Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green

Ecotoxicology and Environmental Safety 193 (2020) 110335

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Extracellular expression of mutant CotA-laccase SF in Escherichia coli and its degradation of malachite green

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Kai-Zhong Xu, Hui Ma, Ya-Jing Wang, Yu-Jie Cai, Xiang-Ru Liao, Zheng-Bing Guan∗ The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Extracellular expression CotA-laccase Escherichia coli Dye decolorization Degradation

In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotAlaccase SF to MG were 39.6 mM and 18.36 s−1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.

1. Introduction Malachite green is a triphenylmethane dye once widely used in industrial production. Malachite green is formed by polymerization of one molecule benzaldehyde and two molecules of dimethylaniline in the presence of concentrated sulfuric acid. Many countries use malachite green as an insecticide and fungicide in aquaculture. However, malachite green will remain in water and fish for a long time and difficult to be degraded. At the same time, malachite green has high teratogenic and carcinogenic effects. Studies have shown that malachite green can inhibit the activity of human glutathione-S-transferase and induce tumor (Stammati et al., 2005). Many countries have decreed against the use of malachite green in industry (Andersen et al., 2018). However, malachite green is cheap and easy to obtain, it can often be detected in water and fish. The traditional methods of malachite green treatment include physical methods and chemical methods. Physical methods are adsorption and membrane filtration (Ling and Mohd Suah, 2017; Xavier et al., 2011). The subsequent treatment of dye after adsorption and concentration is still a problem. What's more, some adsorbents and materials are expensive and difficult to be applied. Chemical treatment methods include photocatalysis, and hydrogen peroxide treatment (Kusvuran et al., 2011; Lu et al., 2018). Secondary pollution and incomplete oxidation are important defects in chemical treatment of dyes. Biological enzyme methods are new dye treatment methods in recent years. The treatment of dyes by biological enzyme has the advantages

∗

of low cost and environmental protection. Many researchers have used enzyme methods to treat malachite green and achieved good results. Ellappan has developed a horseradish peroxidase for decolorization of malachite green from wastewater (Ellappan and Thayumanavan, 2015). Manganese peroxidase has also been proved to be effective in decolorizing malachite green (Yang et al., 2016). Laccase (EC 1.10.3.2) is one of the enzymes related to malachite green degradation (Zhang et al., 2012). CotA-laccase has attracted more and more attention because of its wide range of substrates. Preliminary decolorization of malachite green by CotA-laccase has been reported (Samak et al., 2018). At present, there is no related mechanism about the degradation of malachite green by CotA-laccase. In the previous study, CotA-laccase mutant S208G/F227A (SF) expressed in Escherichia coli has good decolorization effect on Malachite Green (Xu et al., 28AD). The protein produced by E. coli strain is intracellular protein, which requires physical or chemical methods such as sonication and lysozyme treatment to obtain the target protein (Tanji et al., 1998). While traditional cell disruption methods tend to damage fragile enzymes and reduce enzyme activity (Harrison, 1991). It also brings high cost of sonication and long-time consuming. Those methods will hinder the progress of the industrialization of bacterial laccase. Secretion of recombinant protein is more conducive to protein folding (Pines and Lnouye, 1999). It can also reduce the production of inclusion bodies (Kane and Hartley, 1988). What's more, extracellular expression can reduce the cost of purification. Researchers usually use the signal peptide to achieve the secretion of recombinant proteins (Movva et al.,

Corresponding author. E-mail addresses: [email protected], [email protected] (Z.-B. Guan).

https://doi.org/10.1016/j.ecoenv.2020.110335 Received 28 December 2019; Received in revised form 10 February 2020; Accepted 13 February 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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parameters were maintained at 37 °C and 150 rpm agitation. The coexpression strains were grown in 50 ml Luria-Bertani medium at 37 °C. Kan (100 mg/ml) was added into the medium. The strains were induced when the optical density (600 nm) reached 0.4. The strains was induced with IPTG (0.2 mM) (Donovan et al., 1996). Meanwhile, CuSO4 (0.25 mM) and ZnCl2 (0.2 mM) were added into the medium in the induction phase. The strains were induced at 15 °C for 24 h. All enzymes were purified by the nickel ion affinity chromatography. The His6.tag was fused to the N-terminal of the CotA-laccase protein. The purification of CotA-laccase SF by Ni+ affinity chromatography was according to method earlier reported (Xu et al., 28AD). The equilibrating nickel column with phosphate buffer (pH 7.0) contains imidazole (20 mM). The CotA-laccase SF was eluted with different gradient elution buffer solutions [phosphate buffer (20 mM, pH 7.0) containing imidazole (0–500 mM)]. The CotA-laccase SF solutions were desalted by Amicon Ultra-15 (Merck, Shanghai, China). The concentration of CotA-laccase SF protein was determined by Bradford Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) (Zor and Selinger, 1996). The approximate molecular weight of enzyme was determined by comparing the data of the enzyme with the protein marker using the SDS-polyacrylamide gel electrophoresis (Laemmli, 1970).

1980). But signal peptide can only direct proteins to the periplasmic space of E. coli (Choi and Lee, 2004). It still needs to extract proteins from the periplasmic space. It would be much easier to harvest protein if the recombinant protein can be secreted into the medium. It has been reported that organophosphorus acid anhydride hydrolase from Pseudoalteromonas sp. SCSIO 04301 can be secreted directly to the extracellular of E. coli without the help of signal peptide (Xiao et al., 2017). Organophosphorus acid anhydride hydrolase has the function of lipase and peptidase (Legler et al., 2014). What's more, it has been reported that expression of cutinase or lipase in E. coli can cause damage to the cell membrane (Su et al., 2013). The cell membrane is composed of phospholipid bilayer. Phospholipase C (PLC, EC 3.1.4.3) is capable of hydrolyzing C3 phosphate bonds and participate in the dissolution of related segments of cell plasma membrane (Koga and Kusaka, 1970). It has been reported that studies have enhanced the extracellular expression of xylanase by co-expressing phospholipase C (Su et al., 2017). CotA-Laccase SF with high catalytic efficiency for ABTS was obtained in the previous research (Xu et al., 28AD). In this study, we tried to co-express CotA-laccase S208G/F227A (SF) with phospholipase C. This is the first time that bacterial laccase and phospholipase have been co-expressed in E. coli to achieve extracellular secretion. The degradation of malachite green by CotA-laccase SF will be studied in detail. 2. Materials and methods

2.5. Enzyme activity assay

2.1. Chemical reagents

Enzyme activity unit (U): the amount of laccase required to oxidize 1 μmol ABTS in 1 min. Enzyme reaction system: mix 2.4 ml of citrate/ phosphate (20 mM, pH 3.5) and 500 μl of ABTS (0.3 mM) in advance, and take a water bath at 50 °C for 5 min. Add 500 μl of CotA-laccase SF solution. Immediately record the reading at 420 nm with a spectrophotometer. The calculation formula of enzyme activity was as follows equation (Eq. (1)):

2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic ac; ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kanamycin Monosulfate (Kan) and Isopropyl β-D-Thiogalactoside (IPTG) were purchased from TaKaRa (Dalian, China). PrimeSTAR Max DNA Polymerase was purchased from TaKaRa (Dalian, China). Malachite Green (MG) was purchased from Chemsky (Shanghai, China). All chemical reagents were analytically pure.

enzyme activity (U⋅L−1) =

2.2. Strains, plasmid

ΔOD × V1 × n Δt × V2 × ε × 10−6

(1)

where ΔOD was difference between two absorbances, V1 was total volume of reaction system, V2 was volume of enzyme solution in the system, n was dilution ratio of enzyme solution, Δt was the reaction time, ε was molar extinction coefficient. All assays were performed at least in triplicate.

The mutant CotA-laccase S208G/F227A (SF) of B. pumilus W3 was constructed by our laboratory (Xu et al., 28AD). The Phospholipase C (PLC) gene (GenBank: MN822915) of B. cereus CP1 was cloned into the pRSFDuet-1 plasmid. The cloning strain E. coli DH5α was preserved in our laboratory. The expression strain E. coli BL21(DE3) was preserved in our laboratory.

2.6. Optimum pH and pH stability

2.3. Construction of co-expression strains

The optimum pH was measured using ABTS as substrate at 50 °C in citrate/phosphate buffer (20 mM, pH 2–6). The purified enzyme was incubated at 4 °C in Britton-Robinson buffer (40 mM, pH 3–12) for 10 h before measuring the pH stability.

Primers were designed and synthesized by GENWIZ (Suzhou, China) (Table S1). Mutant CotA-laccase SF gene was used as template. Mutant CotA-laccase SF gene was cloned with primers (Table S1). PCR products and pRSFDuet-1/PLC plasmids were digested by endonuclease (EcoR Ⅰ and Hind Ⅲ) respectively. Digestion products were connected by T4 ligase at 16 °C overnight. E. coli DH5α was used as host to recombinant plasmid. Transformation of E. coli was according to the classic method of Huff et al. (1990). It used calcium chloride to obtain the competent cell of E. coli. The competent cells were incubated with plasmids in 42 °C for 90 s. It used the plate containing antibiotics (Kan; 30 μg/ml) to screen the transformed strains. Recombinant plasmids were confirmed by sequencing. The correct plasmids were transformed into E. coli BL21 (DE3) to achieve the recombinant expression according to the same method.

2.7. Optimum temperature and thermal stability The optimum temperature of CotA-laccase SF activity was measured at different temperatures (30–90 °C) and citrate/phosphate buffer (20 mM, pH 3.5) using ABTS as substrate. The enzymes were incubated at different temperatures (4–80 °C) and phosphate buffer (20 mM, pH 7.0) for 2 h before determining the temperature stability. 2.8. Effect of metal ions on CotA-laccase SF activity The effects of metal ions on CotA-laccase SF activity were determined in the presence of different metal ions (K+, Ca2+, Mg2+, Fe2+, Cu2+, Mn2+ and Zn2+). The purified CotA-laccases SF were incubated with metal ions (5 mM) for 10 min at 25 °C before measuring the enzyme activity. The enzyme activity was measured using ABTS as substrate at 50 °C in citrate/phosphate buffer (20 mM, pH 3.5).

2.4. Culture conditions and purification The fermentation was performed in an Erlenmeyer flask (250 mL) containing LB medium (50 mL). The initial pH of the fermentation medium was 7.0. The inoculation amount was 10%. The fermentation 2

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purified CotA-laccase SF protein (5 U/ml) and ABTS (0.3 mM). The reaction mixtures with heat-inactivated CotA-laccase SF were the controls.

2.9. Effect of organic solvents and inhibitors on CotA-laccase SF activity The effects of organic solvents and inhibitors on CotA-laccase SF activity were measured using ABTS as substrate at 50 °C in citrate/ phosphate buffer (20 mM, pH 3.5). The purified CotA-laccase SF were incubated with 10% (v/v) organic solvents (ethanol, methanol, acetone and DMSO) or 0.1 mM of inhibitors (SDS, EDTA, NaN3 and L-Cysteine) for 10 min at 25 °C before determining the enzyme activity. The activity of CotA-laccase observed in the absence of any compound was used as control.

2.14. Detoxification of MG The genotoxicity of MG metabolites was assessed by garlic root tip cells. Garlic root tips were cultured in MG solution (100 mg/L MG, 20 mM phosphate buffer, pH 7.0), MG after degradation solution (100 mg/L MG degraded by CotA-laccase SF, 20 mM phosphate buffer, pH 7.0) and control solution (20 mM, pH 7.0) for 24 h. Cultured garlic root tip cells were observed by optical microscope. The micronucleus rate (MCN rate) of root tip cells was used as genotoxicity index. The calculation of MCN rate was based on equation (Eq. (4)):

2.10. Effect of fermentation conditions on CotA-laccase SF extracellular expression In order to increase the extracellular expression of CotA-laccase SF, the growth conditions and induction conditions (initial cell concentration, IPTG concentration, induction temperature, Cu2+ concentration, surfactants and penetrants) were studied respectively. First, different OD600nm (0.2, 0.4, 0.6, 0.8, 1 and 1.2) were studied in the initial culture condition (IPTG 0.2 mM, 15 °C, Cu2+ 0.25 mM). Different conditions such as IPTG concentration (0, 0.2, 0.4, 0.6, 0.8 and 1.2 mM), induction temperature (10, 15, 20, 25, 30 and 35 °C), Cu2+ concentration (0, 0.25, 0.5, 0.75, 1.0 and 1.25 mM) and chemical penetrant (5 g/L of sorbitol, 5 g/L of glycine, 0.1% volume fraction of triton X-100 and 0.1% volume fraction of tween-80) were carried out respectively on the basis of the previous round. The extracellular CotA-laccase SF activity was the standard of optimization.

N MCN rate (‰) = 1000 × ⎛ 1 ⎞ N ⎝ 0⎠ ⎜

3. Results and discussion 3.1. Cloning and expression Mutant CotA-laccase SF gene and PLC gene were successfully cloned into plasmid pRSFDuet-1. The pRSFDuet-SF-PLC plasmid diagram was as follow Fig. 1. Co-expression plasmid (pRSFDuet-SF-PLC) was transferred into E. coli BL21 (DE3) to achieve the recombinant expression. As a convenient host for expressing heterologous protein, E. coli was usually used to express multiple genes. Similar reports showed that Dsb protein was co-expressed in E. coli to improve the production of horseradish peroxidase (Kondo et al., 2000). Co-expression of phospholipase C to promote extracellular expression of glutamate decarboxylase has been reported (Su et al., 2017). Phospholipase C could destroy cell membrane and cause nonspecific leakage. Similarly, nonspecific leakage was used to improve the production of N-glycosylated protein by E. coli (Ding et al., 2019).

The reaction system containing Britton-Robinson buffer (40 mM, pH 3.78–9.15), MG (0.025 mg/ml) and CotA-laccase SF (5 U/mL). ABTS (0.3 mM) was used as redox mediator. The system was incubated at 25 °C (room temperature). The decolorization rate was calculated spectrophotometrically as the relative decrease in absorbance at maximal absorbance wavelength of MG (Koschorreck et al., 2009). The maximal absorbance wavelength of MG was 620 nm. MG decolorization was calculated by following equation (Eq. (2)):

(A0 − A1 ) A0

3.2. Purification of enzymes (2) The molecular weight of CotA-laccase SF was ~65 kDa (Fig. S2). It had been reported that the molecular weight range of laccase was from 23 to 383 kDa (Ramteke and Jain, 2016). The molecular weight of CotA-laccase SF in this experiment is consistent with that of B. subtilis (Nayanashree and Thippeswamy, 2015) and B. cereus (Wang et al., 2016).

where A1 was the absorbance of the reaction system after incubation for a period of times and A0 was the absorbance of the control group after incubation for equal time. The standard curve of malachite green concentration at 620 nm absorbance was determined at pH 7.0, 25 °C (Fig. S1). The equation of malachite green and absorbance was as equation (Eq. (3)):

y = 0.0192x − 0.0153

, R2 = 0.9989

(4)

where N0 was the total number of cells observed and N1 was the number of cells with micronucleus.

2.11. Decolorization of malachite green

Relative decolorization (%) = 100 ×

⎟

(3)

3.3. Effect of pH on enzyme activity and stability

where x was concentration of malachite green and y was absorbance at 620 nm.

The optimal pH values of CotA-laccase SF (intracellular and extracellular) were about 3.5 using ABTS as substrate (Fig. 2a). The CotAlaccase SF (intracellular and extracellular) still had more than 80% residual activity after incubation for 10 h at pH 6–10 (Fig. 2b). Similar results have been found that bacterial laccase was resistant to alkaline environment (Zhao et al., 2008). The difference in activity of crude and pure enzyme towards pH was shown in Fig. S3. As shown in Fig. S3a, the crude CotA-laccase SF had the highest residual activity at pH 7 after being incubated at 4 °C and Britton-Robinson buffer (40 mM, pH 3–12) for 10 h. The pure CotA-laccase SF showed the highest residual activity at pH 9.0 after being incubated at 4 °C and Britton-Robinson buffer (40 mM, pH 3–12) for 10 h.

2.12. Kinetic analysis of malachite green degradation by CotA-Laccase SF The kinetic analysis of CotA-laccase SF was carried out under the following conditions (pH 7, 25 °C) using different concentrations of MG (25–225 mg/L) as substrate. Kinetic parameters were determined by Michaelis-Menten equation and Lineweaver-Burk plot (Gao and Truhlar, 2002). 2.13. Metabolism analysis of MG degradation by CotA-laccase SF The metabolites from MG by CotA-laccase SF were analyzed through liquid chromatography-mass spectrometry (LC-MS) according to the method of López-Gutiérrez et al. (2013). The reaction mixtures were contained 20 mM phosphate buffer (pH 7.0), MG (25 mg/L),

3.4. Effect of temperature on enzyme activity and stability The CotA-laccase SF (intracellular and extracellular) had the same 3

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Fig. 1. The pRSFDuet-SF-PLC plasmid diagram.

Vantamuri and Kaliwal, 2016). Divalent Ferrous ions had a strong inhibition on CotA-laccase SF activity. This result might be related to the competition of substrate ABTS between Ferrous ion and CotA-laccase SF (Wang and Zhan, 2003).

optimal temperature at 80 °C with ABTS as substrate (Fig. 2c). The residual activities of laccases were more than 40% after incubated 2 h at 60 °C (Fig. 2d). High temperature tolerance had always been an advantage of microbial laccase (Joseph et al., 2014). CotA-laccase may be related to the high temperature tolerance of spores (Setlow, 2012). The difference in activity of crude and pure enzymes towards temperature was shown in Fig. S3. As shown in Fig. S3b, the pure CotAlaccase SF remained 73.46% residual activity after being incubated at 50 °C for 2 h. And the crude CotA-laccase SF only remained 57.10% residual activity after incubation at 50 °C for 2 h. The results indicated that crude CotA-laccases were more unstable at 50 °C compared to the pure CotA-laccase.

3.6. Effect of organic solvents and inhibitors on CotA-laccase SF activity The effects of organic solvents on CotA-laccase SF activity were measured with ABTS as substrate (Table S3). Ethanol and methanol had a weak promoting effect on CotA-laccase SF activity. This might be due to the fact that some organic solvents could enhance the solubility of the substrate and improve the conversion of the enzyme to the substrate (Silbiger and Freeman, 1991). Acetone and DMSO had obvious inhibition on CotA-laccase SF activity. CotA-laccase SF could still maintain 80% activity in the presence of 10% (v/v) acetone. CotA-laccase SF had more than 60% activity in the presence of 10% (v/v) DMSO. This indicated that the CotA-laccase SF in this study had good tolerance to acetone and DMSO. It was consistent with the previous studies that laccase LacA from Cerrena sp. HYB07 showed more than 70% relative activity after incubated with 10% DMSO for 4 h at 25 °C (Yang et al.,

3.5. Effect of metal ions on CotA-laccase SF activity The effects of metal ions were measured with ABTS as substrate (Table S2). Potassium and calcium ions had weak inhibition on CotAlaccase SF activity. Magnesium ion, copper ion and manganese ion all had different promoting effects on CotA-laccase SF activity. Similar results have been reported in other laccases (Aslam et al., 2014; 4

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Fig. 2. Enzymatic properties of CotA-laccase SF. Effect of pH on the activity (a) and stability (b) of the purified CotA-laccase SF using ABTS as substrate. The tests of optimum pH were performed at 50 °C in citrate/phosphate buffer (20 mM, pH 2–6). Enzymes were incubated at 4 °C and Britton-Robinson buffer (40 mM, pH 3–12) for 10 h before measuring the pH stability. Effect of temperature on the activity (c) and stability (d) of the purified CotA-laccase SF using ABTS as substrate and citrate/phosphate buffer (20 mM, pH 3.5). Enzymes were incubated at different temperatures (4–80 °C) for 2 h before determining the temperature stability.

over 0.6 mM, the extracellular expression of CotA-laccase SF decreased significantly (Fig. 3b). This might be due to the toxic effect of high concentration IPTG on bacteria (Kosinski et al., 1992). The optimization of induction temperature indicated that 15 °C was the most suitable induction temperature for CotA-laccase SF expression (Fig. 3c). Inclusion bodies were easy to be produced when the induction temperature was high (Qoronfleh et al., 1992). The results of Cu2+ concentration optimization was shown in Fig. 3d. The extracellular expression of CotA-laccase SF was the highest when the concentration of copper ion reached 0.5 mM. It had been shown that the addition of penetrant was beneficial to the secretion of recombinant protein (Tang et al., 2008). The effect of different kinds of penetrants on the extracellular expression of CotA-laccase SF was shown in Fig. 4e. The extracellular expression of glycine added to the culture medium was better than that of other penetrants (Fig. 3e). Adding Triton X-100 and Tween-80 to the medium did not increase the extracellular expression of CotA-laccase SF, which might be due to the toxic effect of Triton X-100 and Tween-80 on cells and proteins (Janas et al., 2003). Glycine was considered to be the mildest chemical penetrant, which could change the permeability of cell membrane (Oren, 2015). In the previous studies, adding glycine to culture medium could promote the secretion of levansucrase in E. coli (Kang et al., 2004). As shown in Fig. 3f, the extracellular expression of CotA-laccase SF reached the maximum when the concentration of glycine reached 10 g/L. Ultimately, the optimal fermentation conditions were determined as initial induced OD600nm ≈ 0.6, IPTG concentration = 0.4 mM, induction temperature = 15 °C, Cu2+ concentration = 0.5 mM and glycine concentration = 10 g/L. The activity of laccase in the cell reached 1382.73 U/L and that of extracellular

2014). The effects of inhibitors on CotA-laccase SF activity were measured with ABTS as substrate (Table S4). SDS, EDTA, NaN3 and L-Cysteine all inhibited CotA-laccase activity in different degrees. CotA-laccase had more than 60% activity after incubated with EDTA at 25 °C for 10 min. This result might be due to the ability of EDTA to diffuse into the protein and chelate copper ions (Grčman et al., 2001). CotA-laccase SF activity was less than 10% after incubated with NaN3 at 25 °C for 10 min. L-Cysteine could also inhibit the activity of CotA-laccase SF by reducing the disulfide bond of protein (Kawasaki et al., 2010). In this study, CotA-laccase SF had more than 80% activity after incubated with L-Cysteine at 25 °C for 10 min. L-Cysteine and NaN3 had complete inhibitory effect on laccase from B. vallismortis fmb-103 in the previously reported (Zhang et al., 2013). 3.7. Effect of fermentation conditions on CotA-laccase SF extracellular expression In order to improve the extracellular expression of CotA-laccase SF, the fermentation conditions were optimized. As shown in Fig. 3a, optimization of initial induced cell concentration was under initial conditions (IPTG 0.2 mM, 15 °C, and Cu2+ 0.25 mM). CotA-laccase SF activity (intracellular and extracellular) reached the highest level when OD600nm was 0.6. The optimization of IPTG concentration was carried out under repair conditions (OD600nm ≈ 0.6, 15 °C, and Cu2+ 0.25 mM). The extracellular expression of CotA-laccase SF increased with the increase of IPTG concentration when the IPTG concentration was in the range of 0–0.4 mM. When the concentration of IPTG was 5

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Fig. 3. Effect of initial cell concentration (OD600nm) on expression of extracellular CotA-laccase SF (a). Effect of IPTG concentration on expression of extracellular CotA-laccase SF (b). Effect of induction temperature on expression of extracellular CotA-laccase SF (c). Effect of Cu2+ concentration on expression of extracellular CotA-laccase SF (d). Effect of Chemical penetrants on expression of extracellular CotA-laccase SF (e). Effect of glycine concentration on expression of extracellular CotA-laccase SF (f).

3.8. Decolorization of malachite green

reached 1257.22 U/L under the optimal fermentation conditions. The extracellular expression after optimization was 5.48 times higher than that before optimization. The laccase from B. amyloliquefaciens secreted and expressed by Pichia pastoris was reported to be 379.7 U/L (Chen et al., 2015). The reported extracellular expression of CotA-laccase from B. subtilis PCK in E. coli reached 16 IU/ml (Kumar et al., 2015). These results showed that the expression of CotA-laccase SF had a competitive advantage over other laccase's expression.

The effect of pH on the decolorization efficiency was determined at 25 °C for 1 h (Fig. 4). CotA-laccase SF and ABTS system had the best decolorization effect on MG at pH 7.0. Study on the tolerance of CotAlaccase SF to MG was carried out at pH 7.0 and 25 °C. ABTS was used as substrate to detect the residual enzyme activity. CotA-laccase SF still had more than 60% activity after incubated with MG solution (500 mg/ L, pH 7.0) at 25 °C for 5 h (Fig. S4). MG is considered as one of the most difficult environmental pollutants to degrade (Mohanmmad et al., 2018). MG mainly exists in aquaculture wastewater and river water 6

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Fig. 5. Full wavelength scanning of MG reaction mixture. (a) The degradation sample containing with phosphate buffer (pH 7.0), MG (100 mg/L) and CotAlaccase SF (5 U/ml). (b) The control sample containing with phosphate buffer (pH 7.0), MG (100 mg/L) and heat-inactivated CotA-laccase SF. Samples were reacted at 25 °C for 5 h.

Fig. 4. Effect of pH on CotA-laccase SF decolorizing malachite green. The tests of decolorization were performed in Britton-Robinson buffer (40 mM, pH 3.78–9.15) at 25 °C using ABTS (0.3 mM) as a mediator or without ABTS. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The degradation rate of MG by CotA-laccase SF increases with the increase of MG concentration when the MG concentration was under 175 mg/L (Fig. S5). The Vmax of MG degradation by CotA-laccase SF was determined to be 11.94 μmolˑL−1s−1. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s−1. Biological decolorization of MG by Dietzia maris NIT-D was reported the Km of 1317.5 mg/L and Vmax of 166.67 mg dye g cell−1h−1 (Bera et al., 2016). The Vmax and Km of MG by microalgae Cosmarium sp. was found to be 7.63 mg dye g cell−1h−1 (Daneshvar et al., 2007). It was reported that the Km and kcat values of MG by laccase from white-rot fungus Cerrena sp. were 781.9 mM and 9.5 s−1 (Yang et al., 2015). The Km value of MG by CotA-laccase SF was less than that by laccase from white-rot fungus Cerrena sp. The kcat value of MG by CotA-laccase SF was higher than that by laccase from white-rot fungus Cerrena sp. Those results showed that CotA-laccase SF had higher affinity and catalytic efficiency to MG than laccase from white-rot fungus Cerrena sp.

212, 198, 138 and 110 m/z were corresponded to the mass of 4-(Dimethylamino)benzophenone, (4-(methylamino)phenyl)(phenyl)methanone, 4-aminobenzophenone, 4-(dimethylamino)phenol and 4-Aminophenol (Fig. 6b–f). The carbon-carbon bond in the core region of MG might be first broken by CotA-laccase SF to form 4-(Dimethylamino) benzophenone and 4-(dimethylamino)phenol. The detection of 4-(Dimethylamino)benzophenone as an intermediate was consistent with the study of MG biodegradation by Pseudomonas sp. DY1 (Du et al., 2011). Similar results had been reported in the photocatalytic degradation of MG by biomimetic photocatalyst HMS-FePcs (Gao et al., 2008). (4(methylamino)phenyl)(phenyl)methanone, 4-aminophenol and 4-aminobenzophenone detected during the reaction indicated that multiple demethylation of 4-(Dimethylamino)benzophenone and 4-(dimethylamino)phenol by CotA-laccase SF occurred. The degradation of MG accompanied by demethylation has been confirmed by many studies (Du et al., 2013; Ju et al., 2009). It was also reported in previous studies that laccase from Galerina sp. HC1 had potential for lignin demethylation (Ibrahim et al., 2011). Laccase existing effects on N-demethylation of Basic Green 4 was confirmed by the work of Chhabra et al. (2009). MG could be degraded by many microorganisms, but there were few reports on enzyme degradation of MG (Vijayalakshmidevi and Muthukumar, 2014). This was the first detailed study on metabolites of MG degradation by bacterial CotA-laccase. The proposed metabolic pathways of MG by CotA-laccase SF were shown in Fig. 7.

3.10. Metabolism analysis of MG degradation by CotA-laccase SF

3.11. Detoxification of MG

The full wavelength scans of the MG degradation mixture by CotAlaccase SF were performed in Fig. 5. The control sample (MG with heatinactivated CotA-laccase SF) showed maximum absorption at 620 nm. While the degradation sample (MG with CotA-laccase SF) did not show maximum absorption at 620 nm. The disappearance of the visible-color of the MG also indicated that the related chromophore groups of the MG were destroyed (Bai et al., 2013). In order to identify the metabolites, LC-MS was adopted to analysis the degradation sample. The chromatogram of control sample (MG with heat-inactivated CotA-laccase SF) had a conspicuous peak at 5.8–6 min (Fig. S6a). The chromatogram of the degradation sample (MG with CotA-laccase SF) showed a new peak at 5.5–5.6 min (Fig. S6b). The LCMS chromatogram profiles were performed in Fig. 6. The largest peak of control sample performed a mass of 329 m/z, which corresponding to the mass of MG (Fig. 6a). Intermediate metabolites detected at 226,

Micronucleus were formed by the loss of centromere during anaphase of mitosis (Çelik and Aslantürk, 2009). Harmful factors in the environment could lead to the production of micronucleus. Therefore, micronucleus test was often used to detect genotoxicity. The micronucleus test results of garlic root tip cells showed that the MCN rate of root tip cells cultured with MG solution was significantly higher than that cultured with water (Table S5). The MCN rate of root tip cells cultured with MG solution was 46.3‰. By contrast, the MCN rate of root tip cells cultured with MG after degradation solution was 19.50‰. Those results showed that the genotoxicity of MG degraded by CotAlaccase SF decreased. It had been reported that laccase could degrade and detoxify some simple triphenylmethane dyes such as Basic Red 9 (Abadulla et al., 2000). It was correlated with the previous reported that decolorization and detoxification of MG by P. aeruginosa NCIM 2074 (Kalyani et al., 2012). Similar plant toxicology experiments also

whose pH were about 7.0–8.7 (Blume et al., 2010). In this study, CotAlaccase SF can decolorize MG more than 70% at pH 7.0–9.0. This showed that CotA-laccase SF had a good application prospect in the removal of MG from water. 3.9. Kinetic analysis of malachite green degradation by CotA-laccase

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Fig. 6. Identification of metabolites of MG by LC-MS. (a) Malachite green (m/z 329), (b) 4-(Dimethylamino)benzophenone (m/z 226), (c) (4-(methylamino)phenyl) (phenyl)methanone (m/z 212), (d) 4-aminobenzophenone (m/z 198), (e) 4-(dimethylamino)phenol (m/z 138), 4-Aminophenol (m/z 110).

CRediT authorship contribution statement

showed that toxicity reduction of biodegradation products of MG (Ayed et al., 2010; Saravanakumar et al., 2013).

Kai-Zhong Xu: Methodology, Writing - original draft, Data curation, Software. Hui Ma: Software, Validation, Visualization, Investigation. Ya-Jing Wang: Software, Formal analysis. Yu-Jie Cai: Project administration. Xiang-Ru Liao: Supervision, Resources. ZhengBing Guan: Conceptualization, Writing - review & editing, Funding acquisition.

4. Conclusions In this study, CotA-laccase was successfully expressed in E. coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase reached 1257.22 U/L. The results of analysis of MG degradation products by LC-MS showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotAlaccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31472003), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu province 8

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Fig. 7. Proposed mechanism for MG degradation by CotA-laccase SF based on mass spectral analysis.

“Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.

References Abadulla, E., Tzanov, S., Costa, K.H., et al., 2000. Decolorization and detoxification of textile dyes with laccase from Trametes hirsuta. Appl. Environ. Microbiol. 66, 3357–3362. Andersen, W.C., Casey, C.R., Nickel, T.J., et al., 2018. Dye residue analysis in raw and processed aquaculture products: matrix extension of AOAC INTERNATIONAL official method SM 2012.25. J. AOAC Int. 101, 1927–1939. Aslam, M., Aishy, A., Samra, Z., et al., 2014. Identification, purification and characterization of a novel extracellular laccase from cladosporium cladosporioides. Biotechnol. Biotechnol. Equip. 26, 3345–3350. Ayed, L., Chaieb, K., Cheref, A., et al., 2010. Biodegradation and decolorization of triphenylmethane dyes by Staphylococcus epidermidis. Desalination 260, 137–146.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110335. 9

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Laemmli, B., 1970. Cleavage of structural proteins during assembly of head of Bacteriophage-T4. Nature 227, 680–685. Legler, P.M., Boisvert, S.M., Compton, J.R., et al., 2014. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase. Frontiers in Chemistry 2, 46. Ling, Y.Y., Mohd Suah, F.B., 2017. Extraction of malachite green from wastewater by using polymer inclusion membrane. Journal of Environmental Chemical Engineering 5, 785–794. López-Gutiérrez, N., Romero-González, R., Plaza-Bolaños, P., et al., 2013. Simultaneous and fast determination of malachite green, leucomalachite green, crystal violet, and brilliant green in seafood by ultrahigh performance liquid chromatography–tandem mass spectrometry. Food Analytical Methods 6, 406–414. Lu, M., Zhang, Y, Li, B., et al., 2018. Enhanced visible-light photocatalytic decomposition of malachite green over squaraines sensitized SnIn4S8 composites and effect of dye structure on photocatalytic behaviors. J. Mater. Sci. Mater. Electron. 29, 12856–12870. Mohanmmad, M., Maitra, S., Dutta, B., 2018. Comparison of activated carbon and physic seed hull for the removal of malachite green dye from aqueous solution. Water Air Soil Pollut. 229, 445. Movva, N.R., Nakamura, K., Inouye, M., 1980. Amino acid sequence of the signal peptide of ompA protein, a major outer membrane protein of Escherichia coli. J. Biol. Chem. 255, 27–29. Nayanashree, G., Thippeswamy, B., 2015. Biodegradation of natural rubber by laccase and manganese peroxidase enzyme of Bacillus subtilis. Environmental Processes 2, 761–772. Oren, A., 2015. Cyanobacteria in hypersaline environments: biodiversity and physiological properties. Biodivers. Conserv. 24, 781–798. Pines, O., Lnouye, M., 1999. Expression and secretion of proteins in E. coli. Mol. Biotechnol. 12, 25–34. Qoronfleh, M.W., Debouck, C., Keller, J., 1992. Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli. J. Bacteriol. 174, 7902–7909. Ramteke, P.W., Jain, N., 2016. Microbial laccase and its applications in bioremediation. Current Biochemical Engineering 3, 110–121. Samak, N.A., Hu, J., Wang, K., et al., 2018. Development of a novel micro-aerobic cultivation strategy for high potential CotA laccase production. Waste and Biomass Valorization 9, 369–377. Saravanakumar, T., Palvannan, T., Kim, D.-H., et al., 2013. Manganese peroxidase H4 isozyme mediated degradation and detoxification of triarylmethane dye malachite green: optimization of decolorization by response surface methodology. Appl. Biochem. Biotechnol. 171, 1178–1193. Setlow, P., 2012. Dynamics of the assembly of a complex macromolecular structure - the coat of spores of the bacterium Bacillus subtilis. Mol. Microbiol. 83, 241–244. Silbiger, E., Freeman, A., 1991. Continuous Δ1 -hydrocortisone dehydrogenation with in situ product recovery. Enzym. Microb. Technol. 13, 869–872. Stammati, A., Nebbia, C., Angelis, I., et al., 2005. Effects of malachite green (MG) and its major metabolite, leucomalachite green (LMG), in two human cell lines. Toxicol. Vitro 19, 853–858. Su, L., Woodard, R.W., Chen, J., et al., 2013. Extracellular location of thermobifida fusca cutinase expressed in Escherichia coli BL21(DE3) without mediation of a signal peptide. Appl. Environ. Microbiol. 79, 4192–4198. Su, L., Jiang, Q., Yu, L., et al., 2017. Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C. Microb. Cell Factories 16, 24. Tang, J.B., Yang, H-m., Song, S-l., et al., 2008. Effect of Glycine and Triton X-100 on secretion and expression of ZZ–EGFP fusion protein. Food Chem. 108, 657–662. Tanji, Y., Asami, K., Xing, X.-H., et al., 1998. Controlled expression of lysis genes encoded in T4 phage for the gentle disruption of Escherichia coli cells. J. Ferment. Bioeng. 85, 74–78. Vantamuri, A., Kaliwal, B., 2016. Purification and characterization of laccase from Marasmius species BBKAV79 and effective decolorization of selected textile dyes. 3 Biotech 6, 189. Vijayalakshmidevi, S.R., Muthukumar, K., 2014. Biodegradation of malachite green by Ochrobactrum sp. World J. Microbiol. Biotechnol. 30, 429–437. Wang, X.W., Zhan, H.Y., 2003. Effects of Fe2+ ion on the catalytic oxidation by laccase. Biotechnology 13, 13–14. https://doi.org/10.3969/j.issn.1004-311X.2003.03.009. Wang, B., Yan, Y., Tian, Y., et al., 2016. Heterologous expression and characterisation of a laccase from Colletotrichum lagenarium and decolourisation of different synthetic dyes. World J. Microbiol. Biotechnol. 32, 40. Xavier, A., Usha, D., Rajan, J.G., et al., 2011. Studies on the removal of malachite green dyes by adsorption onto activated carbons – a comparative study. Mater. Sci. Forum 671, 165–186. Xiao, Y., Yang, J., Zhang, S., et al., 2017. Expression of a marine bacterial organophosphorus acid anhydrolase in Escherichia coli. Chinese journal of Bioprocess Engineering 15, 1–6. https://doi.org/10.3969/j.issn.1672-3678.2017.03.001. Xu, K.Z., Wang, H.R., Wang, Y.J., et al., 28 October 2019. Enhancement in catalytic activity of CotA-laccase from Bacillus pumilus W3 via site-directed mutagenesis. J. Biosci. Bioeng. https://doi.org/10.1016/j.jbiosc.2019.09.020. In press, Corrected Proof, Available online. Yang, J., Lin, Q., Ng, T.B., et al., 2014. Purification and characterization of a novel laccase from Cerrena sp. HYB07 with dye decolorizing ability. PloS One 9, e110834. Yang, J., Yang, X., Lin, Y., et al., 2015. Laccase-Catalyzed decolorization of malachite green: performance optimization and degradation mechanism. PloS One 10, e0127714. Yang, X., Zheng, J., Lu, Y.-M., et al., 2016. Degradation and detoxification of the triphenylmethane dye malachite green catalyzed by crude manganese peroxidase from

Bai, C., Xiao, W., Feng, D., et al., 2013. Efficient decolorization of Malachite Green in the Fenton reaction catalyzed by [Fe(III)-salen]Cl complex. Chem. Eng. J. 215, 227–234. Bera, S., Sharma, V., Dutta, S., et al., 2016. Biological decolorization and detoxification of malachite green from aqueous solution by Dietzia maris NIT-D. Journal of the Taiwan Institute of Chemical Engineers 67, 271–284. Blume, K.K., Macedo, J.C., Meneguzzi, A., et al., 2010. Water quality assessment of the sinos river, southern Brazil. Braz. J. Biol. 70, 1185–1193. Çelik, T., Aslantürk, Ö., 2009. Investigation of cytotoxic and genotoxic effects of Ecballium elaterium juice based on Allium test. Methods Find. Exp. Clin. Pharmacol. 31, 591–596. Chen, B., Xu, W.Q., Pan, X.R., et al., 2015. A novel non-blue laccase from Bacillus amyloliquefaciens: secretory expression and characterization. Int. J. Biol. Macromol. 76, 39–44. Chhabra, M., Mishra, S., Sreekrishnan, T., 2009. Laccase/mediator assisted degradation of triarylmethane dyes in a continuous membrane reactor. J. Biotechnol. 143, 69–78. Choi, J.H., Lee, S.Y., 2004. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl. Microbiol. Biotechnol. 64, 625–635. Daneshvar, N., Ayazloo, M., Khataee, A.R., et al., 2007. Biological decolorization of dye solution containing Malachite Green by microalgae Cosmarium sp. Bioresour. Technol. 98, 1176–1182. Ding, N., Ruan, Y., Fu, X., et al., 2019. Improving production of N-glycosylated recombinant proteins by leaky Escherichia coli. 3 Biotech 9, 302. Donovan, R.S., Robinson, C.W., Glick, B.R., 1996. Review: optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter. J. Ind. Microbiol. 16, 145–154. Du, L.N., Wang, S., Li, G., et al., 2011. Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity. Ecotoxicology 20 (2), 438–446. Du, L.-N., Zhao, M., Li, G., et al., 2013. Biodegradation of malachite green by Micrococcus sp. strain BD15: biodegradation pathway and enzyme analysis. Int. Biodeterior. Biodegrad. 78, 108–116. Ellappan, K., Thayumanavan, P., 2015. Efficiency of carbohydrate additives on the stability of horseradish peroxidase (HRP): HRP-catalyzed removal of phenol and malachite green decolorization from wastewater. Clean 43, 846–856. Gao, J., Truhlar, D.G., 2002. Quantum mechanical methods for enzyme kinetics. Annu. Rev. Phys. Chem. 53, 467–505. Gao, G., Zhang, A., Zhang, M., et al., 2008. Photocatalytic degradation mechanism of malachite green under visible light irradiation over novel biomimetic photocatalyst HMS-FePcs. Chin. J. Catal. 29, 426–430. Grčman, H., Velikonja-Bolta, Š., Vodnik, D., et al., 2001. EDTA enhanced heavy metal phytoextraction: metal accumulation, leaching and toxicity. Plant Soil 235, 105–114. Harrison, S.T., 1991. Bacterial cell disruption: a key unit operation in the recovery of intracellular products. Biotechnol. Adv. 9, 217–240. Huff, J., Grant, B., Penning, C., et al., 1990. Optimization of routine transformation of Escherichia coli with plasmid DNA. Biotechniques 9, 570–577. Ibrahim, V., Mendoza, L., Mamo, G., et al., 2011. Blue laccase from Galerina sp.: properties and potential for Kraft lignin demethylation. Process Biochem. 46, 379–384. Janas, P., Gustaw, W., Mleko, S., et al., 2003. Effect of detergents on xanthan production during batch and continuous cultivation of Xanthomonas campestris NRRL B-1459. Acta Scientiarum Polonorum Technologia Alimentariz 2, 125–133. Joseph, B., Kuddus, M., Ramteke, P., 2014. Extracellular alkaline thermostable laccase from micrococcus species: partial purification and characterization. Curr. Biotechnol. 3, 145–151. Ju, Y., Yang, S., Ding, Y., et al., 2009. Microwave-enhanced H2O2-based process for treating aqueous malachite green solutions: intermediates and degradation mechanism. J. Hazard Mater. 171, 123–132. Kalyani, D.C., Telke, A.A., Surwase, S.N., et al., 2012. Effectual decolorization and detoxification of triphenylmethane dye malachite green (MG) by Pseudomonas aeruginosa NCIM 2074 and its enzyme system. Clean Technol. Environ. Policy 14, 989–1001. Kane, J.F., Hartley, D.L., 1988. Formation of recombinant protein inclusion bodies in Escherichia coli. Trends Biotechnol. 6, 95–101. Kang, S.A., Lee, J.C., Park, Y., et al., 2004. Secretory production of Rahnella aquatilis ATCC 33071 levansucrase expressed in Escherichia coli. J. Microbiol. Biotechnol. 14, 1232–1238. Kawasaki, M., Miura, Y., Yagasaki, K., 2010. Effects of sulfur amino acids, L-methionine, L-cystine and L-cysteine on lipoprotein lipase and hormone-sensitive lipase in differentiated mouse 3T3-L1 adipocytes. Cytotechnology 62, 225–233. Koga, Y., Kusaka, I., 1970. Involvement of intracellular phospholipase C in autolytic fragmentation of cytoplasmic membranes of Bacillus cereus. Eur. J. Biochem. 16, 407–413. Kondo, A., Kohda, J., Endo, Y., et al., 2000. Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins. J. Biosci. Bioeng. 90, 600–606. Koschorreck, K., Schmid, R.D., Urlacher, V.B., 2009. Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol. 9, 12. Kosinski, M.J., Rinas, U., Bailey, J.E., 1992. Isopropyl-β-D-thiogalactopyranoside influences the metabolism of Escherichia coli. Appl. Microbiol. Biotechnol. 36, 782–784. Kumar, S., Jain, K., Bharadwaj, K., et al., 2015. Multiple genes in a single host: costeffective production of bacterial laccase (cotA), pectate lyase (pel), and endoxylanase (xyl) by simultaneous expression and cloning in single vector in E. coli. PloS One 10, e0144379. Kusvuran, E., Gulnaz, O., Samil, A., et al., 2011. Decolorization of malachite green, decolorization kinetics and stoichiometry of ozone-malachite green and removal of antibacterial activity with ozonation processes. J. Hazard Mater. 186, 133–143.

10

Ecotoxicology and Environmental Safety 193 (2020) 110335

K.-Z. Xu, et al.

5468–5473. Zhao, M., Wei, X.D., Wang, C.L., et al., 2008. Research progress of bacterial laccase. Trans. China Pulp Pap. 23, 107–114. https://doi.org/10.3321/j.issn:1000-6842. 2008.03.025. Zor, T., Selinger, Z., 1996. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236, 302–308.

Irpex lacteus F17. Environ. Sci. Pollut. Control Ser. 23, 9585–9597. Zhang, X., Zhang, S., Pan, B., et al., 2012. Simple fabrication of polymer-based Trametes versicolor laccase for decolorization of malachite green. Bioresour. Technol. 115, 16–20. Zhang, C., Zhang, S., Diao, H., et al., 2013. Purification and characterization of a temperature- and pH-stable laccase from the spores of Bacillus vallismortis fmb-103 and its application in the degradation of malachite green. J. Agric. Food Chem. 61,

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