Purification and heterologous expression of a novel antifungal protein from Bacillus subtilis strain Z-14


 Background: Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and has been confirmed to have strong antifungal activity against R. cerealis. Results: An antifungal protein, termed F2, was isolated from the culture supernatant of Z-14 strain using precipitation with ammonium sulfate, anion exchange chromatography, and reverse phase chromatography. Purified F2 had a molecular mass of approximately 9 kDa, as assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Edman degradation was used to determine the amino acid sequence of the N-terminus, which was NH2-ASGGTVGIYGANMRS. This sequence is identical to a hypothetical protein RBAM_004680 (YP_001420098.1) synthesized by B. amyloliquefaciens FZB42. The recombinant F2 protein (rF2) was heterologously expressed in the yeast host Pichia pastoris, purified using Ni‑affinity column, and demonstrated significant antifungal activity against R. cerealis. The purified rF2 was thermostable, retaining 91.5% of its activity when incubated for 30 min at 100 °C. rF2 maintained its activity under treatment by proteinase K and trypsin.Conclusions: A novel antifungal protein F2 was purified from biocontrol Bacillus subtilis Z-14 strain fermentation supernatant and heterologously expressed in Pichia pastoris to certificate the antifungal activity against R. cerealis and the validity of gene sequence of protein F2. Considering its significant antifungal activity and stable characteristics, protein F2 presents an alternative compound to deal with fungal infections caused by R. cerealis.

During its growth, B. subtilis secretes a wide range of antimicrobial proteins to inhibit the growth of pathogenic microbes [9,10]. Antimicrobial proteins have become an emerging research eld because of their special mechanism of action, lack of serious environmental effects, and their infrequent induction of resistance in their target pathogenic species [11]. The biochemical characteristics and biological functions of puri ed antimicrobial proteins have been studied, or their effect on the histomorphology of the host or pathogen has been observed to clarify their site of action and antimicrobial mechanism [12].
After the physicochemical properties and antimicrobial activities of the puri ed proteins are determined, the genes encoding antimicrobial proteins can be obtained from the original bacteria [13]. The antimicrobial protein gene can then transferred into the affected plant for expression, producing genetically engineered plants with disease resistance. Alternatively, the gene can also be introduced into a plant epiphyte or endophyte to construct high-e ciency biocontrol engineered bacteria [14].
Since Johnson et al. reported that B. subtilis produced antimicrobial substances, many kinds of antimicrobial active substances have been found in different Bacillus strains [15]. Most antimicrobial substances produced by B. subtilis are low molecular peptides synthesized through the non-ribosomal pathway, including cyclic peptides, cyclic lipopeptides, and linear peptides, usually with a molecular weight of about 1000 Da [8]. However, there are also some protein antagonists synthesized via the ribosomal pathway. Identi ed antimicrobial peptides from B. subtilis can be divided into several groups, including bacteriocins, cell wall degrading enzymes (protease, chitinase, and glucanase), pathogenesisrelated proteins, thaumatin-like proteins, non-speci c lipid transfer proteins, and unknown proteins [13,16,17]. Bacisubin (molecular weight = 41.9 kDa), an antifungal protein isolated from B. subtilis B-916, has a strong inhibitory effect on a variety of pathogenic fungi. Bacisubin has ribonuclease and hemagglutination activity, but no protease or protease inhibitory activity [18]. The protein F3A, which was isolated from B. subtilis F3, has high homology with agellin and shows good antimicrobial activity against Monilinia fruticola [19]. These proteins might be important to protect plants from pathogen infection. B. subtilis Z-14, selected from a wheat rhizosphere soil, demonstrated broad spectrum activity against phytopathogenic fungi in vivo and in vitro [20,21]. The present study aimed to purify the novel antifungal protein secreted by strain Z-14, express it heterologously, and perform a preliminary characterization.

Results
Antifungal protein F2 puri cation from Bacillus subtilis strain Z-14 Anion-exchange chromatography was used to isolate antifungal proteins from crude extracts of strain Z-14 fermentation supernatant on a HiTrap diethylaminoethyl (DEAE)-sepharose fast Flow column, which resulted in unadsorbed fraction A and seven adsorbed fractions (B-H; Fig. 1). The antifungal activities of the eight fractions were determined using the test fungus R. cerealis. Only fraction F displayed a strong antifungal activity, with less antifungal activity being detected in fraction G. Reverse phase chromatography was used to further purify fraction F, which resulted in four main peaks (Fig. 2). Among them, antifungal activity was only detected in fraction F2 (Fig. 3) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band with a molecular mass of approximately 9 kDa (Fig. 4).

Amino acid sequence of the N-terminus of F2
The rst 15 amino acids residues from N-terminal segment of the antifungal protein obtained by Edman degradation were ASGGTVGIYGANMRS. After searching the National Center for Biotechnology Information (NCBI) protein database, we observed that the peptide was identical to that of a hypothetical protein RBAM_004680 (YP_001420098.1), which was derived from the genome of B. amyloliquefaciens FZB42 (NC_009725.1) [22]. Except for the amino acid sequence, no other information, such as its function, location, or mass has been reported. Taken together with our results, this indicated that it could Expression of the antifungal protein gene f2 in P. pastoris The antifungal protein gene f2 was ampli ed using primers and successfully inserted into the plasmid pPIC9k. The recombinant plasmid was then transformed into P. pastoris GS115 competent cells. Positive colonies were identi ed by polymerase chain reaction (PCR) and the subsequent antifungal activity was assessed. The fermentation supernatant of the recombinant strain exhibited strong antifungal activity against sheath blight pathogen R. cerealis and subsequent SDS-PAGE analysis demonstrated that a band around 13 kDa (corresponding to the theoretical molecular weight of rF2) was only present in the fermentation supernatant of the recombinant strain; no such band was seen in the supernatant of the strain transformed with the empty vector. Recombinant protein rF2 was puri ed using a Ni-a nity column from a culture induced for 96 h. After puri cation, a single band of approximately 13 kDa was observed in the eluate when analyzed by SDS-PAGE. Protein rF2 was eluted mostly in 20 mmol/l imidazole and almost no protein was found at higher concentrations of imidazole. Furthermore, the eluate samples were assessed for their antifungal activities and the obtained protein band was con rmed as recombinant protein rF2 (Fig. 5).

Effects of proteases, temperature, and on rF2's activity
The effect of pH on the antifungal activity of rF2 was shown in Fig. 6. The highest antifungal activity was detected at pH 7.0, which remained almost unchanged in the pH range 6-8. The activity decreased slightly at pH 5.0, 9.0, and 10.0, but decreased signi cantly by 88% at pH 4.0, and disappeared completely at pH less than 3.0. Protein rF2 preserved most of its antifungal activity after exposure to temperatures of 40-80 °C for 20 min, but lost 8.5% and 21.8% of its total activity when incubated for 30 min at 100 °C and 121 °C, respectively, which demonstrated that protein rF2 possesses strong thermal stability (Table  1). After pepsin treatment, rF2 lost 10.8% of its antifungal activity. However, the antifungal activity remained intact when treated with proteinase K and trypsin at 37 °C for 60 min (Table 2).

Discussion
The continuous use by crop growers of chemical agents for biological control, coupled with their lack of understanding of the mechanism of inhibition by these agents, has contributed to worldwide concerns regarding the use of chemical control of pathogens [23]. In addition, the successful use of a biocontrol agent depends upon being familiar with the biological environment in which the agent is to be used and the production of a stable formulation of the selected biocontrol agent. Thus, the use of natural formulations from gram-positive bacteria, with their advantages of heat and desiccation tolerant endospores, to suppress disease, would help to overcome the disadvantages of other biocontrol agents.
Therefore, choosing the most appropriate biocontrol agent is important to treat complex and devastating pathogens [24]. The use of antimicrobially active species and strains of Bacillus, or their metabolites, represent an alternative method of plant protection [25]. To support the use of known microorganisms or to develop of improved strains to control plant disease, it is necessary to understand the biological control mechanism at the biochemical and molecular levels and to determine the underlying causes of the observed variability in biological control in agro-ecosystems [26]. The present study isolated and characterized a potential antifungal protein from B. subtilis. The protein is hypothesized to function in protecting wheat from infection by harmful pathogens. The antifungal activity of F2 could be used to resist sheath blight disease caused by R. solani, and thus could represent an effective biological control agent to e ciently manage the disease.
Antifungal proteins from Bacillus spp. have been used frequently to suppress various diseases. Over the last 10 years, researchers have identi ed several classes of proteins that inhibit fungal and bacterial growth using in vitro assays. These proteins are distributed widely in animals, plants, bacteria, and even in fungi [11]. The identi ed antimicrobial proteins are divided into several groups, such as ribosome inactive proteins, thaumatin-like proteins, non-speci c lipid transfer proteins, thionines, chitinases, chitinbinding proteins, and pathogenesis-related (PR) proteins [27,28]. These proteins might have vital functions in the protection of plants against infection by pathogens.
To date, few methods have been developed for large-scale protein separation. Extraction using ammonium sulfate precipitation coupled with column chromatography separation is still the main method for protein puri cation [14]. An antifungal protein from B. licheniformis HS10 of approximately 55 kDa was identi ed as a carboxypeptidase after isolation using 30-60% ammonium sulfate precipitation of culture supernatant combined with column chromatography puri cation on DEAE Sepharose Fast Flow, RESOURCE Q, and Sephadex G-75. The puri ed antifungal protein signi cantly inhibited eight kinds of plant pathogenic fungi, and showed stable biological activity after treatment for 30 min at 100 °C and at pH values ranging from 6 to 10 [29].
In the present study, a 9-kDa extracellular thermostable antifungal protein named F2 was demonstrated to have antifungal activity against plant-borne pathogenic fungi. F2 was puri ed using ammonium sulphate precipitation, anion exchange chromatography, and reverse phase chromatography. F2 is active over a wide pH range, and is resistant to high temperature and protease degradation, which is similar to the carboxypeptidase from B. licheniformis HS10 described above [29]. These characteristics and the low costs of producing these antifungal proteins identify them as promising candidates to develop food preservatives, medicines, and commercial biopesticides against pathogenic lamentous fungi.
Unfortunately, the low production yield of these antifungal proteins from Bacillus spp. limits their practical application, despite our detailed understanding of their upstream transcription regulation elements that respond to stressors and environmental signals [30,31]. For their future study and practical applications, it is necessary to produce antifungal proteins such as F2 in higher amounts in a nonsensitive, easily fermentable, 'generally recognized as safe' fungus. Several antimicrobial proteins have been used as the basis to design synthetic proteins and analogs as active ingredients of food preservatives, medicines, and commercial biopesticides [32]. We successfully expressed protein rF2 in P. pastoris and used a nity chromatography with a Ni column to purify it. This con rmed that the gene sequence correctly expressed antifungal protein F2. This e cient synthesis and simple puri cation will lay the foundation for followup applications.
R. cerealis proved to be susceptible to protein F2 secreted from B. subtilis Z-14 in the present study. Taking into account this susceptibility data and the characteristics of protein F2 (resistance against protease degradation, and stable antifungal activity over wide pH and temperature ranges), we believe that F2 represents an alternative compound to treat fungal infections caused by R. cerealis.

Materials And Methods
Strains and culture conditions B. subtilis Z-14 (hereafter referred to as Z-14), which signi cantly reduced the growth of Rhizoctonia cerealis (a wheat sheath blight pathogen), was originally isolated from soil sampled from a wheat rhizosphere [20]. Z-14 was grown on nutritive agar (NA) overnight at 37 °C. An aliquot of the overnight culture was inoculated into 50 ml of fermentation medium (20 g of sucrose; 10 g of tryptone; 2 g of KH 2 PO 4 ; 0.05 g of CaCl 2 ; 0.05 g of MgSO 4 ·7 H 2 O; and 1000 ml of distilled water; pH 7.5) in an Erlenmeyer ask and cultured for 48 h at 37 °C with shaking at 220 rpm.

Antifungal protein extraction from the Z-14 supernatant
The Z-14 culture was centrifuged for 15 min at 10 000 × g and the supernatant was retained and ltered through a 0.22-μm hydrophilic lter (Jinteng, Tianjin, China). The metabolites were precipitated from the supernatant using 80% saturated (w/v) (NH 4 ) 2 SO 4 and stored at 4 °C overnight. The mixture was centrifuged for 15 min at 10 000 × g and the pellet was dissolved in 10 ml of 10 mmol/l Tris-HCl buffer (pH 7.5). To remove the ammonium sulfate, the solution was dialyzed in 2-kDa cut-off dialysis tubing (Sigma-Aldrich, MO, USA) for 48 h at 4 °C, with a buffer change every 4 hours (500 ml each). The dialysates were condensed using vacuum freeze-drying to yield precipitated proteins, which were further puri ed using column chromatography.
Antifungal activity assessment of the metabolites from Z-14 The agar diffusion technique was used to assess the antifungal activity of metabolites from Z-14 against Rhizoctonia cerealis [33]. R. cerealis BD-13 was cultured on potato dextrose agar (PDA) slants at 25 °C for 7 d. Water was then added to the slants, the surface mycelia of which were rubbed gently with a glass rod to harvest the conidia. The fungal spore suspension (10 ml; 1 × 10 6 /ml) was mixed with 100 ml of PDA and poured into Petri dishes (n = 6). After the PDA solidi ed, agar disks were excised to form wells with a diameter of 7 mm, to which were added 30 μl-aliquots of culture extract. The control comprised sterile water. For each sample, three replicates were performed. The plates were incubated for 5 d at 25 °C. The antifungal activity was determined as the diameter of the growth inhibition zone around the wells compared with that of the control well.
Ion exchange chromatography puri cation of antifungal proteins Ion exchange chromatography was carried out using a HiTrap DEAE-sepharose fast Flow column (Amersham Pharmacia, Sweden) equilibrated with 10 mmol/l Tris-HCl buffer (pH 7.5) on the ÄKTA explorer 100 system obtained from Amersham Biosciences (Sweden). The proteins bound to the column were eluted sequentially using 0.05, 0.1, 0.2, 0.3, 0.45, 0.6, and 1.0 mol/l NaCl. The eluate was assessed via its absorbance at 280 nm. Each fraction was dialyzed and adjusted to the same concentration using Tris-HCl buffer. The antifungal activity of each fraction was tested against R. cerealis using the agardiffusion method.
Puri cation of antifungal proteins using reverse phase chromatography The fractions with antifungal activities were collected, and then subjected to reverse phase chromatography on a SOURCE TM 5RPC 4.6/150 column (Amersham Biosciences), which had been equilibrated using 0.06% tri uoroacetic acid (TFA). The material bound to the column was eluted linearly using 60% acetonitrile solution containing 0.05% TFA. Individual fractions were collected, subjected to dialysis, and then vacuum freeze-drying was used to condense the fractions before further analysis.
Purity, molecular mass, and concentration determination of the isolated proteins SDS-PAGE was performed using 0.75-mm-thick gels comprising a 5% stacking gel and a 12% separating gel to assess the purity and molecular mass of the separated protein fractions. The Bradford method was used to determine the protein concentration, using bovine serum albumin as the standard [34].

Antifungal protein N-terminal sequence analysis
The puri ed antifungal protein was separated using SDS-PAGE and then electroblotted onto a polyvinylidene uoride membrane (Bio-Rad, USA) at 60 V for 30 min. The protein on the membrane was then applied to a 491 protein sequencer (Applied Biosystems, USA) to determine its N-terminal amino acid sequence using automated Edman degradation [35].
Construction of the expression plasmid pPIC9K-f2 The amino acid sequence homology of the N-terminus of the isolated protein was analyzed using the NCBI Basic Local Alignment Search Tool (BLAST) online search service to nd similar proteins and related gene sequences. According to the gene sequence and the multiple cloning sites of the expression vector pPIC9K, the primers W-QC: CCGGAATTCATGGTA CGTCGTTTGTCGATC and W-D: ATAAGAATGCGGCCGCTTAGTGGTGGTGGTGGT GGTGTAAACCGTAATAATAAGATAG were designed, which incorporated EcoR I and Not I restriction sites in the PCR amplicon and added a sequence encoding a C-terminal His-tag. The primers were used to PCR amplify the target gene encoding the antifungal protein using Z-14 genomic DNA as the template. The PCR amplicon, encoding a His-tagged protein named f2, was ligated into expression vector pPIC9K via the EcoR I and Not I sites. The ligation products were transformed into Escherichia coli DH5α cells. Positive transformants were selected and screened for presence of the recombinant plasmid. The recombinant plasmid was validated using DNA sequencing.

Recombinant protein expression in Pichia pastoris GS115
Sac I was used to linearize the expression plasmid pPIC9K-f2, which was then transformed into electrocompetent P. pastoris GS115 cells using a model 165-2100 MicroPulser Electroporator (Bio-Rad, USA) following the manufacturer's instructions. The obtained transformant culture was spread on plates comprising yeast potato dextrose (YPD) agar medium containing 2 mg/ml G418. Single colonies that appeared after incubation at 25 °C for 2 days were picked out and detected using PCR with the W-QC and W-D primers. For recombinant protein production, a positive transformant was inoculated into buffered minimal glycerol yeast (BMGY) medium and cultured at 25 °C for 24 h, with shaking at 250 rpm. Centrifugation was used to harvest the cells, which were resuspended in 50 ml of buffered minimal methanol yeast (BMMY) medium in a 500 ml ask. Recombinant F2 (rF2) expression was induced for 96 h and methanol was added every 24 h at 1% (v/v) nal concentration. The fermentation broth was centrifuged at 10 000 × g and 4 °C for 10 min, and the supernatant was collected to measure the antifungal activity and for SDS-PAGE.
Puri cation of the recombinant antifungal protein A His60 Ni Super ow TM Resin & Gravity Column (Clontech Laboratories, CA, USA) was used to purify the HIS-tagged recombinant protein following the manufacturer's protocol. Bound proteins were eluted successively using 20, 50, 100, and 200 mmol/l imidazole. The eluate was dialyzed to remove the imidazole and condensed using vacuum freeze-drying. The precipitate was dissolved in 10 mmol/l Tris-HCl buffer (pH 7.5) and the solution was used to detect the antifungal activity and for SDS-PAGE.
The in uence of proteases, temperature, and pH on the activity of recombinant protein rF2 To analyze the effect of different pHs (range 3.0-10.0) on the antifungal activity of rF2, the pH of the culture ltrate was altered using 2 mol/l NaOH or HCl and incubated for 2 h at 37 °C. Protein rF2 was exposed to a range of temperatures (40, 60, 80, 100 and 121 °C) for 30 min to study its thermal stability.
To detect its stability under protease treatment, rF2 was digested using 1 mg/ml protease K, trypsin, and pepsin (Amresco) for 60 min at 37 °C, respectively. The antifungal activities of the rF2 culture ltrates treated as detailed above were determined according to the method described by Zhao et al. [36]. The formula reported by Wong et al. was used to calculate the relative activity of the treated rF2 protein [37]. Three replicates for each treatment were assessed in three repeated experiments.

Statistical analysis
Replicate data are expressed as the mean ± the standard deviation (SD). The Statistical Product and Service Solutions (SPSS) ver. 17.0 software package was used to perform all the statistical analyses. One-way analysis of variance and Duncan's test (P ≤ 0.05) were used to assess whether the means differed signi cantly.

Declarations Funding
The Hebei Provincial Natural Science Foundation of China (grant numbers C2014204027 and C2019204210) and the Hebei Provincial Key Research and Development Project of China (grant number 20326509D) supported this study. The funding body had no in the design of the study and collection, analysis, and interpretation of the data. investigation and methodology. LC investigation and methodology. ZB supervision and funding acquisition.   Elution pro le of fraction F obtained using reversion phase chromatography The purity and molecular mass of the puri ed antifungal protein F2 on SDS-PAGE Page 17/17

Figure 5
The antifungal activity of recombinant protein F2 (rF2) against R. cerealis Figure 6 Analysis of the antifungal activity of recombinant protein rF2 under different pHs