Identification of bacterial laccase cueO mutation from the metagenome of chemical plant sludge

Abstract

Background

The metagenome contains plenty of genetic resources and can be used to search for the novel gene or mutant.

Results

In this study, the bacterial laccase gene (cueO) with single or multiple mutations was directly cloned based on the metagenome of a chemical plant sludge. An interesting mutation (G276R) was identified from those cloned mutants. The other mutants (G276N, G276Y, and G276K) with improved catalytic efficiency were identified by the saturation mutagenesis on residue G276. The optimal temperature for wild-type CueO enzyme activity was about 70 °C, compared to 60 °C, 50 °C, 50 °C, and 30 °C for the G276R, G276N, G276Y, and G276K mutant enzymes, respectively. The catalytic efficiency (k_cat/K_m) with 8 mmol Cu²⁺ of the G276R, G276N, G276Y, and G276K mutants was 1.2-, 2.7-, 1.3-, and 2.7-fold, respectively, compared to the wild-type enzyme. In addition, the mutants G276R, G276N, G276Y, and G276K oxidized the carcinogen benzo[α]pyrene more efficiently compared to the wild-type enzyme.

Conclusion

All of the results indicate that G276 of CueO plays an important role in enzyme activity, and the useful mutants can be identified based on the metagenome.

Keywords

Bacterial laccase / CueO / G276K / Mutation / Metagenome

References

[1] Bezerra TMDS, Bassan JC, Santos VTDO, Ferraz A, Monti R. Covalent immobilization of laccase in green coconut fiber and use in clarification of apple juice. Process Biochem, 2014, 50(3): 417-423.

[2] Brander S, Mikkelsen JD, Kepp KP. Characterization of an alkali- and halide-resistant laccase expressed in E. coli: CotA from Bacillus clausii. PLoS ONE, 2014, 9(6): e99402.

[3] Brouwers GJ, Vrind JPMD, Corstjens PLAM, Jong VD, Cornelis P, Baysse C. cumA, a gene encoding a multicopper oxidase, is involved in Mn{sup 2+} oxidation in Pseudomonas putida GB1. Appl Environ Microbiol, 1999, 65(4): 1762-1768.

[4] Camarero S, Pardo I, Canas AI, Molina P, Record E, Martinez AT, . Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus. Appl Environ Microbiol, 2012, 78(5): 1370-1384.

[5] Chen B, Xu WQ, Pan XR, Lu L. A novel non-blue laccase from Bacillus amyloliquefaciens: secretory expression and characterization. Int J Biol Macromol, 2015, 76: 39-44.

[6] Claus H. Laccases and their occurrence in prokaryotes. Arch Microbiol, 2003, 179(3): 145-150.

[7] Claus H, Faber G, Konig H. Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl Microbiol Biotechnol, 2002, 59(6): 672-678.

[8] Durao P, Chen Z, Fernandes AT, Hildebrandt P, Murgida DH, Todorovic S, . Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes. J Biol Inorg Chem, 2008, 13(2): 183-193.

[9] Eggert C, Temp U, Eriksson KE. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol, 1996, 62(4): 1151-1158.

[10] Ghindilis AL, Gavrilova VP, Yaropolov AI. Laccase-based biosensor for determination of polyphenols: determination of catechols in tea. Biosens Bioelectron, 1992, 7(2): 127-131.

[11] Guazzaroni ME, Silva-Rocha R, Ward RJ. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening. Microb Biotechnol, 2015, 8(1): 52-64.

[12] Hoegger PJ, Kilaru S, James TY, Thacker JR, Kües U. Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. Febs J, 2006, 273(10): 2308-2326.

[13] Hullo MF, Moszer I, Danchin A, Martin-Verstraete I. CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol, 2001, 183(18): 5426-5430.

[14] Huttermann A, Mai C, Kharazipour A. Modification of lignin for the production of new compounded materials. Appl Microbiol Biotechnol, 2001, 55(4): 387-394.

[15] Ihssen J, Reiss R, Luchsinger R, Thony-Meyer L, Richter M. Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli. Sci Rep, 2015, 5: 10465.

[16] Kalyani D, Tiwari MK, Li J, Kim SC, Kalia VC, Kang YC, . A highly efficient recombinant laccase from the yeast Yarrowia lipolytica and its application in the hydrolysis of biomass. PLoS ONE, 2015, 10(3): e0120156.

[17] Kataoka K, Sugiyama R, Hirota S, Inoue M, Urata K, Minagawa Y, . Four-electron reduction of dioxygen by a multicopper oxidase, CueO, and roles of Asp112 and Glu506 located adjacent to the trinuclear copper center. J Biol Chem, 2009, 284(21): 14405-14413.

[18] Kellenberger E. Exploring the unknown. The silent revolution of microbiology. EMBO Rep, 2001, 2(1): 5-7.

[19] Kellner H, Luis P, Zimdars B, Kiesel B, Buscot F. Diversity of bacterial laccase-like multicopper oxidase genes in forest and grassland Cambisol soil samples. Soil Biol Biochem, 2008, 40(3): 638-648.

[20] Kirsch RD, Joly E. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res, 1998, 26(7): 1848-1850.

[21] Komori H, Higuchi Y. Structural insights into the O2 reduction mechanism of multicopper oxidase. J Biochem, 2015, 158(4): 293-298.

[22] Komori H, Kajikawa T, Kataoka K, Higuchi Y, Sakurai T. Crystal structure of the CueO mutants at Glu506, the key amino acid located in the proton transfer pathway for dioxygen reduction. Biochem Biophys Res Commun, 2013, 438(4): 686-690.

[23] Leis B, Angelov A, Liebl W. Screening and expression of genes from metagenomes. Adv Appl Microbiol, 2013, 83: 1-68.

[24] Li Z, Kessler W, van den Heuvel J, Rinas U. Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. Appl Microbiol Biotechnol, 2011, 91(4): 1203-1213.

[25] Loncar N, Bozic N, Lopez-Santin J, Vujcic Z. Bacillus amyloliquefaciens laccase–from soil bacteria to recombinant enzyme for wastewater decolorization. Bioresour Technol, 2013, 147: 177-183.

[26] Malmstrom BG. Intermediates in the reduction of dioxygen y laccase and cytochrome c oxidase. Adv Exp Med Biol, 1982, 148: 87-94.

[27] Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, . CDD: NCBI’s conserved domain database. Nucleic Acids Res, 2015, 43(Database issue): D222-D226.

[28] Murugesan K. Bioremediation of paper and pulp mill effluents. Indian J Exp Biol, 2003, 41(11): 1239-1248.

[29] Nasoohi N, Khajeh K, Mohammadian M, Ranjbar B. Enhancement of catalysis and functional expression of a bacterial laccase by single amino acid replacement. Int J Biol Macromol, 2013, 60: 56-61.

[30] Nossal NG, Heppel LA. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem, 1966, 241(13): 3055-3062.

[31] Palonen H, Viikari L. Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood. Biotechnol Bioeng, 2004, 86(5): 550-557.

[32] Pardo I, Camarero S. Laccase engineering by rational and evolutionary design. Cell Mol Life Sci, 2015, 72(5): 897-910.

[33] Peter MG, Wollenberger U. Phenol-oxidizing enzymes: mechanisms and applications in biosensors. EXS, 1997, 80: 63-82.

[34] Prins A, Kleinsmidt L, Khan N, Kirby B, Kudanga T, Vollmer J, . The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2). Enzyme Microb Technol, 2015, 68: 23-32.

[35] Rivera-Hoyos CM, David Morales-Alvarez E, Poutou-Pinales RA, Marina Pedroza-Rodriguez A, Rodriguez-Vazquez R, Delgado-Boada JM. Fungal laccases. Fungal Biol Rev, 2013, 27(3–4): 67-82.

[36] Roberts SA, Weichsel A, Grass G, Thakali K, Hazzard JT, Tollin G, . Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli. Proc Natl Acad Sci USA, 2002, 99(5): 2766-2771.

[37] Ruijssenaars HJ, Hartmans S. A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity. Appl Microbiol Biotechnol, 2004, 65(2): 177-182.

[38] Sharma P, Goel R, Capalash N. Bacterial laccases (Article). World J Microbiol Biotechnol, 2007, 23(6): 823-832.

[39] Si W, Wu Z, Wang L, Yang M, Zhao X. Enzymological characterization of Atm, the first Laccase from Agrobacterium sp. S5-1, with the ability to enhance in vitro digestibility of maize straw. PLoS ONE, 2015, 10(5): e0128204.

[40] Singh SK, Roberts SA, McDevitt SF, Weichsel A, Wildner GF, Grass GB, . Crystal structures of multicopper oxidase CueO bound to copper(I) and silver(I): functional role of a methionine-rich sequence. J Biol Chem, 2011, 286(43): 37849-37857.

[41] Solomon EI, Augustine AJ, Yoon J. O2 reduction to H2O by the multicopper oxidases. Dalton Trans, 2008, 252(30): 3921.

[42] Su J, Bao P, Bai T, Deng L, Wu H, Liu F, . CotA, a multicopper oxidase from Bacillus pumilus WH4, exhibits manganese-oxidase activity. PLoS ONE, 2013, 8(4): e60573.

[43] Suzuki T, Endo K, Ito M, Tsujibo H, Miyamoto K, Inamori Y. A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression. Biosci Biotechnol Biochem, 2003, 67(10): 2167-2175.

[44] Thurston CF. The structure and function of fungal laccases. Microbiology, 1994, 140(1): 19-26.

[45] Tian J, Wang P, Huang L, Chu X, Wu N, Fan Y. Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method. Appl Microbiol Biotechnol, 2013, 97(7): 2997-3006.

[46] Tian J, Woodard JC, Whitney A, Shakhnovich EI. Thermal stabilization of dihydrofolate reductase using monte carlo unfolding simulations and its functional consequences. PLoS Comput Biol, 2015, 11(4): e1004207.

[47] Ukkonen K, Mayer S, Vasala A, Neubauer P. Use of slow glucose feeding as supporting carbon source in lactose autoinduction medium improves the robustness of protein expression at different aeration conditions. Protein Expr Purif, 2013, 91(2): 147-154.

[48] Zeng J, Lin XG, Zhang J, Li XZ, Wong MH. Oxidation of polycyclic aromatic hydrocarbons by the bacterial laccase CueO from E. coli. Appl Microbiol Biotechnol, 2011, 89(6): 1841-1849.

[49] Zeng J, Zhu Q, Wu Y, Lin X. Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere, 2016, 148: 1-7.

[50] Zhao H, Zhou F, Qi Y, Dziugan P, Bai F, Walczak P, . Screening of Lactobacillus strains for their ability to bind benzo(a)pyrene and the mechanism of the process. Food Chem Toxicol, 2013, 59: 67-71.

Funding

National High Technology Research and Development Program of China(2013AA102804)