Abstract
The gene encoding a putative triphenylmethane reductase (TMR)-like protein derived from Geobacillus thermoglucosidasius C56-Y593 (named as GtAZR) was synthesized, heterologously expressed in Escherichia coli, and extensively characterized for the first time. The recombinant GtAZR displayed its maximum activity at pH 5.5 and 40 °C. GtAZR was stable at temperatures below 65 °C. It also exhibited a broad pH stability and retained more than 90 % of its initial activities in pH range of 4.5–10.5 after incubating in various buffers for 1 h. Moreover, GtAZR showed significant stability against metal ions and organic solvents. GtAZR displayed broad substrate spectrum toward both azo and triphenylmethane dyes. As a sequence and structural TMR-like protein, GtAZR was characterized as an azoreductase biochemically due to its high specificity for azo dye rather than triphenylmethane dye. Molecular docking and mutagenesis analysis revealed that amino acids Asp-79 and Thr-80 are responsible for its azoreductase activity, which eliminated the steric hindrance caused by His-77 and Tyr-78 at the correspond sites in other structural homologous triphenylmethane reductase. The robust stability and substrate promiscuity of GtAZR made it a promising candidate for practical removal of mixed dye wastewater.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bin Y, Jiti Z, Jing W, Cuihong D, Hongman H, Zhiyong S, Yongming B (2004) Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737. FEMS Microbiol Lett 236(1):129–136. doi:10.1016/j.femsle.2004.05.034
Chengalroyen MD, Dabbs ER (2013) The microbial degradation of azo dyes: minireview. World J Microbiol Biotechnol 29(3):389–399. doi:10.1007/s11274-012-1198-8
Ding H, Gao F, Liu D, Li Z, Xu X, Wu M, Zhao Y (2013) Significant improvement of thermal stability of glucose 1-dehydrogenase by introducing disulfide bonds at the tetramer interface. Enzym Microb Technol 53(6–7):365–372. doi:10.1016/j.enzmictec.2013.08.001
Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A (2006) Comparative protein structure modeling using Modeller. In: Baxevanis AD (ed) Curr Protoc Bioinformatics, vol Supplement 15. Wiley, New York, pp 5.6.1–5.6.30. doi:10.1002/0471250953.bi0506s15
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, Heger A, Hetherington K, Holm L, Mistry J (2014) Pfam: the protein families database. Nucleic Acids Res 42(D1):D222–D230. doi:10.1093/nar/gkt1223
Gouet P, Robert X, Courcelle E (2003) ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31(13):3320–3323. doi:10.1093/nar/gkg556
Gregory P (1993) Dyes and dye intermediates. Kirk-Othmer Encyclopedia of Chemical Technology. Wiley, New York
Jang MS, Lee YM, Kim CH, Lee JH, Kang DW, Kim SJ, Lee YC (2005) Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli. Appl Environ Microbiol 71(12):7955–7960. doi:10.1128/aem.71.12.7955-7960.2005
Johansson HE, Johansson MK, Wong AC, Armstrong ES, Peterson EJ, Grant RE, Roy MA, Reddington MV, Cook RM (2011) BTI1, an azoreductase with pH-dependent substrate specificity. Appl Environ Microbiol 77(12):4223–4225. doi:10.1128/AEM.02289-10
Kim MH, Kim Y, Park HJ, Lee JS, Kwak SN, Jung WH, Lee SG, Kim D, Lee YC, Oh TK (2008) Structural insight into bioremediation of triphenylmethane dyes by Citrobacter sp. triphenylmethane reductase. J Biol Chem 283(46):31981–31990. doi:10.1074/jbc.M804092200
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. doi:10.1038/227680a0
Lang W, Sirisansaneeyakul S, Ngiwsara L, Mendes S, Martins LO, Okuyama M, Kimura A (2013) Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: toward dye decolorization using a packed-bed metal affinity reactor. Bioresour Technol 150:298–306. doi:10.1016/j.biortech.2013.09.124
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. doi:10.1093/bioinformatics/btm404
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26(2):283–291. doi:10.1107/S0021889892009944
Li LT, Hong Q, Yan X, Fang GH, Ali SW, Li SP (2009) Isolation of a malachite green-degrading Pseudomonas sp. MDB-1 strain and cloning of the tmr2 gene. Biodegradation 20(6):769–776. doi:10.1007/s10532-009-9265-z
Liu W, Chao Y, Yang X, Bao H, Qian S (2004) Biodecolorization of azo, anthraquinonic and triphenylmethane dyes by white-rot fungi and a laccase-secreting engineered strain. J Ind Microbiol Biotechnol 31(3):127–132. doi:10.1007/s10295-004-0123-z
Liu G, Zhou J, Meng X, Fu SQ, Wang J, Jin R, Lv H (2013) Decolorization of azo dyes by marine Shewanella strains under saline conditions. Appl Microbiol Biotechnol 97(9):4187–4197. doi:10.1007/s00253-012-4216-8
Lorenzo M, Moldes D, Rodrı́guez Couto S, Sanromán M (2005) Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds. Chemosphere 60(8):1124–1128. doi:10.1016/j.chemosphere.2004.12.051
Misal SA, Lingojwar DP, Gawai KR (2013) Properties of NAD(P)H Azoreductase from alkaliphilic red bacteria Aquiflexum sp. DL6. Protein J 32(8):601–608. doi:10.1007/s10930-013-9522-1
Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791. doi:10.1002/jcc.21256
Novotný Č, Rawal B, Bhatt M, Patel M, Šašek V, Molitoris HP (2001) Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes. J Biotechnol 89(2):113–122. doi:10.1016/S0168-1656(01)00321-2
Pronk S, Páll S, Schulz R, Larsson P, Bjelkmar P, Apostolov R, Shirts MR, Smith JC, Kasson PM, van der Spoel D (2013) GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29(7):845–854. doi:10.1093/bioinformatics/btt055
Ren S, Guo J, Zeng G, Sun G (2006a) Decolorization of triphenylmethane, azo, and anthraquinone dyes by a newly isolated Aeromonas hydrophila strain. Appl Microbiol Biotechnol 72(6):1316–1321. doi:10.1007/s00253-006-0418-2
Ren SZ, Guo J, Cen YH, Sun GP (2006b) Properties of a triphenylmethane dyes decolorization enzyme (TpmD) from Aeromonas hydrophila strain DN322. Acta Microbiol Sin 46(5):823–826. doi:10.3321/j.issn:0001-6209.2006.05.028
Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 77(3):247–255. doi:10.1016/S0960-8524(00)00080-8
Shah MM, Grover TA, Barr DP, Aust S (1992) On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA. J Biol Chem 267(30):21564–21569
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30(12):2725–2729. doi:10.1093/molbev/mst197
Wallace AC, Laskowski RA, Thornton JM (1995) LIGPLOT: a program to generate schematic diagrams of protein–ligand interactions. Protein Eng 8(2):127–134. doi:10.1093/protein/8.2.127
Wang J, Gao F, Liu Z, Qiao M, Niu X, Zhang KQ, Huang X (2012) Pathway and molecular mechanisms for malachite green biodegradation in Exiguobacterium sp. MG2. PLoS One 7(12):e51808
Weisburger JH (2002) Comments on the history and importance of aromatic and heterocyclic amines in public health. Mutat Res 506:9–20. doi:10.1016/S0027-5107(02)00147-1
Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35(suppl 2):W407–W410. doi:10.1093/nar/gkm290
Yang Y, Wei B, Zhao Y, Wang J (2013) Construction of an integrated enzyme system consisting azoreductase and glucose 1-dehydrogenase for dye removal. Bioresour Technol 130:517–521. doi:10.1016/j.biortech.2012.12.106
Zhang C, Diao H, Lu F, Bie X, Wang Y, Lu Z (2012) Degradation of triphenylmethane dyes using a temperature and pH stable spore laccase from a novel strain of Bacillus vallismortis. Bioresour Technol 126:80–86. doi:10.1016/j.biortech.2012.09.055
Acknowledgments
This study was supported by National Natural Science Foundation of China (31200599; 41271335), China Postdoctoral Science Foundation (2012 M521163), Excellent Postdoctoral Science Foundation of Zhejiang Province (Bsh1202087), High Technology Research and Development Program of China (863 Program) (2012AA06A203), National Key Technology Rand D Program (2012BAC17B04), Science and Technology Project of Zhejiang Province (2011C13016; 2013C3303), Environmental Science Project of Zhejiang Province ( 2012B006), Social Development of Science and Technology Project of West Lake District, and Science and Technology Development Plan of Hangzhou City (20130533B36).
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Fen Gao and Haitao Ding contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 160 kb)
Rights and permissions
About this article
Cite this article
Gao, F., Ding, H., Shao, L. et al. Molecular characterization of a novel thermal stable reductase capable of decoloration of both azo and triphenylmethane dyes. Appl Microbiol Biotechnol 99, 255–267 (2015). https://doi.org/10.1007/s00253-014-5896-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-014-5896-z