Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum

Abstract

Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O) by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L−1 of anthracene (PAH). The optimum conditions of pH (4.0–9.0), temperature (5–70 °C), reaction time (10–90 min) and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 °C, 20 °C higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe3+, Hg2+ and Mn2+ and inhibited by NH4+ and Cu2+, but the immobilization protected the enzyme against the deleterious effects of K+ and Mg2+ in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract.

Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases

“…A similar effect was observed for hydroxyquinol 1,2-dioxygenase after its immobilization onto single-walled carbon nanotubes [90]. In turn, after the immobilization of 1,2-dioxygenases from Acinetobacter radioresistens S13, Pseudomonas putida and Mycobacterium fortuitum on nanosponges or sodium alginate gel the optimum temperature for examined enzymes was shifted toward higher values [88,91,92]. Similar results were obtained after the immobilization of catechol 2,3-dioxygenase from Bacillus stearothermophilus and Stenotrophomonas maltophilia KB2 on activated glyoxyl agarose and sodium alginate beads, respectively [18,84].…”

Genomic insights of aromatic hydrocarbon degrading Klebsiella pneumoniae AWD5 with plant growth promoting attributes: a paradigm of soil isolate with elements of biodegradation

“…The cell-free supernatant was used as crude enzyme for determining the enzyme activities in bacterial culture. Catechol 1, 2 dioxygenase (C-1,2D) and Catechol 2, 3 dioxygenase (C-2,3D) were assayed spectrophotometrically by measuring rate of production of metabolites from catechol as described (Silva et al 2013 ).…”

Mycobacterium as Polycyclic Aromatic Hydrocarbons (PAHs) Degrader

“…Silva et al [26] reported that M. fortuitum has an ability to degrade anthracene maximally and increase their metabolic activity by changing various physical conditions, that is, pH and temperature. The other route of PAH degradation is accomplished by the action of monooxygenases.…”