pH stabilization of Escherichia coli penicillin acylase by CMC conjugation

Abstract

The irreversible inactivation of kinetics and the stability of Escherichia coli penicillin G acylase (PGA) at various pH values conjugated by anionic polysaccharide carboxymethyl-cellulose (CMC) were studied. The use of ionic polysaccharides in enzyme technology have many potential advantages, such as water solubility, biocompatibility and non-toxicity. The stabilizing effect of ionic polysaccharides on enzyme molecules is well known by causing the cross links between the free amino group containing amino acid residues on protein molecule and strengthening the electrostatic interactions on enzyme after conjugation. CMC was activated by periodat oxidation to its aldehyde derivative and covalently bound to PGA via Schiffs base formation. The 46% fraction of total enzyme activity and 65% fraction of total protein in PGA solution was conjugated by CMC. The amount of CMC bound protein was 33% of the initial CMC concentration. Native and CMC conjugated PGA were incubated at 40°C and different pH values between (4-9) interval for extended times. The irreversible inactivations of the native and conjugated PGA at pH values studied were obeyed to the first order inactivation kinetics. The highest pH stability of PGA was obtained at pH 8 as fourfold. Cross-linking by CMC did not affect the pH profile and the$k_{cat}$ value of enzyme. However Km and Vm values were decreased after cross-linking. The activation free energy of inactivation (AG,) at different pH values for conjugated PGA were found to be always higher than that for native enzyme. CMC conjugation is improved the catalytic performance of enzyme by increasing the$ k_{cat}/K_m$ ratio.

Özet

90 kDa mol ağırlığında karboksimetilselüloz (CMC) ile konjuge olan Escherichia coli Penisilin G asilaz 'mm sıcaklık ve pH'ya karşı geri aönüşümsüz inaktivasyonu çalışılmıştır. Karboksimetilselüloz periyodat oksidasyonu ile aktive edilmiş ve PGA 'a, Schiff bazı oluşumu ile kovalent olarak bağlanmıştır. PGA ve modifiye olmuş PGA farklı zaman ve farklı pH değerlerinde (pH 4-9) inkübe edilmiştir. PGA 'm pH karşısındaki en yüksek stabilitesi, pH 8 'de 4 kat olarak elde edilmiştir. CMC 'un çapraz bağlanması sonucu PGA enziminin optimal pH ve $k_{cat}$değeri değişmemiştir. Km, ve Vm değerleri ise azalmıştır. Konjuge PGA için farklı pH değerlerinde inaktivasyonun aktivasyon serbest enerjisi $(dDeltaG_i)$ PGA 'nmkine göre daha yüksek bulunmuştur