Purification and kinetics of penicillin G acylase from a mutant strain of escherichia coli ATCC 11105

Abstract

A mutant strain with high penicillin G acylase activity was derived from Escherichia coli ATCC 11105 by chemical mutagenesis, using N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. Penicillin acylase was extracted from the mutant strain and highly purified by DEAE‐cellulose and hydroxyapatite column chromatography followed by preliminary precipitation steps. Vm and Km values of the enzyme (specific activity: 24·81 U mg−1, protein concentration: 0.56 mg cm−3) were found to be 22.73 U cm−3 mm−1 and 3.18 mmold m−3 penicillin G, respectively. The enzyme was shown to be uncompetitively inhibited by excess of substrate. The inhibition by phenylacetic acid was found to be competitive, and 6‐aminopenicillanic acid to be noncompetitive. Inhibition constants for excess of penicillin G, phenylacetic acid and 6‐aminopenicillanic acid were estimated as 74.20, 18.74 and 15.00 mmol dm−3 respectively, at pH 8.0 and 40°C. The activation energy of the enzymatic hydrolysis reaction of penicillin G was found to be 10.78 kcal mol−1. Optimal pH and temperature values of the enzyme were determined as 8.0 and 60°C. Complete loss of the enzyme stability occurred when the enzyme was incubated at pH 10.0 for 2 days or at 50°C for 2 h.

Hydrolysis of Penicillin G by Penicillin G Acylase Immobilized on Chitosan Microbeads in Different Reactor Systems

“…It can be also concluded that 6-APA is a noncompetitive and PAA is a competitive inhibitor. This is in agreement with the results previously reported [12,14,15,25,26]. Jianguo et al [25] determined inhibition constants as K iA = 0.009 M and K iB = 0.013 M for PGA immobilized on poly(vinyl acetate-co-divinyl benzene) beads.…”

“…Jianguo et al [25] reported a K M value of 0.033 M, which is almost the same as the K M value obtained in the present study showing the same affinities for the substrate of the immobilized enzymes. Likewise, some of the previously reported values for K M of the PGA immobilized on different carriers were 4.17 mM [12], 11.36 mM [14], and 3.18 mM [15]. These values are lower than the obtained K M value (32.6 mM) probably due to higher film diffusion resistance to the substrate transport near the chitosan microbeads.…”

“…Erarslan and Guray [14] studied kinetic parameters of PGA immobilized on oxirane-acrylic beads. Erarslan et al [15] determined the kinetic properties of penicillin acylase, derived from a mutant strain of E. coli ATCC 11 105 obtained by chemical mutagenesis. The enzyme proved to be noncompetitively inhibited by an excess of substrate.…”

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

“…Takođe je pokazano i da i slobodnu i imobilisanu PAC inhibira supstrat u višku i oba proizvoda reakcije, pri čemu je 6-APA nekompetitivni, a PAA je kompetitivni inhibitor. Ovi rezultati se slažu sa rezultatima objavljenim u literaturi [29,50,244,246,247]. Za imobilisanu PAC na poli(vinil acetat-ko-diviil benzen) česticama dobijene su vrednosti za K iA i K iB od 0,009 mmol/cm 3 i 0,013 mmol/ cm 3 , redom [246].…”

“…Ove vrednosti su niže nego K iA i K iB vrednosti dobijene u ovom radu, stoga, u ovom slučaju uticaj inhibitora na naš biokatalizator je manji. Takođe, u literaturi su objavljene i vrednosti K iA i K iB za PAC imobilisanu na različite nosače: 100,7 mmol/dm 3 i 68,6 mmol/dm 3 [247], odnosno 76,1 mmol/dm 3 i 90 mmol/dm 3 , redom [50]. Ove vrednosti su veće od K iA i K iB vrednosti koje su dobijene u ovom radu, pa može da se zaključi da je uticaj inhibitora (6-APA i PAA) na naš biokatalizator veći.…”