Cloning and characterization of the gene cahB encoding a cephalosporin C acetylhydrolase from Acremonium chrysogenum

An important problem during the production of cephalosporin C by Acremonium chrysogenum is the hydrolysis of cephalosporin C to deacetylcephalosporin C, since the latter compound has no commercial value and represents an unwa nted side-product. Characterization of the enzymatic process that gives ris e to deacetylcephalosporin C will help to avoid the accumulation of this si de-product. An extracellular cephalosporin C acetylhydrolase (CPC-AH) from Acremonium chrysogenum C10 was purified to near homogeneity. This enzyme ha d a molecular mass of 31 kDa, a pl of 4.0, and showed relatively little aff inity for cephalosporin C (K-m 33.7 mM). We sequenced twenty amino acids at the amino-terminal end; a probe based on this sequence was then used to cl one the cephalosporin acetylhydrolase (cahB) gene. cahB encodes a pre-prote in of 383 amino acids with a deduced molecular mass of 38,228 Da. The seque nced 20 amino acids of the purified protein corresponded to amino acids 107 -127 deduced from the cahB gene, suggesting that mature CPC-AH results from processing of the pre-protein after Gln-106. cahB is located on chromosome VIII of A. chrysogenum C10 and is not linked to the cephalosporin early or late gene clusters. It is expressed as a single 1.4-kb transcript after 72 h of cultivation. Expression declined in batch cultures after 120 h even t hough CPC-AH activity was observed until 144 h. The CPC-AH protein resemble s other wide-spectrum substrate fungal esterases that are functionally rela ted to serine proteases. The cahB gene does not seem to be related to the c ephalosporin biosynthesis genes and encodes an esterase active on several s ubstrates in addition to cephalosporin C.