Quinone-Reducing Activity Of Azoreductases And Their Application In Mediated Decolorization

Biodegradation of azo dyes has been widely studied for years. Azoreductases have been identified from various sources. And genes encoding these azoreductases were used to construct gene-engineered strains. However, compared with wild-type strains, no significantly higher decolorization ability was found with these gene-engineered strains overexpressing cellular azoreductases. Quinone-reducing activity of azoreductases and their application in mediated decolorization of azo dyes were studied in this dissertation.Using NAD(P)H as electron donor, quinone compounds can be reduced by AZR. With menadione as substrate, the optimal pH value and temperature for its quinone reductase activity are 8 and 50℃, respectively. Heat treatment below 50℃has no effect on quinone reductase activity of AZR. The reduction of menadione catalyzed by AZR follows Ping-Pong Bi Bi kinetic mechanism. It was found that AZR can reduce naphthoquinones and anthraquinones including menadione, lawsone, anthraquinone-2-sulfonate and anthraquinone-2,6-disulfonate. Menadione resulted to be the best substrate for AZR of all the quinones assayed. However, no activity was detected with benzoquinone. Compared with NADH, NADPH is a better electron donor. Externally added dicoumarol is a competitive inhibitor of NADPH and menadione, but an uncompetitive inhibitor of methyl red and nitrofurazone. The inhibition constants for them are 87.6, 51.4, 117.7 and 105.9μmol L^-1, respectively.Quinones are reduced by AZR via two-electron reduction. Compared with the control strain, Escherichia coli YB overexpressing cellular AZR demonstrated better oxidative stress resistance.Redox mediators can accelerate the electron transfer from a primary electron donor to a terminal electron acceptor, which may increase the reaction rates by one to several orders of magnitude. Lawsone is an effective accelerator for azo dye decolorization by E. coli YB overexpressing cellular quinone reductase AZR. In presence of 0.2 mmol L^-1 lawsone, 75% amaranth (1 mmol L^-1) was decolorized in 2 h by E. coli YB. Compared to lawsone, menadione is a less effective mediator. In presence of 2.5 mmol L^-1 menadione, 70% decolorization was reached in 12 h by E. coli YB. With lawsone as redox mediator, the optimal carbon source and biomass are glucose and 0.7 g L^-1 respectively for the mediated decolorization by E. coli YB. Repeated mediated decolorization investigation demonstrated that E. coli YB has stable decolorizing ability. Four rounds of repeated decolorization was completed in 12 h. Lawsone can also accelerate the decolorization of azo dyes with complex structures such as acid scarlet GR and reactive brilliant red K-2BP.Decolorization metabolite of acid red B can replace quinone compounds to act as a redox mediator and accelerate azo dye decolorization. Addition of 10% (v/v) acid red B (2 mmol L^-1) decolorization metabolite, more than 80% amaranth was decolorized in 7 h.Decolorization performances of E. coli JM109 and anaerobic sludge are improved when pretreated with methylhydroquinone. After 0.5 mmol L^-1 methylhydroquinone pretreatment for 10 min, in presence of 0.2 mmol L^-1 lawsone, 70% amaranth was decolorized in 3 h by E. coli JM109, while more than 75% amaranth was removed in 11 h by sludge.