Purification and characterization of Stenotrophomonas maltophilia chitinase with antifungal and insecticidal properties

Abstract This study aimed to determine the ability of bacteria to produce the chitinase enzyme, purify, and characterize the enzyme from the isolate with the best activity, and determine the use of this purified enzyme as a biocontrol agent. The chitinolytic bacterium was identified as Stenotrophomonas maltophilia. The chitinase enzyme was purified 1.4 times at a 30% ammonium sulfate concentration with a yield of 40.7%. Following partial purification, the enzyme was purified by ion-exchange chromatography using HiPrep Q XL 16/10 column and HiPrep™ 26/10 desalting column with 25.34% and 18.12% yields, respectively. It was calculated that the purified enzyme had a molecular weight of 52 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The optimum activity of the enzyme was determined at 50 °C and pH 7.0. Enzyme activity was most induced by Fe2+, while it was most inhibited by Zn2+ at 5 mM concentration. Km and V max values of the enzyme for colloidal chitin were calculated as 1.6419 mg/mL and 16.129 U/mg, respectively. The purified chitinase was used as a biocontrol agent against the fungus Fusarium oxysporum and potato beetle Leptinotarsa decemlineata. The enzyme was shown to be effective in reducing the growth of fungus and causing disruption of the chitin structure of potato beetle.


Introduction
Chitin (C 8 H 13 O 5 N) is a complex, white, inflexible, nitrogenous polysaccharide formed by the bonding of N-Acetyl-Dglucosamine monomers (Glc-NAc) with b-1,4 glycosidic bonds, which ranks second among the most abundant polymers in nature. [1,2] It is found in the cell wall of fungi and the outer shell of sea crustaceans (such as crabs, lobsters, and shrimp) and insects, and the mouthparts of cephalopods, including mollusks (toothed tongue), octopus, and squid.
Chitinolytic enzymes are hydrolytic enzymes that break down mono-and oligomers and are classified differently according to their site of action, amino acid sequence, and gene sequence. Chitinolytic enzymes are known as chitinases (EC 3.2.1.14), exo-chitinase (non-reducing end) (EC, 3 Chitinases, endo-chitodextrinases, and chitosanases that catalyze chitin degradation have two major groups based on their site of action. Endo-chitinases such as chitinases, endo-chitodextrinases, and chitosanases break down chitin randomly from internal sites by generating low molecular mass multimers of N-acetylglucosamine. Exo-chitinases classified as exochitinase (reducing end), exo-chitinase (non-reducing end), and b-L-N-acetylhexosaminidases hydrolyze the release of N, N 0 -diacetylchitobiose from the reducing and nonreducing end of chitin and chitodextrins. [3][4][5][6] Chitinases are produced by bacteria, actinomycetes, higher plants, crustaceans, insects, fungi, and some vertebrates. [6,7] Plants generally have chitinase for defense purposes (in the biological control of fungal-borne plant diseases and protection from insect attacks), while crustaceans have it to remove the old cuticle. Apart from this, many organisms produce chitinase enzymes for the morphogenesis of the cell wall/exoskeleton and the degradation of polymers. [6,[8][9][10][11] In biotechnological prosesses, chitinases are used extensively in transforming biological waste into valuable products, in the production of food products, in the treatment of diseases, in agriculture, and various industrial areas, including biocontrol agents, biomaterial production, sweetener, and growth factor production. At the same time, chitinases are preferred for producing single-cell protein, protoplast isolation, biomedical applications, waste management, biopesticide in agriculture against various fungi and insects, and chitooligosaccharides production. [6,9,12] Chitosan fibers from chitooligosaccharides also have applications in wound dressings and wastewater treatment due to their corrosion properties, in photography due to their various optical properties, and in cosmetics due to their fungicidal and fungistatic properties. [1,13] Exo-chitinases, produced by bacteria and archaea and resistant to extreme conditions (pH, temperature, high salt concentrations, etc.), are often preferred in biotechnological processes and are used in studies. Mainly, it has been reported that chitinases belong to the different genera such as Alteromonas, Aeromonas, Arthrobacter, Beneckea, Bacillus, Chromobacterium, Clostridium, Escherichia, Klebsiella, Paenibacillus, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, and Vibrio [6,14,15] and some of them have been used in biotechnological areas. In recent years, some studies have used the chitinase enzyme against fungi and especially insect pests that cause important diseases in the agricultural field. [16][17][18][19][20] In this study, 200 bacterial isolates isolated from various plants (apple, apricot, cherry, peach, and plum) were screened for chitinase production. The best chitinase producer was selected and identified by classical and molecular methods. Chitinase enzyme from the selected isolate was purified and characterized. Finally, the pure enzyme was tested as a biocontrol agent against F. oxysporum, which is responsible for important diseases in agriculture, and L. decemlineata, one of the agricultural pests.

Organisms
All bacterial isolates and F. oxysporium (MK367719) used to determine antifungal chitinase activity were obtained from Erzurum Technical University Molecular Microbiology Laboratory Culture Collection. Potato beetle (L. decemlineata) was obtained from Ataturk University Biodiversity and Application Research Center. Bacterial isolates were grown on Nutrient Agar (NA) medium and incubated at 30 C for 24 h. F. oxysporium was grown on potato dextrose agar (PDA) medium and incubated at 28 C for 72 h.

Preparation of colloidal chitin
The colloidal chitin was prepared by modifying the method suggested by Priya et al. [21] Ten grams of powder chitin were added to 100 mL concentrated HCl and left overnight on the magnetic stirrer. Four hundred ml of 96% ethanol cooled to 4 C was added to the mixture and incubated overnight at 4 C. After centrifugation at 5000 rpm for 20 min, it was washed with distilled water until the pH reached 7.0. The final precipitate was dried at 50 C until it reached the constant weight and stored at room temperature. [21] Screening for chitinolytic bacteria Isolates were inoculated as points for chitinolytic activity on chitinase production agar (CPA) medium containing 4.5 g/L colloidal chitin, 0.3 g/L MgSO 4 .7H 2 O, 3.0 g/L (NH 4 )SO 4 , 2.0 g/L KH 2 PO 4 , 1.0 g/L citric acid monohydrate, 15 g/L agar, and 200 ml/L Tween-80, pH 6.0. [22] Two hundred isolates were incubated on CPA at 30 C for 72 h, and chitinolytic isolates were selected for further investigation, depending on the diameters of the clear chitinolytic zones. [21] Chitinase production One loop of each isolates forming larger zones on CPA were inoculated in chitinase production broth (CPB) medium containing 1% colloidal chitin, 3 g/L (NH 4 ) 2 SO 4 , 0.3 g/L NaH 2 PO 4 , 2 g/L KH 2 PO 4 , 0.3 g/L MgSO 4 .7H 2 O, 0.02 g/L FeSO 4 .7H 2 O, 0.016 g/L MnSO 4 , 0.014 g/L ZnSO 4 0.02 g/L CaCl 2 .2H 2 O, and 200 ml/L Tween 80, pH 5.0. Four isolates incubated in CPB medium at 30 C for 72 h were centrifuged (Universal 320 R Hettich) at 5000 rpm, and chitinase activities were measured from the fermentation broth.
Chitinase activity assay-Determination of reducing the sugar by DNS method The chitinolytic activity was quantified by analyzing the released N-acetyl glucosamine (GlcNAc) from colloidal chitin. [23] The reaction mixture containing 0.5 mL of 2% colloidal chitin in 0.1 M phosphate buffer (pH 7.0) as substrate and 0.5 mL of culture supernatant as enzyme solution was kept at 30 C for 30 min. The reaction was then terminated with 1 mL of DNS solution. DNS solution was prepared by dissolving 0.5 g 3,5-dinitrosalicylic acid and 15 g sodium potassium tartrate in 35 mL distilled water and combining it with 0.8 g of NaOH dissolved in 10 mL distilled water, and adjusting the final volume to 50 mL with distilled water. The resulting mixtures were kept in the boiling water bath for 10 min. After centrifugation at 6000 rpm for 10 min, the absorbance was measured at 540 nm. The amount of released reducing sugar in the samples was calculated according to the standard curve of N-acetyl glucosamine. One unit of chitinase activity was defined as the enzyme releasing one mmol N-acetyl glucosamine of colloidal chitin per min under the reaction conditions. [24] Identification of chitinase-producing isolate Bacterial isolate (AA-439) with the highest chitinase activity was identified by both 16S rRNA gene sequencing using universal forward 27 F (5 0 -AGA GTT TGA TCC TGG CTC 3 0 ) and reverse 1492 R (5 0 -GGT TAC CTT GTT ACG ACT T-3 0 ) primers and conventional identification methods (cell morphology, motility, KOH, protease, amylase, catalase, and hemolysis reactions). The data of the 16S rDNA sequence was deposited in the NCBI database, and the GenBank accession number was received.

Purification of chitinase
Partial purification of chitinase In the first purification step, S. maltophilia was incubated in CPB medium at 30 C for 72 h. Then, fermentation broths were collected and centrifuged at 10,000 rpm for 20 min 4 C to discard bacterial cells, and the supernatant (crude enzyme) was subjected to ammonium sulfate precipitation. The supernatant (100 mL) was treated with ammonium sulfate at 20%, 40%, and 60% saturation with continual stirring at 4 C. Precipitation at each saturation was performed for at least 4 h followed by centrifugation (20,000 rpm, 30 min, 4 C). The precipitated proteins were dissolved with pH 7.0, 50 mM phosphate buffer, and taken into the dialysis bag, and dialysis was performed in the same buffer at 4 C for 12 h by changing the buffer at least three times. [25] Ion exchange chromatography The dialyzed protein solution was concentrated by using AmiconV R Ultra-15 centrifugal filter units (molecular weight cutoff: 10 kDa) at 5000 rpm at 4 C and loaded onto a HiPrep Q XL 16/10 column (GE Healthcare) equilibrated with phosphate buffer (50 mM, pH 7.0). Ion-exchange chromatography was carried out using a low-pressure liquid chromatography system (Bio-Logic LP, Biorad, Hercules, CA). The fractions of 1 mL were collected during elution at a flow rate of 1 mL/min with the linear gradient of NaCl (0-1 M) prepared in phosphate buffer (pH 7.0). All fractions were analyzed for chitinase activity, and the determination of protein concentration was performed according to the Lowry Method using BSA as a standard. [26] Chitinase active fractions were combined and concentrated at 5,000 rpm at 4 C using AmiconV R Ultra-15 centrifugal filter units and then loaded onto the HiPrep TM 26/10 Desalting column (GE Healthcare) to remove the salts. Samples were passed through the column with 0-1.3 M (NH 4 ) 2 SO 4 gradient solution and stored at À20 C until use. All fractions were analyzed for chitinase activity and protein content.

SDS-PAGE profiling of chitinase
The culture supernatant and purified chitinase fractions were analyzed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a discontinuous polyacrylamide gel consisting of stacking (7.5%) and separating (15%) gels according to the literature. [27] A culture supernatant containing approximately 10 mg protein and purified fractions containing 5 mg protein was loaded into the wells. After electrophoresis at 100 V for an hour, the gel was colorized with Coomassie Brilliant Blue R-250. The molecular weights were calculated by comparing the relative mobilities using the Thermo Scientific PageRuler Prestained Protein Ladder ranging from 10 kDa to 180 kDa.
Optimum temperature and temperature stability of chitinase Enzyme activities were measured at various temperatures (10 À 80 C) to find the optimum temperature of the purified chitinase enzyme.
To the temperature stability, enzyme samples were incubated in 100 mM, pH 7.0 phosphate buffer (1:1 ratio) for different times 20, 40, 60, 80, 100, and 120 min at the optimum temperature of the enzyme. At the end of the incubation, the enzyme activities of the samples were measured according to the standard conditions, keeping the enzyme concentration, substrate concentration, and pH constant. Finally, the remaining activities of the enzyme were determined as the percentage of the activity obtained under standard assay conditions.
Optimum pH and pH stability of chitinase The enzyme activities of the purified chitinase were measured at different pH values in 50 mM of sodium acetate (pH 4.0-5.0), potassium phosphate (pH 6.0), Tris-HCl (pH 7.0-8.0), and glycine-NaOH (pH 9.0-10.0) buffers to determine the optimum pH.
To the pH stability, the samples were incubated in sodium acetate, potassium phosphate, Tris-HCl, and glycine-NaOH buffers at a 1:1 ratio at room temperature for 1 h. At the end of the incubation, the enzyme activities of the samples were measured as indicated in the standard assay condition, keeping the enzyme concentration, substrate concentration, and temperature constant. Finally, the remaining chitinase activities were determined as the percentage of the activity obtained under standard assay conditions.
The effect of metal ions, various reagents, and organic solvents on chitinase To examine the effect of metal ions on chitinase activity, enzyme solutions were incubated in the presence of 1 and 5 mM Ca 2þ , Cd 2þ , Co 2þ , Cu 2þ , Fe 3þ , Mg 2þ , Mn 2þ , Ni 2þ , and Zn 2þ ions in phosphate buffer (100 mM, pH 7.0) for 1 h at room temperature at a ratio of 1:1.
To determine the effect of various reagents and solvents, enzyme solutions were incubated in the presence of 1% and 5% (w/v) EDTA, (v/v) H 2 O 2 , (w/v) SDS, and (v/v) Tween 20 and Tween 80 and 5% and 15% (v/v) DMSO, acetone, glycerol, ethanol, isopropanol in 50 mM, pH 8.0 Tris-HCl buffer for 1 h at room temperature. At the end of the incubation, enzyme activities were measured, and relative activities were calculated as the percentage of the activity obtained under standard assay conditions. K m and V max kinetic constants of chitinase enzyme reaction The Michaelis constant (K m ) and the maximum velocity (V max ) of the purified chitinase were calculated using colloidal chitin in the concentration range of 0.05-5 mg/mL under standard assay conditions. [28,29] Biotechnological applications of chitinase enzyme Antifungal activity of chitinase In order to investigate the antifungal activity of chitinase, 20 mL enzyme-impregnated disks were placed 1.5 cm away from F. oxysporum, and the growth of the fungus was observed for 5 days, and the results were recorded. Amphotericin B (10 mg/mL) was used as a positive control, while 0.1 M phosphate buffer was used as the negative control. The antifungal activity of the enzyme was evaluated in terms of hyphae growth as well as spore germination.
Effect of chitinase on spore germination and hyphae growth In order to investigate the effects of chitinase on spore germination, 0.5 mL F. oxysporum fungus spore solution (6 Â 10 3 spores/mL in 0.1% Tween 80), 0.5 mL of potato dextrose broth, and 0.5 mL of chitinase enzyme preparation were mixed in an Eppendorf tube and incubated at 28 C at 150 rpm. Phosphate buffer (0.1 M) was used instead of the enzyme as a control. Every 2 h (up to 24 h) samples were taken and germination of fungal spores was examined under a microscope (10 Â 40). The numbers of germinated and nongerminated spores were recorded. At the same time, 1 mL of fungal spore solution (10 5 spores/mL) was inoculated on a PDA medium and incubated at 28 C. After 24 h, chitinase enzyme-impregnated disks (20 U/mL) were placed on the germinated spores and incubated for 5 days to examine the hyphal growth. [29] Investigation of the effect of chitinase enzyme on potato beetle (L. decemlineata) In order to determine the effect of the enzyme against L. decemlineata, which causes severe damage to potato fields, the insects were first kept in ethyl alcohol for one night, and the alcohol was removed by washing with sterile distilled water. Five microliters of enzyme preparation (20 U/mL) was added to the insects in a glass test tube and incubated at 30 C for 5 days. At the end of this period, the samples were fixed with 5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at 25 C and then dehydrated with ethanol. The samples were dried under the CPD (CO 2 critical-point drying) system. Finally, morphological changes in insects were visualized and photographed under the scanning electron microscope (SEM, Quanta FEG 250) operated at 7 kV. [30] Results and discussion AA-439 isolate was identified based on 16S rRNA gene sequence and its morphological and biochemical characteristics as Stenotrophomonas maltophilia (GenBank ID: MW600524), rod-shaped, motile, cream-colored, Gram-negative, catalase-and protease-positive, amylase, and hemolysis-negative bacterium. It was determined that this bacterial strain could grow in the temperature range of 25-40 C, pH range of 5.0-9.0, and salt range of 2-3%.

Purification of chitinase from S. maltophilia
The extracellular chitinase from S. maltophilia was purified by ammonium sulfate precipitation (20-80%) and then ionexchange chromatography. The purification conditions of chitinase from S. maltophilia is given in

Characterization of chitinase enzyme purified from S. maltophilia
For molecular weight determination, the purified chitinase enzyme from S. maltophilia was analyzed, and the molecular mass of the enzyme was detected to be almost 52 kDa (Figure 2). It has been reported that the molecular weights of chitinases isolated from different sources have varying sizes in the range of 26-200 kDa. [31][32][33] Studies with chitinase enzymes from different Stenotrophomonas species support the result obtained from this study. Chitinase with a molecular weight of 52 kDa from S. maltophilia MUJ, [34]  50 kDa from S. maltophilia N4, [35] and 50 kDa from S. rhizophila G22 [36] enzymes have been reported.
Optimum temperature and temperature stability of chitinase enzyme The effect of temperature on the purified chitinase enzyme was examined at various temperatures (10-80 C), and the optimum temperature for chitinase activity was determined as 50 C (Figure 3(a)).
In order to determine the temperature stability at its optimum temperature, the enzyme solution was kept at 50 C for different times (20,40,60,80, 100, and 120 min). The purified enzyme maintained its activity at 50 C for 80 min, and then there was a sharp decrease in the enzyme activity (Figure 3(b)).
Several chitinase enzymes from Stenotrophomonas isolates showed optimum activity at acidic pH; the optimum pH of the present enzyme from S. maltophilia was found to be neutral rather than acidic. [36,40] The effect of metal ions, various reagents, and organic solvents on chitinase enzyme The effects of various metal ions on the chitinase enzyme are given in Table 2. Among the metal ions tested, it was observed that Fe 2þ ions increased the enzyme activity by 29% and 49% at 1 mM and 5 mM concentrations, respectively. Likewise, Mg þ2 slightly increased the enzyme activity at 1 mM concentration, while it increased 30% at 5 mM concentration. In contrast, Mn 2þ ions at these concentrations did not affect the activity much. While Cd 2þ did not show any effect at 1 mM, it slightly decreased the enzyme activity at 5 mM. Co 2þ , Cu 2þ , and especially Zn 2þ drastically decreased the activity at both 1 mM and 5 mM concentrations.
In different studies, it was seen that the effect of metal ions on chitinases is quite different. [41] Contrary to our study, Zhang et al. [42] reported that chitinase from S. maltophilia C3 was inhibited in the presence of 1 and 5 mM Fe 2þ metal ions. Similar to our results, the chitinase from Bacillus sp. DAU101 was inhibited by 1, 5, and 10 mM Zn 2þ , Cu 2þ , and Hg 2þ , [33] and the chitinase from S. plymuthica HRO-C48 was severely inhibited by 10 mM Co 2þ and Cu 2þ , and chitinase from Streptomyces aureofaciens CMUAc130 was wholly inhibited by Hg 2þ , Cd 2þ , and Ni 2þ . [43] The chitinase enzyme activity decreased after 1 h of pre-incubation in the presence of EDTA, H 2 O 2 , SDS, Tween 20, Tween 80, DMSO, acetone, glycerol, ethanol, isopropanol at the tested concentrations (Table 2). Such reagents and solvents generally inhibit chitinase activity, [44][45][46][47] in some studies, the enzyme was reported to be tolerant to them, [48] or very rarely, reagents such as EDTA, Tween 20, and Tween 80 have been reported to cause minor increases in activity. [35] K m and V max kinetic constants of chitinase enzyme reaction The K m and V max of the purified chitinase enzyme for the hydrolysis of colloidal chitin were calculated from the Lineweaver-Burk plot ( Figure 5) as 1.6419 mg/mL and 16.129 U/mg, respectively. Michaelis-Menten curve of chitinase from S. maltophilia using colloidal chitin as substrate was provided in Figure 6. Several studies reported that the affinity of the chitinase enzyme to the substrate is generally low. [49] De La Cruz et al. [50] reported that K m values of three different chitinases from Trichoderma harzianum for colloidal chitin were 1.0 mg/mL, 0.5 mg/mL, and 0.3 mg/mL. Fu et al. [51] reported higher K m and V max values for purified chitinase from Paenicibacillus barengoltzii for colloidal chitin  [52] reported that the K m value for chitinase from B. licheniformis was 0.23 mg/mL, and the V max value was 7.03 U/mg.

Biotechnological applications of chitinase enzyme
The antifungal activity of the chitinase enzyme According to the results of antifungal activity, it was determined that chitinase inhibited the growth of F. oxysporum at a concentration of 20 U with a zone diameter of 5 mm, and a concentration of 10 U with a zone diameter of 2 mm (Figure 7). Therefore, it was observed that the chitinase showed an inhibitory effect in a dose-dependent manner. These results suggested the sensitivity of F. oxysporum to the chitinase enzyme. Previous studies support the     antifungal activity of chitinase from different sources. Chitinase from S. maltophilia was reported to have a strong antifungal effect against F. oxysporum, [40] chitinase from B. subtilis TV-125A negatively affected the growth of F. culmorum, [53] chitinase from B. cereus had antifungal activity against R. solani and F. oxysporum, [55] and chitinase enzyme obtained from B. pumilus SG2 strongly inhibited the development of F. graminearum. [55] Effect of enzyme on spore germination and hyphae growth of F. oxysporium The antifungal effect of chitinase on spore germination of F. oxysporium was determined by assuming the length of the germ tube. Spore was considered germinated when the length of germ tube of the spore reaches half of the spore length. [56] As seen in Table 3, it was determined that the enzyme significantly inhibited spore germination compared to the control. The antifungal activity of the enzyme on hyphal growth was determined on spores germinated on PDA. The purified enzyme was observed to inhibit fungal hyphal growth slightly. All these results suggest the sensitivity of F. oxysporum to the chitinase enzyme. Previous studies support the antifungal activity of chitinase from different sources. [40,[53][54][55] Investigation of the effect of chitinase enzyme on potato beetle (L. decemlineata) To examine the effect of the purified chitinase on potato beetle (L. decemlineata), insects were treated with the enzyme (Figure 8). At the end of the 5-day treatment, it was observed that the heads and legs of the insects in the test tubes were ruptured. The changes caused by the enzyme on the surface of the insect's wing structure were recorded by SEM, and it was determined that the tight structures of the hexagonal cells on the elytra surface of the insect were disrupted as a result of enzyme treatment ( Figure 9). There is no study on applying the purified chitinase enzyme from S. maltophilia against potato beetle in the literature. However, from previous studies, it has been reported that the chitinase enzyme was applied against the exoskeleton of harmful insects and that effective results were obtained. [19,57,58] In this study, although live insects or larvae were not used, it is shown that the purified chitinase enzyme degrades the structure of the insect's exoskeleton and therefore, can be used as an effective biocontrol agent.

Conclusion
Chitin-degrading enzymes have many uses in industrial and agricultural applications. Since chitin is found in the exoskeleton of insects and the cell wall of fungi, it can be used in agriculture as a biocontrol agent against agricultural pests and fungal pathogens. In the present study, among the 200 isolates, the best chitinase producer was identified as S. maltophilia by classical and molecular methods. Its chitinase enzyme was purified 21.52 times with 18.12% yield by sequential purification steps, and the molecular weight of   the enzyme was calculated as 52 kDa. The enzyme was highly active at 50 C and pH 7. While Fe 2þ and Mg 2þ induced the activity, it was most inhibited by Zn 2þ at 5 mM concentration. All tested reagents and solvents decreased the enzyme activity. The K m and V max values of the enzyme for colloidal chitin were calculated as 1.6419 mg/mL and 16.129 U/mg, respectively.
The purified chitinase was used as a biocontrol agent against the fungus F. oxysporum and potato beetle L. decemlineata. The enzyme effectively reduces fungal germination and hyphae growth and disrupts the chitin structure of the potato beetle.