Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase

Abstract

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier.

Review Article: Immobilized Molecules Using Biomaterials and Nanobiotechnology

“…For enzymes having large dimensions, such as penicillin acylase (PA), the mass transfer is even slower. The immobilization of such enzyme to porous materials can prove tedious using conventional techniques [112].…”

The Art of Immobilization Using Biopolymers, Biomaterials and Nanobiotechnology

“…For enzymes having large dimensions, such as penicillin acylase (PA), the mass transfer is even slower. The immobilization of such enzyme to porous materials can prove tedious using conventional techniques (Van Langen et al, 2002). Wang et al, 2008bWang et al, & 2009a have recently succeeded to immobilize papain and PA using the adsorption technique into the mesocellular siliceous foams (MCFs) using microwave irradiation technology.…”

“…The HRP enzyme was dissolved into the IL 1-butyl-3-methylimidazolium hexafluorophosphate and the IL/HRP phase acts as an efficient biocatalyst and can be easily recycled and reused several times. Due to the immiscibility between the IL and water, the immobilized HRP could be simply recovered by liquid/ liquid phase separation after the biocatalytic reaction van Rantwijk et al, 2003). Although this new method is faster and easier than the classical immobilization of HRP into solid supports, it is not widely applied in the industries because of the ionic liquids' expenses.…”

Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

“…Jedna BPU odgovara količini enzima koja oslobađa jedan mol fenilsirćetne kiseline po minuti iz penicilina G. BPU ima u literaturi različite vrednosti jer ne postoji saglasnost između koncentracije penicilina G, pH i temperature reakcije prilikom izvođenja metode [95].…”

“…"titracijom aktivnog položaja". Na osnovu objavljenih rezultata, 1mol penicilin-acilaze iz E. coli ATCC 11105 odgovara 3660 BPU, što je ekvivalentno katalitičkoj molekulskoj aktivnosti od 61 s -1 [95].…”