Ajit K. Mishra

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.

Oxidation of phenanthrene by a strain ofMicrococcus: Evidence of protocatechuate pathway

A phenanthrene-degrading bacterium, belonging to the genusMicrococcus, was isolated from petroliferous soil. The strain degraded phenanthrene mainly through protocatechuate, as was evidenced by oxygen uptake and enzymatic studies. Moreover, the strain can utilize naphthalene and anthracene without any lag, and as such catechol-2,3-oxygenase may play an important role, most probably as a constitutive enzyme. The mechanism of degradation of these hydrocarbons and other aromatic compounds seems to be controlled by an extrachromosomal mechanism (i.e., through plasmids), as was evidenced by the loss of the phenanthrene-and naphthalene-assimilating property after subculture in nutrient agar and NAH+ phenotype, in benzoate agar medium; this suggests its similarity with other hydrocarbon-assimilating microorganisms in which the degradation is mediated entirely by plasmids.

Isolation ofThiobacillus ferrooxidans from various habitats and their growth pattern on solid medium

Different strains of iron-oxidizingThiobacillus ferrooxidans were grown and purified on solid medium containing Bapco agar, agarose, and carrageenan (Type 1). These strains produced easily countable isolated colonies that could be transferred after 7 days of incubation at 30°C. Increase in viable cell number in relation to growth and iron oxidation was studied by both microscopic count and direct plating method. Colony morphology of different strains growing on solid medium helped in differentiating the colony types.

Removal of sulphur from coal by Thiobacillus ferroxidans and by mixed acidophilic bacteria present in coal

Abstract Pyritic sulphur has effectively been removed from three coal samples by treating with a strain of Thiobacillus ferrooxidans . The acidophilic bacteria present in coals have also been found to efficiently leach pyrite from coal. Addition of an essential bacterial nutrient and a surfactant together with the removal of calcite from coal, favoured the leaching process. Besides pyritic sulphur, significant amount of sulphate sulphur, and a small amount of organic sulphur were removed by the process.

Induction of xylanase in aspergillus ochraceus

Aspergillus ochraceus produced xylanase inductively in washed glucose-grown mycelia incubated with methyl β-d-xyloside. The production of xylan endo-l,4-β-xylosidase (xylanase) was 4.2 times greater than that obtained in xylan medium. The inducer was not metabolized and was most active at a concentration of 0.3 g/L with an incubation period of 16 h. The uptake of the inducer by the mycelia seemed to be energy-dependent. Methyl α-d-xyloside repressed xylanase synthesis. Glucose, cyeloheximide, actinomycin D and eAMP were found to inhibit xylanase induction by methyl β-D-xyloside.

Cytochromes in free-living rhizobia

Difference spectra of the crude cell-free extracts of 22 strains of fast-growing and slow-growing rhizobia, including members of the cowpea group, reveal the presence of cytochromes c, b, aa3, o, and a soluble CO-reactive hemoprotein P-428 in all of them. No strict correlation between the growth properties of the strains and their cytochrome contents are observed. All the pigments are present in varying quantities at all stages of growth ofRhizobium meliloti SU 216 andBradyrhizobium species (Lupinus) RL3001. A progressive increase in the level of the pigments is observed with the progress of bacterial growth until an optimum concentration is reached, whereupon the level tends to decrease. However, the ratio of cytochrome c to cytochrome b increases linearly throughout the growth period. Cytochromes b, c552, aa3, and o are particle bound, whereas cytochrome c550 and P-428 are soluble.

Inhibition of iron oxidation inThiobacillus ferrooxidans by toxic metals and its alleviation by EDTA

Oxidation of ferrous iron and growth ofThiobacillus ferrooxidans are severely affected in the presence of toxic metals, but the iron-oxidizing system of this bacterium is less sensitive to toxic metals than is its growth. Mercury toxicity is much more pronounced than that of uranium and cadmium. Iron oxidation is completely inhibited at 0.1 part/106 of mercury, which is 5000 and 20,000 times less than the minimum inhibitory concentration of uranium and cadmium respectively. The extent of antagonism between EDTA and toxic metal during iron oxidation by this bacterium is specific for a particular metal as well as the concentration used in the medium. Use of EDTA as a chelating agent for alleviation of toxicity during leaching of lowgrade sulfide ores by this bacterium is highly beneficial.

n-Dodecane as a substrate for nitrogen fixation by an alkane-utilizingAzospirillum sp.

Azospirillum sp. ANK-BI-11 was isolated from petroliferous soil. Glucose, nutrient broth, and sugar acids showed better growth thann-alkanes under aerobic conditions. The utilization of glucose was inhibited in the presence ofn-hexane. Microaerobically, succinic acid, pyruvic acid, and lactic acid were the best C-sources for acetylene reduction, whereas glucose was the best source for growth.n-Dodecane, a nonconventional C-source, also showed good response towards acetylene reduction, although growth was not so pronounced here as with glucose but was equal to that of Na-succinate. Optimum pH and temperature for acetylene reduction were between 7.0 and 8.0 and 30°C, respectively. Scanning electron microscopic studies revealed structural alteration in the shapes and sizes of the cells ofAzospirillum sp. when grown onn-hexane andn-dodecane compared with the cells grown on glucose.

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Abstract Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.