The heterologous expression, characterization, and application of a novel laccase from Bacillus velezensis

Journal Pre-proof The heterologous expression, characterization, and application of a novel laccase from Bacillus velezensis

Tao Li, Lin Huang, Yanzhen Li, Zehua Xu, Xiuqi Ge, Yuanfu Zhang, Nan Wang, Shuang Wang, Wei Yang, Fuping Lu, Yihan Liu PII:

S0048-9697(20)30223-0

DOI:

https://doi.org/10.1016/j.scitotenv.2020.136713

Reference:

STOTEN 136713

To appear in:

Science of the Total Environment

Received date:

24 September 2019

Revised date:

11 January 2020

Accepted date:

13 January 2020

Please cite this article as: T. Li, L. Huang, Y. Li, et al., The heterologous expression, characterization, and application of a novel laccase from Bacillus velezensis, Science of the Total Environment (2020), https://doi.org/10.1016/j.scitotenv.2020.136713

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© 2020 Published by Elsevier.

The heterologous expression, characterization, Journal Pre-proof and application of a novel laccase from Bacillus velezensis Tao Lia, b1, Lin Huanga1, Yanzhen Lia, Zehua Xua, Xiuqi Gea, Yuanfu Zhanga, Nan Wanga, Shuang Wanga, Wei Yangb, Fuping Lua, Yihan Liua,* a

Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial

Microbiology, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China b

College of Basic Science, Tianjin Agricultural University, Tianjin 300384, P. R. China

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Tao Li and Lin Huang contributed equally to this work.

* Correspondence: [email protected] (Y. H. Liu). Tel.: +86 022 60601958; Fax: +86 022 60602298.

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Postal address of corresponding author:

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No.29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin 300457, P. R. China

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ABSTRACT

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Laccases have a huge potential in numerous environmental and industrial applications due to the ability to oxidized a wide range of substrates. Here, a novel laccase gene from the identified Bacillus velezensis TCCC 111904 was heterologously expressed in Escherichia coli. The optimal temperature and pH for oxidation by recombinant laccase (rLac) were 80 °C and 5.5, respectively, in the case of the substrate 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 80 °C and 7.0, respectively, in the case of 2,6-dimethoxyphenol (2,6-DMP). rLac exhibited high thermostability and pH stability over a wide range (pH 3.0, 7.0, and 9.0). Additionally, most of the metal ions did not inhibit the activity of rLac significantly. rLac showed great tolerance against high concentration of NaCl, and 50.8% of its initial activity remained in the reaction system containing 500 mM NaCl compared to the control. Moreover, rLac showed a high efficiency in decolorizing different types of dyes including azo, anthraquinonic, and triphenylmethane dyes at a high temperature (60 °C) and over an extensive pH range (pH 5.5, 7.0, and 9.0).

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These unique characteristics of rLac indicated that it could be a potential candidate for applications in treatment of dye effluents

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and other industrial processes.

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Keywords: Laccase, Bacillus velezensis, Enzymatic characterization, Decolorization, Dye effluents

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1. Introduction

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Nowadays, a substantial amount of synthetic dyes with varied structures including anthraquinone, azo, heterocyclic, and triphenylmethane dyes are widely applied in the textile industry, as they have certain advantages over natural pigments, such as fading resistance against exposures to light, chemicals, and water (Abe et al., 2019; Pereira et al., 2019; Wang et al., 2012). The textile industries produce a large amount of wastewater containing spent dyes and some toxic and mutagenic/carcinogenic chemicals every day (Dawkar et al., 2008; Dutta et al., 2020). Millions of liters of untreated dye effluents are discharged into the water bodies in some underdeveloped areas because removal of these dyes is difficult and expensive; this results in severe damage to the aquatic ecosystems for the intense coloration of the water bodies, which reduces the light penetration efficiency and dissolved oxygen levels in the water (Abe et al., 2018; Dawkar et al., 2008; Lu et al., 2012a; Olukanni et al., 2013). Physicochemical processes such as coagulation, adsorption, ozonation, Fenton reaction, etc., have been employed for the

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degradation and removal of dyes from the effluents of textile industries for decades. However, these conventional methods have

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some drawbacks, such as high treatment cost, addition of hazardous chemical additives, complex procedures, and possible secondary pollution to the environment (Robinson et al., 2001; Saratale et al., 2011; Shanmugam et al., 2019). By contrast, the

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enzymatic decolorization method has attracted increasing attention as it has several advantages in the treatment of dye effluents

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over the traditional physicochemical methods, such as lower cost, higher efficiency, simple and eco-friendly procedures, and lower energy requirement (Husain, 2010; Imran et al., 2019; Robinson et al., 2001; Saratale et al., 2011).

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Laccases, a group of copper-containing enzymes that can oxidize numerous substrates, such as aromatic amines, phenolic compounds, lignins, aryl diamines, and some inorganic ions, were first identified and isolated from Rhus vernicifera and have

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been reported to be extensively distributed in bacteria, fungi, and higher plants (Brenelli et al., 2019; Forootanfar and Faramarzi, 2015; Lawrance et al., 2019; Muñoz et al., 1997; Nejad et al., 2019; Trejo-Hernandez et al., 2001; Yaropolov et al., 1994). The

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copper centers in the enzyme’s active site mediate the transfer of four single-electrons from the substrate to two oxygen molecules, thus generating water molecules (Enguita et al., 2004). When laccase oxidizes non-phenolic compounds (e.g. lignin), mediators including ABTS and HBT (1-hydroxybenzotriazole) that act as electron carriers should be added to the redox reaction system mediated by this enzyme due to its relatively low redox potential (Christopher et al., 2014; Munk et al., 2015; Riva, 2006; Siroosi et al., 2018). In the past decades, laccases have been widely applied in diverse industries, such as food industry, textile industry, polymer grafting, laccase-mediated polymer synthesis, and cosmetic and pharmaceutical industries (Bilal et al., 2019; Nemadziva et al., 2018; Pezzella et al., 2015; Slagman et al., 2018). Recently, more and more laccases have been continuously investigated to expand their applications in some industrial sectors that employ harsh conditions, such as high alkaline and temperature conditions (Ausec et al., 2015; Chauhan et al., 2017; Hildén et al., 2009; Mathews et al., 2016; Uthandi et al., 2012). For example, laccases have been proven to effectively degrade dyes used in textile industry, such as anthraquinonic, triarylmethane, azo, and indigoid dyes (Du et al., 2020; Nejad et al., 2019; Rodríguez and Toca Herrera, 2006; Zhuo et al., 2019). Lignolytic fungi, also called white-rot fungi, are the most comprehensively studied laccase-producing microorganisms, and they have been widely employed in the industrial production of laccase due to their striking features, such as low cost of 3

cultivation, extracellular secretion of laccase, and high degradation ability towards lots of xenobiotic compounds (Ikehata et al.,

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2004; Shah and Nerud, 2002). However, the optimal activity of fungal laccases is commonly observed in an environment with low pH and temperature. This hinders the application of fungal laccases in the treatment of textile dye effluents, which typically have high temperatures and a wide pH range. On the contrary, bacterial laccases could function at high temperatures and a wide pH range (Guo et al., 2017; Kumar et al., 2016; Lončar et al., 2016; Sharma et al., 2007). Moreover, the stability of most bacterial laccases is considered to be better than fungal laccases. Additionally, the influence of metal ions on the activities of bacterial laccases is relatively less; indeed, bacterial laccases are not easily influenced by some of the inhibitors (Safary et al., 2016; Sharma et al., 2007; Singh et al., 2011). Bacillus species, which are ubiquitous in nature and able to survive harsh environments including high pH, temperature, and salt concentration, are favored by researchers for obtaining laccases with high activities under harsh conditions, such as high alkaline conditions or high temperatures. A novel laccase derived from Bacillus tequilensis SN4 was

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considered to be highly suitable for industrial applications under extreme conditions due to its thermo-alkali-stable properties

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(Sondhi et al., 2014). A non-blue laccase gene identified in B. amyloliquefaciens was heterologously expressed in Pichia pastoris successfully. This novel enzyme was stable at pH 9.0 for 10 days and remained activated in 200 mM salt solution (Chen et al.,

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2015). Therefore bacterial laccases are apparently more suitable for decolorizing industrial textile dye effluents (Chauhan et al.,

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2017; Sharma et al., 2007).

The objective of this study was to find a novel laccase with high activity under high temperatures and over a broad pH range

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for decolorization of synthetic dyes with a high efficiency. Here, we screened a strain with laccase activity from the forest soil of

investigated.

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2. Materials and methods

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Hainan Island of China. This enzyme was heterologously expressed in Escherichia coli and its characteristics were further

2.1 Strains, vectors, and reagents

E. coli JM109, E. coli BL21 (DE3), and vector pET-22b (+) were conserved in our laboratory. ABTS, 2,6-DMP, azophloxine, Congo red, adizol black B, reactive blue 5, reactive blue 19, crystal violet, indigo carmine, and malachite green were provided by Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes, Pyrobest DNA Polymerase, T4 DNA ligase, pMD18-T vector cloning Kit, DNA Extraction Kit, Plasmid Mini Kit, and Gel Extraction and Purification Kit were ordered from TaKaRa Bio Inc. (Dalian, China). Other chemicals obtained from local suppliers were of analytical grade. 2.2 Strain screening and cultivation conditions The laccase-active strain was obtained from the forest soil of Hainan Island of China. The strain was screened by following the procedure used for the isolation of Klebsiella pneumoniae in our previous report, producing a novel pH-stable laccase (Liu et al., 2017). Briefly, soil sample (10 g) was dispersed in sterile saline solution (100 mL, 0.9% NaCl). Then, the soil suspension (1 mL) was thoroughly mixed with 5 mL Luria-Bertani (LB) liquid medium (yeast extract 5 g/L, tryptone 10 g/L, and NaCl 10 g/L). The enriched cells were serially diluted with sterile saline water and incubated at 37 °C for 24 h on a LB agar plate containing 0.2 mM Cu2+ (copper sulfate). After adding several drops of 0.1% (w/v) syringaldazine (SGZ)/ethanol (absolute) to the bacterial colonies, the colonies positive for laccase activity were picked by observing the appearance of pink color (Wang et al., 2010). The 4

positive colonies were further purified using LB-Cu2+ agar plate, and the colony demonstrating the darkest pure pink color was

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picked and identified by measuring the laccase activity. 2.3 Identification and phylogenetic analysis of the laccase producing strain Total DNA of the isolated cells with laccase activity was extracted to amplify the 16S rDNA by polymerase chain reaction (PCR) using two bacterial universal primers: F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and R: 5′-GGTTACCTTGTTACGACTT3′ (Suzuki and Giovannoni, 1996). The resulting PCR fragments were inserted into the pMD18-T vector. E. coli DH5α competent cells were then transformed with this vector. Afterwards, the 16S rDNA of the positive transformant was sequenced by BGI Company (Beijing, China). The isolated strain was first identified by aligning the sequence of the 16S rDNA with data deposited in the National Center for Biotechnology Information (NCBI) database, and performing phylogenetic analysis by constructing a bootstrap consensus tree

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using the neighbor-joining method with MEGA 6.0 software (Tamura et al., 2013). Further taxonomic analysis was performed

et al., 1994).

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2.4 Cloning and homologous expression of the laccase gene

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according to the instructions provided in Bergey’s Manual of Determinative Bacteriology to confirm the strain identification (Holt

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The full laccase gene was amplified from the genomic DNA using PCR technology with the primers 5′CGCGGATCCGATGGCACTGGAAAAATTTG-3′ (forward primer) and 5′-ACGCGTCGACCTGCTTATCCGTGACGTCC-3′

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(reverse primer) with BamHI and SalI sites (underlined). After double digestion with BamHI and SalI, it was inserted into the BamHI-SalI-linearized pET-22b (+) to construct the plasmid pET-lac. After transforming pET-lac into E. coli BL21 (DE3), the

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recombinant laccase (rLac) with a 6 × His-tag was heterologously expressed in E. coli BL21. Briefly, a positive colony was initially grown at 37 °C for 12 h in 5 mL of LB medium supplemented with ampicillin (100 g/mL) on a rotary shaker with

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constant shaking speed of 220 rpm. Then the starting culture (1 mL) was pipetted into 50 mL LB medium, and incubated until the optical density at 600 nm was 0.6 to 0.8 under the above conditions. Afterwards, the culture was supplied with 1 mM isopropyl-βD-1-thiogalactopyranoside (IPTG) to induce the expression of rLac for 20 h at 16 °C. Meanwhile, E. coli BL21 cells containing the empty plasmid pET-22b (+) were used as control. 2.5 Structure modeling of rLac The 3-D homology model of rLac was built using the Swiss-Model server (http://swissmodel.expasy.org/) and the PyMOL molecular graphic system with the crystal structure of B. subtilis laccase (PDB ID: 2WSD) as the template. 2.6 Purification of rLac The centrifugally (8000 × g, 4 °C, 15 min) collected cells were resuspended in 20 mM Tris–HCl buffer (pH 7.0) containing 20 mM imidazole and 500 mM NaCl and subsequently lysed by sonication in ice bath at 320 W with 4 s strokes and 3 s intervals. The supernatant containing rLac was collected after centrifugation (12,000 × g, 30 min, 4 °C), and injected into a nickelnitrilotriacetic acid (Ni-NTA) agarose gel column (Shenggong, Shanghai, China), which was preequilibrated with 50 mM TrisHCl buffer (pH 7.0). rLac was washed out using elution buffer (20 mM Tris-HCl, 500 mM imidazole and 500 mM NaCl, pH 7.0).

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Finally, the purified rLac was dialyzed and sterilized by filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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(SDS-PAGE) was used to evaluate the molecular mass and purity of rLac. 2.7 rLac activity assay The activity of rLac was assayed according to the method described in our previous study (Liu et al., 2017). Briefly, ABTS and 2,6-DMP were employed in the rLac mediated enzymatic reaction as substrates. The absorbance was monitored at 420 nm using the coefficient of ε420 = 36,000 M-1cm-1 for the oxidation of ABTS prepared with a final concentration of 6 mM in 0.1 M citrate-phosphate buffer (pH 5.5). Similarly, the absorbance was recorded at 470 nm (ε470 = 49,600 M-1cm-1) for the oxidation of 2,6-DMP (1.0 mM) prepared in 0.1 M citrate-phosphate buffer (pH 7.0). One unit of laccase activity was defined as the quantity of laccase capable of oxidizing 1 mol substrate per minute under the assay conditions. 2.8 Characterization of rLac

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The optimum temperature required by rLac to oxidize ABTS and 2,6-DMP was estimated by performing the standard

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reaction at different temperatures, ranging from 30 °C to 90 °C. To evaluate the optimal pH for rLac using 2,6-DMP and ABTS as substrates, the enzymatic reaction was performed in buffers of different pH values, viz. 0.1 M citrate-phosphate buffer (pH 3.0 to

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8.0) and 0.1 M glycine-sodium hydroxide buffer (pH 9.0). The maximum activity was taken as 100% to calculate the relative

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enzymatic activity.

The thermostability of rLac was determined by measuring the residual activity with ABTS and 2,6-DMP as substrates for 0 h

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to 2 h at various temperatures (60 °C , 70 °C, and 80 °C). Similarly, pH stability of rLac was assayed by calculating the residual activity after incubation at 4 °C in buffers with different pH (pH 3.0, 7.0, 9.0) for different durations (0-10 days). The activity of

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rLac without treatment was recorded as 100%.

The effect of metal ions (Cu2+, Na+, K+, Ca2+, Cu2+, Fe3+, Fe2+, Co2+, Zn2+, Mn2+, Ba2+, and Mg2+) and inhibitors (EDTA, SDS, dithiothreitol, and β-mercaptoethanol) on the activity of rLac were assayed by determining the relative activity of the

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L-cysteine,

enzyme in the reaction system containing individual effectors with ABTS as the substrate. The laccase activity measured without any of the effectors was marked as 100%. 2.9 Dye decolorization assay

The decolorization ability of rLac was determined by incubating the enzyme with different types of dyes in the reaction system with ABTS, acetosyringone, and syringaldehyde as the mediators. The final concentration of the dyes were azophloxine (λmax= 530 nm), adizol black B (λmax= 607 nm), reactive blue 19 (λmax= 590 nm), crystal violet (λmax= 587 nm), indigo carmine (λmax= 612 nm), 50 mg/L; reactive blue 5 (λmax= 602 nm), malachite green (λmax= 618 nm), 100 mg/L; and Congo red (λmax= 501 nm), 200 mg/L. The reaction system (5 mL) for decolorization contained buffer of different pH values (0.1 M citrate-phosphate buffer, pH 5.5 and 7.0; 0.1 M glycine-sodium hydroxide buffer, pH 9.0), dyes, purified rLac (80 U), individual dye, and 0.1 mM mediator. The reaction lasted for 6 h at 60 °C and the decolorization ability for each dye was evaluated by calculating the decrease in the maximum absorbance for each dye with the following equation: decolorization (%) = [(initial absorbance) - (final absorbance) / (initial absorbance)] × 100%, which reflected the decrease in concentration due to the oxidation by rLac. 3. Results and discussion 6

3.1 Isolation and characterization of the strain with laccase activity

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Given the advantages of bacterial laccases in industrial applications over those derived from fungi and plant, such as wider pH adaptation, higher pH stability, and thermostability, a considerable number of novel laccases isolated from bacteria have been investigated over the past years, especially those isolated from the genus Bacillus (Chen et al., 2015; Lončar et al., 2013; Lončar et al., 2016; Lu et al., 2012a; Martins et al., 2002; Zeng et al., 2016). We successfully isolated a strain with laccase activity from a soil sample collected from Hainan Island of China. This strain had the following morphological characteristics: Gram-positive, rod-shaped, spore-producing, and rough-surfaced colony (data not shown). The 16S rDNA of this strain was amplified, sequenced, and aligned using BLAST in the NCBI database. The BLAST outcome revealed that this strain shares a high genetic identity with the genus Bacillus (> 99%). Besides, phylogenetic analysis showed that the species most closely related to this strain was B. velezensis (Fig. 1). Therefore, the obtained strain was preliminarily characterized as B. velezensis TCCC 111904 in light of the

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results of 16S rDNA sequence alignment, traditional morphological characterization, and biochemical tests (data not shown).

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3.2 Heterologous expression of laccase gene in E. coli

Nucleotide sequencing of the plasmid extracted from a positive transformant confirmed a successful cloning. The sequence

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of lac has been deposited in GenBank with the accession number MK396097; its open reading frame contains 1536 bp that

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theoretically encode 512 amino acids with a molecular weight of about 58 kDa. As determined by the alignment analysis, the protein sequence has a high identity to other laccases from Bacillus species, including: , B. vallismortis fmb 103 laccase, 77.3%

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identity (Sun et al., 2017); B. subtilis X1 laccase, 75.4% (Chen et al., 2015); B. pumilus ATCC 7061 laccase, 65.9% (Ihssen et al., 2017); and B. licheniformis ATCC 14580 laccase, 64.9% (Koschorreck et al., 2008) (Fig. 2). Based on our literature review and

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analysis, this is the first report of the laccase derived from B. velezensis. The predicted 3-D structure of rLac was built by homologous modeling using the structure of B. subtilis laccase as template (PDB ID: 2WSD) (Fig. 3). The active center of

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laccases from Bacillus genus was highly conserved, and contained four copper atoms, of which one was bound to the T1 or T2 copper center and the other two copper atoms were located in the T3 copper center (Solomon et al., 1996). Similarly, three Cuoxidase domains were also present in the active site of rLac, T1 copper center (H419, C492, H497, and M502), T2 copper center (H105 and H422), and T3 copper center (H107, H153, and H493/H155, H422, and H491). The lysate of cells harboring the plasmid pET-lac showed a significant laccase activity after induction with 0.5 mM IPTG for 20 h at 16 °C, while such phenomenon was not observed in the lysate of E. coli BL21/pET-22b (+). A clear protein band of around 58 kDa was observed after analyzing the constitution of crude protein extracted from the culture of recombinant E. coli containing the laccase gene of B. velezensis TCCC 111904. No analogous protein band was detected in the lysate of the control strain E. coli BL21/pET-22b (+) (Fig. 4a). Next, the crude rLac was purified by Ni-affinity chromatography, and a single band of about 58 kDa was detected on a SDS-PAGE gel (Fig. 4b). Although the laccases isolated from different Bacillus species showed comparable enzymatic characteristics, their molecular weights differed from each other. The molecular weight (MW) of the laccase from novel thermophilic bacterium Bacillus sp. PC-3 was 36 kDa (Sharma et al., 2019), MW of the non-blue laccase from B. amyloliquefaciens LC02 was around 65 kDa (Chen et al., 2015), MW of the CotA laccase from B. subtilis 168 was about 66 kDa (Zeng et al., 2016), MW of the laccase from B. tequilensis SN4 was 32 kDa (Sondhi et al., 2014), and MW of the alkaline 7

laccase from B. halodurans C-125 was around 56 kDa (Ruijssenaars and Hartmans, 2004). Therefore, rLac isolated from B.

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velezensis TCCC 111904 would be a new supplement to the laccase family. The specific activities of purified rLac with ABTS and 2,6-DMP as substrates were 180 U/mg and 380 U/mg, respectively. However, laccases from other Bacillus strains showed lower activities. It was reported that the laccase from B. amyloliquefaciens LC02 exhibited an activity of 20.7 ± 1.2 U/mg towards ABTS (Chen et al., 2015), and laccase from B. tequilensis SN4 demonstrated an activity of 299.4 U/mg towards 2,6-DMP (Sondhi et al., 2014). Though, the laccase from B. pumilus MK001 showed a comparable activity of 182 U/mg towards ABTS (Kumar et al., 2016). 3.3 Enzymatic characterization of rLac rLac displayed its maximum activity for the oxidation of ABTS and 2,6-DMP at 80 °C (Fig. 5a). Moreover, rLac could maintain a good catalytic ability at high temperatures and over a broad temperature range of 60 to 90 °C. The activity of rLac was

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about 80% of its maximum value when oxidizing 2,6-DMP at 90 °C. This dramatic and striking feature of rLac is different from

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those of fungal laccases whose optimum temperature is between 30 to 60 °C, such as the laccase from basidiomycete Cerrena sp., 55 °C (Yang et al., 2015), the laccase from the white rot fungus Cerrena unicolorstrain GSM-01, 45 °C (Wang et al., 2017), the

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laccase from the white rot fungus Trametes sp. F1635, 50 °C (Wang et al., 2018), and the laccase from Aureobasidium

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melanogenum strain 11-1, 40 °C (Aung et al., 2019). The optimum temperatures of some fungal laccases isolated from thermophilic fungus are still evidently low in comparison to those of bacterial laccases, for example, the fungal laccase Lcc9 from

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Coprinopsis cinereahas an optimum temperature of 60 °C (Xu et al., 2019). By contrast, bacterial laccases usually exhibit their best catalytic ability at much higher temperatures, especially those isolated from the genus Bacillus, for example, CotA-laccase

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from B. pumilus strain W3, 80 °C (Guan et al., 2014a); the laccase from a novel strain of B. vallismortis and B. tequilensis SN4, 85 °C (Sondhi et al., 2014; Zhang et al., 2012); the spore laccase from B. licheniformis LS04, 60 °C (Lu et al., 2012b); and the

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laccase from B. licheniformis ATCC 9945a, 90 °C (Lončar et al., 2016). It is no doubt that exceptions exist and the optimum temperature of some laccases from Bacillus species is even lower than those isolated from fungi, such as the laccase from B. subtilis subsp. subtilis str. 168, 25 °C (Yang et al., 2012). The thermostability of rLac was studied by evaluating its residual activity after incubation for different lengths of time at 50, 60, 70, and 80 °C. It was found that the residual activity of rLac was around 80% of its initial value after incubation at 60 °C for 120 min, and it remained over 85% at 50 °C. The stability of rLac evidently decreased after incubation at 70 and 80 °C, but its activity still remained 39.8% and 26.4% of the original value after incubation for 120 min, respectively (Fig. 6a). These results indicate that the thermostability of rLac is superior to some laccases derived from other Bacillus species, such as Bacillus sp. ADR (Telke et al., 2011), B. amyloliquefaciens 12B (Lončar et al., 2013), and B. licheniformis ATCC 9945a (Lončar et al., 2016), and is comparable with laccases from B. tequilensis SN4 (Sondhi et al., 2014), B. subtilis 168 (Zeng et al., 2016), and B. pumilus MK001(Kumar et al., 2016). Thus the outstanding thermostability of rLac isolated from the new strain B. velezensis TCCC 111904 might enable it for direct application in the treatment of hot textile effluents discharged after the dyeing process, which is usually performed at high temperatures (Dawkar et al., 2008; Lončar et al., 2013; Yang et al., 2018). rLac displayed high catalytic activity towards the substrates ABTS and 2,6-DMP over a broad pH range (Fig. 5b). The 8

activity of rLac for oxidation of ABTS was over 50% of its maximum value over the pH range of 5.0 to 7.0, and reached the

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maximum value at pH 5.5, which was consistent with other laccases isolated from B. pumilus W3 (Guan et al., 2014a) and B. vallismortis fmb-103 (Zhang et al., 2012). The optimum pH of rLac towards ABTS was higher than most laccases from other species of the Bacillus genus, such as B. licheniformis ATCC 9945a (Lončar et al., 2016), B. subtilis X1 (Guan et al., 2014b), Bacillus sp. (Guo et al., 2017), B. pumilus MK001 (Kumar et al., 2016), B. amyloliquefaciens 12B (Lončar et al., 2013), B. clausii KSM-K16 (Brander et al., 2014), and Bacillus sp. HR03 (Mohammadian et al., 2010). Besides, the optimum pH of rLac for oxidization of 2,6-DMP was 7.0 (Fig. 5b), which was similar to other laccases from the genus Bacillus (Guan et al., 2014a; Guan et al., 2014b; Guo et al., 2017; Lu et al., 2012b). Thus, it is necessary to investigate the optimum pH when oxidizing different substrates using laccase. Additionally, this interesting and outstanding feature could broaden the applications of rLac in treating different kinds of dye effluents having various pH values. Most of the laccases from the genus Bacillus exhibit this feature, which

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was attributed to the conformation variation of the enzyme in different pH environments. Our study further confirmed that the

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optimum pH for rLac in catalyzing particular xenobiotic compounds depend on its structure (Xu, 1997). Besides, the stability of rLac in buffers of different pH values (pH 3.0, 7.0, and 9.0) was investigated by estimating its

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residual activity after incubation for 0 to 10 days at 4 °C (Fig. 6b). rLac was quite stable over a broad pH range, and its residual

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activity was still up to 65.5% of the initial value after incubation for 10 days at pH 3.0. This is a striking feature in comparison to other laccases from Bacillus strains, which become unstable at pH 3.0. The laccase from B. licheniformis LS04 lost 77.12% of its

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initial activity after incubation at pH 3.0 for 24 h (Lu et al., 2013). The stability of laccase from B. subtilis cjp3 was also not good at pH 3.0, only 20.56% of the initial activity remained after incubation for 1 h (Qiao et al., 2017). The laccase activity of B.

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pumilus MK001 was almost completely lost after 10 days incubation at pH 4.0 (Kumar et al., 2016). Thus rLac was quite stable in the acidic environment in comparison to the other laccases isolated from the genus Bacillus.

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Surprisingly, the residual activity of rLac gradually increased in the first two days of incubation in a buffer of pH 7.0, demonstrating a maximum increase of 2.79-fold in comparison to its initial activity. Although the residual activity of rLac decreased slowly with increase in the incubation time, it remained high, at 242% of the initial value after 10 days of incubation. Similar phenomena were also observed for laccases from other Bacillus strains, such as the activity of laccase from B. pumilus W3 remained at approximately 130% of its original activity after incubation for 10 days at pH 7.0, and the laccase from B. licheniformis LS04 exhibited a residual activity of up to 123% of the initial activity (Guan et al., 2014a; Lu et al., 2012b). Not all bacterial laccases showed significantly increased activity after incubation at pH 7.0, and activities of some laccases, such as the recombinant laccase (CotA) from B. pumilus MK001 (Kumar et al., 2016) and laccase from B. amyloliquefaciens LC02 (Chen et al., 2015), reached below 100% of their initial activities after incubation. The stability of rLac at pH 9.0 was almost the same to that at pH 3.0, and its residual activity was retained at 67.6% of the initial value after 10 days of incubation at pH 9.0. By contrast, fungal laccases, such as laccase from Paraconiothyrium variabile (Forootanfar et al., 2011) and Cladosporium cladosporioides NCIM1340 (Halaburgi et al., 2011), are only stable in acidic to neutral environments. Thus, the thermostability and pH tolerance of rLac would broaden its industrial applications. 3.4 Influence of metal ions or inhibitors on activity of rLac 9

As presented in Table 1, most of the selected metal ions did not significantly affect the activity of rLac, except for Mn2+,

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2+

3+

2+

Fe , Fe , and Co , which showed more severe inhibition at a high concentration. The residual activity of rLac was only 4.5%, 22.5%, 33.2%, and 42.3% in presence of 5 mM Mn2+, Fe2+, Fe3+, and Co2+ in the enzymatic reaction system, respectively. Similar phenomena have also been observed for the laccases from P. variabile in the presence of Mn2+ (Forootanfar et al., 2011) and B. tequilensis SN4 in the presence of Fe2+ (Sondhi et al., 2014). The activities of some laccases are more easily affected by Mn2+, for example, the activity of laccase from B. safensis sp. strain S31 remains at only 13.2 ± 0.5% of the control after adding 1 mM Mn2+ in the reaction system (Siroosi et al., 2018). In a high salinity solution with over 100 mM NaCl, most fungal laccases lose their activities because of their intrinsic sensitivity towards halides (Jimenez-Juarez et al., 2005). The possible reason for the occurrence of this inhibition effect on the laccase activity could be that the high concentration of chloride disrupts the transfer of electrons from substrate to T1 copper or

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from T1 copper to T3 copper, which eventually influences the oxidation-reduction reaction mediated by the laccase. However,

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rLac showed great tolerance against high concentration of NaCl, and it retained 50.8% of its residual activity in the solution with 500 mM NaCl compared to the control. Similar results have also been reported for other bacterial laccases, such as B. pumilus W3

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(Guan et al., 2014a), and B. vallismortis fmb-103 (Zhang et al., 2012). Therefore, laccases from different sources have remarkably

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different tolerance against NaCl (Shafiei et al., 2019; Wang et al., 2019). Hence, the high-salinity tolerance of rLac would be much more advantageous in treating the textile effluents, which usually contain high concentration of NaCl (Rodrigues et al., 2009).

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Besides, EDTA also showed a significant inhibitory effect on the activity of rLac, especially at the high concentration of 5 mM (Table 1). The reason for the inhibition effect of EDTA against laccase was probably due to the chelation of the copper ions 2+

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by EDTA at the T1 copper center (Kaushik and Thakur, 2013). This result further revealed the importance of Cu

activity.

in laccase

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As shown in Table 1, the influence of some reported inhibitors (SDS, L-cysteine, dithiothreitol, and β-mercaptoethanol) on the activity of rLac was also investigated. Similar to the laccase from B. vallismortis fmb-103 (Zhang et al., 2012), SDS affected the activity of rLac slightly. However, 0.5 mM of dithiothreitol and β-mercaptoethanol and 1 mM of L-cysteine completely deactivated rLac, which was also observed for other bacterial and fungal laccases (Guan et al., 2014a; Hamid et al., 2011; Zhang et al., 2012). Conversely, the performance of laccases from different Bacillus strains is not always the same when being incubated with the same inhibitors. Low concentration of SDS (0.1 mM) greatly inhibited the activity of laccase from B. safensis sp. strain S31, which remained only 1.3% of the control (Siroosi et al., 2018). 3.5 Dye decolorization by rLac Nowadays, synthetic dyes are used in many industrial units, including the textile processing industry, food and drugs production, and paper manufacturing industry (Waring and Hallas, 2013). Direct discharge of the dye effluents containing many non-biodegradable and toxic chemicals in water bodies t would lead to serious damage of our ecosystem and environment (Forootanfar et al., 2011). Thus, daily treatment of thousands of tons of dye effluents has been huge challenge for us. Laccases, polyphenol oxidases belonging to the family of oxidoreductases, are capable of degrading a series of recalcitrant organic pollutants, including synthetic dyes (Bilal et al., 2019; Majeau et al., 2010; Sondhi et al., 2018). Given their high catalytic 10

efficiency, wide substrate specificity, and environment friendly feature, laccases have been widely studied and applied in the dye

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degradation process in the last few decades (Nguyen and Juang, 2013). Most of the reported laccases have been isolated from fungi, and are unstable in alkaline and high temperature environments (Sharma et al., 2007). This limits the application of fungal laccases in dye effluents treatment as the effluents are discharged with a high temperature and alkaline pH (Kapdan and Alparslan, 2005). By contrast, bacterial laccases display a much higher thermostability and pH stability in comparison to the fungal laccases, and have a high potential for application in the treatment of dye effluents treatment (Santhanam et al., 2011; Sun et al., 2017; Yang et al., 2018). Hence, it spurred many researchers to find novel bacterial laccases and evaluate their enzymatic characteristics and practical applications (Dawkar et al., 2008; Guan et al., 2014a; Lu et al., 2012b; Qiao et al., 2017; Sun et al., 2017; Wang et al., 2010; Yang et al., 2018; Zhang et al., 2012). Here, we isolated a novel rLac with high pH stability and thermostability from a new strain B. velezensis TCCC 111904 (Fig. 6), and we investigated its decolorization efficiency at high temperature (60 °C) and over

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a wide pH range (pH 5.5, 7.0, and 9.0) (Fig. 7). Three mediators (ABTS, acetosyringone, and syringaldehyde) and eight dyes

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including three azo types of dyes (azophloxine, Congo red and adizol black B), two anthraquinonic dyes (reactive blue 5, reactive blue 19), and three triphenylmethane types of dyes (crystal violet, indigo carmine, and malachite green) were employed in the

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decolorization test, which was conducted in different pH environments (pH 5.5, 7.0, and 9.0) (Fig. 7). The results indicated that

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the extent of decolorization did not reach over 27% when only rLac was used for dyes decolorization in the absence of mediators (data not shown). However, rLac could effectively decolorize all of the eight dyes over a wide pH range of 5.5 to 9.0 using ABTS

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as the mediator, and the decolorization rates ranged from 42% to 94% (Fig. 7a). Compared to the decolorization efficiency of rLac against the eight dyes at pH 9.0, rLac was much more effective in decolorizing the dyes at pH 5.5 and pH 7.0 using ABTS as

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mediator except for reactive blue 5 (Fig. 7a). However, rLac was more powerful in decolorizing the eight dyes using acetosyringone and syringaldehyde as mediators in the buffers of pH 7.0 and pH 9.0 (Fig. 7b and Fig. 7c). Therefore, it could be

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concluded that rLac from the newly identified B. velezensis TCCC 111904 can decolorize multiple dyes over an extensive pH range of 5.5-9.0 using an appropriate mediator for specific synthetic dyes. By contrast, fungal laccases can best carry out decolorization in only an acidic environment. The laccase from Trametes hirsuta could decolorize indigo carmine only in acidic environment (Rodrigues et al., 2009). The optimum pH for laccase from basidiomycete Cerrena sp. was 3.5, but its dye decolorization ability was exhibited at pH 6.0 (Yang et al., 2015). The laccase from white rot fungus Cerrena unicolor GSM-01 showed a potent decolorizing ability against bromothymol blue, Evans blue, methyl orange, and malachite green at pH 3.0, demonstrating decolorization efficiencies of 50% to 85% (Wang et al., 2017). In conclusion, rLac showed a superior ability for dye decolorization in comparison to the fungal laccases, which could only carry out decolorization in acidic conditions. Apart from the high efficiency of decolorization in alkali conditions, rLac exhibited another anadvantage in the decolorization process, i.e., the ability to decolorize for a long duration at a higher temperature (60 °C) compared to the laccases from other species of Bacillus genus, such as B. amyloliquefaciens LC02, 40 °C (Chen et al., 2015), B. pumilus W3, 50 °C (Guan et al., 2014a), B. vallismortis fmb-103, 37 °C (Zhang et al., 2012), B. subtilis X1, 50 °C (Guan et al., 2014b), Bacillus sp. A4, 40 °C (Guo et al., 2017), B. amyloliquefaciens 12B, 50 °C (Lončar et al., 2013), B. licheniformis LS04, 40 °C (Lu et al., 2013), and Bacillus sp. ADR, 40 °C (Telke et al., 2011). As the effluent discharged after the dyeing process usually has a high temperature, it is of great 11

importance to immediately and efficiently decolorize the hot effluents upon their release using a laccase with high thermostability.

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A great deal of energy would be saved if the hot water could be used again after decolorization (Lončar et al., 2013). Given that most of the textile effluents are alkaline and discharged at high temperature (Kapdan and Alparslan, 2005; Pereira et al., 2010), use of rLac with high efficiency of decolorization in neutral to alkaline pH at a high temperature (60 °C) in the treatment of alkaline effluent containing synthetic dyes would be more advantageous than fungal laccases. 4. Conclusion The novel laccase rLac isolated from Bacillus velezensis TCCC 111904 displayed high thermostability and high pH stability over a broad pH range. Besides, it also showed a high efficiency in decolorization of azo, anthraquinonic, and triphenylmethane dyes at high temperature over a broad pH range. Additionally, rLac showed great tolerance against high concentration of NaCl. These unique characteristics indicated that it could be a potential candidate for application in the treatment of dye effluents and

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other industrial processes.

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Acknowledgments

This work was supported by the Tianjin Natural Science Fund (17JCYBJC23700), the National Key R&D Program of China

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[2017YFD0201405-04], the Tianjin Correspondent Program of Science and Technology of China (19JCTPJC52200), the Natural

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Science Foundation of Tianjin Municipal Education Commission [2017KJ183], and the Foundation of Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin

Conflicts of Interest

References

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The authors declare no conflict of interest.

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Decolorization of dyes by a novel sodium azide-resistant Spore laccase from a halotolerant bacterium, Bacillus safensis sp. strain S31. Water Sci. Technol. 77, wst2018281. Slagman, S., Zuilhof, H., Franssen, M. C. R., 2018. Laccase-mediated grafting on biopolymers and synthetic polymers: a critical review. ChemBioChem 19, 288-311. Solomon, E. I., Sundaram, U. M., Machonkin, T. E., 1996. Multicopper Oxidases and Oxygenases. Chem. Rev. 96, 2563. Sondhi, S., Kaur, R., Kaur, S., Kaur, P. S., 2018. Immobilization of laccase-ABTS system for the development of a continuous flow packed bed bioreactor for decolorization of textile effluent. Int. J. Biol. Macromol. 117, 1093-1100. Sondhi, S., Sharma, P., Saini, S., Puri, N., Gupta, N., 2014. Purification and characterization of an extracellular, thermo-alkalistable, metal tolerant laccase from Bacillus tequilensis SN4. PLoS One 9, e96951. Sun, J., Zheng, M., Lu, Z., Lu, F., Zhang, C., 2017. Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application. Process Biochem. 55, 77-84. Suzuki, M. T., Giovannoni, S. J., 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625-630. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30, 2725-2729. 14

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Telke, A. A., Ghodake, G. S., Kalyani, D. C., Dhanve, R. S., Govindwar, S. P., 2011. Biochemical characteristics of a textile dye Journal Pre-proof degrading extracellular laccase from a Bacillus sp. ADR. Bioresour. Technol. 102, 1752-1756. Trejo-Hernandez, M. R., Lopez-Munguia, A., Quintero Ramirez, R., 2001. Residual compost of Agaricus bisporus as a source of crude laccase for enzymic oxidation of phenolic compounds. Process Biochem. 36, 635-639. Uthandi, S., Prunetti, L., De Vera, I. M. S., Fanucci, G. E., Angerhofer, A., Maupin-Furlow, J. A., 2012. Enhanced archaeal laccase production in recombinant Escherichia coli by modification of N-terminal propeptide and twin arginine translocation motifs. J. Ind. Microbiol. Biotechnol. 39, 1523-1532. Wang, C., Zhao, M., Li, D., Cui, D., Yangi, H., 2010. Isolation and characterization of a novel Bacillus subtilis WD23 exhibiting laccase activity from forest soil. Adv. Mater. Res. 113-116, 725-729. Wang, H., Huang, L., Li, Y., Ma, J., Wang, S., Zhang, Y., Ge, X., Wang, N., Lu, F., Liu, Y., 2019. Characterization and application of a novel laccase derived from Bacillus amyloliquefaciens. Int. J. Biol. Macromol. Wang, S. N., Chen, Q. J., Zhu, M. J., Xue, F. Y., Li, W. C., Zhao, T. J., Li, G. D., Zhang, G. Q., 2018. An extracellular yellow laccase from white rot fungus Trametes sp. F1635 and its mediator systems for dye decolorization. Biochimie 148, 46-54. Wang, S. S., Ning, Y. J., Wang, S. N., Zhang, J., Zhang, G. Q., Chen, Q. J., 2017. Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01. Int. J. Biol. Macromol. 95, 920-927. Wang, W., Zhang, Z., Ni, H., Yang, X., Li, Q., Li, L., 2012. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase. Microb. cell fact. 11, 75. Waring, D. R., Hallas, G. The chemistry and application of dyes: Springer Science & Business Media, 2013. Xu, F., 1997. Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J. Biol. Chem. 272, 924-928. Xu, G., Wang, J., Yin, Q., Fang, W., Xiao, Y., Fang, Z., 2019. Expression of a thermo- and alkali-philic fungal laccase in Pichia pastoris and its application. Protein Expression Purif. 154, 16-24. Yang, J., Ng, T. B., Lin, J., Ye, X., 2015. A novel laccase from basidiomycete Cerrena sp.: Cloning, heterologous expression, and characterization. Int. J. Biol. Macromol. 77, 344-349. Yang, Q., Zhang, M., Zhang, M., Wang, C., Liu, Y., Fan, X., Li, H., 2018. Characterization of a novel, cold-adapted, and thermostable laccase-like enzyme with high tolerance for organic solvents and salt and potent dye decolorization ability, derived from a marine metagenomic library. Front. Microbiol. 9, 2998. Yang, S. S., Liu, Z. W., Yi, X. P., Zhang, A. L., Zhang, T. Y., Luo, J. X., Zhang, Z. H., Shen, J. C., Yin, H. X., Chen, L. P., 2012. Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties. Gene 491, 49-52. Yaropolov, A. I., Skorobogat’ko, O. V., Vartanov, S. S., Varfolomeyev, S. D., 1994. Laccase. Appl. Biochem. Biotechnol. 49, 257-280. Zeng, J., Zhu, Q., Wu, Y., Lin, X., 2016. Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere 148, 1-7. 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. Zhuo, R., Zhang, J. W., Yu, H. B., Ma, F. Y., Zhang, X. Y., 2019. The roles of Pleurotus ostreatus HAUCC 162 laccase isoenzymes in decolorization of synthetic dyes and the transformation pathways. Chemosphere 234, 733-745.

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Tables

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Table 1. Influence of metal ions or inhibitors on activity of rLac. All experiments were carried out independently for three times, and the values represent mean ± SD.

Metal ions/inhibitors

Concentration (mM)

None

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KCl

0.5

100.3±2.1

5

93.6±0.9

0.5

100.3±1.2

5

97.8±1.3

0.5

17.7±1.8

MnCl2

100±1.5

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CaCl2

Relative activity (%)

5 0.5

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CuCl2

4.5±0.6

MgCl2

-p

5 0.5

0.5 5

BaCl2

FeSO4

SDS

EDTA

NaCl

105.6±1.6 96.0±1.2 102.7±2.0

79.6±1.7

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CoCl2

0.5

90.1±1.0

93.1±0.9

5

33.2±1.2

0.5

93.9±2.2

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FeCl3

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ZnCl2

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5

93.4±2.3

5

42.3±1.5

0.5

104.4±0.8

5

94.9±1.3

0.5

77.2±2.1

5

22.5±1.1

0.5

86.9±0.9

5

83.1±2.0

0.5

71.3±1.4

5

25.1±1.8

0.5

94.6±2.6

5

91.0±1.6

10

81±1.0

100

68.9±0.9

500

50.8±1.7 16

1000

0

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Dithiothreitol

β-Mercaptoethanol

L-Cysteine

0.1

29.2±1.1

0.5

0

1

0

0.1

32.8±1.6

0.5

0

1

0

0.1

75.3±2.7

0.5

27.1±1.0 0

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Figure captions

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Figure 1. Phylogenetic analysis of 16S rDNA of B. velezensis TCCC 111904 with other related Bacillus strains. The MEGA 6.0 software was used to build the bootstrap consensus tree by the neighbor-joining method. The percentage of bootstrap sampling, derived from 1000 replications was suggested by the numbers at branch points. Figure 2. Amino acid sequence alignment of rLac and other laccases from the Bacillus genus. The protein sequences of the other four laccases from B. vallismortis fmb 103, B. pumilus ATCC 7061, B. subtilis X1, and B. licheniformis ATCC 14580 were downloaded from the NCBI website. The alignment was performed using DNAMAN software, and similar and identical amino acids are highlighted in solid grey and black, respectively.

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Figure 3. Predicted 3-D structure of rLac. The model was built with the PyMOL molecular graphics system according to its sequence. The amino acid residues in the three copper centers, Type 1 (His419, Cys492, His497, and Met502), Type 2 (His105

-p

respectively. The red balls stand for Cu2+ in the active site of rLac.

ro

and His422), and Type 3 (His107, His153, and His493/His155, His424, and His491) are highlighted in green, blue, and yellow,

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Figure 4. Expression and analysis of molecular weight of rLac by SDS-PAGE. (a) Lane M: protein marker; Lane 1: crude protein

The band of rLac is marked with black arrow.

lP

extracted from E. coli BL21/pET-lac; Lane 2: crude protein extracted from E. coli BL21/pET-22b (+). (b) Lane 1: purified rLac.

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Figure 5. Influence of temperature (a) and pH (b) on the activity of rLac isolated from B. velezensis TCCC 111904. The activity of rLac was analyzed following the standard assay with ABTS or 2,6-DMP as substrate under each condition. All experiments were

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conducted in triplicate, and the values represent mean ± SD. Figure 6. Thermostability (a) and pH stability (b) of the rLac isolated from B. velezensis TCCC 111904. The stability of rLac was analyzed following the standard activity assay with ABTS or 2,6-DMP as substrate under each treatment condition, and it is shown as the percentage of the residual activity compared to the initial value. All experiments were performed in triplicate, and the values represent mean ± SD. Figure 7. Decolorization of different dyes by the rLac isolated from B. velezensis TCCC 111904 with mediators ABTS (a), acetosyringone (b), and syringaldehyde (c) at pH 5.5, 7.0, and 9.0. The decolorization of each dye was performed by incubating 80 U of rLac at 60 °C. Dye symbols from 1 to 8 in the horizontal coordinates represent azophloxine, Congo red, adizol black B, reactive blue 5, reactive blue 19, crystal violet, indigo carmine, and malachite green, respectively. All experiments were carried out in triplicate, and the values represent mean ± SD.

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Fig. 7

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Conflict of Interest Statement

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The authors declare that they have no known competing financial interests or personal relationships that

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could have appeared to influence the work reported in this paper.

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Graphical abstract

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Journal Pre-proof A new strain Bacillus velezensis TCCC 111904 with laccase activity was identified The novel laccase from Bacillus velezensis TCCC 111904 was expressed in E. coli The recombinant laccase (rLac) displayed high thermostability and pH stability rLac showed great tolerance against high concentration of NaCl

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rLac efficiently decolorised azo, anthraquinonic, and triphenylmethane dyes

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