Production Of 7-ADAC By Microtransformation

Since the 1990's, reseachers have paid much attention to development of β-lactam antibiotics, especially to semisynthetic cephalosporins. According to their structures, the starting compounds of semisynthetic cephalosporins can be divided into two groups: 7-AC A and 7-ADCA. Among those semisynthetic producs, biologists and chemists are interested in 3-deacetyl cephalosporin intermediates. As the product results from transforming 7-ACA, 7-ADAC has an active hydroxy at C3 which can be conveniently changed into many other active derivatives.To date, 7-ADAC can be produced from 7-ACA by chemical synthesis or by enzyme catalysis. Because of the disadvantages of chemical method, it is important to develop enzyme method. This paper studied the Cephalosporin-C-deacetylase(CAH) in both Bacillus subtilis and Rhodotorula glutinis. Because the CAH in R. glutinis has a higher activity than that in B. subtilis, we did research in screening of R. glutinis and obtained a strain with high CAH activity. At the same time, we optimized the media composition, fermentation conditions and reaction conditions. Under the optimum conditions, the reaction can be finished in an hour and the yield of transforming is above 99% (mol). After purification, the purity of the product is 98%, and the total yield of the process is about 88%(mol).Besides, DNA coding fragment of CAH was amplifed by PCR from genornic DNA of B. subtilis 6633, cloned into pUC 18 and sequenced. The recombinant plasmid for expressing fusing CAH was constructed. The high level expressing E. coli strain of CAH was obtained! and expressed fusion protein was proved by SDS-PAGE. The amount of the expressed protein was 21.3% of the total protein.