Mutated cephalosporin hydroxylase and its application in deacetylcephalosporanic acid synthesis

Abstract

The present invention relates to a mutant hydroxylase with increased activity and greater substrate specificity towards phenylacetyl-7-ADCA derivatives for the production of phenylacetyl-7-HACA derivatives, which carries one or more amino acid modification at residue positions when compared with the wild type hydroxylase from the following group of residues, Proline at position 7, Alanine at position 40, Threonine 51, Methionine at position 53, Glutamic acid at position 82, Arginine at position 91, Threonine at position 96, Glycine at position 108, Isoleucine at position 149, Valine at position 171, Alanine at position 177, Arginine at position 182, Methioinine at position 184, Isoleucine at position 193, Phenylalanine at position 195, Glutamine at position 209, Alanine at position 210, Valine at position 226, Methionine at position 233, Leucine at position 236, Alanine at position 237, Alanine at position 241, Valine at position 249, Arginine at position 250, Serine at position 251, Glycine at position 255, Glutamic Acid at position 258, Serine at position 260, Phenylalanine at position 267, Alanine at position 280, Valine at position 307 and Asparagine at position 313.. The invention further provides a process for the preparation of deacetyl cephalosporanic acid from the corresponding deacetoxy cephalosporanic acid using an enzyme of the present invention. The invention also provides the method for producing and processing of such enzymes.

Description

technical field [0001] The present invention relates to modified / mutated cephalosporin hydroxylases from Streptomyces clavuligerus with better hydroxylation activity and increased specificity for compounds of formula (II) Compounds include, but are not limited to, phenylacetyl-7-aminodeacetoxycephalosporanic acid (phenylacetyl-7-ADCA) and desacetoxycephalosporin C (DAOC). [0002] [0003] wherein R represents but is not limited to hydrogen, phenylacetyl, phenoxyacetyl, aminoadipyl, glutaryl or any other suitable group. [0004] The present invention also describes a biological process for the preparation of cephalosporanic acids of the general formula I by hydroxylation of the corresponding compound of formula (II) using the mutated cephalosporin hydroxylases of the invention, [0005] [0006] wherein R represents the same groups as described above. The process is carried out in the presence of alpha-ketoglutarate and ascorbic acid. [0007] The invention further de...

AI Technical Summary

Problems solved by technology

The cloned hydroxylase gene was used in the hydroxylation reaction of cephalosporin compounds; however, the activity of the enzyme was not satisfactory for industrial manufacture