CA1041447A - Production of 7-amino-cephem compounds by comamonas and pseudomonas - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
7-Amino-cephem compounds are produced from 7-substituted-cephem compound by enzymatic action derived from microorganisms Comamonas sp.
SY-77-1 FERM-P 2410 or Pseudomonas ovalis ATCC 950.

Description

i.

g~o~ ~97 ~ ~

.... ... . . . ~
~ : This invention relates to a process for manufacture of 7-amino~
. ~ .
cephem compounds. More particularly, this invention partains a microbio-logical production process of 7-amino-cephem compounds of the ~ormula, S
H.N

~ CH~X ~ t COOH

wher~ein X represents hydroqen atom, hydro~y, acetoxy or nuc-eophllic r~sidual group, from cephalosporin analogues. '~
We have found that a microorganlsm belonglng to genus Comamonas ~'' strain SY-77-l separated from soil samples has an activity to produce 7-~
ami~no-cephem'compounds from à compound of the ~ormula '~

wherein~R~Is~-C~ H~j~or:-C COOH group and X has the same meanlngs herein-by;~s lttlng an~ ~de 1 nkaqe.thereof.~
r ~ croorg ' m~dcscrlbod~h~rcinabov ~has tt, Follow'ng:t .n ~ l~propertl~s,~
' :A. Morphol y~
' E~ Observations'~ere taken:by.optical or electric'~mi~roscope on bouillon:~

~ agar slant culture at 30~C.
1) Form and size: short rod, round end, 0.1 - 0.3 x 0.3 - 0 5 ~.

2) Metamorphosis: single or short chain, no capsule.

3) Motility: yes, flagellate.

4) Spore: no sporulation.

5) Gram's'stain: negative.

6) Acid-fastness: negative. - ~;
B. Growth conditdtons on several media:
1) Bouillon agar plate (30C., 24 hours):
colony round; convex rizing; smooth surface; wavy edge; white to slight yellowish white; slightly sticky; translucent to opaque;
no dispersion.
2) Bouillon agar slant (30C., 24 hours):
good growth; linear growth; smooth surEace; lustrous; moist;
opalescent to faint yellowish white surface; translucentlto opaque;
medlum no color change.
3) Bouillon (30C., 1 - 3 days): ~ -homogeneously turbid; trace precipitation; thready suspension by shaking; no film and ring.
:~ ?
4) Bouillon gelatine stab (30C., 24 hours): ' ;
linear growth along stab line, especially on middle to bottom.
5) Soy bean agar slant (30C., 24 hours): ` ;
good growth; the same as on Bouillon agar, smooth surface; lustrous.
6) Potato agar slant~
good growth; white colony surface; smooth surface; slightly sticky;
translucent to opaque; moist; lustrous; no pigment formation.

7) Litmus mllk (30C., 25 days):
almost no llquefaction. ~ ' ~

8) Glucose bouillon agar slant (30C., 24 hours): -: ~: . . :.
~ better growth as compared with bouillon agar slant; siightly sticky. ~

, ~ :
~ ~ 2 : ~: : ,, ~:.,, :.:.:, ~ ' ~ ~ . . ...
~, : ( ''~' ~ ' ` ',:'' .:

3L~ 7

9) Glucose nitrate agar slant (30C., 1-3 days):
almost no growth.

10) Tyrosine agar slant ~30C., 48 hours):
growth; medium slightly turns to brown.

11) Manitol-yeast extract agar plate (30C., 48 hours):
good growth; white to faint yellow; smooth surface; moist;
convex; medium no color change.

12) Bouillon agar plate, 7~ NaC1 added ~30C., 10 days):
almost no growth.

13) Glycelin-peptone agar plate (30C., 3 days):
good growth; smooth surface; lustrous; convex; medium turns brown.

14) Milk agar plate (30C., S days)~
i: . .
no casein hydrolysis. '

15) Taurocholate agar plate (30C., 15 days):

no growth.

C. Physiological properties:

1) Nitrate reduction : - -2) Denitrification : ~

3) Methyl red test : - (yellow) 4) Voges-Proskauer's reaction : +

5) Indol formation : -6) Hydrogen sulfate formation : -7) Starch hydrolysis : -8) ~tilization of citrate : -~

9) Utilization of inorganic nitrogen source: no utilization of nitrate.

10) Pigment formation : depend on media. ,~

11) Urease : -.:
12) Oxidase : +

13) Catalase- : +

14) Gelatin liquefaction : -... ~ , ~ , 43L4~7 - 15) Arginine hydrolysis

16) Gluconate oxidation - `

17) Growth range growth pH 5 - 12; 5 - 42C
optimUm growth pH 7 0 - 3 5; 28 - 35C ~;

18) Aerobic or anaerobic aerobic

19) 0-F test no change on 0-type and F-type test ; ~-

20) Fermentation of carbohydrate acid formation gas formation -L-arabinose D-xylose ~ ~ -~
D-glucose ~ - - .-D-mannose D-fractose - -D-galactose - ~ ~ ~
Maltose - - ; ;

Sucrose - - ~
:: ; .: ::. : ' :
Lactose - -Treharose ~ - - ;
Sorbitol Mannitol : Inositol Starch ~ - - I
When the taxonomical situation of the microorganism strain SY-77-1 having the above mentioned mycological properties was examined with reference to ~, . :
anual for the Identif1cation of Medical Bacteria, by Cowan and Steel (Ed . -1965)"~and~"Bergey's Manual of Determinative Bacteriology (7th Ed 1961)"
comparlng~with type~aulture~of relateive strain, it was identified that the 5train~ bslongsd~to~the genus Comamonas Accordingly, the inventors compared the ~t~.n Af-7l~l wi.A the ~y~e cul~ure obtal _ d fro= AmerlFan Ty~s ~ltur~

':

, ~a)4~4~7 . Collection (Al'CC) to re~ogniz~ that t:he strain was simil~r to Com~monas terrigena but was different therefrom in the point that the strain SY-77-1 had the strong activity for splitting an amide-linkage of the compound ~II].
From the above, it may be recognized that the strain SY-77-1 is a new strain belonging to the genus Comamonas and refered to Comamonas sp. SY-77-1. This strain has been deposited under the numbex "FERM-P 2410" in the Research Institute for Microbiological Industry and Technology, Agency of Industrial Science and Technology, Japan, and also has been deposited in the US Department of Agriculture, ARS, Northern Utilization Research and Develop~
ment Division and has been assigned the numerical clesignation NRRL B-8070~
. We have also found that Pseudomonas ovalis ATCC 950 has the same activity, Thus, the present inventlon is a process for manufacturing 7 amino-cephem -.
compounds of the formula [I] (hereinafter designates as amino compound tI]) comprising deacylating a compound of ~II] by treatment in the preseace of an aqueous medium with microbial culture or preparation thereof haviang an activit~, for deacylating the compound lII] to form compound [I] de~ived from the cult-ure of deacylating enzyme producing microbial strain.
- . . . : :
- An object of the present invention is to provide a novel process for :

. ~ ~producing 7-amino-cephem compounds from analogous compounds of cephalosporin C
mlcroblologlcally.
Another object of the present invention.is to provide the important ,, ~ , . .
intermediates for production of pharmaceutically valuable cephalosporin . derivatives~

: :: In the deacylating reaction of the present invention, microorganisms , .`
- . . , . :~belonging to genus Comamonas or genus P udomonas having the deacylating ~ ~.
activity-to form the compound 1I] from the compound lIIj can be use.
ln order to obtain higher activity to form amino compound lI] from th~
compound-~ ~II] (hereinafter the said activity will be designated as N-deacylat-ing activity), known:mutation methods for trèating bacteria such as selective treatment~fo stralns~by~ultra violet, X-ray and mutagenic agent,~and~selectlon of media and ermentation condition may~advantageously applied.~

4i~g7 Examples of the microorganisms having N-deacylating activity hereinabove are;

Comamonas sp. SY-77-1 FERM-P 2410 and Psudomonas ovalis ATCC 950 :
The production of amino compound [I] using the microorganisms having N-deacylating activity can be achieved by culturing the said microorganisms ~-and treating the compound [IIl with the microbial culture or preparation thereof.
', :. ; ! ` :
The cultivation of the microorganisms can be advantageously carried oot by aerobic condition, more preferablly by submerged aeration culture.
Nutrient media may include assimilable source of carbon, assimirable source of nitrogen and salts. Culturing temperature is preferablly at 25 - 37~C.
and the culturing period is generally 2 - 10 days and at the time when the N-deacylating activity reaches maximum, the cultivation should naturally ~ ;
be terminated.
The thus obtained microbial culture or preparation thereof can be used for N-deacylating reaction on compound [IIl. The preparation of the micro-bial culture ~eans the treated cultured mass to increase the N-deacylating ac~ivity for preferable production of amino compound lI], and includes, for . :. .
example, due to an endo-enzymatic property of N-deacylating activity, washed microbial cells collected from culture mass, cell-free extract such as ground ~ ~ :
or ~onicated cells, cell-lysate treated by buffer solution or cetylpylidinium- -ehloride, puriPied or partially purified N-deacylating enzyme obtained from cell-free extract by known methods sueh as salting out, chromatography or the like, immobililized ~nzyme preparation or microcapsulQ corltainlng microbial cells or preparation thereof havinq N-deacylating ~ctivity.
The~mlcrocapsule may preferablly~be a three dlmensional struceure ~gel structure)~consisting of semipermeable membrane wall polymer, whlch~
dses no t inactivate N-deacylating activity, covering the core-substance of :~4~L~47 N-deacylating active preparation.
Examples of encapsulation m~thods are, for example, orif ice method such that the core substance is dissolved or dispersed in water miscible solvent dissolving the semipermeable membrane forming wall polymer in aqueous medium (Japan. Pat. Open. No. 49-25128); complex emulsion method in aqueous vehicle such method that dissolving the wall polymer in water immiscible solvent which has a boiling point lower ~an that of water and a vapour pres~ure higher than that of water, which does not inactivate the N-deacylat-ing activity, thereafter dispersing a core substance therein and the said dispersed solution is dropped to suspend into aqueous solution containing surface active agent or protective colloid, then removing the solvent to form microcapsule (Japan, Pat. Publ. No. 47-43803, Japan. Pat. Open. No. 49-4~679);
microcapsulation of solvent removal by non-solvent emulsion, for example , dispersing or dissolving a core substance in organic solvent being dissolved wall polymer therein, emulsifying said solution in a vehicle shich is poorly miscible with the solvent of said wall polymer solution to prepare an emulsion, adding to the emulsion a non-solvent for the wall polymer, the non-solvent J ~. . ' ~ , for the wall polymer, the non-solvent being miscible with the solvent for wall polymer but miscible or poorly miscib1s with the vehicle and bs1ng inactivste the N-deacylating activity thereby to form microcapsules (French Pat. 2,166,062) and the complex emulsion method in lipophylic vehicle, for example, dissolv-ing the wall polymsr to form a solvent~ said soIvsnt being poorly miscible with vehicle, miscible with water, having boilin~ point lower than that of water and being inactivate N-deacylating ~ctivity, dissolving or disysrsing a core substance in the solution, emulsifying dispersing the said resulting solution in a vehic1s comris1ng 1iquid psraffin or~a~silicon;oil, and~rsmov- ~ ;
ng~the organic so1vent to~form mlcrooap~ule (U.S.Pst. 3,714,065).
The~N-deacy1ating reaction of compound~ is~gensral1y pr~cegded in~aque-OUS medium, preferablly at pH 6 - 8. Alternatively water insoluble micro-b1al cells or preparatlon thereof 15 ussd 1n the form of~suspension,;or ln )4~447 the Eorm of column whcrein thc c~m~und ~II] is N-deacylated wl~l, passing the aqueous solution of compound [II] through the said column.
Reaction period is generally 3 - 30 hours and it should be terminated when the production of amino compound ~Il is reached maximum. Reaction tempera- ~ ;
ture may be at 20 - 45C., preferablly at 30 - 37DC. Substrate concentration is depend on the N-acylating activity and it may be generally 0,1 - 5~.
The starting material compounds [II] of the present invention can be prepared by known process. For example, the compound [II] having-COOH group in R can be prepared from the compound of the formula ~

-OOC '~ S ' , ~ , ,.
~ ~I N ~ C~I(CH2~3C0NH If ~ [III]
3 ~ CH2X
~0011 ~ '' wherein X has the same meaning hereinabove, or salt thereof by the action -;l of D-amino acid oxidase derived from microorganisms Trigonopsis variabiris etc. in aerobic condition (British Pat., 1,272,769); the compound EIII] or ,i salt thereof is treated with activated cells of Trigonopsis variabiris or ', Fusarium sp. IJapan. Pat. Open. No. 47-39595)O
The compound ~II] having -COCOOH group in R can be prepared from the compound ~ in the presence of catalase act1vity and D-amino ac1d oxi~
~ dase activity. -,~ The substituent X in formula [IIl represents hydrogen atom, hydroxy, ' acetoxy or nucleophilic residual group, the said group can be formued from I cephalosporin C by known method. For example a compound [III] having hydrogen atom in group X is prepared by catalitic reduction in the presence of palla-! -, dium-earbon ~U.S.Pat. 3,124,576); a compound [III] having hydroxy group in I I group X is prepared by an action of esterase (Japan. Pat. Publ. No. 42-7553);
"'~ ~: : ' . ;~ . a compound ~III] having nucleophilic residual group in group X is prepared by `

treatment with weak basic heterocyclic~tertiary amine such as pyridine l~a~an.
Pat. Publ. No. 38-26179) or by the other methods (Japan. Pat. Publ. Nos. 39-~ ~ 17926, 4l-47l4, 43-5010, 42-1305, 46-14735, 46-13025, 46-15951 and 40-9155). ~ -~

J~ 8-~4~447 The compound lII] can be u~ed ln the form of water soluble salt such as sodium, potassium or ammonium salt.
The thus formed amino compound ~I] can be isolated and purified by the known methods such as column chromatography, ion-exchanye, precipita-tion or the like.
The following examples illustxate the invention but not construed as limiting.
,; ,.
Assay method of amino compound [I]:
- Aliquot of reaction mixture is ad~usted at pH 2.5 by 1 N-HCl and after the said solution is washed three times with half amount of hutyl `~ acetate, adjusted the solution at pH 7.5 by 1 N-NaOH. Further an aliquot amount thereof is reacted with phenylacetyl chloride and assayed on Eacillus subtilis PCI 211 for 16 hours at 37C.
Thin layer chromatography (TLC):
Carrier: silica gel pre-coated.
, i Developer I: n-BuOH:Acetic acid:water ~3:1:1) ... . . .
Il: n-BuOH : Acetic acid : pyridine : water ~15:3:10:12) III. n-BuO~3 : Acetic acid : formaldehide : water (3:1:5:1 .1 : ~ , .
~ Examples 1 - 5.
.. ~ . ~ -100 ml. of an aqueous medium (pH I0.0) consisting of meat extract 0.5~, peptone 1~, yeast extract 0.1~, qlutarlc acid 0.05~ and NaCl 0.5~ were introduced into a 500 ml. Erlenmyer flask, sterilized at 120C. for l5 minutes, inoculated with Comamonas sp. SY-77-1 FERM-P 2410 and rotary shake cul-tured .. . . .
1 at 30C. for i8 days. After cultivation, cultured broth was centrifuged at~

:.
12,000 r.p.m. for 10 minutes under cooling to collect bacterial cells. The said mass was sus~ende~3 in 50 ml. of Ool mole phosphate buffer solution (pl3 7~.0). Thexcin were~added 50 ml.~of 0.1~mole phosphate bufEer (pH 7.0) contain-lng the each 3~ of 3-acetoxymethyl-7-14-carboxybutariamide)-ceph-3-em-4-carb~xylic acid an~l incubated at 37C. for l0 l~ours to ob~ain the amino com~
ound II~].~Cultivation~of~acteria in the same manner;wa~ repeated and each ~ Eoll~lgg ~ubFt~

34~L4~7 3-methyl-7-(4-carboxybutaneamide)-ceph-3-em-4-carboxylic acidi 3-hydroxymethyl-7-E4-carboxy b,utaneamide)-ceph-3-em-4-carboxylic acid;
N-[7-(4-carboxybutaneamide)-ceph-3-em-3-yl methyl]-pyridinium-4-carboxylate;
and 7-t4-carboxYbutaneamide)-3-(5-methYl-1,3,4-thiadia2ol-2-yl)-thiomethyl-ceph-3-em-4-carboxylic acid.
Results are showinq in the table below, Example Amino compound [1] (R=-COOH) -X formation TLC
ratio % _ ' ,'; Rf solvent system :,' . . _ ._.,.
1 H 72 0.32 I ~, 2 -OCOC~13 53 0.15 I ' ' 3 -OH 64 0.33 II

~ 4 -N~cN!> 58 O.23 III ,, ,, 5 -S ~ ~ CH3 50 0.17 , ''~
., :, - :
,, r~h amino c~ ~ound~ we-~ identified by thin lay r chromatogr~hi wi~h ''~

j ninhydrin developer, bltra eiolet and bioassay.

Jf Examples 6 - 9.

~' ' ' In Examples l - 5, 3-acetoxymethyl-7-14-carboxy-butaneamide)-ceph-, 3-em-4-carboxylic acid and others were replaced by the following compounds ~, and obtained the r~sult hereinbelow.

-methyl-7-~5-carboxy-5-oxopentaneamido)-ceph-3-em-4-carboxyl1c acid;

~1~ 3-acetoxymethyl-7-(5-carboxy-5-oxopentaneamid~-ceph-3-~m-4-~ carboxylic acid;

'~ . , 3-hydroxymethy1-7-(5-carboxy-5-oxopentancamido)-ceph-3-em-4-', carboxylic acid and !~ . .
'~ N-t7-(5-carbo~y-5-oxopentaneamido)-ceph-3-em-3-yl-methylE-pyridinium-4-carboxylate.

,! ~ , ~ 10-:( ' ~ ' ' t,' ~ , :' ; . ' .
~: : ; ': . ~ .. , ' ` ' ' ' .

4~L7 Example Amino compound [Il ( R - -COCOO~i) ~~~~ ~~- - ~formation TLC
ratio % _ _ _ 6 ¦ _ 65 ~ 3z ~ Sol~ent system 7 -OCOCH3 50 0.l5 I
~ -OH ~ 5B 0 23 ¦ TII

Example l0 - 13.
l00 ml. of an aqueous medium (pH 7.0) consisting of meat extract 0.5%, peptone l~, yeast extract 0.l~, glutaric acid 0.05~ and NaCl 0.5~ were introduced into a 500 ml. Erlenmyer flask, sterilized at 120C. for 15 minu- `
tes, inoculated with Pseudomonas ovalis ATCC 950 and rotary shake cultured at 30C. for 8 days. After cultivation, cultured broth was centri~uged at 12,000 r.p.m. for l0 minutes under cooling to collect bacterial cells. The said cultured cells were suspended in 50 ml. of 0.l mole phosphate buffer (pH 7.0). Cultivation were repeated in the same manner and prepared cell suspenion. The cell suspensions were incubated at 37C. for l0 hours with ` each substance (2~ in 0.l mole phosphate buffer solution~showing in the ~ table below. Results were shown in the table.
-~ ~ Compound ¦ I I ]
i ~ 3-methyl-7-(4-carboxybutaneamido)-ceph-3-em-4-carboxylate;
3-hydroxymethyl-7-(4-carboxybutaneamido)-ceph-3-em-4-carboxylate;
3-acetoxymethyl-7-t4-carboxybutaneamido)-ceph-3-em-4-carboxylate;
~ and N-~r7-(4-carboxybutaneamido)-ceph-3-em-3-ylmethyl[-pyridinium-! ';m ~ carboxylate;

_ ...
amino compound [I]
Example _ ~ . .
X ~ Rf on TLC -H 0.33 (solvent system I) 11 -OH 0.33 ( " " II) 12 -OCOCH3 0.15 1 " " I) U ~ ¦ 0.22 ( .
Example 14.
Comamonas sp. SY-77-1 FERM-P 2410 was cultured in the same manner as in Example 4 to obtain 6 1. of cultured broth. Cells were collected by .
centrifuge at 12,000 r.p.m. for 10 minutes and washed. The thus obtained cells were suspended in 0.1 mole phosphate buffer (pH 7.0) to prepare 300 ml.
suspension. Therein was added 15 ml. of cation surface active ~gent, stirred at room temperature for 30 minutes, then centrifuged at 10,000 r.p.m. for 10 minutes to obtain 288 ml. of supernatant.

.;~ .
~he supernatant was dialyzed in 0.1 mole phosphate buffer solution ~: . . . .
~ (pH 7.0) using cellopha~ tube at 4C. ~or 48 hours. 300 ml. of dialysate .!1, having N-deacylating activity were obtained.~ ~ ~
'1='' ' ' ' . ' . ' , The thus obtained dialysate is included in the scope of preparation i, 1, , .
of microbial culture o the present invention and it may be prepared as micro~

~ capsulated preparation, powdered preparation by drying, lyophili~ation or .. 1 . :. . ;.
a~monium sulfate precipitation.

~ Also Pseudomonas ovalis can be treated in the same manner.

;{~ ;3~g. of cellulose~acetate (triacetate form) were dissolved in 100 ml. of me,~hiylenechloride. 3 q. of cultured cells of Comamonas ~. SY-77-1 FE~N-P 24}0, obtained by the same manner as in Example 1, suspended in 4~ 7 distilled water, were dispersely emulsified therein. The thus obtalned emulsion was stirred, dispersed in 800 ml. of 2% gelatin aqueous solution at room temperature and further stirred until evaporized the methylene chloride to obtain microcapsule (diameter O.l 0.3 mm.) containing Comamonas ~. SY-~ . ~
77-l PERM-P 2410. This microcapsule can be used for the production of amino compound lI] as hereinbelow exemplified.

Example l6.

Example 15 was repeated except that 2 g. of cellulose acetate, 70 ml. of methylene chloride and 2 g.-of Pseudomonas ovalis ATCC 950 were used.

Nicrocapsulescontaining the bacteria we~ obtained.

~; Example 17;
:' ' Example 15 was repeated, escept that 20 ml~ of supernatant having .. . .
N-deacylating activity obtained in Example 14 were used instead of 3 g. of , Comamonas cells suspended in 20 ml. of water to obtain the m~crocapsule of `, the same size.

- Example 18. ~ ;~

' 21.9 g. (wet weight) of microcapsule obt~lned ir ~Aamplc 15 w^ro -~

,l packed in the column with jacket (2 X 9.6 cm. V=30 ml.) and washed with O.l mole phosphate buffer ~pH 1.0). 840 ml. solution of 3-acetoxymethyl~1-(4-carboxybutanamide)-3-cephem-4-carboxylic acid in O.l mole phosphate buffer ~9.92 mg./ml.) were charged at SV about 0.4. 865 ml. of eluate containing 7-ACA were obtairled ~formation ratio 84.5%) and then concentrated up to 80 ml.

after adjusting pH 3.2 The concentrate was allow to stand for overnight at "I , .
4C. to precipitate the 7-ACA. (Yield 4.6 g., purity 86.2%).

Example ~l9.

Example lB;was repeated, except`that microcapsules containing ~ 1 , . . .
; Co~amonas sp. SY-77-l FERM-P 2410 wore~replaced by the microcapsules contain-ing Pseudomonas ovalis ATCC 950 obtained in Example 16 and 1% solution of the ub~tances llsted~below were used instead~of sub~trate in Exa=ple 18 to obtain amlno~compounds lll~ hereinbelow.

,. ., , ~ , ,, , .,,, , , ', , ~ , . : . . . ..

~ Substancc Yleld of compound ~I]

3-methyl-7-(4-carboxybutaneamide)-3-cephem-4-carboxylate: 84.2 3-hydroxymethyl-7-(4-carboxybutaneamide)-3-cephem-4-carboxylate:
83.1~ ,, , N-[7-(4-carboxybutaneamide)-3-cephem-3-yl-methyl~
pyridinium-4-carboxylate: 79.2% ' .
Example 20.
:..
In Example 18, the microcapsules containing Comamonas sp. ,, SY-77-1 FERM-P 2410 were replaced by the microcapsules containing super-nat,ant having N-deacylating activity obtained in Example 17, and the substrate was replaced by 1% solution of 3-acetoxymethyl-7-(4-carboxybutaneamide)-3-cephem-4-carboxylate, 3-methyl-7-(5-carboxy-5-oxopentaneamide)-3-cephem-, ' 4-carboxylate and 3-hydroxymethyl-7-~5-carboxy-5-oxopentaneami~e)-3-cephem-,, i, 4-carboxylate to obtain amino compound [I] of yieid 87.5~, 78.8% and 79.5%, respectively.
i ~ - -- - ` - .
;', . ,' . ' ' . . ',"
- , 1 ~ - . ' . , :, ... . . . . . . .
., . .~ :

,, . . . , ::
,. . .
:::, `
.~` ' ' ~ ` . ''` :, : . - : : , : .
s~ . ~ , ~ : ' ' `: ' `- .

~ 14 - ' :., , ~ , , , .:

. :~
. ~ . . , ! : : ; ~ .

. , , :
~ ~ .
'`,' ~ ` ` '. :

Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for production of 7-amino-cephem compounds of the formula (I) wherein X represents hydrogen atom, hydroxy, acetoxy or nucleophilic residual group which comprises treating an aqueous solution of the compounds of the formula (II) wherein R represents -COOH or -COCOOH group and X has the same meaning as above with microbial culture or preparation thereof derived from Comamonas sp. SY-77-1 (FERM-P 2410) or Pseudomonas ovalis ATCC 950 in an aqueous medium or from mutants thereof having the activity for deacylating a compound of general formula (II) to give a compound of general formula (I).

2. A process according to Claim 1 wherein the microbial culture or preparation derived therefrom is used in microencapsulated form.

3. A process according to Claim 1 or 2 wherein the process is carried out at a pH 6-8.

4. A process according to Claim 1 or 2 wherein the treatment is carried out at a temperature of 20 to 45°C.

5. A process according to Claim 1 or 2 wherein the treatment is carried out at a temperature of 30 to 37°C.

6. A process according to Claim 1 or 2 wherein the substrate concentration used is 0.1 to 5% w/v.

7. A process according to Claim 1 or 2 wherein the starting material is in the form of a sodium, potassium, or ammonium salt.