Process for modifying the enzyme 7.beta.-(4-carboxybutanamide) cephalosporinacylase and purifying said enzyme in a single chromatographic step

A procedure for modifying a native 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme to produce a modified 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme in host cells, said native enzyme having alpha and beta subunits, said method comprising the steps of:

• (a) conducting site directed mutagenesis in a gene encoding said native 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme by inserting in the gene a nucleotide sequence encoding a chain of at least six histidines at a site in the gene encoding the N-terminal region of the alpha subunit that is 3' to a sequence coding for Leu-Ala-Glu-Pro such that (i) the modified enzyme with the chain of at least six histidines has an activity and enzymatic specificity which are not significantly changed from that of the native enzyme, and (ii) the chain of at least six histidines is present in an area of the modified enzyme in which it can interact with a metal chelate on a chromatographic support;
• (b) fusing said mutagenized gene with a promoter for the expression of mutagenized gene to form a fusion gene construct;
• (c) transforming host cells with the fusion gene construct;
• (d) growing the transformed host cells in a suitable culture medium and under conditions allowing expression of the modified 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme comprising the chain of at least six histidines;
• and
• (e) recovering the modified 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme with the chain of at least six histidines from the culture medium by a process comprising centrifuging to separate the transformed cells from the culture medium, disrupting the transformed host cells to form a cell extract and purifying the cell extract by chromatography on a chromatographic support containing a metal chelate.