Esterase free enzymes

Abstract

A process for decreasing esterase activity present in the presence of D-amino acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and D-amino acid oxidase activity and/or having esterase activity and glutarylacylase activity by treatment with phenylmethylsulphonyl fluoride; immobilized biocatalyts in spherical particle form obtainable by treating microorganism cells having enzyme activity with a primary or secondary amine containing polymer, an organic solvent which is able to form a two-phase system with water and a bifunctional agent; and the use of that process and biocatalysts in the production of 7 -aminocephalo-sporanic acid, 7 -amino- 3 -hydroxymethyl- 3 -cephem- 4 -carboxylic acid and cephalosporin antibiotics.

1 . A process for decreasing esterase activity present in the presence of D-amino acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and Damino acid oxidase activity and/or glutarylacylase activity comprising treating a mixture having esterase activity and D-amino acid oxidase activity and/or glutarylacylase activity with phenylmethylsulphonyl fluoride.  
     
     
         2 . A process for the production of glutaryl-7-aminocephalosporanic acid comprising the steps 
 i) treating a mixture having esterase activity and D-amino acid oxidase activity with phenylmethylsulphonyl fluoride,    ii) reacting cephalosporin C with a mixture obtained in step i) to obtain glutaryl-7-aminocephalosporanic acid; and isolating glutaryl-7-aminocephalosporanic acid obtained, if desired.    
     
     
         3 . A process for the production of 7-aminocephalosporanic acid comprising the steps 
 i) treating a mixture having esterase activity and glutarylacylase activity with phenylmethylsulphonyl fluoride,    ii) reacting glutaryl-7-aminocephalosporanic acid with a mixture obtained in step i) to obtain 7-aminocephalosporanic acid; and isolating 7-aminocephalosporanic acid obtained in step ii), if desired.    
     
     
         4 . A process for the production of spherical particles from microogranism cells having an enzyme activity, comprising the steps 
 i) treating microorganism cells with a primary or secondary amine containing polymer,    ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells,    iii) treating a mixture obtained in step ii) with a bifunctional agent; and isolating the spherical particles obtained.    
     
     
         5 . Spherical particles containing microorganism cells which contain enzyme activity obtainable from a process comprising the steps 
 i) treating microorganism cells having enzyme activity with a primary or secondary amine containing polymer    ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells,    iii) treating a mixture obtained in step ii) with a bifunctional agent; and isolating the spherical particles obtained.    
     
     
         6 . Spherical particles obtained by a process comprising the steps as defined in  claim 5 .  
     
     
         7 . A process for the production of glutaryl-7-aminocephalosporanic acid comprising the steps 
 i) treating microorganism cells having D-amino acid oxidase activity with a primary or secondary amine containing polymer,    ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells,    iii) treating a mixture obtained in step ii) with a bifunctional agent; and isolating the spherical particles obtained, and if desired, treating a mixture having esterase activity in the presence of D-amino acid oxidase activity with phenylmethylsulphonyl fluoride, or treating spherical particles obtained in step iii) having esterase activity in the presence of D-amino acid oxidase activity with phenylmethylsulphonyl fluoride,    iv) reacting cephalosporin C with spherical particles obtained in step iii) to obtain glutaryl-7-aminocephalosporanic acid, and isolating glutaryl-7-aminocephalosporanic acid, if desired.    
     
     
         8 . A process for the production of glutaryl-7-aminocephalosporanic acid which comprises reacting cephalosporin C with spherical particles obtainable by step iii) as defined in  claim 7  to obtain glutaryl-7-aminocephalosporanic acid, and isolating glutaryl-7-aminocephalosporanic acid, if desired.  
     
     
         9 . A process for the production of 7-aminocephalosporanic acid wherein glutaryl-7-aminocephalosporanic acid obtained according to  claim 2 , step ii) or  claim 7 , step iv) is converted into 7-aminocephalosporanic acid.  
     
     
         10 . Use of a process as claimed in  claim 4  or use of spherical particles as claimed in  claim 5  in the production of 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin C or from glutaryl-7-aminocephalosporanic acid.  
     
     
         11 . A process for the production of 7-aminocephalosporanic acid comprising the steps 
 i) treating microorganism cells having glutarylacylase activity with a primary or secondary amine containing polymer,    ii) contacting an organic solvent which is able to form a two-phase system with water with a mixture of microorganism cells,    iii) treating a mixture obtained in step ii) with a bifunctional agent; and isolating the spherical particles obtained,    iv) if desired, treating a mixture having esterase activity in the presence of glutarylacylase activity with phenylmethylsulphonyl fluoride, or treating spherical particles obtained in step iii) having esterase activity in the presence of glutarylacylase acylase with phenylmethylsulphonyl fluoride, if desired,    v) reacting glutaryl-7-aminocephalosporanic acid with spherical particles obtained in step iii) or obtained in step iv) to obtain 7-aminocephalosporanic acid; and isolating 7-aminocephalosporanic acid obtained, if desired.    
     
     
         12 . A process for the production of a cephalosporin wherein 7-aminocephalo-sporanic acid or 7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid produced according to the present invention according to any one of claims  3  or  9  is used as an intermediate.  
     
     
         13 . Product produced by a process according to any of  claims 1  to  4 ,  7  to  9  or  11  to  12 .