A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purified to homogeneity from Rhodotorula glutinis 38B1, whose resting cells have been previously reported as useful for the conversion of 7-ACA derivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667–2672, 1996]. The purified enzyme was a dimer comprised of identical subunits with a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low Km and high kcat values as substrates, as well as some acetyl esters with relatively long-chain alcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most active at 35°C and pH 5.5, and was inhibited by several serine enzyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked “hybrid type” oligosaccharide, and most of the enzyme activity was found in the purified cell wall fraction. The enzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.

Through the screening of yeasts and bacteria, some strains of Agrobacterium radiobacter have been found to efficiently convert 7-aminocephalosporanic acid (7-ACA) to deacetyl 7-ACA. The enzyme responsible for the conversion was constitutively formed by the bacterium. The 7-ACA deacetylating enzyme was purified from a cell-free extract of A. radiobacter IFO 12607 to homogeneity. The purified enzyme was a tetramer, composed of identical subunits with a molecular mass of 26 kDa, and showed activity toward some derivatives of 7-ACA, aryl acetates, monoacetin and triacetin, but was not active towards alkyl acetates and cephalosporin C. The Km and kcat values of the enzyme for 7-ACA were 8.24 mM and 81 s−1, respectively. Serine inhibitors and sulfhydryl reagents were both found to inhibit the activity. An amino acid sequence, GDSLT, which is the active center motif of an arylesterase, was identified in the amino terminal region. These results indicate that the enzyme belongs to the family of arylesterase (EC 3.1.1.2), and is a new 7-ACA-deacetylating enzyme.

A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form deacetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carrying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino acid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esterases including Bacillus cephalosporin C deacetylase, but has the pentapeptide motif sequence GDSLT (amino acid position 9–13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalytic properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence led to complete loss of the enzyme activity. Thus, the Ser-11 residue within the GDSLT motif sequence was determined to construct the catalytic center. These results together with those of our previous studies indicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12607 is a new member of the family of lipolytic serine esterases containing the GDSLT sequence as their catalytic center.

When 7-aminocephalosporanic acid (7-ACA) was used as a single carbon source in the enrichment culture medium for screening 7-ACA-degrading microorganisms, pink yeast colonies appeared frequently, and these were identified as Rhodotorula glutinis. These intact R. glutinis cells converted (i) 7-ACA to deacetyl-7-ACA (7-ADACA) and (ii) monochloroacetyl-7-ACA to monochloroacetyl-7-ADACA at sufficiently high levels to be of commercial interest. Acetylation of 7-ADACA to 7-ACA, the reverse reaction of hydrolysis in an organic medium with methyl acetate as an acetyl donor, was also demonstrated.