Heterologous expression of an acidophilic multicopper oxidase in Escherichia coli and its applications in biorecovery of gold
© The Author(s) 2017
Received: 23 March 2017
Accepted: 13 April 2017
Published: 22 April 2017
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 188.8.131.52) from the Proteus hauseri ZMd44 is induced by copper ions, and has evolved to participate in the mechanism of copper transfer.
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.
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.
The enzyme multicopper oxidase (MCO) is a type of laccase (EC 184.108.40.206) that has important industrial applications owing to its oxidizing and degrading activities on a wide variety of aromatic compounds (Mayer and Staples 2002; Claus 2003; Rodgers et al. 2010). For instance, laccases are used in the process of paper-pulp bleaching to degrade lignin (Larsson et al. 2001). Furthermore, MCO, as one of laccases, has a high demand in different manufacturing processes such as wine production, medical analysis, electrochemical detection (Li et al. 2015), bioremediation (Santhanam et al. 2011), and gold nanoparticle preparation (Guo et al. 2015).
Proteus hauseri strain ZMd44 is a gram-negative bacterium with outstanding performance in biodecolorization of azo-dyes (Ng et al. 2013), and has been used in the microbial fuel cell (MFC) system (Chen et al. 2010, 2012). In addition, its MCO-laccase activity of 357 U/L at optimal cultivation condition was induced by copper (Zheng et al. 2013). Furthermore, this MCO-laccase participates in the transport of copper ions from the medium to the cells (Grass and Rensing 2001). By whole genome sequencing, it has been found that P. hauseri ZMd44 possesses LacA and LacB (Wang et al. 2014). However, genetic engineering of any laccase or multicopper oxidase from the Proteus genus has not been illustrated in the literature thus far. Owing to the insufficient enzyme production by naturally occurring microbes, heterologous expression of the laccase genes via E. coli cloning is urgently required.
Genetic heterologous expression may be influenced by different factors, including protein structure (Gopal and Kumar 2013), effect of bacterial strain (Moreira et al. 2014), and the use of different expression vectors (Rosano and Ceccarelli 2014). In general, the most common plasmid used in recombinant engineering of E. coli is the pET system, owing to its strong expression levels. In this study, three different vectors, pET22b, pET28a, and pET32a, are used to produce recombinant MCO. All the vectors contain the same T7 promoter, and the resulting proteins are fused to a 6-Histidine tag for protein affinity purification. While pET28a is a vector in which recombinant protein would be overexpressed intracellularly, the pET22b vector includes a signal peptide, pelB, so that the recombinant protein is secreted to the periplasam. Finally, vector pET32a contains a fusion protein, TrxA, which works as a chaperone to assist in protein folding and to enhance soluble protein expression (Baneyx and Mujacic 2004).
Over the past decades, the number of consumers using electronic devices manufactured by the semiconductor industry is rapidly increasing, resulting in the accumulation of a huge amount of electronic waste (Natarajan and Ting 2014, 2015). Routine recovery of gold (Au) from industrial metal waste is expensive. Further, biotechnology industries are taking eco-friendly approaches to recover metals from waste, which have significantly contributed to control the pollution in the environment. Previous studies showed Proteus spp. (Chen et al. 2010, 2012) and Shewanella spp. (Ng et al. 2014, 2015) with good performance in biodecolorization and metal absorption, but they never been used of genetic approach. Therefore, the use of genetically engineered MCO for bioremediation is a promising and cost-effective solution.
Until now, only few research studies have explored the heterologous expression of MCO in E. coli. This is the first attempt to determine the optimal vector to express MCO. Additionally, the optimal reaction conditions, including temperature, pH, and ion effect, were assessed. Finally, the use of the recombinant MCO in Au adsorption and gold nanoparticle (Au@NPs) preparation, and its catalytic activity, were also explored.
Cloning and construction of recombinant LacB in E. coli
List of strains, plasmids, and oligonucleotide primers in this study
Strains, plasmid, or primer
E. coli strains
F− recA1 endA1 hsdR17(r k − m k + ) supE44 thi-1 gyrA relA1
F− ompT hsdS B (r B − m B − ) gal dcm (DE3)
Ampr T7 promoter trxA-tag His-tag T7 terminator lacI f1 pBR322 origin E. coli expression vector
Kanr T7 promoter His-tag T7 terminator lacI f1 pBR322 origin E. coli expression vector
Ampr T7 promoter pelB His-tag T7 terminator lacI f1 pBR322 origin E. coli expression vector
Culture and heterologous expression of recombinant MCO
The expression host BL21 (DE3) harboring the recombinant ZMd44-lacB vector was cultivated in LB medium with corresponding antibiotics and agitation (200 rpm) at 37 °C. Once the cultures reached a biomass with OD600 between 0.6 and 0.8, the isopropyl β-d-1-thiogalactopyranoside (IPTG) inducer and the key factor CuSO4 were added to the cultures at a final concentration of 0.1 mM and 0.5 mM, respectively. At this point, the incubation temperature was changed to 22 °C with the same agitation speed. After 12 h in culture, the recombinant bacteria were placed in an incubator at 22 °C without agitation for an additional 12 h. For cell density analysis, sample was taken out from the broth to measure the optical density by a spectrophotometer at a wavelength of 600 nm (VersaMaxTM microplate reader, Molecular Devices, CA). The OD600 values were converted to biomass in terms of g/L via a calibration between optical density and dry cell weight. All the experiments were run in triplicate and designated as (A) +IPTG and Cu, (B) +IPTG, and (C) without induction, taking into consideration different cell fractions (i.e., W = whole cell, S = supernatant, P = pellet, and M = periplasm).
Determination of MCO activity and protein concentration
MCO activity was determined by a spectrophotometric method based on the use of ABTS [2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] as substrate. To this end, a 200 μL reaction mixture containing 100 μL of 2 mM ABTS, 95 μL of 50 mM reaction buffer (pH 3.0), and 5 μL of enzyme solution was prepared. Enzyme activity was monitored with a spectrophotometer (VersaMax™ microplate reader, Molecular Devices, CA) set up at a wavelength of 420 nm (i.e., OD420) for the ABTS substrate. Reaction rates were calculated using a molar extinction coefficient of 36 mM/cm. One unit was defined as the amount of enzyme that oxidized 1 μmol of substrate per minute (min). Protein concentrations were determined by the Bradford method (Bradford 1976), using bovine serum albumin as the standard.
Protein expression determined by SDS-PAGE
Proteins were run in gels prepared with 0.1% SDS, using 12% separating gel and 4% stacking gel. Tris–glycine buffer (pH 8.3) containing 0.1% SDS was used as electrode buffer. Samples at same concentration in terms of OD = 5 were treated with buffer and heated at 100 °C for 5 min before loading onto the gel. Electrophoresis was run from the cathode to the anode at a constant current of 20 mA per slab at room temperature in a Biorad mini gel electrophoresis unit. Proteins were visualized by staining with Coomassie blue R-250. Stained SDS gels were scanned on the Image scanner Labscan 6.0 (GE Healthcare). Subsequently, band intensities were quantified by densitometry, using the Quantity One 4.6.2 analysis software (BioRad).
Biochemical characterization of LacB
LacB activity was analyzed using 2 mM ABTS in 50 mM sodium citrate buffer (pH 2.2), and the reaction was incubated at a temperature range of 37–65 °C. The crude enzyme of MCO was analyzed in 50 mM buffer at variable pHs. Different concentrations of CuSO4, CuCl2, and NaCl were included in the ABTS substrate at a range of 0–2 mM.
Purification of recombinant LacB
The supernatant of the recombinant LacB solution produced from the pET28a-lacB vector in BL21(DE3) cells was loaded onto the AKTA system (GE, USA), using a His-trap affinity column for purification. The purified enzymes were put into a micro-centrifuge tube at 30-kDa molecular weight cut-off (GE, Millipore), to remove the imidazole and concentrate the solution for Au adsorption.
Application of recombinant LacB on Au adsorption
Adsorption of free Au ions
Solutions containing 100 ppm of AuHCl4 and 50 ppm of purified recombinant LacB were prepared. Two milliliters of both solutions were mixed and agitated for 1 h at 70 rpm. Following agitation, 4 mL of the solution was transferred into the Amicon® device equipped with a 3 kDa molecular cut-off filter and centrifuged for 25 min. The resulting filtrate was used to measure the Au concentration by inductively coupled plasma with atomic emission spectroscopy (ICP-AES) (Thomas 2001).
Adsorption of Au@NPs
A solution containing 1 mM AuHCl4 and 38.8 mM sodium citrate was prepared as follows: 60 mL of AuHCl4 solution was heated until boiling, followed by the addition of 6 mL of sodium citrate solution. Once the color of the solution changed from yellow to purple, the solution was left to cool at room temperature. This solution was the Au nanoparticle solution. Next, 300 μL of Au nanoparticle solution and 200 ppm of purified recombinant LacB were mixed together and agitated for 1 h at 70 rpm. Following agitation, 200 μL of the solution was loaded into a 96-well plate to monitor the optical density with a spectrophotometer set up at a wavelength of 350–750 nm (VersaMax™ microplate reader, Molecular Devices, CA).
Characterization of MCO-Au
Zeta potential (Malvern Zetasizer Nano ZS, UK) was used to measure the isoelectric points of MCO or MCO with Cu (MCO+Cu) following Au adsorption. The zeta potential was measured in 100 mM phosphate buffer. Each sample was analyzed at least by triplicate. MCO+Cu and MCO+Cu after Au adsorption were further analyzed for their kinetic parameters against ABTS. The ABTS concentrations ranged from 0.01 to 2 mM at pH 2.2. The Michaelis–Menten kinetics was assumed and fitted by Lineweaver–Burk plot, using Sigmaplot 10.0 software.
Nucleotide sequence accession number
The sequences of lacB of P. hauseri ZMd44 have been deposited in the GenBank database under accession number JF718783.
Results and discussion
Cloning Mco-lacB from P. hauseri ZMd44
The full-length 1578 bp gene sequence encoding LacB, which included a signal peptide of 27 amino acids, was obtained from genomic annotation of the biodecolorizing bacterium, Proteus hauseri Zmd44 (Ng et al. 2013; Chen et al. 2010). Although the native multicopper laccase activity has been reported in P. hauseri (Zheng et al. 2013) and P. mirabilis (Olukanni et al. 2010), the heterologous expression of such kind of enzyme in E. coli has never been explored. Therefore, we cloned and overexpressed lacB in the E. coli for the first time. LacB exhibited activity after expression in the pET22b vector but it is not satisfying. LacB was shown to be a simple monomer by SWISS-MODEL simulation (Fig. 1d); therefore, this study focussed on the heterologous expression of LacB in different vectors.
Mco-lacB expression in different vectors
Comparison of the catalytic kinetic parameters of MCO on ABTS from different microorganisms
k cat /K m
Bacillus tequilensis SN4
pH 5.5 at 85 °C
Sondhi et al. (2014)
pH 7.4 at 55 °C
Ye et al. (2010)
Thioalkalivibrio sp. ALRh
pH 5 at 50 °C
Ausec et al. (2015)
Pseudomonas sp. 593
pH 5 at 55 °C
Yang et al. (2016)
Bacillus sp. SL-1
pH 4.5 at 50 °C
Safary et al. (2016)
pH 2.2 at 50 °C
pH 2.2 at 50 °C
Application and characterization of recombinant MCO-lacB for Au adsorption
The initial working conditions of MCO and MCO+Cu (i.e., cultured with Cu) for adsorbing Au were determined at pH 7.75 and 7.73, respectively. However, the zeta potential of both samples following Au adsorption was determined at different pHs. These results are shown in Fig. 5c. The isoelectric points of MCO and MCO+Cu were determined at pH 5.29 and pH 5.51, respectively. This showed that the recombinant proteins were anionic and the Au adsorption on the protein was through electrostatic interactions. The isoelectric points of MCO and MCO+Cu upon Au adsorption were determined at pH 6.21 and 6.15, respectively. At this point, the proteins were still anionic, but the isoelectric point shifted to a higher pH, especially for MCO. We suggest that Au ions compete for the same binding site within the protein, affecting the chelation of the copper ion by the MCO and causing structural changes. Higher isoelectric points showed that, at the same pH, there were fewer negative charges of the protein after Au adsorption. This is consistent with the fact that Au possesses protons combined to the proteins, which partially neutralize the protein’s negative charges.
We optimized the heterologously expression of MCO and investigated the extent of its applications. Among the three different vectors analyzed in this study (i.e., pET28a, pET22b, and pET32a), pET28a, carrying the lacB gene, showed the best results regarding expression levels, cell growth, and enzyme activity at different pHs. The optimal pH of the recombinant MCO was 2.2 at an incubation temperature of 55 °C with the addition of 0.5 mM of copper. We also found that the addition of chloride ion strongly inhibited MCO activity. This is the first attempt to explore the ability of MCO from P. hauseri to adsorb Au or Au@NPs, which may become a novel application for bioremediation in the future.
IS and SI designed the experiment and analyzed the data, SI performed most of experiments in genetic section, and YJ did the major part of gold adsorption. IS and SI wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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This work was support by the Ministry of Science and Technology (MOST 105-2221-E-006-225-MY3 and MOST 105-2621-M-006-012-MY3) in Taiwan.
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