Initial steps in the degredation of 1, 3, 5-trihydroxybenzene by Bacillus sp. BPG-8

Acharya, Kashmira P. (1986) Initial steps in the degredation of 1, 3, 5-trihydroxybenzene by Bacillus sp. BPG-8. Masters thesis, Memorial University of Newfoundland.

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Abstract

Gram postitve, sporulating, aerobic rods (Bacillus BPG-8) isolated from local soil sample were found to grow on 1,3,5-trihydroxybenzene (Phloroglucinol, PG) when this substance was used as a sole source of carbon and energy. The optimal growth was reached at a substrate concentration of 0.1%; the pH optimum for the gtowth in mineral salt medium was pH 5.5 and the optimum temperature was 25°C. Phloroglucinol reductase (PGR), the initial enzyme in the metabolic pathway appeared to be an inducible enzyme. High elevated levels of this enzyme were prsent in cells grown on either PG alone or on other carbon substrates (like succinate, pyruvate etc.) mixed with PG. Besides PG, the isolate also grew on pyrogallol and orcinol as sole sources of carbon. The oxidation of PG by cell suspension in minimal salt medium was easily demonstrable and the cell suspension was found to metabolize about 100 mg of the substrate in about five hours. The generation time of BPG-8 was calculated to be 3.75 hours. -- The PGR required electron donors such as reduced NADP⁺ and NAD⁺ for the reduction of the substrate. Metal ions such as Zn²⁺, Fe²⁺, Mn²⁺, Mg²⁺ and Cu²⁺ did not stimulate the PGR activity. Some metal ions inhibited the activity to varying degrees. The PGR activity was inhibited by potassium phosphate, sodium chloride, sodium phosphate, imidazole and poly buffers. -- The PGR and resorcinol hydroxylase (RH) activities were unseparable by ammonium sulphate treatment, gel filtration and ion exchange column chromatography. The activity of PGR was always higher than that of RH in all enzyme preparations. The Km values for PGR was 2 x 10⁻⁴ M and molecular weight was found to be 155,000 daltons. The PGR and RH activities from BPG-8 reached a peak in about 18 hours. The pH optimum for the enzyme activity was found to be 7.4. -- Freezing and thawing had little or no effect on the PGR and RH from BPG-8. When the crude extract stored at 4°C for two days, a 90% loss of the RH activity resulted. Increasing concentrations of glycerol to (15%) offred protection to the PGR and RH. -- The spectral changes observed during chemical reduction of PG by sodium borohydride indicated the formation of dihydrophloroglucinol. Evidence is presented to show that enzymic reduction of PG in the presence of NADPH forms a product with a similar spectrum. -- The data presented suggest that BPG-8 may carry an enzyme complex with two separate activities, namely PGR and RH.

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