Characterization of the aflatoxin B1 degradation role of laccases in Stenotrophomonas acidaminiphila
Hong Jiang¹, Zhengjun Du¹, Yingying Qian¹, Nan Chen¹, Mengyu Cai¹, Qingru Fei¹, Jingjing Chen², Yu Zhou¹
1. State Key Laboratory of Tea Biology and Utilization, School of Tea and Food Science Technology, Anhui Agricultural University, Hefei 230036, Anhui Province, China;
2. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu Province, China
Received: 7 March 2020; Revised: 28 March 2020; Published online: 15 April 2020
Foundation item: Supported by the National Natural Science Foundation of China (31671949), by the Anhui Natural Science Foundation for Distinguished Young Scholars (1608085J08) and by the National Key R & D Program of China (2017YFC1600902)
Corresponding author: Jingjing Chen, Tel/Fax:+86-551-65786701;E-mail:jingjinc@jiangnan.edu.cn; Yu Zhou, E-mail:microbes@ahau.edu.cn.

Abstract: [Objective] Characterization of the aflatoxin B1 (AFB1) degradation roles of the laccases screened from Stenotrophomonas acidaminiphila CW117.[Methods] Two laccase genes lc1 and lc2 from strain CW117 genome were screened, and their AFB1 degrading activity was examined in vitro by heterologous expressed proteins of rLC1 and rLC2 in E. coli BL21. On the basis of in vitro test, two laccase-deficient strains CW117^△lc1 and CW117^△lc1-lc2 were constructed by homologous recombination method by using suicide plasmid pK18mobsacB, and the laccases (lc1 and lc2) AFB1 degradation role on strain CW117 was validation in vivo.[Results] Laccase rLC1 showed the AFB1 degradation activity in vitro, but rLC2 did not show degradation activity. Degradation activity of rLC1 was improved by redox mediators of 2, 2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), acetosyringone or syringaldehyde. The degrading activity of mutants CW117^△lc1 and CW117^△lc1-lc2 showed similar degradation activity to the wild-type strain CW117 in most evaluation time-points.[Conclusion] Laccase LC1 from S. acidaminiphila showed AFB1 degradation activity, and the degradation activity could be enhanced by redox mediators as previous study. However, the laccases' contribution to AFB1 degradation in strain CW117 was minimal, and other degradation pathways existed in the strain.
Keywords: detoxificationlaccaseaflatoxin B1degradation genemutant
微嗜酸寡养单胞菌中的漆酶对黄曲霉毒素B1降解脱毒的生物活性
姜宏¹, 杜郑君¹, 钱莺莺¹, 陈楠¹, 蔡梦宇¹, 费晴如¹, 陈静静², 周育¹
1. 安徽农业大学茶树生物学与资源利用国家重点实验室, 安徽 合肥 230036;
2. 江南大学食品科学与技术国家重点实验室, 江苏 无锡 214122
收稿日期：2020-03-07；修改日期：2020-03-28；网络出版日期：2020-04-15
基金项目：国家自然科学基金（31671949）；安徽省****基金（1608085J08）；国家重点研发计划（2017YFC1600902）
通信作者：陈静静, Tel/Fax:+86-551-65786701;E-mail:jingjinc@jiangnan.edu.cn; 周育, E-mail:microbes@ahau.edu.cn.

摘要：[目的] 探究微嗜酸寡养单胞菌中的漆酶对AFB1的降解活性，并确定漆酶在菌株CW117降解代谢AFB1过程中的贡献。[方法] 从微嗜酸寡养单胞菌基因组中，共筛选到两个漆酶基因lc1和lc2，并用大肠杆菌BL21外源表达蛋白rLC1和rLC2，在体外检测其对AFB1的降解活性。同时参考前人报道，研究了氧化性辅剂对漆酶AFB1降解的促进作用。在体外实验基础上，利用自杀质粒pK18mobsacB，以同源重组方法构建了两株漆酶缺失株CW117^△lc1和CW117^△lc1-lc2，验证了漆酶基因（lc1和lc2）对AFB1体内降解作用。[结果] 体外实验显示，重组酶rLC1具有AFB1降解活性，氧化性辅剂ABTS、AS或SA可显著地提高rLC1降解活性，但rLC2未显示降解活性。突变株CW117^△lc1和CW117^△lc1-lc2对AFB1仍显示了较高的降解活性，且在大多数降解时间点与野生株CW117无显著差异。[结论] 微嗜酸寡养单胞菌CW117菌株中，LC1在体外显示了AFB1的降解活性，且降解活性可以被氧化性辅助因子增强，LC2未显示体外降解活性；体内试验发现，漆酶基因lc1和lc2对菌株CW117降解AFB1的贡献较小，该菌株还存在其他降解途径。
关键词：解毒漆酶黄曲霉毒素B1降解基因突变菌株
The aflatoxins' chemical structures consist of a coumarin and bifuran rings; the natural contaminants are mainly produced by fungi Aspergillus flavus and Aspergillus parasiticus^[1]. As well-known, aflatoxin B1 (AFB1) is recognized as the most potent and prevalent aflatoxin; the extremely toxic effects of mutagenic, carcinogenic and teratogenic on humans and livestock pose a serious health and economic hazards^[2-3].
At present, various microbial species (or genera) have been isolated and characterized for aflatoxins degradation, and the microbial transformation (or degradation) has been considered as one of the most effective means to aflatoxins detoxification. The most investigated microbial species including Armillaria spp.^[4-6], Pleurotus spp.^[7], Pseudomonas spp.^[8], Bacillus spp.^[9-11], Flavobacterium spp.^[12–13], Mycobacterium spp.^[14–15], Rhodococcus spp.^[15–17] and Stenotrophomonas spp.^[18]. Besides the degrading microbial strains characterization, aflatoxins degrading enzymes (including degrading genes) equally received great attention. For instance, manganese peroxidase (MnP)^[19], aflatoxin-oxidase (AFO)^[6] and pyridoxamine 5′-phosphate oxidases (PNPOxs)^[20] were determined as aflatoxins detoxification enzymes. Among these aflatoxins detoxification enzymes from microbial strains, the laccases were the most focused and recognized degrading agent for aflatoxin detoxification^[21-23]. Laccases belong to the superfamily of multicopper oxidases and widely present in bacteria and fungi^[24-26], and laccase catalyze ring cleavage of aromatic compounds, and are generally considered as an important biotransformation candidate to environmental pollutants^[27]. In previous studies, laccases from Pleurotus pulmonarius^[21], Pleurotus eryngii^[22-23] and Trametes versicolor^[28] were determined as the aflatoxin detoxification agents. After degraded by laccases from the microbial species, the biotoxicities (or mutagenicity) of degraded products were significantly reduced or even disappeared^[29]. Degradation results in previous studies indicated that laccases were the general recognized detoxification agent to aflatoxin degradation.
Our previous study found that strain S. acidaminiphila CW117 showed efficient degrading activity to aflatoxin B1, and the 24 h degrading ratio higher than 85%. One laccase gene lc1 from strain CW117 were screened and heterologous expressed by E. coli BL21. However, AFB1 degrading test showed that rLC1 showed relatively low degrading activity in vitro^[30]. In view of the results, the following possibilities might be included: (i) laccases were the sole bioagents (enzymes) for AFB1 transfomation, but the heterologously produced rLC1 proteins in E. coli system were not appropriate folded (or mismodified) and resulted in low degradation activity; (ii) Other than laccases, some cofactors (e.g., coenzymes or redox mediators) in strain CW117 can significantly assist the degradation ability of laccases, but the cofactors (or coenzymes) are absent in E. coli system; (iii) Other efficient degrading enzymes (or other degrading agents) are responsible for AFB1 degradation, and laccases were only a secondary degradation agent in strain CW117. In this study, other laccase genes were further screened, and the AFB1 degradation characteristics of all laccase genes were investigated by methods of in vitro and in vivo, and the laccase degradation role was examined.
1 Materials and methods 1.1 Chemicals, bacterial strains and plasmids The AFB1 degrading strain S. acidaminiphila CW117 was previous isolated and preserved in our laboratory. AFB1 standard was obtained from Pribolab Pte. Ltd (Bejing, China), other analytical reagents were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Chromatographically grade solvents (i.e., methanol, acetonitrile) were purchased from TEDIA (Shanghai, China). The analytical grade (purity > 98%) of ABTS [2, 2-azino-bis-(3-ethyl benzothiazoline-6-sulphonic acid)], AS (acetosyringone) and SA (syringaldehyde) were obtained from Sigma-Aldrich (Shanghai, China). The strains, plasmids and the PCR primers used in this study are shown in Table 1. The antibiotics (i.e., gentamycin and kanamycin) used for mutant screening were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and the antibiotic solutions were prepared by diluting in distilled water at the final concentration of 50 μg/mL for each. Bacterial culture media of Luria-Bertani (LB), nutrient broth (NB) and nutrient agar (NA) are the products of Difco (Lawrence, Kansas, USA). The affinity chromatography resin (ProteIniso^? GST Resin) used for heterologous expressed protein purification is the TransGen Biotech (Beijing, China) product. The DNA polymerase (PrimerSTAR Max DNA Polymerase), restriction endonuclease (XbaⅠ and Hind Ⅲ) and DNA ligase (solution Ⅰ) used in this study are the products of TaKaRa (Dalian, China). Agarose gel DNA recovery kit is the Tiangen (Beijing China) product, and the plasmid MiniPrep kit is product of AxyPrep (Axygen, CA, USA).
Table 1. Thestrains, plasmids and primers used in this study

Strains, plasmids or primers	Characterization	Reference or restriction sites
Strain CW117
pGEX/lc1	LC1 expression strain	This study
pGEX/lc2	LC2 expression strain	This study
CW117?lc1	The gene lc1 deletion mutant of CW117	This study
CW117?lc1-lc2	The gene lc1 and lc2 deletion mutant of CW117	This study
Escherichia coli Trans1-T1 phage resistant	Receptive cells	TransGen Biotech, Beijing, China
Escherichia coli BL21	Expression cells	TransGen Biotech, Beijing, China
pK18mobsacB	Allelic exchange vector, KanR	[40]
lc1-F*	CGGGATCCATGGCCGCCGCGTTGCC	BamHⅠ
lc1-R	CCGCTCGAGTCACCGCGCCATCCACAC	XhoⅠ
lc2-F	CGGAATTCATGAAAAACGATTTCCTTTCCG	EcoRⅠ
lc2-R	CCGCTCGAGTCATGCCTCGATCCTCACT	XhoⅠ
lc1-US-F	GCTCTAGAGCGTCGGTATCGGTATGCG	XbaⅠ
lc1-US-R	ACTGGCGGGTGGACCTGTA
lc1-DS-F#	gtacaggtccacccgccagtGCCCAGGTTGAAACTGTCG	Lowercase indicates adapter sequence for overlapping PCR
lc1-DS-R	CCCAAGCTTATCCCGCTGTCGGTCCTGT	Hind Ⅲ
ver1-F	CCTGCCCGGCCTAATCCA	lc1 deletion identification primer
ver1-R	TCCCGCCTGAGCGAATGG	lc1 deletion identification primer
lc2-US-F	GCTCTAGACCGGAATCAGGTGGGTATCG	XbaⅠ
lc2-US-R	GGGAACTCCATCGGCCGC
lc2-DS-F	gggcggccgatggagttcccAGACGACGACCCGATCCCT	lowercase indicates adapter sequence for overlapping PCR
lc2-DS-R	CCCAAGCTTCGCGTCCAGCGACCACTC	Hind Ⅲ
ver2-F	CGGGTGGCTGGACTCGT	lc2 deletion identification primer
ver2-R	CACCACGGTGAGATGGAATG	lc2 deletion identification primer
*Underlined sequences represent the digestion site.

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1.2 Laccase genes cloning, expression and AFB1 degradation During genome sequence analysis, two laccase genes (i.e., lc1 and lc2) were screened from complete genome sequence (NZ_CP012900.1) of strain S. acidaminiphila ZAC14D2_NAIMI4_2 which showed the closest taxonomic affiliation to strain CW117. Laccase genes were amplified by using CW117 genomic DNA and the primer pairs of lc1-F/R and lc2-F/R with PrimerSTAR Max DNA Polymerase (Table 1). The PCR condition was 5 min denaturing at 98 ℃, followed by 30 cycles of 5 s denaturing at 98 ℃, 5 s annealing at 61 ℃, 5 s extension at 72 ℃, and a final 10 min extension at 72 ℃. The purified PCR product and expression plasmid pGEX-4T-1 were digested by BamHⅠ (or EcoRⅠ) and XhoⅠ at 37 ℃ overnight, respectively. The digest mixture contained 1.0 μL XhoⅠ, 1.0 μL BamH Ⅰ (or EcoRⅠ), 1 μg of purified lc1 or lc2 (or 1 μg pGEX-4T-1) and 5 μL of 10×H buffer (TaKaRa, Dalian, China), and then supplemented distilled water to 50 μL. The digested lc1 (or lc2) fragment and expression plasmid was purified using an agarose gel DNA recovery kit according to manufacturer's instructions, and the purified lc1 or lc2 fragment was ligated to digested pGEX-4T-1 by incubation with solution Ⅰ DNA ligase for 1 h at 16 ℃. The ligation mixture consists of 2 μL digested pGEX-4T-1, 4 μL digested lc1 or lc2 and 6 μL solution Ⅰ DNA ligase. After ligation, 5 μL of the recombinant plasmid (pGEX/lc1 or pGEX/lc2) was transformed to E. coli Trans1-T1 by heated shock and identified by PCR sequencing. The positive clone was enriched in AmpR LB broth which containing 50 μg/mL ampicillin, and the recombinant plasmid was extracted by an Plasmid MiniPrep kit according to manufacturer's instructions and transformed to E. coli BL21 by heated shock.
The laccase transformant (containing pGEX/lc1 or pGEX/lc2) was incubated in Amp^R LB brothat 16 ℃ with agitation of 150 r/min; until the culture OD₆₀₀ reached 0.6, about 0.2 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and followed by additional 4 hours incubation. The recombinant proteins of rLC1 and rLC2 were purified by ProteIniso GST resin according to manufacturer's instructions. The purity of obtained rLC1 (or rLC2) recombinant protein was examined by SDS-PAGE (BioRad, Hercules, USA), and the protein concentration was evaluated by spectrophotometrically method^[31]. The method of Cai et al.^[30] was used to determine the rLCs' AFB1 degradation activity, and the method of Loi et al.^{[21, 23]} with minor modification was used to evaluate the redox mediators' (i.e., ABTS, AS and SA) assistant effects on AFB1 degradation. Briefly, 0.1 mL of purified rLC1 (1.0 mg/mL) and 0.1 mL redox mediator (50 mmol/L) were added to 0.3 mL AFB1 working solution (40 μg/L) to make the final degradation mixture. The redox mediator and AFB1 working solution were prepared by glycine- hydrochloric acid buffer (pH 4.0). The redox mediator (without rLC1) degradation test and rLC1 (without redox mediator) degradation test were selected as controls. The degradation tubes were incubated for 24 h at 37 ℃, and AFB1 residues were analyzed by using HPLC^[32].
1.3 Construction of mutant CW117^?lc1 and double mutant CW117^?lc1-lc2 The protocols of recombinant plasmid pK18mobsacB_US-DS construction and the laccase mutants CW117^?lc1and CW117^?lc1-lc2 screening (as shown in Figure 1) were followed as the methods of previous^[33-35]. The upstream (lc1-US) and downstream (lc1-DS) DNA fragments which ?anking the lc1 open reading frame (ORF) were amplified by PCR using the PrimerSTAR Max DNA Polymerase. A primer pair of lc1-US-F and lc1-US-R, and a primer pair of lc1-DS-F and lc1-DS-R were employed for lc1-US and lc1-DS in PCR amplification, respectively (Table 1). The PCR condition of lc1-US (or lc1-DS) were 5 min denaturing at 98 ℃, followed by 30 cycles of 5 s denaturing at 98 ℃, 5 s annealing at 61 ℃, 5 s extension at 72 ℃, and a final 10 min extension at 72 ℃. After PCR amplification, both PCR products were purified using an agarose gel DNA recovery kit according to manufacturer's instruction. The overlap PCR was performed using the primer pair of lc1-US-F and lc1-DS-R (Table 1) with the PrimerSTAR Max DNA Polymerase (TaKaRa, Dalian, China), the purified PCR products of lc1-US and lc1-DS (1 μL for each) were selected as DNA templates. The overlap PCR conditions of lc1-US-DS were 5 min denaturing at 98 ℃, followed by 30 cycles of 5 s denaturing at 98 ℃, 10 s annealing at 61 ℃, 10 s extension at 72 ℃, and a final 10 min extension at 72 ℃.

Figure 1 Schematic description of recombinant plasmid pK18mobsacB_US-DS construction and gene mutant screening. A: pK18mobsacB_US-DS construction; B: genemutant screening.

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The purified overlap PCR product (lc1-US-DS) and suicide plasmid pK18mobsacB were digested with Hind Ⅲ and XbaⅠat 37 ℃ overnight, respectively. The digest mixture contained 1.0 μL XbaⅠ, 1.0 μL Hind Ⅲ, 1.0 μL purified lc1-US-DS (or 1 μg pK18mobsacB) and 5 μL of 10×M buffer (TaKaRa, Dalian, China), then supplemented distilled water to 50 μL. The digested lc1-US-DS fragment and suicide plasmid was purified using an agarose gel DNA recovery kit, respectively. The purified lc1-US-DS fragment was ligated to digested pK18mobsacB by incubation with solution Ⅰ DNA ligase 1 h at 16 ℃. The ligation mixture consists of 2 μL digested pK18mobsacB, 4 μL digested lc1-US-DS and 6 μL solutionⅠ DNA ligase. After ligation, 5 μL of the ligation product were transformed to E. coli Trans1-T1, and screened by KanR LB agar which containing 50 μg/mL kanamycin. Positive clones were identified by PCR sequencing, and the successful recombinant plasmid was defined as pK18mobsacB_lc1-US-DS. The positive clone was enriched by Kan^R LB medium, and extracted by a Plasmid MiniPrep kit. The recombinant plasmid pK18mobsacB_lc1-US-DS was transformed into strain CW117 (wild-type) by electroporation method as previous^[36]. After transformation, cell solution was evenly coated on the Kan^R nutrient agar (NA). Single colony on Kan^R NA screening plates was inoculated into 5 mL Kan^R NB, and incubated at 37 ℃, with agitation of 180 r/min. Until the culture OD₆₀₀ reached 0.6, bacteria cells (0.5 mL) were collected by centrifugation (5000×g), and washed twice by sterile NB, and resuspended in 0.5 mL NB. The serial NB diluted bacteria suspensions were coated on the NA containing 13% sucrose (named "sucrose screening agar"), and the positive clones in sucrose plate were rescreened by Kan^R NA which containing 50 μg/mL kanamycin. The resulting colonies which grown on sucrose plate but sensitive to 50 μg/mL kanamycin were considered as the mutant candidate of CW117^?lc1. The mutant candidate was identified by PCR sequencing with PrimerSTAR Max DNA Polymerase, and the primer pair of ver1-F and ver1-R (Table 1). The PCR conditions were 5 min denaturing at 98 ℃, followed by 30 cycles of 5 s denaturing at 98 ℃, 5 s annealing at 55 ℃, 10 s extension at 72 ℃, and a final 10 min extension at 72 ℃.
The construction and screening protocols of double gene lc1 and lc2 mutant CW117^?lc1-lc2 were followed the construction and screening methods of CW117^?lc1, and the mutant CW117^?lc1 was selected as starting strain (original strain) for CW117^?lc1-lc2 construction. The PCR primer pairs of lc2-US (lc2-US-F, lc2-US-R), lc2-DS (lc2-DS-F, lc2-DS-R) and gene lc2 deletion verification (ver2-F and ver2-R) were shown as Table 1.
1.4 The AFB1 degradation tests by wide-type strain and mutants Strains of CW117, CW117^?lc1 and CW117^?lc1-lc2 were inoculated on NA, and single colonies were selected, inoculated and incubated in 5 mL NB at 37 ℃, with agitation of 180 r/min. Until the culture OD₆₀₀ absorbance reached 0.6, the fresh culture (0.5 mL) transferred to 50 mL NB containing 20 μg/L AFB1. The degradation tests were performed at 37 ℃, with agitation of 180 r/min. The 50 mL NB containing 20 μg/L AFB1 without bacteria inoculation was used as control. During degradation, AFB1 residues at 0th, 12th, 24th, 36th, 48th, 60th and 72th hour were determined by HPLC following the method as previous^[31].
1.5 The AFB1 degradation tests by culture supernatant of wide-type strain and mutants Fresh culture (0.5 mL) of CW117, CW117^?lc1 or CW117^?lc1-lc2 was inoculated into 50 mL NB, incubated at 37 ℃ with agitation of 180 r/min for 48 h. The bacterial culture (CW117, CW117^?lc1 or CW117^?lc1-lc2) was centrifuged at 5000×g for 10 min at 4 ℃, and the supernatant and cell pellet were separated. Culture supernatant was prepared by passing the supernatant through a sterile filter (0.22 μm, Millipore, Billerica, USA). During the degradation test, 25 mL culture supernatant was mixed with 25 mL PBS (pH 6.8) which containing 40 μg/L AFB1.The degradation tubes were incubated at 37 ℃ without agitation. During degradation, AFB1 residues at 0th, 1th, 3th, 6th, 9th, 12th and 24th hour were determined by HPLC following the method as previous^[31].
1.6 Data analysis There were three biological repeats in the experimental data. One way ANOVA and t-test (^*P < 0.05) were used to analyze the data by Graphpad prism (5.0) software.
2 Results 2.1 Laccase genes cloning, expression and protein purification As shown in Figure 2, gene lc1 (780 bp) was cloned by the primer pair of lc1-F/R and the PCR condition, and recombinant plasmid pGEX/lc1 was successfully constructed and transformed to E. coli BL21. Similarly, gene lc2 (1773 bp) was cloned by the primer pair of lc2-F/R and the PCR condition, and recombinant plasmid pGEX/lc2 was also successfully constructed and transformed to E. coli BL21 (Figure 3). After heterologous expressed in E. coli BL21, the recombinant proteins of rLC1 and rLC2 were purified by affinity chromatography by using ProteIniso^? GST Resin. The size of GST tagfrom pGEX-4T-1 is about 26 kDa, and the protein sizes of LC1 and LC2 were about 27.1 kDa and 65.08 kDa, respectively; thereby, the recombinant protein sizes of rLC1 and rLC2 are about 53 kDa and 91 kDa, respectively. As shown in Figure 4, a single protein band of 45–60 kDa was observed in lane 2, and a single protein band of 80–100 kDa was observed in lane 3 in SDS-PAGE, and the results indicated that genes lc1 and lc2 were successfully expressed in E. coli BL21, and electrophoretic homogeneous rLC1 and rLC2 were obtained by ProteIniso^? GST Resin.

Figure 2 Gene cloning and heterologous expression (pGEX/lc1) of laccase gene lc1 in strain E. coli BL21. A: the PCR fragment of gene lc1. B: lc1 PCR product and pGEX plasmid double digestion (M: DNA marker; lane 1: lc1 double digestion; lane 2: lc1 PCR product; lane 3: pGEX-4T-1 double digestion products; lane 4: pGEX-4T-1). C: lc1 PCR verification from recombinant plasmid pGEX/lc1 in E. coli Trans1-T1. D: lc1 PCR verification from recombinant plasmid pGEX/lc1 in E. coli BL21.

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Figure 3 Gene cloning and heterologous expression (pGEX/lc2) of laccase gene lc1 in strain E. coli BL21. A: the PCR fragment of gene lc2. B: lc2 PCR product and pGEX plasmid double digestion. M: DNA marker; lane 1: lc2 double digestion; lane 2: lc2 PCR product; lane 3: pGEX-4T-1 double digestion; lane 4: pGEX-4T-1. C: lc2 PCR verification from recombinant plasmid pGEX/lc2 in E. coli Trans1-T1. D: lc2 PCR verification from recombinant plasmid pGEX/lc2 in E. coli BL21.

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Figure 4 SDS-PAGE analysis of rLC1 and rLC2 purified protein. M: protein marker; lane 1: purified protein of GST tag; lane 2: purified protein rLC1; lane 3: purified protein rLC2.

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2.2 Degradation activity of laccase, and the redox mediators assistance to rLC1 The preliminary test showed that 24 hours AFB1 degradation ratio of rLC1 was about 12% in degradation condition of this study, but degradation activity was not observed on rLC2. As indicated by Loi et al.^{[21, 23]}, redox mediators can enhance the AFB1 degradation activity of microbial laccases. In this study, the redox mediators of ABTS, AS and SA were tested for aflatoxin degradation assistance to rLCs. As a result, the three redox mediators of ABTS, AS and SA equal significantly enhanced the AFB1 degradation activity of rLC1; the most significant improvement was observed on ABTS as the additive, which improved the degradation ratio from 10.4% to 27.0% (Figure 5). However, the redox mediator ABTS also showed the AFB1 degradation activity (5.6%) which was not observed on the other two redox mediators (Figure 5).

Figure 5 Aflatoxin B1 degrading activity of rLC1 supplemented with different redox mediators. A: the effect of ABTS. B: the effect of AS. C: the effect of SA. **: P < 0.01, and ***: P < 0.001, compared with the control (Laccase group).

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2.3 Mutants CW117^?lc1 and CW117^?lc1-lc2 construction By the PCR primer pairs of lc1-US and lc1-DS, the expected fragments of upstream (US, 1006 bp, Figure 6-A) and downstream (DS, 860 bp, Figure 6-B) flanking the gene lc1 were successfully cloned. Then, the US and DS fragments were further lingated by overlap PCR with the primers of lc1-US-F and lc1-DS-R, and the overlap PCR product of lc1-US-DS fragment was 1886 bp. After the purification and sequencing validation, the purified PCR product lc1-US-DS was digested with Hind Ⅲ and XbaⅠ, and ligated to digested suicide plasmid pK18mobsacB. The recombinant plasmid pK18mobsacB_lc1-US-DS was successfully constructed and validated in E. coli Trans1-T1 by PCR sequencing (Figure 6-C). After electroporation, the mutant CW117^?lc1 was produced by 13% sucrose and screened by Kan^R NA, and validated by PCR sequencing. The PCR fragment on the successful mutant CW117^?lc1 was expected as 2202 bp, but the PCR fragment on wild-type CW117 was 2683 bp by using the same primer pair of ver1-F and ver1-R (Table 1). The screening results of this study showed that the band sizes (about 2.7 kb) of lanes 5, 6, 10 were consistent to wild-type CW117; while, the band sizes (about 2.2 kb) of lanes 1, 4 and 8 were considered as mutant CW117^?lc1candidates (Figure 6-D). The screening results indicated that the monoclonal strains represented by lanes 5, 6, 10 probably restored to wild type, and the monoclonal strains represented by lanes 1, 4 and 8 might be the expected mutants. PCR sequencing results further validated the colonies from lanes 1, 4 and 8 was the positive mutant CW117^?lc1.

Figure 6 The gene lc1 mutant construction from wide-type strain CW117. A: the upstream PCR fragment of gene lc1. B: the downstream PCR fragment of gene lc1. C: the PCR fragment of lc1-US-DS from pK18mobsacB_US-DS. D: the PCR validation on gene lc1 mutant screening. M: DNA marker; lanes 1, 4, 8: positive candidates; lanes 5–6: negative colonies; lane 10: wild-type CW117.

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By the PCR primer pairs of lc2-US and lc2-DS, we can see that the upstream and downstream fragments flanking the gene lc2 were successfully amplified and connected together (Figure 7-A, B and C). The PCR fragment on the successful mutant CW117^?lc1-lc2 was expected as 1637 bp, but the PCR fragment on starting strain CW117^?lc1 without lc2 deficiency was 3410 bp using the same primer pair of ver2-F and ver2-R (Table 1). The screening result showed that the band size of lane 2 were consistent to starting strain CW117^?lc1; while, the band size of lane 1 was considered as mutant CW117^?lc1-lc2 candidate (Figure 7-D). PCR sequencing validated the clone from lane 1 was the positive mutant CW117^?lc1-lc2.

Figure 7 The genes lc1-lc2 double mutant construction from staring strain CW117?lc1. A: the upstream PCR fragment of gene lc2. B: the downstream PCR fragment of gene lc2. C: the PCR fragment of lc2-US-DS from pK18mobsacB_US-DS. D: the PCR validation on gene lc1-lc2 double mutant screening. M: DNA marker; lane 1: positive candidate; lane 2: staring strain CW117?lc1.

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2.4 The AFB1 degrading activity of wide-type and mutants The degradation ratios of wild-type strain CW117 and the mutants (CW117^?lc1 and CW117^?lc1-lc2) were about 23% after 12 h incubation (no significant difference). After 24 h incubation, the degradation ratio of wide-type increased to 42.2% which a little higher than that of mutant CW117^?lc1 (35.9%) and mutant CW117^?lc1-lc2 (34.7%); however, the three strains (wide-type and two mutants) showed no significant difference from each other after 48 h incubation (Figure 8-A).

Figure 8 The AFB1 degradation dynamics of wide-type and mutants. A: the degradation dynamics of bacterial strains. B: the degradation dynamics of culture supernatant. Error bars indicate Mean±SD.

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2.5 The AFB1 degradation activity of culture supernatant from wide-type and mutants In previous study, we found that the AFB1 degradation agent(s) mainly distributed in culture supernatants (cell-free) the strain CW117^[30]; thereby, the culture supernatants from the wide-type strain and mutants were further evaluated here. Similar as the AFB1 degradation dynamics by bacterial strains (i.e., CW117, CW117^?lc1and CW117^?lc1-lc2), the degradation activity by culture supernatants from wild type and mutants showed no significant difference from each other. The culture supernatants from three type strains (wide-type, lc1 mutant and double genes mutant) degraded above 50% AFB1 within 3 h incubation, and degradation ratios increased to 80% within 9 h incubation (Figure 8-B).
3 Discussion The degradation result of culture supernatant of CW117 was similar to the degradation characteristics of several previous studies, which equally showed the aflatoxin degrading agents in culture supernatant. For instance, the AFB1 degradation ratios by culture supernatant of Bacillus shakletonii L7^[11], Bacillus velezensis DY3108^[37], Stenotrophomonas maltopholia 35-3^[18], Mycobacterium FA4^[14] and Rhodococcus erythropolis^[16] were 77.9%, 81.97%, 78.7%, 80% and 66.8% within 72 hours, respectively. The supernatants of Candida versatilis CGMCC 3790^[38] and Tetragenococcus halophilus CGMCC 3792^[39] also showed degradation activity to AFB1, the degradation ratios by the two strains' culture supernatant were 29.61% and 14% in 60 minutes, respectively. The similar degradation characteristics of the microbial strains might indicate the same degrading mechanism.
The redox mediators improvement on rLC1 of this study were much lower than the study of Lac2 from Pleurotus pulmonarius^[21], which obtained an efficient degradation of AFB1 by adding 10 mmol/L AS (degradation ratio of 90%), SA (degradation ratio of 72%) and ABTS (degradation ratio of 81%) as redox mediators. Similarly, the additive improvements on rLC1 in this study were lower than the study of laccase Ery4 from Pleurotus eryngii^[23], which obtained AFB1 degradation ratios of 73%, 68%, and 39% by adding 1 mmol/L AS, 1 mmol/L SA and 10 mmol/L ABTS, respectively. The original laccases of Lac2 and Ery4 also showed more efficient degradation than rLC1 of this study.
Substrate AFB1 in the culture medium could be degraded completely by any of the wide-type or mutant strain with increasing of incubation time. This result indicated that gene lc1 showed degrading activity to AFB1, but the laccases (i.e., lc1 and lc2) could not be considered as the primary degradation agent in Stenotrophomonas sp. CW117.
The AFB1 degradation ratio of Lac2 purified from Pleurotus pulmonarius was 23% within 72 h, this degradation activity was similar to rLC1 of this study^[21]. However, some other laccases, especially of laccases from several fungi strains showed more efficient aflatoxins degradation activity than rLC1. For instance, the laccase Ery4 from Pleurotus eryngii showed an AFB1 degradation ratio of 74% within 72 h^[23]; the purified laccase (1 U/mL) from Aspergillus niger showed degradation ratio of 87.3% to AFB1 within 72 h^[26]; the laccase from Trametes versicolor showed a degradation ratio of 67% to AFB1 within 48 h^[29].
These studies suggested that laccases have been recognized as an effective aflatoxin degradation agent and widely distributed in microbial strains. For some degrading strains (e.g., Pleurotus pulmonarius and Stenotrophomonas sp. of this study), the laccases degradation activity was not as efficient as their host strains. However, whether laccase(s) were the sole degradation agents in these degrading strains are not investigated further, the exact reasons for the degradation difference between laccase(s) and the host strains still unknown. In this study, when the laccase genes lc1 and lc2 were deleted, the mutant CW117^?lc1and CW117^?lc1-lc2 showed the similar degradation activity to wide-type strain CW117. The validation results of lc1 and lc2 degradation test in vivo indicated that laccases are not the sole degrading agents (even not the primary degrading agent) in Stenotrophomonas sp. CW117.
Degradation activity of culture supernatants were much higher than those of bacterial strains on the first 12 h. As we know, during the strains degradation test, first 12–24 hours are required for bacterial growth, but degrading agent(s) already existed in culture supernatants at the beginning of the test. The culture supernatant degradation tests equally showed that the laccase genes were not the sole (or even not the critical) degrading agent in Stenotrophomonas sp. CW117.
4 Conclusion Laccases are the well-known aflatoxins degrading agent and widely distributed in microbial strains. In several previous studies, the aflatoxin degradation activities of laccases were not as efficient as the host strains, but the exact reasons were not investigated. In this study, two laccase genes (lc1 and lc2) were isolated and evaluated for AFB1 degradation by heterologous expression. Compared to host strain CW117, laccase rLC1 showed much lower AFB1 degrading activity and rLC2 showed no activity on AFB1 transformation. The degradation results of mutants CW117^?lc1and CW117^?lc1-lc2 indicated that laccases are not the sole degrading agents (even not the critical factor) in Stenotrophomonas sp. CW117. And the finding of this study should be significant to further mechanism investigations on aflatoxins degradation.

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