The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

Abstract

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.

FIG. 1

Reduction of amaranth (A) or Mordant yellow 3 (B) under anaerobic conditions by whole cells of Sphingomonas sp. strain BN6 (●) or BN6(pRJR34) (■). The cells were grown aerobically in a mineral medium with glucose (10 mM) and naphthalene-2-sulfonate (0.5 mM) [plus 10 μg of tetracycline per ml for BN6(pRJR34)]. When the cells reached the late-exponential growth phase, 25 ml of the cultures was transferred into serum bottles (100-ml volume) with a nitrogen atmosphere, and glucose (10 mM) and Mordant yellow 3 (1 mM) or amaranth (1.3 mM) were added. At the indicated intervals, aliquots were taken, cells were removed by centrifugation, and the concentrations of the azo dyes were determined by high-performance liquid chromatography.

FIG. 2

Reduction of amaranth by cell extracts from E. coli K38(pEE1001) under aerobic (□, ▵) and anaerobic conditions (■, ▴). The reaction mixtures contained (per milliliter) 50 μmol of NaK phosphate buffer (pH 7.7), 0.4 μmol of NADH, 0.1 μmol of FAD, and 0.05 μmol of amaranth. The reactions were started by the addition of 20 μl of a cell extract from E. coli K38(pEE1001) (88 μg of protein), and the decrease in absorbance was simultaneously determined spectrophotometrically at λ = 340 nm (■, □) and λ = 520 nm (▴, ▵). For the reaction under anaerobic conditions, all solutions were made anaerobically by repeated gassing with N₂, and the reaction was performed in gastight cuvettes.