Process for the preparation of deacetylcephalosporin C

Deacetylcephalosporin C is converted to cephalosporin C by contact with an acetylesterase produced by Aureobasidium pullanans strain 1F0 4466.

This invention relates to a new process for the preparation of 
deacetylcephalosporin C by an enzymatic deacylation of cephalosporin C. 
More particularly, it relates to a new process for the preparation of 
deacetylcephalosporin C or its salt by contact of cephalosporin C or its 
salt with new esterase of a strain belonging to the genus Aureobasidium. 
A process of this invention is expressed by the following reaction scheme: 
##STR1## 
The object compound (I) of the present process, i.e. deacetylcephalosporin 
C is known as a valuable and important intermediate for the syntheses of 
therapeutically useful cephalosporin compounds. 
It is already known that deacetylcephalosporin C can be produced by 
deacetylation of cephalosporin C with esterases of various microorganisms, 
but those processes can not be said to be sufficient and useful for the 
industrial manufacture of the same. 
This invention has been completed by extensive study of the inventors based 
on their new finding that the esterase produced by a strain belonging to 
the genus Aureobasidium has an activity to convert cephalosporin C by 
deacetylation into deacetylcephalosporin C in good yield, i.e. in a 
conversion rate of almost 100%. 
According to this invention, deacetylcephalosporin C can be prepared by 
contact of cephalosporin C or its salt, in an aqueous medium, with the 
esterase of a strain belonging to the genus Aureobasidium until 
substantially all the cephalosporin C is deacetylated and isolation of 
deacetylcephalosporin C thus produced in solid form from the aqueous 
medium. 
Preferred salts of cephalosporin C and deacetylcephalosporin C may be an 
alkali metal salt (e.g. sodium salt, potassium salt) or an alkaline earth 
metal salt (e.g. magnesium salt, etc.). 
The strain belonging to the genus Aureobasidium to be used in the present 
invention may include all of the microorganisms belonging to the genus 
Aureobasidium which produce an esterase for hydrolyzing the ester linkage 
at 3rd position of cephalosporin C to produce deacetylcephalosporin C and 
is widely distributed in nature or kept in the culture collections. 
Accordingly, said strain can be isolated from natural sources such as a 
soil sample or can also be selected from the collected cultures available 
in public facilities for culture collection. 
As the representative example of said microorganism, there is exemplified 
Aureobasidium pullulans IFO 4466, which is available from one of the 
culture collection, i.e. Institute for Fermentation, Osaka, Japan. This 
invention may also include, within its scope, the use of mutants produced 
from said microorganism by conventional mutation means such as X-rays, 
ultraviolet radiation, treatment with mutagenic agents (e.g. nitric acid, 
N-methyl-N'-nitro-N-nitrosoquanidine) and the like. 
According to the process of this invention, it is to be noted that contact 
of cephalosporin C with the esterase of the microorganism can be conducted 
by various modes of the present process, which will be apparent from the 
following description. 
As one mode of the present process, it is to be understood that contact of 
cephalosporin C with the esterase of the microorganism of this invention 
is conducted by the contact of cephalosporin C with the cultured broth 
which is obtained by conventional cultivation of the strain belonging to 
the genus Aureobasidium or the processed material thereof, in an aqueous 
medium. 
Cultivation of the microorganism to be used in this invention can be 
generally conducted in a conventional manner, and it may be advantageously 
carried out under stirring with aeration. As a culture medium to be used, 
there may be used a nutrient one containing sources of assimilable carbon 
and nitrogen and inorganic salts. The preferred sources of carbon are, for 
example, glucose, sucrose, lactose, glycerol and starch. The preferred 
sources of nitrogen are organic nitrogen sources, for example, meat 
extract, peptone, gluten meal, corn meal, cotton-seed meal, soybean meal, 
corn steep liquer, yeast extracts, casaminic acid and amino acids, as well 
as inorganic nitrogen sources, for example, ammonium salts (e.g., ammonium 
nitrate, ammonium phosphate, etc.). If desired, mineral salts, for 
example, calcium carbonate, sodium or potassium phosphate, magnesium salts 
and copper salts, and various vitamins can also be used. 
Suitable pH of the culture media, suitable cultivation temperature and 
suitable cultivation time vary with the kind of the microorganisms to be 
used. A desirable pH usually lies in a range of pH 5 to 8. The temperature 
is usually selected from about 20.degree. C. to about 30.degree. C., 
preferably about 25.degree. C. The cultivation time is usually selected 
from 15 hours to 72 hours, preferably from 24 hours to 50 hours. 
The cultured broth per se thus obtained and its processed material can be 
employed for the preparation of deacetyl cephalosporin C. The processed 
material of the cultured broth means any preparation, which is prepared 
from the said cultured broth in a conventional manner for elevating the 
enzyme activity and is capable of deacetylating cephalosporin C to 
deacetylcephalosporin C. 
The estrase activity usually exists in mycelia. Therefore, the following 
preparations can be exemplified as a processed material of the cultured 
broth. 
(1) Raw mycelia; separated from the cultured broth in conventional manners 
such as filtration and centrifugation 
(2) dried mycelia; obtained by drying said raw mycelia in conventional 
manners such as lyophilization and vacuum drying 
(3) cell-free extract; obtained by destroying said raw or dried cells in 
conventional manners (e.g. autolysis of the mycelia using an organic 
solvent, grinding the mycelia with alumina, sea sand, etc. or treating the 
mycelia with super sonic waves) 
(4) enzyme solutions; obtained by purification or partial purification of 
said cell-free extracts in conventional manners (e.g. column 
chromatography) 
(5) immobilized mycelia or enzyme; prepared by immobilizing said mycelia or 
enzyme in conventional manners (e.g. a method using acrylamide, glass 
bead, ion exchange resin, etc.). 
The reaction comprising contact of cephalosporin C with the enzyme can be 
conducted in an aqueous medium such as water or a buffer solution, that 
is, it can be usually conducted by dissolving or suspending the cultured 
broth or its processed material in an aqueous medium such as water or a 
buffer solution containing cephalosporin C. 
Preferable pH of the reaction mixture, concentration of cephalosporin C, 
reaction time and reaction temperature may vary with properties of a 
cultured broth or its processed material to be used. Generally, the 
reaction is carried out at pH 3 to 7, preferably pH 3.5 to 5, at 
20.degree. to 40.degree. C., preferably 25.degree. to 37.degree. C. for 2 
to 50 hours. 
In the reaction mixture, the concentration of cephalosporin C as a 
substrate may be preferably selected from a range of 0.1 to 100 mg/ml. 
As an alternative way for contact of cephalosporin C with the esterase, 
there is provided another mode of the present process which may be more 
convenient in the industrial scale production. That is, cephalosporin C in 
a cultured broth of a cephalosporin C-producing microorganism per se, 
without isolating cephalosporin C from said broth, is directly brought 
into contact with mycelia in a cultured broth of the esterase-producing 
strain belonging to the genus Aureobasidium to produce 
deacetylcephalosporin C. 
For this purpose, there may preferably be employed a mixed culture of a 
cephalosporin C-producing microorganism and the esterase-producing strain 
belonging to the genus Aureobasidium. The cephalosporin C-producing 
microorganism includes Cephalosporium acremonium, which is known and 
available to the public (e.g. Cephalosporium acremonium ATCC 11550) and 
the like. 
The mixed culture may preferably be conducted firstly by culturing a 
cephalosporin C-producing microorganism (e.g. Cephalosporium acremonium) 
in a medium, and then by inoculating, at an appropriate time, the 
esterase-producing strain belonging to the genus Aureobasidium into the 
said cultured broth of a cephalosporin C-producing microorganism and 
further continuing the cultivation till cephalosporin C thus accumulated 
in the broth is converted into deacetylcephalosporin C. In other words, 
the mixed culture may preferably be started by inoculating the 
esterase-producing strain belonging to the genus Aureobasidium to a 
cultured broth of a cephalosporin C-producing microorganism at an 
appropriate time during the cultivation period of cephalosporin 
C-producing microorganism. 
In the mixed culture step as explained above, it is to be noted that 
inoculation of the esterase-producing strain belonging to the genus 
Aureobasidium is preferably conducted by adding a culture broth of said 
strain to a cultured broth of a cephalosporin C-producing microorganism. 
It is further to be noted that an appropriate time for innoculating the 
esterase-producing strain may be set up in consideration of kinds of 
strains of both of the microorganisms and the cultivation conditions and 
actually according to pre-experiment for detecting a preferable 
conditions, and it can be said to be usually preferable time when mycelia 
of a cephalosporin C-producing microorganism grows and begins to increase 
in the medium and cephalosporin C per se begins to be produced and 
increased in the cultured broth. And, the time for inoculating the 
esterase-producing strain may be appropriately set up 2 to 6 days, 
preferably 3 to 5 days after the beginning of the cultivation of a 
cephalosporin C-producing microorganism. 
The medium for the mixed culture contains substantially the same 
ingredients as those exemplified for the culture medium for the strain 
belonging to the genus Aureobasidium as mentioned above. 
Suitable pH of the medium is usually adjusted within a range of 5-7 
preferably 5-6. The temperature of the mixed culture is usually selected 
from about 20.degree.-30.degree. C., preferably about 25.degree. C. The 
suitable cultivation time is usually at least more than two days. 
The object compound thus produced in the reaction mixture or the mixed 
cultured broth may be isolated and purified in a conventional manner used 
in cephalosporin chemistry. As such a manner, there are exemplified 
methods of purification with appropriate solvents, concentration under 
reduced pressure, lyophilization, crystallization, recrystallization and 
treatment with anionic or cationic exchange resin or macroporous nonionic 
adsorption resin and the like. 
As explained in details hereinabove, the inventors of this invention found 
out a new and effective process for the preparation of 
deacetylcephalosporin C which comprises contacting cephalosporin C or its 
salt with the esterase of a strain belonging to the genus Aureobasidium, 
particularly Aureobasidium pullulans IFO 4466. Following the above study, 
the inventors of this invention continued to make an extensive study for 
identifying the properties of the esterase of the genus Aureobasidium used 
in this invention and confirming the novelty of the same over esterase 
known in a pertinent prior art, so that they concluded that the esterase 
of the genus Aureobasidium is a new enzyme which is capable of hydrolyzing 
cephalosporin C to deacetylcephalosporin C. 
In this respect, as a pertinent prior art, there can be cited U.S. Pat. No. 
3,912,589 issued on Oct. 14, 1975, in which there is disclosed the 
esterase of the genus Rhodotorula which is capable of hydrolyzing 
cephalosporin C to deacetylcephalosporin C, and it is to be noted that the 
genus Rhodotorula is a close microorganism to the Aureobasidium of this 
invention in view that each of them is a kind of microorganisms belonging 
to "yeast".

The following is the detailed explanation properties of the esterase 
produced by a strain of the genus Aureobasidium pullulans IFO 4466, which 
is a representative strain belonging to the genus Aureobasidium, and the 
relevant matters. 
Preparation of the enzyme 
The esterase was prepared by means of isolation and purification from a 
cultured broth of Aureobasidium pullulans IFO 4466, and the details of the 
preparation is referred to Example 3 as explained hereinafter. 
As clearly seen from said Example 3, the esterase includes two components 
of enzymes, which are capable of hydrolyzing cephalosporin C to 
deacetylcephalosporin C, and is characterized by the property that the 
said esterases are capable of hydrolyzing an acetic ester, but are not 
capable of hydrolyzing an ester besides acetic ester, which will be 
apparent in the following. Accordingly, the esterase produced from 
Aureobasidium pullulans IFO 4466 should be named as so-called 
"acetylesterase", and so the inventors of this invention names two 
components of the esterases of this invention as Acetylesterase 4466-I and 
Acetylesterase 4466-II for convenience sake in the following explanation. 
Properties of the esterase 
The Acetylesterase 4466-I and Acetylesterase 4466-II have the following 
enzymatic properties. 
(1) Substrate Specificity (Acetylesterase 4466-I and 4466-II): 
Hydrolyzable: Acetic ester 
Not hydrolyzable: Ester besides acetic ester 
The Substrate Specificity was determined by the following method. 
The substrate (5 mg) as listed below was inserted into a test tube and 
dissolved in 0.05 M acetate buffer solution (pH 4.5) (1 ml). To the 
solution was added the Enzyme solution A (i.e. Acetylesterase 4466-I) (0.1 
ml) or B (i.e. Acetylesterase 4466-II) (0.1 ml) as prepared in the Example 
3. 
After the reaction mixture was incubated on a shaken at 25.degree. C. for 
an hour, hydrolysis of the substrate was determined in the following 
manner. 
(i) Analysis 1 (Thin layer chromatography) 
Test solution (1 .mu.l) and authentic sample were spotted on a silica gel 
plate, respectively. The plate was developed with 70% aqueous n-propanol 
and dried. Detection was carried out by ultraviolet absorption. 
(ii) Analysis 2 (Gas chromatography) 
Test solution was analyzed by gas chromatography in the following 
condition. 
Column: Se. 30 lm 
Carrier gas: N.sub.2 (15 ml/minutes) 
Column temperature: 80.degree. C. 
Injection temperature: 120.degree. C. 
Detector temperature: 80.degree. C. 
Detector: FID (Air 1.0 kg/cm.sup.2 G, H.sub.2 1.0 kg/cm.sup.2 G). 
The results are shown in the following table. 
______________________________________ 
Hydrolysis 
Acetyl- Acetyl- 
esterase esterase Analysis 
Substrate 4466-I 4466-II Method 
______________________________________ 
Cephalosporin C 
++ ++ Analysis 1 
7-Aminocephalosporanic 
++ ++ " 
acid 
Cephapirin ++ ++ " 
Methyl acetate + + Analysis II 
Ethyl acetate + + " 
Methyl propionate 
- - " 
Methyl butyrate 
- - " 
Ethyl formate - - " 
______________________________________ 
(Note)- 
++: Completely hydrolyzed 
+: Partly hydrolyzed 
-: Not hydrolyzed 
(2) Effect of pH 
(A) Substrate: Cephalosporin C 
(a) Optimum pH: (Acetylesterase 4466-I and 4466-II) Below pH 4 
As shown in FIG. 1 of accompanying drawings, optimum pHs of Acetylesterase 
4466-I (0--0) and Acetylesterase 4466-II (.DELTA.--.DELTA.) are below pH 
4. 
Enzyme activities at various pH were measured by the following method. 
(i) Buffer solution 
pH 3.5-6.0:0.05 M acetate buffer solution 
pH 5.0-8.0:0.1 M phosphate buffer solution 
pH 7.0-9.5:0.1 M tris-hydrochloride buffer solution 
(ii) Reaction 
To the enzyme solution (0.5 ml), were added warm (30.degree. C.) buffer 
solution (4 ml) at various pH as mentioned above and 5% cephalosporin C 
sodium dihydrate aqueous solution (0.5 ml). The reaction mixture was 
incubated on a shaker at 30.degree. C. for an hour. Subsequently, produced 
deacetyl cephalosporin C and unreacted cephalosporin C were determined by 
high pressure liquid chromatography. 
(b) Stable pH range (Acetylesterase 4466-I and 4466-II): pH 4.0-6.0 
As shown in FIG. 2 of accompanying drawings, Acetylesterase 4466-I (0--0) 
and Acetylesterase 4466-II (.DELTA.--.DELTA.) are stable in a range of pH 
4.0-6.0. 
The stability test was carried out by the following method. 
To the enzyme solution (1 ml), was added a buffer solution (9 ml) at 
various pH as mentioned above. The enzyme solution (10 ml) was heated at 
60.degree. C. for an hour. Subsequently, residual enzyme activity of the 
acetyl esterase was determined according to the Determination Method I as 
mentioned hereinafter. 
(B) Substrate: Ethyl acetate 
Optimum pH (Acetylesterase 4466-I and 4466-II): Around pH 5.25 
As shown in FIG. 5 of accompanying drawings, optimum pH of Acetylesterase 
4466-I (0--0) and Acetylesterase 4466-II (.DELTA.--.DELTA.) are around pH 
5.25 in case of using ethyl acetate as substrate. 
Enzyme activity at various pH were measured by the following method. 
To ethyl acetate (5 .mu.l), were added warm (30.degree. C.) buffer solution 
(1 ml) at various pH as mentioned above and the enzyme solution (300 
.mu.l). The reaction mixture was incubated on a shaker at 30.degree. C. 
for an hour. Subsequently, produced ethanol and unreacted ethyl acetate 
were determined by gas chromatography. 
(3) Effect of Temperature 
(a) Optimum temperature (Acetylesterase 4466-I and 4466-II): Around 
37.degree. C. 
As shown in FIG. 3 of accompanying drawings, optimum temperature of 
Acetylesterase 4466-I (0--0) and Acetylesterase 4466-II (.DELTA.--.DELTA.) 
are around 37.degree. C. 
Enzyme activities at various temperature were measured by the following 
method. 
To the enzyme solution (0.5 ml), were added 0.05 M acetate buffer solution 
(pH 4.0) (4 ml), which was warmed at prescribed temperature, and 5% 
cephalosporin C sodium dihydrate aqueous solution (0.5 ml). The reaction 
mixture was incubated on a shaker at prescribed temperature for an hour. 
Subsequently, produced deacetyl cephalosporin C and unreacted 
cephalosporin C were determined by high pressure liquid chromatography. 
(b) Stable temperature range (Acetylesterate 4466-I and 4466-II): Below 
60.degree. C. 
As shown in FIG. 4 of accompanying drawings, Acetylesterase 4466-I (0--0) 
and Acetylesterase 4466-II (.DELTA.--.DELTA.) are stable below 60.degree. 
C. 
The stability test was carried out by the following method. 
Enzyme solution (pH 4.0) was allowed to stand at prescribed temperature 
(25.degree.-70.degree. C.) for an hour. Subsequently, residual enzyme 
activities were determined according to Determination Method I as 
mentioned below. 
(4) Determination of Km 
(In case that the substrate is cephalosporin C) 
Acetylesterase 4466-I: Km=11.5 mM 
Acetylesterase 4466-II: Km=13.7 mM 
Km values of Acetylesterase 4466-I and Acetylesterase 4466-II were 
determined in the following method. 
To 0.05 M acetate buffer solution (pH 4.0) containing cephalosporin C 
sodium dihydrate in a various concentration (0.2-5%) (4.5 ml), was added 
enzyme solution (0.5 ml). 
The reaction mixture was incubated on a shaker at 30.degree. C. for an 
hour. Subsequently, produced deacetyl cephalosporin C and unreacted 
cephalosporin C were determined by high pressure liquid chromatography. Km 
values of each esterase were calculated in a conventional manner. 
(5) Effect of metal ion 
Enzyme activities of Acetylesterase 4466-I and Acetylesterase 4466-II are 
not affected by metal ion such as Cu.sup.2+, Mn.sup.2+, Mg.sup.2+, 
Zn.sup.2+, Ni.sup.2+, Ca.sup.2+, Ba.sup.2+, Fe.sup.3+. 
Effect of metal ion on the said acetylesterase was determined by the 
following method. 
To the enzyme solution (0.5 ml), were added 0.05 M acetate buffer solution 
(pH 4.0) containing one of metal ion as mentioned above (5.times.10.sup.-4 
mol) and 5% cephalosporin C sodium dihydrate aqueous solution (0.5 ml). 
The reaction mixture was incubated on a shaker at 30.degree. C. for an 
hour. Subsequently, produced deacetylcephalosporin C and unreacted 
cephalosporin C were determined by high pressure liquid chromatography. 
As a control, there was used 0.05 M acetate buffer solution (pH 4.0) 
containing no metal ion in the procedure as mentioned above. 
Enzyme activity of esterases can be measured by the following method. 
Determination Method I 
(a) Reaction 
To the enzyme solution (0.5 ml), is added warm (30.degree. C.) 0.05 M 
acetate buffer solution (4 ml) and 0.05 M acetate buffer solution (0.5 ml) 
containing cephalosporin C sodium dihydrate (50 mg/ml). The reaction 
mixture is incubated on a shaker at 30.degree. C. for an hour. 
Subsequently, produced deacetyl cephalosporin C and unreacted 
cephalosporin C are determined by high pressure liquid chromatography in 
the following conditions, respectively. 
Stationary phase: Polygosil 60-10 C.sub.18 (trade mark, made by Sumitomo 
Chemical Co., Ltd.) 
Mobile phase: A mixture of methanol, water and acetic acid (100:900:5) 
Column height: 30 cm 
Column temperature: Ambient temperature 
Detector: Ultraviolet absorption at 250 nm 
Flow rate: 1.0 ml/minutes 
Enzyme activity (mg/hour.ml or mg) is expressed as deacetylcephalosporin C 
(mg) thus produced per an enzyme preparation (1 ml or 1 mg) in the above 
reaction condition. 
(b) Calculation method of specific activity 
(i) Determination of protein content 
Protein content in an enzyme preparation can be determined according to 
Lawry method [cf. Journal of Biological Chemistry 193,265 (1951) ] using 
bovine serum albumin as standard sample. 
(ii) Calculation of specific activity 
Specific activity can be calculated in the following equation. 
##EQU1## 
From the observation of the properties as explained hereinabove, it can be 
said that the acetylesterases of this invention have very unique 
properties, especially having a capacity of hydrolyzing cephalosporin C to 
deacetylcephalosporin C, very narrow substrate specificity (i.e. the said 
acetylesterases are capable of hydrolyzing only acetic ester, but are not 
capable of hydrolyzing the other ester) and unique optimum pH (i.e. said 
acetylesterases have considerable acidic pH) in comparison with known 
esterases. 
Further, in comparison of the acetylesterase of this invention with a known 
esterase which is capable of hydrolyzing cephalosporin C to 
decetylcephalosporin C, i.e. the esterase produced by the genus 
Rodotorula, as the representative thereof, Rodotorula glutinis IFO 1125, 
it was observed that the acetylesterases of this invention (hereinafter 
referred to A.E.) are quite different from the esterase produced by the 
genus Rodotorula (hereinafter referred to R.E.), for example, in the 
following points. That is: 
optimum pH of A.E. is below pH 4, while that of R.E. is around pH 5 to 6; 
optimum temperature of A.E. is around 37.degree. C., while that of R.E. is 
around 50.degree. C.; and 
stable pH range of A.E. is around pH 4.0 to 6.0, while that of R.E. is 
around pH 6.0 
According to the extensive study and analysis of the results as mentioned 
above, the inventors of this invention concluded that Acetylesterase 
4466-I and Acetylesterase 4466-II are new enzymes. 
The following Examples are given for the purpose of illustrating the 
present invention. 
EXAMPLE 1 
An aqueous medium (pH 7.0) (100 ml) containing 1% of glucose, 0.3% of 
peptone and 0.3% of beef extract was poured into each of four 500 ml 
Erlenmeyer flasks and sterilized at 120.degree. C. for 20 minutes. To each 
of the media was added a loopful of slant culture of Aureobasidium 
pullulans IFO 4466. The organism was grown on a shaker at 25.degree. C. 
for 2 days. 
Further, the same aqueous medium as mentioned above (20 liters) was poured 
into 30 liters jar-fermenter and sterilized at 120.degree. C. for 20 
minutes. To the medium was added whole volume of the cultured broth as 
obtained above. The organism was grown at 25.degree. C. for 2 days. 
The cultured broth thus obtained was used in the following preparations of 
deacetylcephalosporin C. 
(1) (Preparation of deacetyl cephalosporin C using mycelia of Aureobasidium 
pullulans) 
The wet mycelia (10 g) which is obtained by centrifugation of the above 
cultured broth was added to an aqueous solution (300 ml) containing 
cephalosporin C sodium.dihydrate (25 mg/ml). The reaction mixture was 
stirred at pH 4.5 and 30.degree. C. for 10 hours until cephalosporin C in 
the mixture disappeared. The resultant mixture was filtered. The filtrate 
was adjusted to pH 7.0 and concentrated under reduced pressure to give 
crystals, which was allowed to stand overnight at 5.degree. C. To the 
crystals was added a small volume of 75% aqueous ethanol and the mixture 
was triturated. 
The crystals were obtained by filtration and dried in vacuo to give 
crystalline deacetyl cephalosporin C (3.8 g). 
IR spectrum of the crystals as obtained above was identical with that of 
the authentic deacetylcephalosporin C. 
(2) (Preparation of deacetylcephalosporin C using enzyme extracts from 
mycelia of Aureobasidium pullulans) 
To the wet mycelia (10 g) was added a small volume of chloroform. The 
mixture was allowed to stand at ambient temperature for 30 minutes. To the 
mixture was added distilled water (20 ml). After allowing to stand at 
ambient temperature for a day, the mixture was centrifuged to give enzyme 
extracts (20 ml). The enzyme extracts was added to an aqueous solution 
(300 ml) containing cephalosporin C sodium.dihydrate (25 mg/ml). The 
reaction mixture was stirred at pH 4.5 and 30.degree. C. for 10 hours 
until cephalosporin C in the mixture disappeared. To the resultant mixture 
was added activated charcoal (3 g) and the mixture was filtered. The 
filtrate was adjusted to pH 7.0 and concentrated under reduced pressure to 
give crystals, to which was added a small volume of 75% aqueous ethanol 
and the mixture was triturated. The crystals were separated by filtration 
and dried in vacuo to give crystalline deacetylcephalosporin C (3.6 g). 
IR spectrum of the crystals as obtained above was identical with that of 
the authentic deacetylcephalosporin C. 
(3) (Preparation of deacetylcephalosporin C using immobilized mycelia) 
The frozen and thawed mycelia (24 g) of Aureobasidium pullulans IFO 4466 
was added to an aqueous solution (20 ml) containing acrylamide (7.5 g) and 
N,N'-methylenebisacrylamide (0.4 g). To the mixture was added 5% aqueous 
N,N,N',N'-tetramethylethylenediamine (5 ml) and 1% aqueous ammonium 
persulfate (5 ml) at 4.degree. C. in nitrogen atmosphere. The reaction 
mixture was polymerized in a glass tube at 4.degree. C. for an hour. The 
polyacrylamide gel thus produced was pushed out and crushed with a 
crusher. The crushed gel was packed in a column (volume: 100 ml) and 
washed with 0.1 M acetate buffer solution and cold water (1 liter). An 
aqueous solution (1 liter) containing cephalosporin C sodium.dihydrate 
(1.6 mg/ml) was passed through the column as prepared above in a rate of 
SV=0.5. After it was confirmed that no cephalosporin sodium.dihydrate 
existed in the passed solution, the passed solution was adjusted to pH 
7.0, concentrated under reduced pressure and allowed to stand overnight at 
5.degree. C. To the residue was added 75% aqueous ethanol and mixed 
sufficiently to precipitate crystals, which were separated and dried in 
vacuo to give crystalline deacetylcephalosporin C (0.9 g). 
IR spectrum of the crystals was identical with that of the authentic 
deacetylcephalosporin C. 
EXAMPLE 2 
An aqueous medium (pH 7.0) (100 ml) containing 3% of soybean meal, 2% of 
sucrose, 1% of glucose, 1% of corn steep liquor and 0.5% of calcium 
carbonate was poured into each of four 500 ml Erlenmeyer flasks and 
sterilized for 20 minutes at 120.degree. C. On each of the media was 
inoculated a loopful of slant culture of Cephalosporium acremonium ATCC 
11550. The organism was grown on a shaker at 30.degree. C. for 5 days. 
On the other hand, an aqueous medium (pH 7.0) (20 liters) containing 3% of 
peanut powder, 1% of soybean meal, 2% of corn steep liquor, 2% of methyl 
oleate, 0.5% of ammonium sulfate, 0.6% of DL-methionine, 2% of beet 
molasses, 2% of glucose and 0.8% of calcium carbonate was poured into 30 
liters jar-fermenter and sterilized at 120.degree. C. for 20 minutes. On 
the medium was inoculated whole volume of the cultured broth as obtained 
above. The organism was grown at 25.degree. C. for 3 days. 
Further, an aqueous medium (100 ml) (pH 7.0) containing 1% of glucose, 0.3% 
of peptone and 0.3% of beef extract was poured into each of four 500 ml 
Erlenmeyer flasks and sterilized at 120.degree. C. for 20 minutes. On each 
of the media was inoculated a loopful of Aureobasidium pullulans IFO 4466. 
The organism was cultured at 25.degree. C. for 2 days. The whole volume of 
this cultured broth was inoculated on the 3 days-cultured broth of 
Cephalosporium acremonium as obtained above and the mixed cultured broth 
was incubated at 25.degree. C. for 2 days. Subsequently, the mixed 
cultured broth was analyzed by bioautography and bioassay using Bacillus 
megaterium. The result of the analysis showed the fact that all of the 
cephalosporin C in the cultured broth was converted to 
deacetylcephalosporin C and yield of deacetylcephalosporin C was 0.5 
mg/ml. 
EXAMPLE 3 
The mycelia (350 ml) were collected by centrifuge from the cultured broth 
(10 liters) of Aureobasidium pullulans IFO 4466 which was prepared in 
substantially the same manner as described in Example 1. To the mycelia, 
was added one-fifth volume of chloroform. 
The mixture was stirred for 30 minutes. Twice volumes of water were added 
to the mixture. The aqueous mixture was allowed to stand at 25.degree. C. 
for 24 hours. 
The mixture was filtered to give Enzyme extract I (1000 ml) and adjusted to 
pH 4.0 and then filtered to remove insoluble materials. The filtrate was 
subjected to a ultrafiltration using a membrane (Molecular weight: 0.1 
million cut) to give a concentrate (80 ml), which was lyophilized to give 
a crude powder. The crude powder (4 g) was dissolved in 0.05 M acetate 
buffer solution (pH 4.0) (20 ml) and passed through a column of 
CM-Sephadex C-25 (trade mark: made by Pharmacia AB, internal diameter of 
the column: 40 mm, height of the column: 675 mm) and the column was 
developed with the same acetate buffer solution as mentioned above 
(velocity: 64 ml/hour). The fraction (440 ml) containing the object 
compound was collected and passed through a column of DEAE-Sephadex A-25 
(trade mark: made by Pharmacia AB, internal diameter of the column: 40 mm, 
height of the column: 785 mm). The column was washed with 0.05 M acetate 
buffer solution (pH 4.0) (1000 ml) and developed with (0.05 M-0.25 M) 
acetate buffer solution (pH 4.0) in a linear gradient manner (velocity: 64 
ml/hour) to give Fraction A (360 ml) and Fraction B (980 ml). 
The fraction A had 1.7-hold higher specific activity than the Fraction B. 
The Fraction A and Fraction B were concentrated by ultrafiltration and 
passed through a column of Sepharose 4B (trade mark: Pharmacia AB, 
internal diameter of the column: 29 mm, height of the column: 535 mm), 
respectively. Each column was developed with 0.05 M acetate buffer 
solution (pH 4.0) to give Enzyme solution A (72 ml) from the Fraction A 
and Enzyme solution B (32 ml) from the Fraction B, respectively. 
Each specific activity of the enzyme preparations as prepared above is 
given as a relative specific activity in the following table. In this 
respect, it is to be noted that said "relative specific activity" is 
indicated with a figure calculated on the criterion that the activity of 
the Enzyme Extract I shall be taken as a FIG. 1. 
______________________________________ 
Relative Specific 
Enzyme Preparation 
Activity 
______________________________________ 
Enzyme Extract I 
1 
Fraction A 110 
Fraction B 64 
Enzyme Solution A 
167 
Enzyme Solution B 
105 
______________________________________