Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent

Journal Pre-proof Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent

Zhao-Lin Ji, Shuai Peng, Li-Li Chen, Yang Liu, Chun Yan, Feng Zhu PII:

S0141-8130(19)38150-4

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.216

Reference:

BIOMAC 14244

To appear in:

International Journal of Biological Macromolecules

Received date:

9 October 2019

Revised date:

11 December 2019

Accepted date:

24 December 2019

Please cite this article as: Z.-L. Ji, S. Peng, L.-L. Chen, et al., Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.216

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© 2018 Published by Elsevier.

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Identification and characterization of a serine protease from Bacillus licheniformis W10: A potential antifungal agent Zhao-Lin Ji, Shuai Peng, Li-Li Chen, Yang Liu, Chun Yan, Feng Zhu*

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College of Horticulture and Plant Protection, Joint International Research Laboratory of

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Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University,

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Yangzhou, Jiangsu 225009, China

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ABSTRACT

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*Correspondence: [email protected] (F.Z.)

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Bacillus licheniformis W10 is a strain of biocontrol bacteria that was obtained from plant

rhizosphere screening. In this study, we purified, identified, and carried out bioinformatics

analysis of the W10 antifungal protein from Bacillus licheniformis. Mass spectrometry analysis

was carried out by passing the antifungal protein through a high-resolution time-of-flight mass

spectrometer. Mascot searches of the tandem mass spectrometry data identified this antifungal

protein as a serine protease, and the 1347 bp gene encoding this protein was cloned.

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Bioinformatics analysis of this protein indicated that it contains 448 amino acid residues, has a

molecular weight of 48794.16 Da and an isoelectric point of 6.04, and is a hydrophilic protein. In the secondary and tertiary structure of this protein, the proportion of α-helices and β-folds is

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similar, and the protein possesses a Peptidase_S8 conserved domain. Using BApNA as a

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substrate, it was found that the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF)

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can inhibit the W10 antifungal protein. PMSF concurrently reduced the inhibitory effects of the

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antifungal protein on Botrytis cinerea, showing that the W10 antifungal protein possesses serine

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protease activity. The W10 antifungal protein has good thermal stability. The study implies

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potential of this enzyme for biocontrol of fungal plant pathogens.

bioinformatics

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Keywords: Bacillus licheniformis, antifungal protein, isolation, identification, serine protease,

1. Introduction

Chemical pesticides are still the primary method used for the prevention of plant disease, and

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pesticide treatment is integrated into plant disease management. However, the long-term,

large-scale, and widespread use of chemical pesticides not only increases the risk of pesticide

residue and environmental pollution but also leads to the development of drug resistance,

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resulting in decreased efficacy. In addition, this also disrupts the biocontrol mechanisms of

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probiotics on pathogens, resulting in uncontrolled pathogen proliferation. These factors seriously

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threaten the sustainable development of agriculture and its related economy and severely affect

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human health [1]. Due to environmental conservation and food security requirements, many

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researchers are searching for new plant disease control methods. Among these methods, the

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development of new and environmentally friendly microbial pesticides for biological control is

pollution [2].

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an important route for controlling postharvest diseases of fruits and reducing chemical pesticide

Biocontrol microorganisms are the source of microbial pesticides and mainly include fungi,

bacteria, and actinobacteria [3–5]. As biocontrol bacteria proliferate rapidly, are easy to culture,

and can colonize and transfer in plants, they have become an important biocontrol resource.

Studies have shown that biocontrol bacteria are mainly Bacillus, Pseudomonas, Agrobacterium,

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and Serratia, of which Bacillus and Pseudomonas are extensively studied [6] and Bacillus,

Agrobacterium, and Pseudomonas are predominantly used [7]. At present, many types of

biocontrol bacteria have been commercialized, the most well-known being Agrobacterium

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radiobacter K84 from Australia. In addition, other commercialized strains include the Bacillus

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subtilis BS2208 wettable powder from Wuhan Tianhui Biological Engineering Co., Ltd.,

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Genfuxiao (B. subtilis and Pseudomonas fluorescens) from Kunming Aolym Biotech Co. Ltd,

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and Shudekang (Bacillus, Salmonella) developed by Nanjing Agricultural University (Jiangsu,

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China).

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Several members of the group are known to produce mycolytic enzymes, antimicrobials and

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siderophores as principal antifungal agents [8]. Although glucanases and chitinases are

considered as major mycolytic enzymes, several recent studies have also identified role of

proteases in fungal biocontrol. Protease refers to a group of enzymes whose catalytic function is

to hydrolyse peptide bonds of proteins. Proteolytic enzymes can attack the cell wall of

phytopathogenic fungi, causing cell lysis and subsequent death [9]. Currently, proteases are

divided into four major groups based on the character of their catalytic active site and conditions

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of action, including aspartic proteinases, cysteine proteinases, serine proteinases, and

metalloproteinases [10]. For example, an alkaline extracellular serine protease secreted by

Aureobasidium pullulans PL5 played a role in the biocontrol activities against some postharvest

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pathogens of apple and peach, such as Monilinia laxa, Botrytis cinerea and Penicillium

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expansum [11]. Several studies have also demonstrated that proteolytic enzymes produced by the

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Trichoderma harzianum and Pseudomonas aeruginosa M-1001 play an important role in

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biological control of fungal pathogens [12,13]. A 20 kDa serine protease purified from

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Streptomyces sp. A6 exhibited biocontrol efficacy against the fungal pathogen [14].

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Overproduction of extracellular serine protease by Stenotrophomonas maltophilia strain W81

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improved biological control of Pythium ultimum [15]. Therefore, antifungal action of protease

suggests their potential in agriculture for control of fungal phytopathogens. Although proteases

have been reported from several fungal biocontrol agents, information on protease from Bacillus

licheniformis is very limited. And there are rarely reports on their potential antifungal activity.

Bacillus licheniformis is widely distributed in nature. This bacterium has many physiological

characteristics and possesses many special functions, such as the production of endospores to

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resist various adverse environments [16] and the production of various antimicrobial substances,

such as lipopeptides, peptides, phospholipids, polyenes, amino acids, and nucleic acids, which

can inhibit various animal, plant, and human pathogens. Bacillus licheniformis also possesses

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potent protease [17], lipase [18] amylase [19], glucanase [20], and chitinase [21] activity and is

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thus widely used in medical, pesticide, foodstuff, feed processing, and environment purification

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applications.

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Bacillus licheniformis is a typical plant growth-promoting bacterium, and its effector

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mechanisms include nutrient and space competition with pathogens, antibiotic effects,

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bacteriolytic effects, induction of plant resistance, and promotion of plant growth. Examples

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include B. licheniformis NJWGYH 833051, which can colonize the leaves and rhizosphere of

tomato and play a role in biocontrol [22]; B. licheniformis GZ-3, discovered by Ma et al. [23],

which not only shows good antagonism against Botrytis cinerea and Fusarium oxysporum but

also has inhibitory activity against many tested pathogenic fungi; and a B. licheniformis species

that was isolated from fermented grains and possesses bacteriolytic activity against different

Gram-positive bacterial strains [24]. Root treatment with B. licheniformis TG116 can increase

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the activity of defense enzymes such as peroxidase (POD), polyphenol oxidase (PPO), and

phenylalanine

ammonia-lyase

(PAL)

in

cucumber

leaves

while

slightly

decreasing

malondialdehyde (MDA) content to a new equilibrium and inducing systemic resistance in

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cucumbers [25]. Ansari et al. [26] found that B. licheniformis B642 and P. fluorescens FAP2 can

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interact in biofilms, thereby increasing plant growth and photosynthesis.

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Bacillus licheniformis W10 is a bacterial strain with biocontrol potential that our laboratory

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obtained from rhizosphere screening. It can inhibit many types of fungal plant pathogens, impede

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hyphal growth, sporulation, conidial germination and germ tube elongation, and disrupt mycelial

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morphology [27]. In addition, B. licheniformis W10 has good colonization ability, can induce

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disease resistance, exerts growth promoting effects [28], and demonstrates good indoor [29,30]

and field efficacy [31,32]. However, the biocontrol mechanism of W10 is still not completely

understood, though it is through the production of an antifungal protein. In this paper, we studied

the W10 antifungal protein and employed protein purification and mass spectrometry analysis to

determine the type of protein as well as its physiochemical properties and related biological

information in order to provide a theoretical foundation for the further application of this

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biocontrol bacterium.

2. Materials and Methods

2.1. Strains and Materials

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Bacillus licheniformis W10 (CGMCC No.14859) and B. cinerea were from our laboratory.

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Tris-base was obtained from Oxoid Limited (UK). Acrylamide, bisacrylamide, and sodium

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dodecyl sulfate (SDS) were obtained from Sangon Biotech (Shanghai) Co., Ltd. Plant gel was

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obtained from Sigma Aldrich Inc. (USA). Other reagents were locally produced and were

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analytical grade.

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2.2. Culture Medium, Commonly Used Solutions, and Buffers

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Nutrient broth (NB) culture medium (g L-1): 5.0 polypeptone, 5.0 sodium chloride, 3.0 beef

extract, pH 7.2–7.4. Nutrient agar (NA) culture medium: 15.0 g of agar powder was added to the NB culture medium. Luria-Bertani broth liquid (LB) culture medium (g L-1): 10.0 tryptone, 5.0 yeast extract, 10.0 sodium chloride, pH 7.2–7.4. LB broth agar (LA) culture medium (g L-1): 10.0

tryptone, 5.0 yeast extract, 10.0 sodium chloride, 15.0 agar powder, pH 7.2–7.4. Potato dextrose agar (PDA) culture medium (g L-1): 200.0 fresh deskinned potatoes, 20.0 sucrose, and 15.0 agar

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powder. Skim milk culture medium (g L-1): 2.0 KCl, 1.0 MgSO4·7H2O, 0.48 CaCl2·2H2O, 0.06

NaHCO3, 0.001 FeCl3, 40.0 NaCl, 10.0 skimmed milk, 15.0 agar, pH 7.5.

Staining solution (1 L): 500 mL anhydrous ethanol, 2.5 g Coomassie brilliant blue R250, in a

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final volume of 1 L after dissolving in ddH2O. The solution was fully mixed before filtering. The

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filtrate was stored in a brown glass bottle for subsequent experiments. Destaining solution (1 L):

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500 mL ethanol, 80 mL acetic acid, top up to 1 L with ddH2O, mix evenly. Tris-HCl buffer 0.05

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mol L-1 pH 6.8 (1 L); 6.0 g Tris-base was topped up to 1 L with deionized water and concentrated

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hydrochloric acid was used to adjust the pH to 6.8.

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2.3. W10 Antifungal Protein Sample Preparation and SDS-PAGE Electrophoresis

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Ammonium sulfate precipitation was employed [33]. The W10 strain was cultured in NB culture medium for 72 h (28°C, 180 r/min) before centrifugation at 4°C and 8000 r min-1 for 10

min. The supernatant was passed through a filter (pore size: 0.45 µm) and ammonium sulfate

was added to the filtrate to 30% saturation and left to stand at 4°C overnight. The solution was centrifuged at 4°C and 8000 r min-1 for 10 min, following which the precipitate was collected. The precipitate was dissolved in a suitable volume of 0.05 mol L-1 pH 6.8 Tris-HCl buffer and

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loaded into a dialysis bag (retains molecules with a molecular weight of 8000–10000 Da), and

dialysis with the same buffer was carried out at 4°C. The solution outside the dialysis bag was

changed every 8 h until the inorganic salt and micromolecules in the dialysis bag had decreased

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to the lowest value. The solution in the dialysis bag was then collected. A bacteria filter (pore

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size: 0.22 µm) was used to remove impurities in the dialysis solution to obtain the crude protein.

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The SDS-PAGE resolving gel and stacking gel were prepared, and 10 µL crude protein was

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loaded on the gel. Twelve percent resolving gel (20 mL): 6.6 mL ddH2O, 5.0 mL 1.5 mol L-1

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Tris-HCl pH 8.8, 8.0 mL 30% Acr-Bis, 200 µL 10% SDS, 200 µL 10% AP, and 8 µL

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tetramethylethylenediamine (TEMED). Five percent stacking gel (5 mL): 3.4 mL ddH2O, 630

µL TEMED.

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mL 1.0 mol L-1 Tris-HCl pH 6.8, 830 µL 30% Acr-Bis, 50 µL 10% SDS, 50 µL 10% AP, and 5

After electrophoresis, the prepared staining solution was used for staining on a horizontal

shaking incubator with gentle shaking at room temperature for 45 min. Following that, the

staining solution was discarded and a suitable volume of destaining solution was added. The

destaining solution was changed every 45 min until the bands were clear, following which the

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gel was photographed.

2.4. Purification of W10 Antifungal Protein Samples

The HiPrep 16/60 Sephacryl S-100 High Resolution column (GE Healthcare Bio-Sciences AB,

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Sweden) was used for purification of the antifungal protein through the ÄKTA purifier (protein

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low-pressure chromatography system) (Amersham Biosciences AB, Sweden). The elution buffer

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(mobile phase) was 0.05 mol L-1 pH 6.8 Tris-HCl, and elution was carried out at room

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temperature at a flow rate of 0.8 mL min-1. The eluates from different peaks were collected and

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used for activity detection and mass spectrometry.

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2.5. Enzyme assay

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Detection of W10 protein enzyme activity by revised anson assay [34], one unit of denatured

hemoglobin degradation activity was defined as the amount of enzyme that released 1 mmol of

tyrosine per hour under standard assay conditions. A serine protease enzyme-linked

immunosorbent assay kit was used to determine the content of serine protease in liquid samples

of B. licheniformis W10.

2.6. Antifungal Activity Testing

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The zone of inhibition method [35]was employed, in which B. cinerea hyphal blocks were

inoculated onto the center of PDA plates, and 50 µL of Tris-HCl buffer (pH 6.8) and 0.48 mg

protein sample or purified protein were added to symmetrical sites 25 mm away from the

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pathogen. The fungi were cultured for 48 h at 25°C to observe for fungistatic activity. The

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sample treatment was repeated in triplicate.

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2.7. Mass Spectrometry Identification

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The purified protein band was excised, destained (50 mmol L-1 NH4HCO3/acetonitrile=1:1

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solution), and dried before enzymatic digestion was carried out inside the gel (0.01 μg μL-1

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trypsin in 25 mmol L-1 NH4HCO3 buffer overnight in a 37°C waterbath) followed by extraction

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(50% acetonitrile and 0.1% trifluoroacetic acid aqueous solution) and drying [36]. After

enzymatic digestion, an AB SCIEX 5800 mass spectrometry system (AB SCIEX, USA) was

used for matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry.

After obtaining the mass information of the polypeptide ions, polypeptide ions with specific

mass-to-charge ratios (m/z) were selected, and collision-induced dissociation was used to

fragment the polypeptide ions into smaller fragments. Following that, the tandem mass

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spectrometry search function (MS/MS Ions Search) in the Mascot analysis software (Matrix

Science London, UK) was used for searching the NCBInr database (version: 2016-08-21)

according to the m/z of the fragment ions.

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2.8. Protease Activity Analysis of the W10 Antifungal Protein

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The filter paper method [37]was used to collect 5 g of bacterial sample, which was added to

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45 mL sterile water before shaking for 30 min at 168 r min-1. Five milliliters of the supernatant

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was collected and heated for 10 min at 85°C. After heating, the supernatant was serially diluted

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and smeared onto the skim milk agar. The agar plates were cultured at 28°C for 48 h to observe

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for protease activity. The sample treatment was repeated in triplicate.

(MIC)

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2.9. Preparation of spore suspension and determination of minimum inhibitory concentration

The spore suspensions of B. cinerea were obtained from its 10-day-old cultures, mixed with sterile distilled water to obtain a homogenous spore suspension of 1 × 108 spore mL-1 [38]. The

minimum inhibitory concentrations (MICs) of the W10 protein were investigated by twofold

dilution method against B. cinerea [39]. The W10 protein was added to potato dextrose broth

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(PDB) to final concentrations of 0.50, 0.25, 0.13, 0.06, 0.03, 0.02 and 0.01 mg/mL, respectively. A 15 μL spore suspension of B. cinerea was inoculated in the test tubes in PDB medium and

incubated for 3–6 days at 28 °C.

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2.10. Inhibitory Effects of PMSF on W10 Antifungal Protein Activity

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Phenylmethylsulfonyl fluoride (PMSF) is a reversible serine protease inhibitor that inhibits

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the effects of serine proteases by binding the serine residues in proteins. PMSF (100 mM) of 0.25

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µL–6 µL (intervals of 0.25 µL) was added to 24 samples of 0.48 mg W10 antifungal protein (9.5

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mg/mL). And the final concentration of PMSF is from 0.5 mM to 12 mM. The zone of inhibition

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method was employed, in which B. cinerea hyphal blocks (diameter: 6 mm) were inoculated at

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the center of the PDA plates, and 50 µL of Tris-HCl buffer and 0.48 mg protein sample or

purified protein were added to symmetrical sites 25 mm away from the pathogen. The fungi were

cultured for 48 h at 25°C to observe for fungistatic activity. The sample treatment was repeated

in triplicate.

2.11. Effect of temperature and pH on stability and activity of the W10 protein

The effect of temperature and pH on stability and antifungal activity of the W10 protein was

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determined as described by Wang et al. [40]. To analyze the acid-base stability of W10 protein,

the antifungal protein was exposed to pH ranging from 4 to 12 for 1 h. The effect of the thermal

stability was investigated when temperatures were 20–100°C for 30 min. After incubation at

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different temperatures and different pH, the antifungal activity of W10 protein was determined.

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Three repetitions were included in each treatment, and each trial was repeated three times.

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2.12. Serine Protease Activity Analysis of the W10 Antifungal Protein

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Alfa-N-benzoyl-L-arginine p-nitroanilide is a specific substrate for serine protease hydrolysis.

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Spectrophotometry [41] was employed with BApNA as the substrate to measure the inhibitory

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effects of the serine protease inhibitor PMSF on the W10 antifungal protein. The neutral protease

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inhibitor EDTA and Triton X-100 were used as controls. The absorbance wavelength of 405 nm

was used to analyze whether the W10 antifungal protein possesses serine protease activity.

2.13. Cloning and Amino Acid Sequence of the W10 Antifungal Protein

Specific primers were designed for PCR amplification according to homologs found in the Mascot search. The primers used were: F: 5’-AATGCCGTTACAGCCCGCTCAT-3’ and R: 5’-GTAAGTGCCATTG TGATTCCTCC-3’ and were synthesized by Sangon Biotech (Shanghai)

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Co., Ltd. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min, followed by 30

cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30

s, followed by a final extension step at 72°C for 2 min before holding at 4°C.

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Agarose gel electrophoresis was used to detect the PCR products before they were sent for

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sequencing by Sangon Biotech (Shanghai) Co., Ltd. DNAMAN was used to analyze the gene

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sequence obtained from sequencing and determine its open reading frame (ORF). DNAMAN

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was then used to translate the correct ORF into an amino acid sequence, and this sequence was

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aligned with reported serine proteases.

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2.14. Bioinformatics Analysis of the W10 Antifungal Protein

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The ExPASy online software ProtParam (http://us.expasy.org/tools/protparam.html) was used

to analyze the physiochemical characteristics of the antifungal protein. The ProtScale online

program (http://www.expasy.org/tools/protparam.html) was used to analyze hydrophobicity,

TMHMM software (http://www.cbs.dtu.dk/services/TMHMM/) was used to analyze the

transmembrane domains of the W10 protein, SignalP (http://www.cbs.dtu.dk/services/SignalP/)

was used for signal peptide prediction, the SMART server (http://smart.embl-heidelberg.de/) was

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used

to

identify

functional

domains

in

the

W10

protein,

NetPhos

2.0

Server

(http://www.cbs.dtu.dk/services/NetPhos/) was used to analyze the protein phosphorylation site

modification,

the

SOPMA

(https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automa

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t.pl?page=npsa_sopma.html) was used to analyze the secondary structure of the protein, and the

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SWISS-MODEL server (http://www.expasy.ch/swissmod/ SWISS-MODEL.html) was used to

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construct a tertiary structure map.

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3.1. Crude W10 Antifungal Protein

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3. Results

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The W10 crude protein was extracted from the B. licheniformis W10 culture medium filtrate

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using ammonium sulfate precipitation. Plate inhibition tests showed that the W10 crude protein

could significantly inhibit the growth of B. cinerea (Fig. 1A). SDS-PAGE of the W10 crude

protein showed that the extracted crude protein contained two proteins of different sizes, which

were near the 44.3 kDa and 20.1 kDa markers (Fig. 1B). As the size of the band near the 44.3

kDa protein band was similar to the antifungal protein previously reported by our laboratory, this

was used for further protein purification.

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3.2. W10 Antifungal Protein

The ÄKTA low-pressure chromatography system was used to purify the extracted W10 crude

protein. According to Fig. 2A, three elution peaks were present, of which the area of peak I was

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the greatest. The eluates from the various elution peaks were collected. After freeze-drying and

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concentrating, the collected fractions were used for SDS-PAGE. The results showed that only a

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band appeared for the eluate of peak I, which was similar in size to the band in the

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electropherogram of the crude protein, and no protein bands were detected for the eluates of peak

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II and III (Fig. 2B). From this, it is evident that the eluate from peak I is the purified protein.

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Fungistatic activity testing was carried out on the eluate from peak I. We found that the purified

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protein had high activity and could significantly inhibit B. cinerea (Fig. 2C), demonstrating that

the purified protein is the W10 antifungal protein.

3.3. Serine Protease Activity of the Purified W10 Antifungal Protein

As shown in Table 1, the active protease from B. licheniformis W10 was purified to 27.83

folds using gel permeation chromatography with a final yield of 8.17%. The W10 purified

protein possesses serine protease activity. As shown in Fig. 3B, the W10 purified protein could

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produce an obvious transparent halo on the skim milk culture medium, thus confirming protease

activity. In Fig. 3A, different amounts of the serine protease inhibitor PMSF were added to the

purified W10 antifungal protein. As the amount of added PMSF increased, the inhibitory effects

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of the antifungal protein towards B. cinerea gradually decreased, but inhibition remained

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generally unchanged when the amount of PMSF added exceeded 3.5 μL. The results showed that

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the W10 antifungal protein possesses serine protease activity. Further study is needed to examine

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why complete inhibition did not occur with PMSF.

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By using BApNA as a substrate and neutral protease inhibitors (EDTA and Triton X-100) as

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controls, we measured the inhibitory effects of the serine protease inhibitor PMSF on the W10

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antifungal protein. As shown in Fig. 3C, the inhibitory effects of PMSF on the enzyme activity of

the W10 purified protein were significant, while EDTA and Triton X-100 did not exhibit strong

inhibitory effects on the enzyme activity of the W10 purified protein. This shows that the W10

antifungal protein possesses serine protease activity.

3.4. W10 protein stability

The W10 antifungal protein solution still has a strong antifungal activity against B. cinerea

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after treatment at different temperatures of 20, 40, 60, 80, and 100°C (Fig. 4A), indicating that

the W10 protein has good thermal stability. After treatment at different pH, the protein has

antifungal activity in the range of pH 6-12, the activity is highest at around pH 7, and the

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fungistatic ability is lost below pH 5 (Fig. 4B). It shows that W10 protein loses its fungistatic

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activity under strong acid environment and still has good stability under strong alkaline

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environment.

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3.5. Determination of minimum inhibitory concentration (MIC)

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Next, the minimum inhibitory concentrations (MICs) of the W10 protein were investigated by

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twofold dilution method against B. cinerea. As shown in Table 2, the minimum inhibitory

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concentrations (MICs) defined as the lowest concentration of W10 protein that resulted in

complete growth inhibition of B. cinerea was found to be 0.03 mg/mL.

3.6. Mass Spectrometry Analysis and Identification of the W10 Antifungal Protein

Data obtained from mass spectrometry were used to search the NCBInr database. The results

obtained showed that the concordance score of two candidate proteins exceeded the threshold

value of 73, and the reliability was significant (P<0.05). This indicated that our protein is highly

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similar to these two proteins (Fig. 5). Table 3 lists the data of these two highly similar proteins.

Therefore, from the mass spectrometry analysis results, we believed that the antifungal protein

that was purified in this study is a serine protease.

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3.7. Cloning and Amino Acid Sequence Analysis of the W10 Antifungal Protein

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PCR amplification was carried out using the W10 genomic DNA as a template according to

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primers designed from the mass spectrometry data and reported serine protease genes. A 1840 bp

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band was detected under electrophoresis (Supplementary Fig. 1) and sent to Sangon Biotech

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(Shanghai) Co., Ltd. For sequencing. DNAMAN was used to analyze the sequence, and a

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complete ORF was found. The W10 antifungal protein gene sequence was registered in GenBank

residues.

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with an accession number of MH141931. This gene encodes for a protein with 448 amino acid

The top 12 amino acid sequences of serine proteases from different Bacillus species in terms

of similarity were obtained from searching the NCBIr database. After multiple sequence

alignment using DNAMAN, we found that the total similarity of the amino acid sequence of the

antifungal protein in our study was 89.96% (Fig. 6). MEGA5 was used to construct phylogenetic

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trees (Fig. 7), which indicated that our antifungal protein had the highest homology with Bacillus

pumilus, which is consistent with the mass spectrometry identification results. This further

confirmed that the cloned gene is the serine protease gene.

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3.8. Bioinformatics Analysis of the W10 Antifungal Protein

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3.8.1. Antifungal Protein Amino Acid Composition, Hydrophobicity, Signal Peptide Prediction,

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and Transmembrane Domain Analysis

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Using the ProtParam tool, it was found that the W10 antifungal protein contains 448 amino

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acid residues, does not contain pyrrolysine and selenocysteine, and is rich in aliphatic amino

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acids such as isoleucine, valine, leucine, methionine, alanine, proline, and glycine. The molecular

Table 1).

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weight is 48794.16 Da and isoelectric point is 6.04 (Supplementary Fig. 2A; Supplementary

Hydrophobicity is an amino acid characteristic and is an important factor that determines the

three-dimensional spatial conformation of proteins. Understanding the hydrophobicity/

hydrophilicity of amino acid sequences has some role in predicting protein structure and function.

ProtScale was used for analysis, with y-axis coordinates of 0 and above representing

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hydrophobic amino acids and those with coordinates of below 0 representing hydrophilic amino

acids. The x-axis shows the amino acid positions. From the figure, we can see that most amino

acids in the W10 antifungal protein have a value below 0 (Supplementary Fig. 2B). Therefore,

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the W10 antifungal protein is a hydrophilic protein. SignalP was used for signal peptide

ro

prediction, and we found that the amino acid sequence does not contain a signal peptide

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-p

sequence or signal peptide cleavage site and value<0.5 (Supplementary Fig. 2C). Therefore, we

lP

believed that no signal peptide is present in the W10 antifungal protein.

na

Transmembrane domains are major regions in which membrane proteins bind to membrane

ur

lipids. TMHMM software was used to analyze the transmembrane domains of the W10

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antifungal protein. As shown in Supplementary Fig. 2D, where the x-axis represents the amino

acid location, that there was no amino acid that was above the outside line (the inside line

represents amino acids). These results indicated that the W10 antifungal protein does not contain

transmembrane domains.

3.8.2. Prediction and Analysis of Domain and Phosphorylation Sites of the W10 Antifungal

Protein

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The SMART server was used to search for functional domains in the W10 antifungal protein,

and many domains were found. However, the major domain is the Peptidase_S8 domain in

amino acids 152–437 (Supplementary Fig. S3A). The Peptidase_S8 domain is a characteristic

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catalytic domain in the B. subtilis protease family. As this family belongs to the serine protease

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family, this once again confirms that our protein is a serine protease. According to the NCBI

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-p

alignment and analysis of domains, we found that the W10 protein domain is similar to many

lP

known proteins (Supplementary Fig. 3B). Furtherfore, NetPhos 2.0 Server was used to analyze

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the protein phosphorylation site modification. The results suggest that there are lots of serine

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phosphorylation sites in W10 protein (Supplementary Table 2).

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3.8.3. Secondary and Tertiary Structure Analysis of the W10 Antifungal Protein

The secondary and 3-D structures of W10 protein were analyzed by SOPMA and

Swiss-Model. The result showed that W10 protein contains 33.93% alpha helices, 40.4% random

coils, 15.85% Beta strands and 9.82% beta turns (Supplementary Table 3). The 3-D structure of

W10 protein had the highest similarity (40.05%) with the template 3afg.2.A, subtilisin-like serine

protease (Fig. 8). Therefore, the results indicates that W10 protein may possess serine protease

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activity.

4. Discussion

Our study confirmed that the antifungal protein produced by B. licheniformis W10 is a serine

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protease with a molecular weight of 48794.16 Da and an isoelectric point of 6.04. Our previous

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study reported that the B. licheniformis W10 antifungal protein had a molecular weight of

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46049.2 Da and an isoelectric point of 6.71 [42]. The reason for this difference may be because

lP

SDS-PAGE and isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) were

na

previously used to determine the molecular weight and isoelectric point of the protein, while the

ur

present study determined these two values based on the amino acid sequence. The results of this

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study should thus be more accurate. In our study, BApNA was used as the substrate and neutral

protease inhibitors (EDTA and Triton X-100) were used as controls to verify that the W10

antifungal protein possesses serine protease activity. We also used different amounts of PMSF to

treat the antifungal protein and observe the inhibitory effects of the antifungal protein on B.

cinerea. The results showed that although PMSF can attenuate the inhibitory effects of the W10

antifungal protein on B cinerea, it cannot completely abolish its effect. One reason for this is that

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PMSF is unstable in an aqueous solution, as half of it will be degraded within 30 min and its

inactivation rate will increase at 25°C. The second reason for this is that the extensibility of

PMSF in culture medium is not as good as the serine protease. The third reason may be that there

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are other active amino acid regions that are fungistatic in addition to the serine protease activity

-p

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found that its inhibitory rate did not reach 100%.

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center. Tang et al. [43] identified a serine protease by using PMSF for confirmation and also

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Serine proteases are basic proteins with serine residues in the activity centers that interact

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with the carbonyl groups in the substrate [44]. A pH range of 8 to 10 is usually suitable for its

ur

activity. In this study, The W10 antifungal protein has good stability under strong alkaline

Jo

environment. Serine proteases have broad physiological effects in living organisms, and more

than one-third of known proteases are serine proteases [45,46]. Serine proteases can degrade

BApNA and are specifically inhibited by PMSF [47]. Serine proteases regulate protease

precursors through activation or inhibition and also play important roles in cell differentiation,

coagulation, embryonic development, tissue reconstruction, and pathogen invasion [48,49]. Sang

et al. [50] studied the effects of nematophagous fungal serine proteases on fungal infection in

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nematodes and found that alkaline serine protease has stronger catalytic and nematicidal activity

than neutral proteases. Shan et al. [51] found that Galeruca daurica serine protease (GdSP)

expression was significantly increased after treatment with high and low temperature stress.

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Sanjeev et al. [52] reported that serine proteases demonstrated efficacy in hemostasis and

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thrombus formation. It can thus be seen that serine proteases have broad research and application

re

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value. A study found that the C-terminal domain of serine proteases is involved in its catalytic

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function [53], which is consistent with our functional domain analysis result. In addition, a

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previous study claimed that the C-terminal domain of serine proteases is rich in cysteine [54],

ur

which differs from the antifungal protein in our study. As there is a large difference in the

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three-dimensional structure, the contrary result might be that their serine protease does not show

homology to the amino acid sequences of bacterial serine proteases [55].

Functional domain analysis of the W10 antifungal protein identified many domains, but the

major domain is the Peptidase_S8 domain consisting of amino acids 152–437. This domain is a

characteristic catalytic domain in the B. subtilis serine protease family, which is the second

largest family of serine proteases [56] that was first isolated and identified from B. subtilis [57].

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Subsequently, these proteases were discovered in Bacillus amyloliquefaciens, B. licheniformis,

and other Bacillus species. In recent years, studies on subtilisin have mainly focused on stability

in fungal phenotypes, the pathogenicity of pathogens, and other biological functions. However,

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there are also researchers who are aiming to develop subtilisin for the prevention and treatment

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of diseases caused by insects and parasites. Kabanov et al. [58] discovered that the subtilisin-like

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proteinase of B. pumilus 3–19 subtilisin-like proteinase will disrupt Pseudomonas aeruginosa

lP

biofilms. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of

na

egress, suggesting that the interruption of the life cycle at this stage may effectively inhibit the

ur

propagation of infection [59]. Fan et al. [60] employed genetic engineering to fuse the

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subtilisin-like protease CDEP-1 and the chitin gene to form a hybrid protease. Strains that

express this hybrid protease have greater pathogenicity and insecticidal activity than the

wild-type and strains overexpressing the native protease.

We carried out bioinformatics analysis of the W10 antifungal protein and the results showed

that this protein contains 448 amino acids and has a molecular weight of 48794.16 Da and an

isoelectric point of 6.04. There was no signal peptide and no transmembrane domain, and the

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protein contains a catalytic domain that is characteristic of subtilisins and possesses serine protease activity. In the secondary structure, the proportions of α-helices and β-folds are similar,

and this protein is a hydrophilic protein. These results provide a theoretical foundation for further

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studies of the biocontrol mechanisms of the W10 antifungal protein-serine protease, including its

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fungistatic mechanisms, functional domains, mining of receptors that interact with the antifungal

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protein in plants and pathogens, and application of the antifungal protein.

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In conclusion, the present study indicated the biocontrol potential of antifungal protease from

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B. licheniformis W10. Furthermore, The W10 antifungal protein has good thermal stability,

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suggesting that this antifungal protein can be used as a potential antifungal agent against

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phytopathogens for agricultural applications.

Acknowledgements

This work was supported by the earmarked fund for Modern Agro-industry Technology

Research System (CARS-31-2-02) and the Qing Lan Project of Yangzhou University.

Appendix A. Supplementary data

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The Supplementary Material for this article can be found online:

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55. L.X. Zhang, Protein engineering of serine protease, Chem. Life 12 (1992) 6–10. (in Chinese) 56. G.Y. Cheng, Characterization of a spore-associated protease and anextracellular serine protease from Thermoactinomyces sp. CDF, Hubei: Wuhan Univ. 2010. (in Chinese) 57. A.V. Guntelberg, M. Ottesen, Purification of the proteolytic enzyme from Bacillus subtilis, Compt. Rend. Trav. Lab. Carlsberg Sér. Chim. 29 (1954) 36–48. 58. D. Kabanov, N. Khabipova, L. Valeeva, M. Sharipova, A. Rogov, S. Kuznetsova, I. Abaseva, A. Mardanova, Effect of subtilisin-like proteinase of Bacillus pumilus 3-19 on Pseudomonas aeruginosa biofilms, Bionanoscience 9 (2019) 515–520. 37

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Table 1. Purification of extracellular protease from B. licheniformis W10. Total activity

Total protein

Specific activity

Purification

Yield

(AU)

(mg)

(AU/g)

(fold)

(%)

1.53×10-2

241.30

0.06

1

100

1.15×10-2

109.74

0.14

2.33

75.16

4.21×10-3

9.33

0.45

7.50

27.51

1.25×10-3

0.75

1.67

27.83

8.17

Crude Ammonium sulphate

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Purification steps

Gel permeation

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Dialysed sample

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precipitation

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sephacryl S-200

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Table 2. MIC of W10 protein 0.50

0.25

0.13

0.06

0.03

0.02

0.01

0

Spore germination (100%)

0

0

0

0

0

7.8

9.1

100

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Protein concentration（mg/mL）

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Table 3. Similar proteins found in the Mascot search. Accession

Mass

Score

48756

453

gi | 504071043 serine protease

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[Bacillus velezensis]

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gi | 489423124 serine protease 48706

-p

[Bacillus subtilis group]

124

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Note: Table 3 is the search information of two similar proteins found in Figure 5.

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Figure legends

Fig. 1. Extraction and activity testing of the W10 crude protein. (A) Antagonistic effects of the

W10 crude protein on Botrytis cinerea. Left: Water control; right: crude protein. (B) SDS-PAGE

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electropherogram, M: Premixed Protein Marker (Low); 1: W10 crude protein.

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Fig. 2. Purification and activity testing of the W10 crude protein. (A) Elution chromatogram of

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the crude protein after ÄKTA purification. (B) SDS-PAGE electropherogram of the purified

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protein, M: Premixed Protein Marker (Low); 1–5: peak I eluate; 6–7: peak II eluate; 8–10: peak

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right: purified protein.

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III eluate. (C) Antagonistic effects of the W10 crude protein on B. cinerea. Left: water control;

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Fig. 3. Serine protease activity analysis of the W10 antifungal protein. (A) Inhibitory effects of

the purified protein after different amounts of PMSF treatment on B. cinerea 1: W10 purified

protein 2: PMSF-treated W10 purified protein. (B) Protease activity analysis of purified proteins.

Left: water control; right: purified protein. (C) PMSF specifically inhibits serine protease activity

in the purified protein.

Fig. 4. Effect of temperature (A) and pH (B) on antifungal activity of W10 protein.

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Fig. 5. Mascot search results of the MS/MS data.

Fig. 6. Alignment map of serine protease amino acid sequences from different Bacillus species.

Fig. 7. Phylogenetic trees constructed using serine protease amino acid sequences. The GenBank

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accession numbers of the sequences used for alignment are included in the parentheses. The

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bootstrap value is labeled at the branch, and the ruler shown is a nucleotide substitution rate of

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0.02.

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Fig. 8. Homologous simulation of W10 protein three-dimensional structure by the

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SWISS-MODEL.

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Fig. 1

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Author Contribution Statement

Author Contributions

ZLJ and FZ designed and supervised the study. ZLJ, SP, LLC, YL and CY carried out the experiments. FZ was

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involved in the research discussions and helped to finalize the manuscript. ZLJ wrote the manuscript. All

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authors read and approved the final manuscript.

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