Surface Display Of A Typical Triphenylmethane Reductase And The Mining Of A Novel Dual-Functional Dye Reductase As Well As Their Substrate Recognition Mechanism

The large amount, high temperature and complex composition of printing and dyeing wastewater poses a severe challenge to the current biological treatment method. Therefore, an efficient, robust, low-cost and broad spectrum wastewater treatment scheme is highly desirable. This study aimed to construct cell surface display systems with dye degradation enzymes, which can not only solve the problem of dye transmembrane transport, but also can reduce the cost of wastewater treatment, as well as provide a theoretical basis for the practical applications of dye degradation enzymes. Meanwhile, a novel reductase capable of degrading both triphenylmethane and azo dyes was discovered through genome mining, and its substrate recognition mechanism was partially clarified. The main results of this study are as follows:1) The recombinant triphenylmethane reductase, CsTMR, is a homodimer, and its optimum pH and temperature are 8.5 and 55℃. The enzyme was stable after treatment at 45℃for half an hour and maintained more than 50% of its initial activity after incubation at the pH ranging from 5.0 to 10.0. Overall, the recombinant CsTMR is a promising candidate for surface display mediated dye degradation due to its reasonable activity and stability.2) The N-terminal of ice nucleation proteins (InaPb-N) from Pseudomonas borealis was used as an anchoring protein to construct the engineered E. coli for surface displaying CsTMR. About 85% of the foreign protein have been successfully displayed on the outer membrane of E. coli. The decolorization rate of surface displayed CsTMR toward malachite green reached 640 μmol min-1 g"1 DWC. The stability of the surface displayed CsTMR showed significant improved than its free form, especially for the long-term stability. However, the attempt to generate a InaPB-N mediated glucose 1-dehydrogenase (DS255) surface display system was failed, which was meant to construct a coenzyme regeneration system to continuous provide coenzyme for CsTMR.3) The coat protein cotG was used as a carrier molecule to construct engineering spores of Bacillus subtilis str.168 for displaying CsTMR and DS255, respectively. Compared with their purified form, these two spore displayed enzymes showed robust stability against broad pH and temperature range. The engineered spores were employed to construct whole cell coenzyme regeneration system for dye removal. The results showed that a higher proportion of spore displayed DS255 is desired to achieved a higher dye degradation rate.4) A probable gene encoding triphenylmethane reductase from Geobacillus thermoglucosidasius C56-YS93 was obtained through database mining using the amino acid sequence of CsTMR as a probe. This novel reductase was designated as GtAZR according to its substrate specificity. The recombinant GtAZR showed maximum activity at pH 5.5 and 40℃, and was stable at temperatures below 65℃. The enzyme also exhibited markable tolerance to a wide range of pH and organic solvents. In addition, GtAZR showed promiscuous substrate specificity, which can degrade both triphenylmethane and azo dyes. Generally, the robust stability and broad spectrum of substrate specificity made it an excellent candidate in the practical treatment of mixed dye wastewater.5) A preliminary exploration for the substrate recognition mechanism of CsTMR and GtAZR was conducted through homology modeling, molecular docking, molecular dynamics simulations and site-directed mutagenesis experiments. The results showed that the 79th,80th and 148th residues in GtAZR might be involved in the recognition of methyl red, while the 146th residue in CsTMR might be involved in recognition of malachite green. This study partially clarified the substrate recognition mechanism of GtAZR from structural perspective, which provided new ideas to manipulate the substrate specificity of dye degradation enzyme.