ABSTRACT
The role of sugars, polyhydroxy compounds, phenylacetic acid and 6-aminopenicillanic acid in stabilization of immobilized penicillin G acylase (IMPGA) was studied. The loss in the activity of IMPGA at 50 degrees C, 2 h, after incorporation of sucrose and mannitol at 0.1 M concentration was 16 and 18% respectively; the loss in the activity of the enzyme under these conditions in the absence of stabilizing agents was 40%.
Subject(s)
Carbohydrates/pharmacology , Enzyme Stability , Enzymes, Immobilized/chemistry , Hot Temperature , Penicillin Amidase/chemistry , Polymers/pharmacologyABSTRACT
Penicillin V acylase from Fusarium sp. SKF 235 culture filtrate was purified to homogeneity. The enzyme was a glycoprotein and composed of single polypeptide chain with molecular weight of 83,200 Daltons. The pH and temperature optima were 6.5 and 55 degrees C, respectively. The KM for penicillin V was 10 mM but the enzyme was inhibited by penicillin V at concentrations above 50 mM. Products of reaction, 6-aminopenicillanic acid and phenoxyacetic acid inhibited the enzyme competitively and noncompetitively with Ki values of 18 mM and 45 mM, respectively. The enzyme specifically hydrolyzed penicillin V, cephalosporanic acid V and penicillin V sulphoxide. Other phenoxy acetyl amides studied were not hydrolysed. It is proposed that phenoxyacetyl moiety alone is not recognized by the penicillin V acylase and in addition, the beta-lactam structure contributes in formation of enzyme-substrate complex.
Subject(s)
Amidohydrolases/analysis , Binding, Competitive , Cephalosporins/metabolism , Culture Media , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/chemical synthesis , Fusarium/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Molecular Weight , Penicillanic Acid/analogs & derivatives , Penicillin Amidase/antagonists & inhibitors , Penicillin V/analogs & derivatives , Phenoxyacetates/chemistry , Substrate Specificity , TemperatureABSTRACT
Enzymatic parameters such as pH, temperature and substrate concentration were studied for the hydrolysis of 7-PADCA by penicillin G acylase. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. Km value of soluble and immobilized enzyme for 7-PADCA was 2.3 x 10(-5) M and 7.5 x 10(-5) M, respectively. At 7-PADCA concentration of 5% and an IME: 7-PADCA ratio of 1:2.5, the hydrolysis was complete in 110 min.
Subject(s)
Cephalosporins/metabolism , Enzymes, Immobilized , Hydrogen-Ion Concentration , Hydrolysis , Penicillin Amidase/metabolism , TemperatureABSTRACT
The growth of Candida sp. 115 was investigated on the constituents of penicillin G hydrolysis reaction mixture. Neither penicillin G nor 6-aminopenicillanic acid was degraded or utilised for growth. The yeast accepted phenylacetic acid, sodium acetate and glucose as growth substrates. Phenylacetic acid was metabolised via p-hydroxy phenylacetic acid, which was the only accumulated metabolite. The enzymes responsible for hydroxylation of phenylacetic acid were induced by phenylacetic acid and sodium acetate.
Subject(s)
Acetic Acid , Acetates/metabolism , Candida/growth & development , Culture Media , Glucose/metabolism , Hydrolysis , Penicillin G/metabolism , Phenylacetates/metabolismABSTRACT
Fermentation parameters for the production of penicillin G acylase by Escherichia coli NCIM 2400 have been evaluated. The bacterium produced the enzyme intracellularly when grown in nutrient broth containing PAA. PAA stimulated the enzyme synthesis by 8-10 fold and reduced the lag period. The optimum concentration of PAA for induction was 20 mM and addition of PAA prior to inoculation gave maximum production of PGA. Glucose, lactose, sorbitol, acetate and lactate even at 0.1% concentration catabolically repressed the enzyme formation. Peptone was the best utilised 'N' source for the enzyme production. Phosphate and yeast extract were found to be essential for both the growth and for enzyme biosynthesis. Temperature between 22-24 degrees C was optimum and under ideal condition E. coli NCIM 2400 produced 0.45-0.55 U/ml of penicillin G acylase.
Subject(s)
Amidohydrolases/biosynthesis , Escherichia coli/enzymology , Fermentation , Hydrogen-Ion Concentration , Hydrolysis , Penicillin Amidase/biosynthesis , Penicillin G/metabolism , Phenylacetates/pharmacologyABSTRACT
Binding of penicillin amidase from E. coli 436 to aniline-, benzylamine- and phenylethylamine-Sepharose was studied. Binding of the enzyme to aniline-Sepharose was exclusively due to hydrophobic interactions. Benzylamine-Sepharose binds the enzyme due to affinity interactions in the absence of ammonium sulphate and due to hydrophobic interactions in the presence of ammonium sulphate. A conformational change in the penicillin amidase molecule due to ammonium sulphate there by exposing the side chain binding site as a hydrophobic core is suggested.