Cyclohexyl meccrylate, Dimethylaminoethyl meccrylate, Glycidyl meccrylate, Tetrahydrofurfuryl methacrylate, Hydroxyethyl meccrylate, etc. Acid esters: Vinyl ketones such as methyl vinyl ketone and ethyl isopropenyl ketone: Vinylidene compounds such as vinylidene chloride and vinylidene bromide. Acrylamide derivatives: vinyl acetate, etc.
Fatty acid vinyl derivatives such as vinyl caprate; thiofatty acid derivatives such as methyl thioacrylate and vinyl thio-Rate; There are heterocyclic vinyl compounds such as imidazole, methylvinylimidazole, vinylpyrazole, vinylnaphthalene, vinylthiazole, vinylpyridine, methylvinylpyridine, and 2,4-dimethyl-6-vinyltriazine.

The crosslinking polymerizable monomers that constitute this copolymer include divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene, divinylethylbenzene, trivinylbenzene, divinyldiphenyl, divinyldibenzyl, divinylphenyl ether, divinyl Diphenylamine, divinyl sulfone, divinyl ketone, divinylpyridine, divinylquinoline, diallyl phthalate, diallyl maleate, diallyl fumarate, diallyl carbonate, diallyl oxalate, diallyl adipate, diallyl tartrate, diallylamine, triallylamine, triallyl phosphate, tri- triallyl carballylate, N,N'-ethylene diacrylamide, NIN'-methylene dimethacrylamide, ethylene glycol dimethacrylate,
Polyethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, 1.3 butylene glycol diacrylate, trimethylpropane triacrylate,
Included are pentaerythritol tetraacrylate, triallylisocyanurate, 1,3.5-triacryloylhexahydro-L, 3.5-triazine, diarylmelamine, and the like.

The content of the crosslinking polymerizable monomer is 2 monomers to 80% by weight, preferably 5 monomers to 70% by weight, and more preferably 8 monomers to 60% by weight.

If the content of the crosslinking polymerizable monomer is too low, the degree of swelling condensation will increase and the mechanical strength will decrease.

Furthermore, if the degree of crosslinking increases too much, it becomes difficult to form micropores that provide sites for enzyme adsorption, and the rate of diffusion of liquid into the copolymer decreases.

The copolymer used in the present invention has an average pore diameter of 40λ to 900
Although it is a structure with a large number of holes within the range of 0 people,
The average pore diameter is more preferably in the range of 50 to 4,000 pores, and even more preferably in the range of 60 to 2,500 pores.

If the average pore size is too small, enzymes will enter the pores and cannot be adsorbed, which is disadvantageous, and the diffusion rate will be significantly reduced. If the pore size is too large, the surface area that contributes to adsorption will be small and the mechanical strength will be reduced. This results in disadvantages such as a decrease in

Its pore size distribution is also an important factor regarding enzyme adsorption capacity.
When the average pore diameter is d, the volume of pores with a pore diameter of 0.5 d or more and less than 2 d is 60% or less of the total pore volume, preferably 50%.
The following is desirable.

There is no particular limit to the lower limit of this value, but since the largest number of pores are around the average pore size, it will inevitably be 20% or more, but generally it is 3.
It is 0%.

This wide pore size distribution indicates the presence of large pore channels that facilitate the penetration of the enzyme solution and the presence of branched micropores that provide enzyme adsorption seats, which are responsible for the extremely large enzyme adsorption capacity. It is considered that

Furthermore, milk yield also has a significant relationship with enzyme adsorption ability.

That is, when the weight fraction of the crosslinking polymerizable monomer with respect to the total polymer is z%, the total pore amount per 1 g of dry polymer is 0.0.
It is preferably 5V'Zd or more and 1.5V'Z- or less, and more preferably o, t5v'i- or more and 1.3. %' ZTI
It is desirable that it is below Ll.

If the amount of milk is too small, it will not be possible to provide a sufficient adsorption surface, and if the amount of milk is too large, it will not only reduce the mechanical strength of the copolymer, but also reduce the amount of adsorption per copolymer in the green volume. On the contrary, it lowers it.

Next, the method for measuring porosity characteristics adopted in the present invention will be described.

The average pore size, pore size distribution, and milk yield specific surface area were measured using a mercury intrusion porosimeter.

In this method, mercury is injected into a porous material, and the amount of pores is determined from the amount of mercury infiltrated.The pore size is also determined based on the principle that the diameter of a pore is inversely proportional to the pressure required to inject mercury into that pore. It is something to be measured.

Details of this method can be found in Kaisho Fine Particle Measurement.
ent) Clyte ° Or ° Junior and G.M.
Daaraabaal (C]yde, Orr, Jrand J
, M., Dal Iaval Ie) co-author, The Macmillan Company, New York.
-Illan Company, New York) 1
959.

With this method it is possible to measure up to 35-40 holes.

In the present invention, a pore is defined as a pore communicating from the surface with a pore diameter of 40 Å or more, and the milk yield and surface area are also values derived from the pore.

In addition, the average pore diameter is defined as the value when the value of dv/d log r becomes the maximum value.

Here, r is the pore diameter, and ■ is the cumulative pore amount measured with a porosimeter.

Another value that is an indicator of porosity is bulk specific gravity.

The inventors measured the bulk specific gravity using the following method. That is, first, a column with a glass filter is filled with resin, water is sufficiently poured, and the empty volume (m) of the portion filled with resin at that time is determined.

After ffl, the sample was sufficiently dried and weighed, and the weight of both was calculated.

The method for producing the above-mentioned porous copolymer has already been described in detail in the above-mentioned prior application, and therefore will be omitted here.

A part of this will be clarified in Experimental Examples 1 and 2 below.

In addition, as a carrier that can be used in the present invention,
Mention may also be made of the copolymers disclosed in Publication No. 25517.

This product combines acrylonitrile or mechacrylonitrile and polyvinyl hetamine with an aromatic hydrocarbon solvent that is inert to them and acts as a solvent, and can swell the produced copolymer, as well as the nitrile and polyvinyl hetamine. - and a molecular weight t x that can form a homogeneous liquid phase with the solvent.
This is a porous crosslinked copolymer obtained by polymerizing in the coexistence of a monovinyl linear polymer of t o' to 1×10 5 and extracting and removing the linear polymer from the monovinyl copolymer.

In the present invention, a 7-aminocephem compound (compound [
The immobilized enzyme used in the production of I]) can be obtained by bringing the porous copolymer into contact with a liquid containing the amide bond-degrading enzyme.

The contact method includes a method in which a solution containing an amide bond-degrading enzyme is passed through a column packed with a porous copolymer (in particular, spherical particles obtained by suspension polymerization method); Any method may be used in which the enzyme-containing solution is stirred and mixed in a reaction tank, and then the resin that has adsorbed the enzyme is separated.The key is to contact the enzyme under conditions that do not cause loss of enzyme activity. It is carried out efficiently and an extremely highly active immobilized enzyme is obtained.

During the above contact, glutaraldehyde, hexamethylene diisocyanate, bisazobenzidine, N.

By allowing a crosslinking agent such as N'-ethylene bismaleimide to be present, an immobilized enzyme in which the enzyme is firmly adsorbed and immobilized by the porous copolymer can be obtained.

The mechanism of action of these crosslinking agents is not completely clear, but rather than creating a chemical bond between the enzyme and the porous material, they bind the enzymes together, making them more difficult to escape than the pores of the porous material. It is estimated that

When the immobilized enzyme obtained as described above is allowed to act on the compound [■], the objective compound CI) of the present invention can be obtained.

As the format of this action, either column format or batch format can be adopted.

That is, a liquid containing compound (II) may be passed through a column of immobilized enzyme, or a liquid containing compound (n) may be brought into contact with the immobilized enzyme in a container with stirring.

The reaction temperature at that time is preferably 20 to 45°C, more preferably 30 to 37°C, considering the stability of compound CI).

Furthermore, from the viewpoint of the stability of compounds (II) and CI), the reaction is preferably carried out in neutral to slightly acidic conditions, and is
If the reaction is carried out in the presence of an H buffer, decomposition during the reaction can be suppressed.

The method of the present invention is significantly superior to the method described in JP-A-50-101584 and other conventional methods in the following respects.

■ During the production of immobilized enzymes, enzyme adsorption is performed efficiently, the operation is easy, and there is little enzyme loss.

■ The activity density per unit weight of the immobilized enzyme obtained is extremely high.

■ The resulting immobilized enzyme has a very long half-life of activity due to continuous reactions, and combined with the great strength of the porous copolymer, the number of repeated uses of the immobilized enzyme is extremely low.

■ The enzyme activity per unit of adsorbed protein of immobilized enzyme is extremely high.

These advantages (1) to (2) enable shortening of the reaction time and reduction in the size of the reaction vessel in the production of the 7-aminocephem compound (compound [■]), and their significance is particularly important.

In general, cephalosporin compounds are extremely unstable even in the physiological pH range, and cannot be used in microbial conversion processes, i.e., under mild conditions, unless the reaction time or reaction temperature is reduced. This is because the production rate of , compound CI) decreases, and the substrate (compound [■]) during the reaction tends to decompose.

For these reasons, the production method of the present invention for obtaining compound (1) from compound (II) using the immobilized enzyme (enzyme conjugate) of the present invention makes an extremely important contribution industrially. .

Experimental examples and examples are shown below.

Display the weight of Yama. Experimental example l (Production of porous copolymer) Reflux condenser, stainless steel two-blade stirrer,
In a 3-g three-ring flask equipped with a thermometer, add 55 g of freshly distilled acrylonitrile (1 divinylbenzene)
(purity 56φ, contains 44φ vinyl ethylbenzene as an impurity) 45g, acetophenone 130g, decalin 120g, azobisisobutyronitrile 1g, 2,2'
-Azobis(2,4-dimethylvaleronyl-IJ)1
g to make a homogeneous solution.

Furthermore, partially saponified polyvinyl alcohol (viscosity 23 cp
Add 1,270 g of distilled water in which s, degree of saponification 88) and sulfur chloride l-IJum L2.5.9 were dissolved.
The reaction was heated at 45°C for 1 hour, at 50°C for 2 hours, and further at 70°C for 4 hours while stirring at a rotation speed of 100 rpm.

The amount of residual monomer was quantified and the polymerization rate was 98% or higher.

The produced resin has a good spherical shape, with a diameter of 60 to 12
It was in the range of 0μ.

After performing wet classification using a sieve, unreacted monomers and added liquid were removed with methanol.

A portion of the obtained resin was taken and dried under reduced pressure at 60°C for 18 hours to prepare a sample for a porosimeter.

The remaining resin was thoroughly washed with water. The bulk specific gravity of this resin is 0.21, the average pore diameter is 1200, and the pore volume is 2.28.
ml/g, surface area 220rn''/9.

Also, the milk yield between 600 and 2400 pores is 0.87.
ml/'9.

Hereinafter, this resin will be referred to as R1.

Experimental Example 2 (Production of porous copolymer) Hydroxyapatite 10 was placed in the flask used in Experimental Example 1.
g, 1.0 g of hydroxyethyl cellulose, 20 g of calcium chloride, and 200 g of distilled water ([!] were added, stirred at 70°C to make the solution uniform, and then lowered the liquid temperature to 30°C.

While stirring this aqueous solution at 30 Orpm, 50 g of acrylonitrile, 10 g of ethylene glycol dimethacrylate, 40 g of styrene, and 300 g of chlorobenzene were added.
A homogeneous solution of 0.25 g of tert-butyl pervalerate, 0.75 g of benzoyl peroxide was added all at once.

30 minutes at 30℃, 1 hour at 40℃, 2 hours at 50℃, 6
The reaction was carried out at 0°C for 2 hours, at 70°C for 2 hours, and further at 80°C for 2 hours.

The performance of the resin obtained after thorough extraction and water washing was as follows.

Particle size 90-260μ, average pore size 500, pore volume 2.0
2rrt17'El, the pore volume based on 250 to 1000 diameter pores is 1. , 03 yrtl/g, surface area 260 m"/'ji.

This resin will be referred to as R2 hereinafter.

Experimental Example 3 (Preparation of amide bond-degrading enzyme) 500 TL of a liquid medium (pH 7.0) containing 0.5 dilution of meat extract, 0.30 φ of salt, and 1 dilution of peptone was dispensed into a 51-volume Erlenmeyer flask, and incubated at 120°C. (2) After steam sterilization for 5 minutes, Comamonas spicies 5Y-77-1 (Feikoken Bacteria No. 2410) was inoculated and cultured with rotary shaking at 30°C for 1 day.

The entire volume of the culture solution was transformed into a liquid medium containing 2% casein, 0.05% yeast extract, 0.4% C3L, 0.5% sodium glutamate, and 0.1% glucuric acid (1)) (9,0)
Dispense L Oe into a 206-volume jar fermenter and add 1
Inoculated into 20°G steam sterilized for 30 minutes and heated at 30'C.
The cells were cultured for 3 days with 100% aeration/min and stirring at 300 rpm.

After culturing, the culture was centrifuged under cooling to collect bacteria.

The wet bacterial cells i, o o g obtained in this way were reduced to 0
．． The surfactant cation FB[F] (
Add 25 ml of NOF (product name) and stir at room temperature for 1 hour.

The bacterial cell residue is centrifuged under cooling to obtain 500 ml of enzyme solution.

The recovery rate of enzyme by this operation was 93%.

The enzyme solution thus obtained is called El.

Experimental Example 4 (Preparation of amide bond-degrading enzyme) In Experimental Example 3, Adecatol 5O-120■ (trade name, Kadenka Co., Ltd.) was used in place of the cation FB[F], and the enzyme recovery rate was 92%.

This enzyme solution is called R2.

Experimental Example 5 (Immobilization of enzyme) Add 1500 ml of enzyme solution E and 7.5 parts of 25% glucuraldehyde to 130 g of resin R, and stir at 30°C for 3 hours.

After stirring, filter and add 0.1 M IJ acid buffer (pH 7,
0) to obtain a wet carrier.

It was confirmed that 95φ of the enzyme in the enzyme solution used was adsorbed to this.

This immobilized enzyme is called R,-El-G.

Experimental Example 6 (Immobilization of Enzyme) An immobilized enzyme was obtained in exactly the same manner as in Experimental Example 5 except that glutaraldehyde was not used.

80% adsorption of enzyme was confirmed.

This immobilized enzyme is called R1-El.

Experimental Example 7 (Immobilization of Enzyme) An immobilized enzyme was prepared in exactly the same manner as in Experimental Example 5 except that R2 was used as the resin.

Enzyme adsorption was 100%.

This immobilized enzyme is called R2-El-G.

Example 1 The immobilized enzyme R1-El-G obtained in Experimental Example 5 was packed into a column with an outer shell (2 x 3.5 cIrL, V = 42 m), and O, L M phosphate buffer (pH 7, After washing, 1% 3-acetoxymethyl-7(4-carboxybutanamide)cef-3-em-4-carboxylic acid 3
0e at a constant flow rate (SVVS2).

It took approximately 240 hours to complete the spill. The results are shown in the attached drawing, and the production rate of 7-ACA remains at 90% even after 200 hours.
% or more.

After adjusting the total effluent 30e to pH 3.2, it was heated to t at 4 °C.
In the evening, 60 g of 7-ACAI (purity: 94.0) was obtained.

Example 2 Using R1-El obtained in Experimental Example 6 as the immobilized enzyme, 3-acetoxymethyl-7(4-carboxybutanamide) cef-3-M4-carboxylic acid solution of Part 1 was
The same procedure as in Example 1 was carried out except that 7-ACAl 5
．． 2 g (purity: 92.7) was obtained.

Actual Examples 3 to 8 In Example 1, 3-methyl-7-(4-carboxybutanamide) was used instead of 3-acetoxymethyl-7(4-carboxybutanamide) cef-3-em-4-carboxylic acid. Cef-3-M-4-carboxylic acid (actual example 3),
3-hydroxymethyl-7-(4-carboxybutanamide) cef-3-em-4-carboxylic acid (Example 4),
3-methyl-7-(5-carboxy-5-oxopencunamide) cef-3-em-4-carboxylic acid (Example 5)
, 3-acetoxymethyl-7-(5-carboxy-5-oxopentanamide) cef-3-em-4-carboxylic acid (Example 6), 3-hydroxymethyl-7-(5-carboxy-5-oxopentane Amido) Cef-3-M-
Using 4-carboxylic acid (Example 7) and N-(7-(5carboxy-5-oxopentanamido)cef-3-em-3-ylmethyl]pyridinium-4-carboxylate (Example 8), the following The 7-amino-cephem compounds listed in Table 1 were obtained.

(Support) Developing solvent Solvent system I; n-butanol:acetic acid:water= 3:1:1 〃 ■;.

-Butanol: dipyridine acetate: water -15:3:10:
2/zl[;n-butanol:acetic acid:bormaldehyde:water=3:1:5:1 Actual cases 9 to 15 Immobilized enzyme R2-El-G obtained in Experimental Example 7 and the following compounds ( II), an experiment was conducted in the same manner as in Example 1.

In Example 9, the production rate of 7-ACA after 200 hours was 93 pieces, and 165.
9 (purity 93%) was obtained.

[Brief explanation of drawings]

The figure shows the relationship between the production rate of compound [1] and reaction time in Example 1.

Claims (1)

[Claims] 1 General formula [■] In the formula, R represents a -C00H group or a -Co, COOH, group, and X represents a hydrogen atom, a hydroxy group, an acetoxy group, or a nucleophilic residue. From the compound represented by (referred to as compound (II)), a 7-aminocephem compound (referred to as compound [I]) represented by the general formula (I), where X is the same as that of formula (II'), is extracted from a microorganism. For chemical production, an amide bond-degrading enzyme capable of producing compound (1) from compound (■) is synthesized with the general formula (11[
) (In the formula, Y represents a hydrogen atom or an alkyl group.) A polymer mixture containing a cyano-containing monomer represented by the formula (Y represents a hydrogen atom or an alkyl group) and a crosslinking polymerizable monomer of 2 to 80 weight units is copolymerized. The resulting copolymer is characterized by allowing an immobilized enzyme adsorbed on the porous copolymer having an average pore diameter of 40 to 9000 pores to act on the compound (If) in an aqueous medium. A method for producing a 7-aminocephem compound represented by general formula (1). 2 An amide bond-degrading enzyme having the ability to produce compound CI) from compound (II) is adsorbed onto a porous copolymer in the presence of a crosslinking agent, expressed by the general formula CI) according to claim 1. A method for producing a 7-aminocephem compound. 3. A method for producing a 7-aminocephem compound represented by the general formula (, 1) according to claim 2, wherein the crosslinking agent is glutaraldehyde. 4. The porous copolymer has a pore size distribution such that the volume of pores of 0.5 dla to less than 2 d is 60 φ or less of the total pore volume, and the weight of the crosslinkable monomer relative to the total monomer When the fraction is Z%, 0.054 per 1 g of dry copolymer
"l-1,5s-'71 ral (7) General formula CI according to claim 1 or 2 having a pore volume)
A method for producing a 7-aminocephem compound represented by 5. 7- represented by the general formula (1) according to claim 1, 2 or 4, wherein the cyano-containing monomer is acrylonitrile and the crosslinking polymerizable monomer is divinylbenzene. Method for producing aminocephem compounds. 6 The shea-containing monomer is acrylonitrile, and the crosslinking polymerizable monomer is ethylene glycol dimecacrylate, represented by the general formula [■] according to claim 1, 2, or 4. A method for producing a 7-aminocephem compound. 7. React the immobilized enzyme and compound (II) for 20 to 45 minutes.
℃, neutral to slightly acidic in the presence of a pH buffer,
A method for producing a 7-aminocephem compound represented by the general formula [1] according to claim 1, which is carried out in an aqueous medium.