Purification, substrate specificity, and N-terminal amino acid sequence analysis of a beta-lactamase-free penicillin amidase from Alcaligenes sp.

A beta-lactamase-free penicillin amidase from Alcaligenes sp. active against various beta-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.

Bioconversion of progesterone by the activated immobilized conidia of Aspergillus ochraceus TS.

Progesterone was transformed to its 11alpha-hydroxy derivative (100% e.e) by the activated immobilized conidia of Aspergillus ochraceus TS. The immobilized preparation retained 79% of free conidial activity. The immobilized conidia, activated by nutrients, exhibited an increase in 11alpha-hydroxylation, and it was free of the side product 6beta, 11alpha-dihydroxy progesterone. The half life and turnover of immobilized and activated immobilized conidia were 14 and 12 days and 187 and 416 micromoles of the product/g of conidia respectively. The pH and temperature profiles of the free conidia remained unaltered after immobilization and activation. Some germination of conidia inside the matrix owing to incubation with nutrients was detected by scanning electron microscopy.

Beta-lactamase-free penicillin amidase from Alcaligenes sp.: isolation strategy, strain characteristics, and enzyme immobilization.

Isolation and characterization of a beta-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1. 11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme.

Microsomal glutathione S-transferase (GST) isozymes in Aspergillus ochraceus TS: induction by 3-methylcholanthrene.

Induction of microsomal glutathione S-transferase isozymes in Aspergillus ochraceus TS is reported. Among the inducers of mammalian drug-metabolising enzymes 3-methylcholanthrene, benzo(a)pyrene and polychlorinated biphenyl enhanced the microsomal GST activity quite considerably. On the other hand phenobarbital, beta-naphthoflavone and trans-stilbene oxide were almost without any effect. To our knowledge this seems to be the first report on induction of microsomal GST in lower eukaryotes. A significant increase (7.9 fold) in GST activity was observed with the cells induced by 3-methylcholanthrene. Moreover, three new GST isozymes were found to be present in 3-methylcholanthrene induced microsomes. The isozymes are basic in nature. The GST isozymes were found to be catalytically distinct from each other.

Onset of new catalytic activity in immobilized spores of Aspergillus ochraceus TS due to in situ germination: C17-C20 lysis accompanies 11 alpha-hydroxylation of steroid.

The change in product pattern during transformation of progesterone by calcium alginate entrapped spores of Aspergillus ochraceus TS (A. ochraceus TS) due to germination in situ is reported. While progesterone was transformed exclusively to its 11 alpha-hydroxy derivative by both free and immobilized spores of A. ochraceus TS, the latter germinated in situ by yeast extract furnished 11 alpha-hydroxyprogesterone and C19 steroids during transformation under identical conditions. The characterization of metabolite(s) and the pathway proposed demonstrated that delta 1-dehydrogenation and lysis of C17-C20 bond are apparently two independent reactions. The change in product profile due to activation of immobilized spores is believed to be caused by accumulation of compatible solutes in the biocatalyst which had relatively low water content.

Novel oxidative cleavage of C17-C20 bond in pregnane by a Pseudomonas sp.

Oxidative cleavage of the C17-C20 bond in progesterone by Pseudomonas sp. is reported. Transformation occurred under in vivo conditions. The bioconverted products were characterized as 1,4-androstadien-3,17-dione and 17 beta-hydroxy-1,4-androstadien-3-one. The steroid nucleus was not further degraded although the test organism had the capacity to induce dehydrogenation at C1 of this alpha, beta-conjugated steroid.

Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS.

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M(r) of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pI values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.

A121--an antifungal compound from Streptomyces species.

A non-polyene antifungal antibiotic, A121, produced by an unidentified strain of Streptomyces species was isolated and purified to homogeneity. This antibiotic was found to be active against a number of filamentous fungi including some plant and human pathogens. On the basis of its infrared spectrum, proton magnetic resonance, mass spectra, and some chemical derivatives, a plausible chemical constitution of this antibiotic is proposed. Its antibiotic activity apparently requires the presence of an alpha-ketol system in the molecule.

Effect of inducers on metabolism of benzo(a)pyrene in vivo and in vitro: analysis by high pressure liquid chromatography.

The characterisation of metabolites formed from benzo(a)pyrene (BP) by Aspergillus ochraceus TS and effect of inducers on BP metabolism are reported. The high pressure liquid chromatographic profile of BP metabolites was similar to that of mammalian microsomes furnishing diols, quinones and phenols. The production of BP-4,5-dihydrodiol (K-region diol) by Aspergillus ochraceus TS seems to be novel and provides first report on BP metabolism by eukaryotic fungi. In control, phenols and quinones were produced in excess over dihydrodiols while the induced preparation showed the reverse order. Presumably the induction effecting production of excess dihydrodiols influenced the synthesis of epoxide hydrolase. In addition, a differential increase in BP metabolism was observed with inducers of narrow and broad specificity.

Characterization of progesterone 11 alpha-hydroxylase of Aspergillus ochraceus TS: a cytochrome P-450 linked monooxygenase.

The monooxygenase of Aspergillus ochraceus TS capable of 11 alpha-hydroxylation of progesterone has been resolved into three components and characterized as (i) cytochrome P450, (ii) NADPH-cytochrome P450-reductase and (iii) phosphatidyl choline. The 11 alpha-hydroxylase was observed to be NADPH dependent, and hydroxylation was enhanced by a NADPH regenerating system. This fungal monooxygenase has many features in common with that of mammalian liver microsomes. The role of mammalian cytochrome P450 inducers were tested for induction of 11 alpha-hydroxylase in Aspergillus ochraceus TS. The reductase has been partially purified.

Induction of benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: evidences of multiple forms of cytochrome P-450.

The filamentous fungus Aspergillus ochraceus TS produces an inducible microsomal cytochrome P-450 linked monooxygenase which is capable of hydroxylating benzo(a)pyrene in presence of O2 and NADPH. The addition of Benzo(a)pyrene, 3-Methyl cholanthrene, beta-Naphthoflavone and other aryl hydrocarbons during the induction period causes dramatic improvement in the kinetics of benzo(a) pyrene hydroxylation as was evidenced by large decrease in Km and increase in Vmax values. On the other hand, treatment with Phenobarbital, Polychlorinated biphenyl and Progesterone has no significant effect on the kinetics of benzo(a)pyrene hydroxylation although a significant induction of NADPH-Cyt C reductase activity was observed in all the three cases. Again, both Phenobarbital and 3-Methyl cholanthrene induced microsomes exhibit the characteristic reduced metyrapone difference spectra. These findings together with the results obtained with flavone on the metabolism of benzo(a)pyrene by various microsomal preparations suggest a parallel induction of multiple forms of cytochrome P-450 as observed in mammalian liver under identical condition.

Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: assay and characterization of the enzyme system.

Microsomal preparations of Aspergillus ochraceus TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and 02 and exhibits a pH optimum of 8.0-8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistent with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)-pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.

An improved 11 alpha-hydroxylation of progesterone by Aspergillus ochraceus TS.

1. The stereospecific hydroxylation of progesterone exclusively to its 11 alpha-hydroxy derivative by Aspergillus ochraceus TS is reported. 2. There is no secondary metabolite (6beta, 11 alpha-dihydroxyprogesterone) formed even when the transformation was attempted with different concentrations of the substrate (200 microgram/ml-200 mg/ml) for prolonged with Zn2+ at a concentration of 50 microgram/ml identical results were obtained. 3. Similar results were also obtained at different pH values of the culture medium.