Some properties of protease extract derived from Pseudomonas fluorescens and its antifungal activity against Fusarium spp.

Yati Sudaryati Soeka¹, Joko Sulistyo²

Introduction

Community awareness of environmental problem increasingly high and pressure from experts and environmentalists to make enzyme technology as one of alternative to substitute the different chemical process in industry sector [1]. The use of chemical fertilizers and pesticides has caused an incredible harm to the environment. These agents are both hazardous to animals and humans and may persist and accumulate in natural ecosystems [2]. An answer to this problem is replacing chemicals with biological approaches, which are considered more environment friendly in the long term.

One of the emerging research area for the control of different phytopathogenic agents is the use of biocontrol plant growth promoting rhizobacteria (PGPR), which are capable of suppressing or preventing the phytopathogen damage [3]. Their applicability as biocontrol agents has drawn wide attention because of production of secondary metabolites such as siderophores, antibiotics, volatile compounds, HCN, enzymes and phytohormones [4].

Pseudomonas fluorescens is considered as biological biocontrol agent against root diseases colonized by Fusarium spp [5]. This fungus affects all parts of plants namely roots, stems, shoots, leaves and spikes. Fusarium spp. are a widespread cosmopolitan group of fungi and commonly colonize aerial and subterranean plant parts, either as primary or secondary invaders. Fusarium spp.

can cause direct damages such as seedling foot and stalk rots, or indirect damages resulting from seedling blight or reduced seed germination; however, the most important diseases that may cause severe reduction in yield and quality [6]. They can be utilized in low-input sustainable agricultural applications, such as biocontrol, on account of their ability to synthesize secondary metabolites with antibiotic properties [7]. Antagonistic activity was also observed for Pseudomonas spp. in the rhizosphere has been recognizes as major factor in the suppression of many phytopathogens. Several antibiotic-like substance have been identified, including bacteriocins and phenazine antibiotics, but one of the most important mechanism responsible for the suppression of plant pathogens for Pseudomonas spp. is siderophore mediated competitions for iron. Pseudomonas comprise a large group of the active biocontrol strains as a result of their general ability to produce a diverse array of potent antifungal metabolites [8]. The aim of research is to know the ability of P. fluorescens bacteria, a collection of to produce protease that will be used to suppress a wilt disease of Fusarium solani and Fusarium oxysporum on

Materials and methods

Isolates preparation

Isolate of P. fluorescens was a collection of Microbiology Division of Research Center for Biology, Indonesian Institute of Sciences, Cibinong, Indonesia had been cultivated in media Nutrient Agar and incubated at o solani InaCC F76 and F. oxysporum InaCC F78 were grown in Potato Dextrose Agar (PDA) [9].

Determination of protease activity on selective media

Selective media containing 0.5% skim milk, 0.5% pepton, 2% agar at pH 8.0 was used to determine the capability of bacteria on hydrolysis protein contained in the media during incubation at 37ºC for 24h. A positive result was qualitatively determined by indication of a clear zone surround bacterial colony [10].

Enzyme production

Bacterial cell production was carried out by growing bacteria on NA slant media, at room temperature for 3 days, and furthermore adds with a sterilized distilled water. An optical density (OD) of bacterial suspension was measured using spectrophotometer at 600 nm to obtain OD of 0.5. Protease production was carried out according to the method of Cappuccino and Sherman [11] by inoculating the production media for protease with bacterial suspension and incubated at room temperature for 2 days on a shaker at 120 rpm. As much as 2 mL of sample of bacterial culture was collected every day, and a filtrate was separated from a sludge, and then centrifuged with at 8000 rpm for 5 min. Supernatant was collected and used as crude enzyme solution and furthermore examined for its protease activity.

Determination of protease activity

As much as 0.2 mL of enzyme solution was reacted with 0.2 mL 0.1% azocasein in buffer solution of glycineNaOH 0.05 M at pH 8.0 and incubated at temperature 40°C for 20 min. A reaction was stopped by adding with 0.6 mL of 10% trichloroacetic acid (TCA), and then centrifuged at 10,160 g for 5 min and the filtrate was separated from the sludge. Tyrosine that was deliberated in the filtrate was determine by reading its OD at 280 nm. One unit of protease activity was defined as amount of enzyme that can produce 1 μg tyrosine in the reaction condition [12].

Effect of pH, temperature, and metal ion against protease activity

The effect of pH against protease activity was determined by reacting the enzyme solution with azocasein as substrate at the highest activity of the enzyme concentration in a different buffer of glycine-NaOH 0.05 M at pH 7.5; 8.0; 8.5; 9.0; 10.0 and 11.0. The effect of temperatue against protease activity was determined by reacting the enzyme solution with azocasein as substrate at the highest activity of enzyme concentration at the at temperature of 30-70°C, and to determine the stability of enzyme against pH and temperature then the respective enzyme solution were incubated at optimum pH and temperature for 10 min and furthermore were determined 2+ their residual activities. The effect of metal ions of Ca , 2+ + + 2+ 2+ Mn , K , Na , Cu , Hg in the form of salts CaCl , 2 MnCl , KCl, NaCl, CuCl , CoCl , ZnCl , and HgCl as an 2 2 2 2 2 activator as well as an inhibitor against protease activity were determined by reacting the enzyme solution in the reaction mixture containing 1% of skim milk and 1 mM of metal ions and the reaction mixture without metal ion was used as control.

Results and discussions

P. fluorescens showed that it could degrade protein of the skim milk in the media (Figure 1). Skim milk contains casein was used as the only carbon and nitrogen source to enrich the medium with nutrient for microbial growth. Casein hydrolysate was used as an inducer for protease production of Pseudomonas 1A4R [13, 14]. Casein is a milk protein consists of phospho protein bound calcium to form insoluble calcium calsenic in water to form a whitish coloid in a solid media [15]. P. fluorescens could degrade protein as well lipid in the skim milk since it could produce lipase as well [16]. Protease activity on agar media containing skim milk had been shown with presence of the clear zone surrounding colony. According to Patil et al. [17] the skim milk agar media was suitable to use for selection of microbial producing protease qualitatively as well as microbial secretting extracellular proteolitic enzymes that could be recognized with the formation of the clear zone surrounding colony. P. fluorescens produced several extracellular enzymes such including protease, hydrolase, lechitinase and lipase [18- 20]. Gradually decreasing of enzyme activity was observed along with increasing of incubation time, indicating role of enzyme as primer metabolite [21]. Protelytic activity of P. fluorescens that had been incubated for 6 days was range between 47.64 to 83.07 U/mL, and the optimum yield (83.07 U/mL) could be achieved after incubation for 2 days. However, Figure 2 showed that enzyme production dealing with proteolytic activity apparently fluctuated along with incubation on the day-3 up to day-6 due to extrinsic factors that could

Almost all biological reactions are accelerated or supported by a macromolecule referred as enzyme. Enzyme as catalysts that increase the rate of virtually all the chemical reactions within cells. Most biological reactions are catalyzed by proteins. In the absence of enzymatic catalysis, most biochemical reactions are so slow that they would not occur under the mild conditions of temperature and pressure that are compatible with life. Enzymes accelerate the rates of such reactions by well over a million-fold, so reactions that would take years in the absence of catalysis can occur in fractions of seconds if catalyzed by the appropriate enzyme. Cells contain thousands of different enzymes, and their activities determine which of the many possible chemical reactions actually take place within the cell [22]. Many studies of P. fluorescens have been concerned with the purification and properties of deleterious extracellular heat‐stable proteases. The amount of protease and the degree of proteolysis which could cause gelation of milk protein have not been determined. Isolation was carried out with use medium contains casein, as a good substrate to isolate bacteria that produce protease enzyme and induce an alkali protease enzyme synthesis [23, 24]. Enzymes are biological catalysts that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyze a wide range of commercially important processes. Various environmental factors are able to affect the rate of enzyme-catalyzed reactions through reversible or irreversible changes in the protein structure. The effects of pH and temperature are generally well understood [25]. The effect of temperature at 30- 70ºC against enzyme activity was range between 18.05- 79.58 U/mL and the enzyme stability was range between 11.73-50.54 U/mL. The enzyme showed optimum activity at temp 30ºC was 79.58 U/mL and showed stable was at 50,42 U/mL. The enzyme activity and stability were decreased at temp 35ºC to 70ºC (Figure 3). Wang and Jayarao [26] had shown that protease activity of P. fluorescens was higher at temp 22ºC rather than that of 7ºC and 32ºC. Decreasing enzyme yield at temp greater changes or degradation at higher temp. Temperature plays an important role in both the synthesis and excretion of the enzyme through the cell membrane for extracellular enzymes. The temperature requirement for growth may not necessarily be the same for optimum enzyme production as observed in this study where the growth temp was at 35°C while that of enzyme production was at 45°C. High enzyme yields achieved on a wide pH and temp range is an advantage for proper regulation of these parameters under large-scale production. [27]. The effect of pH against protease activity range between 33.39- 72.32 U/mL. Optimum activity at pH 8.5 was 72.32 U/mL with stability enzyme was 47.00 U/mL. The enzyme activity and its stability initiated to decrease at pH 9 (Figure 4). The optimum enzyme activity against pH depends on experimental condition, such as buffer that was used, substrate particle, and its enzyme [28]. It should be noted that the optimum pH of an enzyme may not be identical to that of its normal intracellular surroundings. This indicates that the local pH can exert a controlling influence on enzyme activity [25]. Protease was classified have range of acid (pH 2.0-6.0), neutral (pH 6.0-8.0) and base (pH 8.0-13.0) [29; 30]. The genus Pseudomonas is well known for its production of both

Oh et al. [31] reported protease production in P. aeruginosa that was active in the pH 7.0-9.0 range, with optimum activity at pH 8.0. Koka and Weimer [32] reported metalloprotease production in P. fluorescens, with an optimum activity at pH 5.0 and incubation temperature of 35ºC. Protease optimum activity of P. fluorescens RO98 was at pH 5.0 and 35ºC, while the enzyme stability was at 15ºC-55ºC and pH 4.5-9.0. The majority species of Pseudomonas have the optimum pH of 6.5-8.0 [33-35]. P. fluorescent could be classified as alkali bacteria. Reaction rate of enzyme increased in alkali and then decreased after a maximum pH had been reached. Zambare et al. [36] reported that had fermented P. aeruginosa MCM-327, where the maximum production of protease was obtained at pH 8.0, which corresponds to over 80% of the activity observed in the present study. The optimum enzyme yield was reached at a wide ranges of pH and temperature give advantage to regulation this parameter with accurate for a big scale and can also influence characteristic, morphology and physiological of organisms [27]. Metal ions give a significantly effect on enzyme activity as a vital nutrition for all life organisms and usually protein-bonded that plays an important role in structure and function of protein, probably can increase activity or inhibit a reaction of enzyme-substrate [37, 38]. Table 1, showed that protease activity of P. fluorescent activated by divalent cations of CaCl², MnCl², while divalent cations of CuCl², CoCl², ZnCl^2, HgCl² and monovalent cations of KCl and NaCl were as enzyme inhibitor. General type of protease that produced by P. fluorescens is a metal protease [39].

Tabel 1. The effect of metal ion supplementation against enzyme activity.

Metal Protease Activator/ supplementation Activity Inhibitor

(U/mL)

Control 60.03 2+ Ca 62.12 Activator 2+ Mn 64.23 Activator + K 48.45 Inhibitor + Na 53.23 Inhibitor Cu2+ 43.87 Inhibitor 2+ Co - Inhibitor 2+ Zn - Inhibitor 2+ Hg - Inhibitor Metal ion very important for all of life form, it is needed in form of monovalent and divalent cations as activators to increase the specific enzymes activity. However, ionthe ions can also act as inhibitors on the certain + + 2+ concentration [40]. Inhibition by ions of K , Na , Cu , 2+ 2+ 2+ Co , Zn , and Hg , protease activity may be caused by interaction of protein sulphur group that change enzyme conformation so that decrease protease catalytic capacity + 2+ showed that ions of Ag and Ca of 1 mM were enzyme 2+ 2+ 2+ 2+ 2+ + activators, while ions of Ba , Cd , Co , Cu , Hg , K , + 2+ 2+ Na , Sr , and Sn were as protease inhibitors against some bacterial isolates and the results had been reported 2+ by Baehaki et al. [43] showed that metal ions of Mn (5 2+ mM) and Ba (5 mM) were the protease strong activator on Staphylococcus epidermidis that could increase + + enzyme activity, while ions of Na (1 mM), K (1 mM), 3+ 2+ 2+ Fe (1 mM and 5 mM), Zn (5 mM), and Ca (1 M) were as the enzyme inhibitors. It was reported by Siddalingeshwara et al. (2010), protease of Bacillus sp. showed the optimum activity at pH 10 and temp 65°C and 3+ 2+ 2+ 2+ the metal ions of Fe , Cu , Mn and Ca were inhibitors. Zhang [24] reported that protease of P. fluorescens showed optimum activity at pH 7.0-8.5 and temp 45-50°C and its activity could be increased by ions 2+ 2+ 2+ Ca and Zn , however inhibited by ion Co . The biotechnological applications of proteases are rapidly growing as the proteases with novel properties and wide substrate specificities were documented in recent years. Proteases capable of functioning at extreme pH (3-11), temperature (40-80°C) and in organic solvents are considered as robust proteases and suitable for industrial applications. Currently, thermo-, organic solvent stable, alkaline proteases were preferred for various industrial and therapeutic applications [44]. P. fluorescens bacteria can give advantage effect against development and growth of plants as PGPR which are capable of suppressing or preventing the phytopathogen damage [3]. Phytopathogenic fungi, as the most common plant pathogens, are capable of infecting different types of plant tissues. Among the main aims in agriculture is finding adequate strategies for their suppression. One of these strategies is biological control of plant diseases that relies on the use of natural antagonists of phytopathogenic fungi [45]. A special place among the natural antagonists of phytopathogenic fungi belongs to rhizobacteria that show beneficial effects on PGPR [46]. Pseudomonas species are capable of inhibiting the phytopathogenic fungi that belong to genus Fusarium [47]. Majority species of Fusarium are pathogenic and cause the damage in plants, thus can cause crop failure including F. solani that frequently cause a disease on leaves of paddy, tomatoes, sugarcane, soybean and banana. The food damage due to the diseases caused by F. solani on potaoes usually called as dry rot. The dry rot attacks a bark of crop and the damage frequently occurred upon harvesting. Due to the soil-borne nature of the diseases caused by Fusarium species the use of chemical methods for the control of disease is rarely successful. Inconsistencies in biocontrol under varying environmental conditions have been a common limitation of soil-borne pathogens. The present research was conducted to evaluate the efficacy of indigenous Pseudomonas isolates against these pathogens. Figure 5 fluorescens toward strains F. solani. The hydrolytic enzymes get great attention because they play a significant role in controlling diseases by excreting cell wall hydrolases [48]. Several rhizobacteria, including genera of Pseudomonas, are known to produce a battery of hydrolases such as chitinase which help in the maceration of cell walls of those plant pathogens [49-52]. The selected isolates were evaluated for their antagonistic effect against Fusarium. It showed that protease extract of P. fluorescens could inhibit the growth of F. solani InaCC F76 (Figure 5A) and F. oxysporum InaCC F78 (Figure 5B), thus the fungi could not grow well and thrive by covering the entire surface of the media, although both fungi had been grown for more than a week. The potential antagonistic activity of the bacteria P. fluorescens could be correlated with the highest protease activity of the bacteria. The involvement of antifungal activity compounds produced by P. fluorescens in the inhibition of fungal growth was confirmed by the ability of cell-free culture filtrate of this strain to inhibit the hyphal growth of Fusarium sp. Conducted tests revealed the direct influence of P. fluorescens on the growth rate of the tested fungus under study. Studies shows that prolonging the bacterial culturing time and at the same time increasing the amount of secondary metabolites affect the inhibition the growth of the fungus. Selective activity of P. fluorescens against of phytopathogens has been described in many research papers. The researchers report that the antifungal properties of Pseudomonas largely depend on the capability of secretion of secondary metabolites, notably lytic enzymes and antibiotics [53]. Koche et al [54] found that P. fluorescens was most efficient in inhibiting the mycelial growth. Whereas, Toua et al [55] demonstrated restricting the growth of the two species of F. oxysporum by five strains of P. fluorescens. Moreover conidial germinartion and germ tube elongation were inhibited and reduced. The results of the experiments by [53] indicated the importance synthesized by P. fluorescens of salicylic acid in reducing of fungal phytopathogens. The inhibition of mycelial growth amounted to successively for F. greaminarum, F. avenaceum and F. solani in the range 80-100%, depending on the tested Pseudomonas strain [53]. The inhibitory properties of P. fluorescens obtained could be probably due to the production by own strain of secondary metabolites and/or lytic enzymes that can degrade cell wall. Although [53] reported that fungal lysis need not necessarily be caused by lytic enzymes capable of decomposition of glycosidic bonds-chitinase and β 1,3 glucanase but also by other substances which are manufactured by bacteria from the Pseudomonas which include intensively secreted siderophores, hydrogen cyanide as well as exogenous proteases.

fluorescens on growth of F. solani InaCC F76 (A) and F. oxysporum InaCC F78 (B).

Conclusion

The results obtained in this study pointed out the possible use of strains of Pseudomonas as biocontrol agents against Fusarium sp.. However, further research is needed under pot culture as well as field conditions to elucidate the mechanism of action of the potential antagonistic bacteria in detail. Our investigation confirmed more or less pronounced antifungal activity of all tested Pseudomonas fluorescens. Regarding the Fusarium species, the highest sensitivity to antibiotic-producing Pseudomanas isolates was observed for F. oxysporum and F. solani. Our findings impose that the studied Pseudomonas isolates have potential in controlling plant diseases caused by Fusarium spp., whereby the bacterial isolates with the highest inhibitory potential will be selected for further experiments. Conducted research confirmed fungistatic properties of P. fluorescens against F. oxysporum and F. solani strains and prove that growth inhibition of the fungi depends not only on the biological properties and age of the bacterial culture and also susceptibility of the fungus to bacterial metabolites. Therefore, promising method to crop protection against Fusarium sp. may be the application of P. fluorescens as the biocontrol agents. However, antagonist Pseudomonas with the antagonistic activity in vitro may not act in vivo due to environmental conditions and competition with other microorganisms. Therefore, it is important that biocontrol potential under field conditions should be further evaluated.

Acknowledgments

We are grateful to the Research Center for Biology, Indonesian Institute of Sciences, Cibinong, Indonesia to provide financial support and lab facilities to carry out the present investigation.

References

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