Immobilized recombinant penicillin

Recombinant penicillin G amidase 4-Hydroxy-D-phenylglycine Immobilized recombinant penicillin G amidase... 

The enzyme mixture of 20 ml containing immobilized recombinant penicillin G amidase as the enzyme, 10% hydroxyethyl ester of 4-hydroxy-D-phenylglycine, 4% cefprozil (amine source), and 8% enzyme (immobilized recombinant penicillin G amidase, equivalent to 32 IU/ml of enzyme) was made up without buffer. The above prepared ester solution (6.9 ml) was mixed with water (2 ml) and adjusted to pH 7.5 with 10 N NH4OH. Then the amine source (0.8 g) was added to it and the pH adjusted to 7.5 with 1 N NH4OH and the volume to 18.4 ml. Then the mixture was cooled to 5-15°C and solid enzyme (1.6 g 640 IU) was added to it. The pH was not maintained at 7.5 and fell about 0.6 units during the reaction. The reaction mixture was analyzed by HPLC on a C18 Reverse Phase column. The mobile phase was 10% acetonitrile/0.3% H3P04. The isomers of cefprozil appeared at 2.9 minutes (cis) and at 5.1 minutes (trans). The final product was obtained with a maximum yield of 92-96%. The whole experiment was completed in 25-50 min. 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

A hybrid protein comprising of protein A and /J-lactamase has also been constructed using recombinant DNA technology [165]. The hybrid enzyme bound specifically and strongly to IgG-Sepharose and the immobilized preparation was superior in its ability to hydrolyse penicillin G as compared to the covalently immobilized enzyme. 

The setting up of new biotechnology companies (Section 1.4.2) was the key event for the development of biopharmaceuticals based on recombinant technologies. Antibiotic manufacture had been established before this (Section 1.3). Antibiotics are still the most important strategy against bacterial infections. The world production of antibiotics is estimated at over 60000 tons per aimum, valued at more than 30 billion per year [128, 129]. For the synthesis of semisynthetic penicUlins and cephalosporins, the fermentation products penicillin G and cephalosporin, respectively, are hydrolyzed by immobilized enzymes to yield the acid form with the intact P-lactam ring as the active principal 6-APA, 7-ACA, or 7-aminodeacetoxy cephalosporanic acid (7-ADCA). 

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