CONTRIBUTIONS OF IMMOBILIZED PENICILLINASE TO THE ANALYTICAL CHEMISTRY OF PENICILLINS (CONTINUOUS-FLOW).

摘要

Scope and Method of Study. Synthetic routes for the covalent coupling of penicillinase on silica were investigated. Silylation of the glass carrier, using four amino silanes, was accomplished from anhydrous toluene as well as from 95% ethanolic solution. The effect of these silanes on the immobilization of penicillinase was evaluated by measuring the initial rates of enzyme catalyzed hydrolysis. Four immobilized penicillinase reactors were constructed and evaluated for use in a continuous-flow system, using a flow through pH detector. Characterization of the immobilized penicillinase reactor with respect to pH, buffer concentration, sample size, temperature, and stability were performed. The immobilized enzyme reactor was used in a continuous-flow system for the determination of penicillins in tablets, injectables, and fermentation broths. A new flow through cell coupled with pH detection was constructed for use in the above continuous-flow system.;Findings and Conclusions. The use of short, rigid silanes, such as (aminophenyl)trimethoxysilane, for the immobilization of penicillinase gave maximum activity on glass. Silylation of a glass matrix was easily achieved from 95% ethanolic solution, as compared to the slow refluxing conditions from anhydrous toluene. The Single Bead String Reactor (SBSR) with enzyme immobilized on glass beads, after an ammonium bifluoride etching process, gave the maximum activity of penicillinase. The immobilized penicillinase retained 97% of its initial activity after ten months of usage. The geometry of the design allowed minimum dispersion of the sample plug in a continous-flow system. There was no back pressure associated with the SBSR. The SBSR was used in a continous-flow system for the determination of penicillins in tablets, injectables, and fermentation broths. The results agree very closely with High Performance Liquid Chromatography (HPLC) and chemical assays of Eli Lilly and Company. The new flow through cell with pH detection was superior to that the capillary pH electrode because it did not suffer from problems of streaming potentials.