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
Among different matrices prepared, ampicilloic acid-polymer matrix offered 86.7% adsorption, 95% elution and 82.4% overall recovery of penicillinase. The structure of both the side chain and penicilloic or cephalosporoic acid moieties contribute to the affinity interactions.
Subject(s)
Adsorption , Bacillus cereus/enzymology , Cephalosporins/chemistry , Chromatography, Affinity/methods , Fermentation , Ligands , Penicillanic Acid/analogs & derivatives , Penicillinase/analysisABSTRACT
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
Production of beta-galactosidase by Sclerotium rolfsii NCIM 1084 was studied under submerged fermentation conditions. The enzyme was produced extracellularly and constitutively on glucose. The enzyme production was enhanced when galactose, raffinose, cellobiose, sucrose, xylose, maltose, cellulose and pectin were used as carbon sources. Cellulose and diammonium hydrogen phosphate were best carbon and nitrogen sources, respectively. Surfactants such as Sag, Paraffin oil, Tween 20 and Tween 80 increased the enzyme production. Maximum yield of beta-galactosidase obtained was 3.8-4.2 nkat/ml. The optimum pH, optimum temperature and molecular weight of the beta-glactosidase were 2.7, 60 degrees C and 2,21,000 daltons, respectively. The enzyme is an aryl beta-glactosidase and did not hydrolyse lactose. The Km value for o-nitrophenyl beta-D-galactoside was 3.7 mM. Galactose and 2-mercaptoethanol inhibited the enzyme.
Subject(s)
Ascomycota/enzymology , Carbon/metabolism , Cellobiose/chemistry , Cellulose/chemistry , Fermentation , Galactose/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Lactose/metabolism , Maltose/chemistry , Molecular Weight , Pectins/chemistry , Polysaccharides/chemistry , Quaternary Ammonium Compounds/chemistry , Raffinose/chemistry , Sucrose/chemistry , Surface-Active Agents/chemistry , Temperature , Xylose/chemistry , beta-Galactosidase/analysisABSTRACT
The Schiff's base formation between 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid and p-dimethylaminobenzaldehyde (PDAB) was investigated. The factors that affect the reaction such as concentration of PDAB, time and pH were studied and optimised for estimation of these intermediates.
Subject(s)
Benzaldehydes/chemistry , Calibration , Cephalosporins/chemistry , Hydrogen-Ion Concentration , Indicators and Reagents/chemistry , Kinetics , Penicillanic Acid/analogs & derivatives , Schiff Bases/chemistryABSTRACT
Cephalosporin acylases have application in the production of 7-aminocephalosporanic acid which forms a key raw material for the preparation of semisynthetic injectable cephalosporins. The enzymes are of industrial importance and hyperproducing genetically engineered strains have been constructed. Different aspects of these enzymes such as subunit structure, post translational modification, primary structure, substrate specificity and their importance in pharmaceutical industry are discussed.
Subject(s)
Amino Acid Sequence , Cephalosporins/chemical synthesis , Fermentation , Penicillin Amidase/chemical synthesisABSTRACT
Coproduction of alpha-amylase, beta-amylase, amyloglucosidase, cellulase, xylanase, pectinase and beta-galactosidase by Sclerotium rolfsii was studied on various polysaccharides. Starch induced alpha-amylase, beta-amylase, amyloglucosidase and beta galactosidase; cellulose induced cellulase, xylanase, pectinase and beta-galactosidase; and pectin induced pectinase and beta-galactosidase. None of the enzymes studied except beta-galactosidase were induced on xylan. Group controlled mechanism for production of carbohydrases by Sclerotium rolfsii is suggested.
Subject(s)
Enzyme Induction , Fermentation , Fungi/metabolism , Glycoside Hydrolases/biosynthesis , Polysaccharides/metabolismABSTRACT
Various beta-lactam compounds and structurally related moieties were examined as substrates of beta-lactamase from Bacillus cereus 5/B NCTC 9946. The enzyme was specific for penicillins and none of the cephalosporins were hydrolysed. Electronic environment of allylic carboxy group in dihydrothiazine ring restricts the acceptance of cephalosporins as substrates. The efficiency of hydrolysis of penicillins is dependent on dense resonating electronic environment of phenyl ring present in the side chain, flexibility of the side chain and the distance between the phenyl ring and carbonyl group in the side chain.
Subject(s)
Hydrolysis , Molecular Structure , Penicillins/chemistry , beta-Lactamases/metabolismABSTRACT
Penicillin G acylase was immobilized on numerous cation exchange resins and hydrophobic adsorbents. Amberlite XAD-7 was the matrix of choice among the matrices studied for the immobilization of enzyme. Binding of 96.8% and expression of 82.6% of the penicillin G acylase was achieved on XAD-7. Penicillin G acylase immobilised on XAD-7 was used for 80 cycles for the production of 6-PA in a stirred tank reactor.
Subject(s)
Acrylic Resins , Enzymes, Immobilized , Penicillanic Acid/metabolism , Penicillin Amidase , PolystyrenesABSTRACT
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
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.