Heterologous expression of an acidophilic multicopper oxidase in Escherichia coli and its applications in biorecovery of gold

Abstract

Background

Copper oxidase is a promising enzyme for detection of oxidation, which can function as a biosensor and in bioremediation. Previous reports have revealed that the activity of the multicopper oxidase (MCO, EC 1.10.3.2) from the Proteus hauseri ZMd44 is induced by copper ions, and has evolved to participate in the mechanism of copper transfer.

Results

From P. hauseri ZMd44, a full-length, 1497-base-pair gene, lacB, encoding 499 amino acids without signal peptide, was cloned into Escherichia coli (E. coli) to obtain high amounts of MCO. The use of the pET28a vector yielded better enzyme activity, which was approximately 400 and 500 U/L for the whole cell and soluble enzyme extracts, respectively. The crude enzyme showed activity at an optimal temperature of 55 °C and it remained highly active in the range of 50–65 °C. The optimal pH was 2.2 but the activity was significantly inhibited by chloride ions. This MCO has great potential for Au adsorption (i.e., 38% w/w) and the Au@NPs were directly adsorbed on enzyme’s surface.

Conclusion

An acidophilic MCO from bioelectricity generating bacterium, P. hauseri, is first cloned and heterologously expressed in E. coli with high amounts and activity. This MCO has great potential for Au adsorption and can be used as a biosensor or applied to bioremediation of electronic waste.

Keywords

Multicopper oxidase / Proteus hauseri / Recombinant protein / Copper effect / Au adsorption

References

[1] Ausec L, Črnigoj M, Šnajder M, Ulrih NP. Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp. Appl microbiol biotechnol, 2015, 99(23): 9987-9999.

[2] Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechol, 2004, 22(11): 1399-1408.

[3] Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C. Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry, 2002, 41(23): 7325-7333.

[4] Bradford MM. A rapid and sensitive method for the quantitation of protein utilizing the principle of protein–dye binding. Anal Biochem, 1976, 72(1–2): 248-254.

[5] Brown S. Metal-recognition by repeating polypeptides. Nat Biotechnol, 1997, 15: 269-272.

[6] Brown S, Sarikaya M, Johnson E. A genetic analysis of crystal growth. J Mol Biol, 2000, 299(3): 725-735.

[7] Chen BY, Zhang MM, Chang CT, Ding Y, Lin KL, Chiou CS, Hsueh CC, Xu H. Assessment upon azo dye decolorization and bioelectricity generation by Proteus hauseri. Bioresour Technol, 2010, 101(12): 4737-4741.

[8] Chen BY, Wang YM, Ng IS, Liu SQ, Hung JY. Deciphering simultaneous bioelectricity generation and dye decolorization using Proteus hauseri. J Biosci Bioeng, 2012, 113(4): 502-507.

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

[10] Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J, 2013, 32(6): 419-425.

[11] Grass G, Rensing C. CueO Is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochem Biophys Res Commun, 2001, 286(5): 902-908.

[12] Guo S, Li H, Liu J, Yang Y, Kong W, Qiao S, Huang H, Liu Y, Kang Z. Visible-light-induced effects of Au nanoparticle on laccase catalytic activity. ACS appl mater inter, 2015, 7(37): 20937-20944.

[13] Kepp KP. Halide binding and inhibition of laccase copper clusters: the role of reorganization energy. Inorg Chem, 2015, 54(2): 476-483.

[14] Larsson S, Cassland P, Jonsson LJ. Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microb, 2001, 67(3): 1163-1170.

[15] Li D, Lv P, Zhu J, Lu Y, Chen C, Zhang X, Wei Q. NiCu alloy nanoparticle-loaded carbon nanofibers for phenolic biosensor applications. Sensors, 2015, 15(11): 29419-29433.

[16] Li J, Lawton TJ, Kostecki JS, Nisthal A, Fang J, Mayo SL, Rosenzweig AC, Jewett MC. Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase. Biotechnol J, 2016, 11(2): 212-218.

[17] Mayer AM, Staples RC. Laccase: new functions for an old enzyme. Phytochemistry, 2002, 60(5): 551-565.

[18] Moreira GM, Cunha CE, Salvarani FM, Goncalves LA, Pires PS, Conceicao FR, Labato FC. Production of recombinant botulism antigens: a review of expression systems. Anaerobe, 2014, 28: 130-136.

[19] Naqui A, Varfolomeev SD. Inhibition mechanism of Polyporus laccase by fluoride ion. FEBS Lett, 1980, 113(2): 157-160.

[20] Natarajan G, Ting YP. Pretreatment of e-waste and mutation of alkali-tolerant cyanogenic bacteria promote gold biorecovery. Bioresour Technol, 2014, 152: 80-85.

[21] Natarajan G, Ting YP. Gold biorecovery from e-waste: an improved strategy through spent medium leaching with pH modification. Chemosphere, 2015, 136: 232-238.

[22] Ng IS, Zheng X, Chen BY, Chi X, Lu Y, Chang C. Proteomics approach to decipher novel genes and enzymes characterization of a bioelectricity-generating and dye-decolorizing bacterium Proteus hauseri ZMd44. Biotechnol Bioproc Eng, 2013, 18: 8-17.

[23] Ng IS, Chen T, Lin R, Zhang X, Ni C, Shun D. Decolorization of textile azo dye and congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01. Appl Microbiol Biotechnol, 2014, 98(5): 2297-2308.

[24] Ng IS, Isaac NC, Zhou Y, Wu X. Cultural optimization and metal effects of Shewanella xiamenensis BC01 growth and swarming motility. Bioresour Bioprocess, 2015, 2: 28-37.

[25] Olukanni OD, Osuntoki AA, Kalyani DC, Gbenle GO. Decolorization and biodegradation of Reactive Blue 13 by Proteus mirabilis LAG. J Hazard Mater, 2010, 184(1–3): 290-298.

[26] Rodgers CJ, Blanford CF, Giddens SR, Skamnioti P, Armstrong FA, Gurr SJ. Designer laccases: a vogue for high-potential fungal enzymes?. Trends Biotechnol, 2010, 28(2): 63-72.

[27] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 2014, 5: 172.

[28] Safary A, Moniri R, Hamzeh-Mivehroud M, Dastmalchi S. A strategy for soluble overexpression and biochemical characterization of halo-thermotolerant Bacillus laccase in modified E. coli. J Biotechnol, 2016, 227(227): 56-63.

[29] Santhanam N, Vivanco JM, Decker SR, Reardon KF. Expression of industrially relevant laccases: prokaryotic style. Trends Biotechnol, 2011, 29(10): 480-489.

[30] Sondhi S, Sharma P, Saini S, Puri N, Gupta N. Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4. PLoS ONE, 2014, 9: e96951.

[31] Tatsuo M, Hironari M, Yukiko S, Ichiro K, Misa H, Saori S, Noriho K, Masahiro G. Proteins and protein-rich biomass as environmentally friendly adsorbents selective for precious metal ions. Environ Sci Technol, 2007, 41(4): 1359-1364.

[32] Thomas R. A beginner’s guide to ICP-MS. Spectroscopy, 2001, 16: 38-42.

[33] Wang N, Ng IS, Chen PT, Li Y, Chen YC, Chen BY, Lu Y. Draft genome sequence of the bioelectricity-generating and dye-decolorizing bacterium Proteus hauseri strain ZMd44. Genome Announc, 2014, 2(1): e00992-e009913.

[34] Wu Z, Hong H, Zhao X, Wang X. Efficient expression of sortase A from Staphylococcus aureus in Escherichia coli and its enzymatic characterizations. Bioresour Bioprocess, 2017, 4: 13-22.

[35] Yang S, Long Y, Yan H, Cai H, Li Y, Wang X. Gene cloning, identification and characterization of the multicopper oxidase CumA from Pseudomonas sp. 593. Biotechnol Appl Bioc, 2016

[36] Ye M, Li G, Liang WQ, Liu YH. Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression. Appl Microbiol Biot, 2010, 87(3): 1023-1031.

[37] Zheng X, Ng IS, Ye C, Chen BY, Lu Y. Copper ion-stimulated McoA-laccase production and enzyme characterization in Proteus hauseri ZMd44. J Biosci Bioeng, 2013, 115(4): 388-393.

Funding

Ministry of Science and Technology, Taiwan(105-2221-E-006-225-MY3)