The Applied Studies On Immobilization Of Penicillin Acylase From Alcaligenes Faecalis

6-aminopenicillanic acid (6-APA) is a precursor for the synthesis of semi-synthetic penicillins and a critical element in the production of semi-syntheticβ-lactam antibiotics. Immobilized penicillin acylase (IPA) is capable of catalyzing the hydrolysis of penicillin G to produce 6-APA and has now been widely used in relevant pharmaceutical industry. Penicillin acylase from Alcaligenes faecalis (AfPGA) shows a promising perspective on industrial application for its great enzymatic characters as high-affinity to substrate, superior thermo/pH stableness and resistance to organic solvents. In this work, AfPGA is immobilized on a commercial oxirane carrier, Eupergit CM, and modified by mercaptoethanol after immobilization, which improves operational and thermo stabilization ofIPA. The optimum conditions of enzymatic coupling reaction turn out to be as follows:1M phosphate potassium buffer pH8.0, loading enzyme about 500-550 U/g wet carrier, shaking gently (150rpm) for 36-48 hours at 30℃. The apparent activity of the immobilized enzyme prepared in conditions above (enzyme loading 433 U/g wet carrier, reacting for 36 hours) is 177 U/g wet carrier and the yield is about 41%.The optimal pH and temperature of Immobilized AfPGA are 9.0 and 47℃respectively. The immobilized enzyme is stable in the range of pH 4-9 and at the temperature below 40℃. All enzymatic characterizations indicate the IPA a superior to free AfPGA. The yield of 6-APA derived from Penicillin G hydrolysis catalyzed by immobilized AfPGA is 60.5%, with 4% initial penicillin G concentration. The comparison of Eupergit CM with amine carrier ZH-HA using for AfPGA immobilization tells that the IPA on ZH-HA gets higher activity than those on Eupergit CM and its neutral optimal pH suggests a better utilization factor in a practical usage; yet the IPA on Eupergit CM is more thermo-stable than peer.