Biochemical characterization of a novel azo reductase named BVU5 from the bacterial flora DDMZ1: application for decolorization of azo dyes

Abstract

One of the main mechanisms of bacterial decolorization and degradation of azo dyes is the use of biological enzymes to catalyze the breaking of azo bonds. This paper shows the expression and properties of a novel azo reductase (hybrid-cluster NAD(P)-dependent oxidoreductase, accession no. A0A1S1BVU5, named BVU5) from the bacterial flora DDMZ1 for degradation of azo dyes. The molecular weight of BVU5 is about 40.1 kDa, and it contains the prosthetic group flavin mononucleotide (FMN). It has the decolorization ability of 80.1 ± 2.5% within 3 min for a dye concentration of 20 mg L⁻¹, and 53.5 ± 1.8% even for a dye concentration of 200 mg L⁻¹ after 30 min. The optimum temperature of enzyme BVU5 is 30 °C and the optimum pH is 6. It is insensitive to salt concentration up to a salinity level of 10%. Furthermore, enzyme BVU5 has good tolerance toward some metal ions (2 mM) such as Mn²⁺, Ca²⁺, Mg²⁺ and Cu²⁺ and some organic solvents (20%) such as DMSO, methanol, isopentyl, ethylene glycol and N-hexane. However, the enzyme BVU5 has a low tolerance to high concentrations of denaturants. In particular, it is sensitive to the denaturants guanidine hydrochloride (GdmCl) (2 M) and urea (2 M). Analysis of the dye substrate specificity shows that enzyme BVU5 decolorizes most azo dyes, which is indicating that the enzyme is not strictly substrate specific, it is a functional enzyme for breaking the azo structure. Liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS) revealed after the action of enzyme BVU5 that some intermediate products with relatively large molecular weights were produced; this illustrates a symmetric or an asymmetric rapid cleavage of the azo bonds by this enzyme. The potential degradation pathways and the enzyme-catalyzed degradation mechanism are deduced in the end of this paper. The results give insight into the potential of a rapid bio-pretreatment by enzyme BVU5 for processing azo dye wastewater.