Production, purification, immobilization and application of penicillin acylase from Acremonium sclerotigenum

Abstract

Penicillin acylase has been the target of research because of its importance and wide usage in the field of biotechnology. In the present study, the production of penicillin acylase was reported from the fungus Acremonium sclerotium isolated from industrially contaminated soil. The crude enzyme was purified by ammonium sulphate saturation (50%) followed by hydrophobic interaction chromatography using Octyl Sepharose. The purified enzyme showed temperature optima at 40 C and pH optima of 5.0 with 11.24-fold purification. The thermal stability profile of the enzyme showed a t1/2 of 45 min and 20 minutes at 55 C and 60 C respectively. The Kmand Vmax values were found to be 13.32mM and 333.33 mol mg 1minute respectively. The Kcat and Kcat/Km values of the penicillin acylase were 1351.83sec-1 and 102.179 mM-1sec -1 respectively. Penicillin acylase was immobilized by entrapment method. The immobilized enzyme was able to retain 68.85% of its initial activity after immobilization. Km and Vmax value of the immobilized enzyme was also calculated to study its substrate efficiency. The entrapped enzyme was reusable up to 5th cycle and was found to be more stable than soluble enzyme. The potential of the immobilized enzyme was exploited for 6-amino penicillanic acid synthesis. Maximum conversion of penicillin G to 6 -amino penicillanic acid was recorded at 50 C, pH 6 with 50 mM (w/v) penicillin G (substrate). 6-amino penicillanic acid production was also scaled up to 1 litre and the molar conversion yield was 87.84%. The percentage purity of the sample was 54.33% and the productivity was and the productivity was 17.41mg/L/h. 6-amino penicillanic acid was further characterized by HPLC, U. Vis spectroscopy, Fluorescence and FTIR spectroscopy.