Timir B. Samanta

Characterization of progesterone 11α-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.

11α-hydroxylation of progesterone by cell free preparation of Aspergillus ochraceus TS

Abstract Hydroxylation of progesterone at C 11 by cell-free preparation of A. ochraceus TS has been studied. The hydroxylating enzyme (11α-hydroxylase) is inducible in character and located in post-mitochondrial supernatant. The reaction in vitro is effected in presence of sodium meta-periodate indicating the hemoprotein nature of the enzyme. The in vitro hydroxylation stimulated by cyanide and inhibited by metyrapone suggested that this microbial hydroxylation is possibly mediated by cytochrome P-450.

Induction of benzo(a)pyrene hydroxylase in aspergillusochraceus 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.

Effect of inducers on metabolism of benzo(a)pyrene invivo and invitro: 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.

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

Abstract. Progesterone was transformed to its 11α-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 11α-hydroxylation, and it was free of the side product 6β, 11α-dihydroxy progesterone. The half life and turnover of immobilized and activated immobilized conidia were 14 and 12 days and 187 and 416 μmoles 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.

β-Lactamase-Free Penicillin Amidase from Alcaligenes sp.: Isolation Strategy, Strain Characteristics, and Enzyme Immobilization

Abstract. Isolation and characterization of a β-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.

Novel catalytic activity of immobilized spores under reduced water activity

Abstract Onset of a new catalytic function during transformation of progesterone by immobilized spores of Aspergillus ochraceus TS under reduced water activity is reported. The pathway of transformation, which furnished 1,4-androstadien-17β-ol-3-one and 1,4-androstadien-3, 17-dione due to cleavage of C 17 C 20 bond, is different from normal reaction sequence.

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 Mr of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pl values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.

Influence of substituents at C11 on hydroxylation of progesterone analogs by Bacillus sp.

Transformation of progesterone analogs viz., progesterone, 11 alpha-, 11 beta-hydroxyprogesterones and 11-ketoprogesterone by Bacillus sp. is reported. Both progesterone and 11-ketoprogesterone were hydroxylated while the C(11) epimeric alcohols of progesterone remained unaltered under the conditions used. The major bioconverted products obtained from progesterone and 11-ketoprogesterone were characterized as 6 beta- and 14 alpha-hydroxyprogesterones and 14 alpha-hydroxy-11-ketoprogesterone respectively by mass and NMR spectra. The conversion of 11-ketoprogesterone to its 14 alpha-hydroxy derivative by microbe is unprecedented and novel. Moreover, hydroxylation at 6 beta- and 14 alpha-positions of progesterone by Bacillus sp. is significant. In conclusion, the present data showed that the substituents at 11-position of steroid play important role on hydroxylation by microbe.