Abstract
Chitinolytic activity and antibiosis are gaining prominence in various biotechnological fields. Dead fungal biomass (DFB) was used as a mycostimulator of chitinase production and antibiosis by Aspergillus fumigatus. The presence of DFB stimulated the synthesis of various secondary metabolites by A. fumigatus that were detected by gas chromatography-mass spectrometry analysis such as 6,8-Di-C-á-glucosylluteolin; bistrimethylsilyl N-acetyl eicosasphinga-4,11-dienine; curan-17-oic acid, 19,20-dihydroxy-, methyl ester, (19S)-; spiro[5à-androstane-3,2′-thiazolidine; retinal; Androsta-1,4-dien-3-one; Panaxydol; Costunolide; Cyclo-(glycyl-L-tyrosyl); and 2-amino ethane thiolsulfuric acid. Chitinase activity was 42.9 Units/mL with the presence DFB, where it was 10.3 Units/mL without DFB. The maximum activity of chitinase was observed at 1.5 g of dead fungal biomass, at 4 h, 50 °C and pH 6. Thermodynamic properties showed ∆H° and ∆S° values of 126 KJ mol-1 and 432 J mol-1 K-1, respectively, indicating an endothermic reaction up to 60 °C. Deviation in ∆G° values confirmed that the reaction at 10 to 20 °C is a nonspontaneous reaction, and at 30 to 60 °C the reaction has a spontaneous nature. DFB encouraged the antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Aspergillus fumigatus, Mucor circinelloides, and Candida albicans with 2.3, 2.2, 2.8, 0.8, 0.7, and 2.2 mm inhibition zones, respectively.
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Mycostimulator of Chitinolytic Activity: Thermodynamic Studies and its Activity against Human and Food-Borne Microbial Pathogens
Marwah M. Bakri,a Aisha M. H. Al-Rajhi,b Emad Abada,a Olfat M.A. Salem,c Abdel-Rahman M. Shater,a Mohamed S. Mahmoud,d and Tarek M. Abdel Ghany e,*
Chitinolytic activity and antibiosis are gaining prominence in various biotechnological fields. Dead fungal biomass (DFB) was used as a mycostimulator of chitinase production and antibiosis by Aspergillus fumigatus. The presence of DFB stimulated the synthesis of various secondary metabolites by A. fumigatus that were detected by gas chromatography-mass spectrometry analysis such as 6,8-Di-C-á-glucosylluteolin; bistrimethylsilyl N-acetyl eicosasphinga-4,11-dienine; curan-17-oic acid, 19,20-dihydroxy-, methyl ester, (19S)-; spiro[5à-androstane-3,2′-thiazolidine; retinal; Androsta-1,4-dien-3-one; Panaxydol; Costunolide; Cyclo-(glycyl-L-tyrosyl); and 2-amino ethane thiolsulfuric acid. Chitinase activity was 42.9 Units/mL with the presence DFB, where it was 10.3 Units/mL without DFB. The maximum activity of chitinase was observed at 1.5 g of dead fungal biomass, at 4 h, 50 °C and pH 6. Thermodynamic properties showed ∆H° and ∆S° values of 126 KJ mol-1 and 432 J mol-1 K-1, respectively, indicating an endothermic reaction up to 60 °C. Deviation in ∆G° values confirmed that the reaction at 10 to 20 °C is a nonspontaneous reaction, and at 30 to 60 °C the reaction has a spontaneous nature. DFB encouraged the antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Aspergillus fumigatus, Mucor circinelloides, and Candida albicans with 2.3, 2.2, 2.8, 0.8, 0.7, and 2.2 mm inhibition zones, respectively.
DOI: 10.15376/biores.17.3. 4378-4394
Keywords: Mycostimulator; Chitinase; Antibiosis; Fungi; Thermodynamic
Contact information: a: Biology Department, Faculty of Science, Jazan University, Jazan 82817, Saudi Arabia; b: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University P.O. Box 84428, Riyadh 11671, Saudi Arabia; c: Biology Department, Aldarb College, Jazan University, Jazan; d: Sanitary and Environmental Engineering Institute (SEI), Housing and Building National Research Center (HBRC), 1770, Giza, Egypt; e: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 11725, Egypt; *Corresponding author: tabdelghany.201@azhar.edu.eg
INTRODUCTION
The cell wall of fungi consists of many components, the most common of which are cellulose and chitin. The latter is considered the main structural component of most fungal mycelia. The composition of chitins were characterized previously as polymers of β-(1,4)-linked-N-acetylglucosamine that are present in un-branched chains in the two large groups of Basidiomycota and Ascomycota (Elieh-Ali-Komi and Hamblin 2016). In nature, chitin represents the next most abundant polysaccharide after cellulose and hemicellulose. In addition to its occurrence in fungi, it is also found in algae, insects, and different vertebrates (Rana and Farhana 2019). However, most fungi produce many enzymes (Abdelghany and Bakri 2019; Abdelghany et al. 2018, 2020), from which chitinase can play an essential role as a defense mechanism against other fungi or to obtain nutrients via degradation of substrates containing chitins. Chitinase also has applications in industrial process, agricultural field, food processing (da Silva et al. 2005; Nofal et al. 2021a,b,c; Al-Rajhi et al. 2022a,b). Other applications of chitinases were reported in recent studies. For example, chitinase may be applied in the bioconversion of several wastes resulted from industrial and food process; also it is used in biocomposting of chitin-containing waste and depolymerizing chitosan (Gilvanova et al. 2022). Microbial chitinase has been shown to contribute in the biocontrol process against pathogenic fungi including Fusarium graminearum, Magnaporthe oryzae, Rhizoctonia solani, Botrytis cinerea and Puccinia species (Ekundayo et al. 2022). The presence of chitin induces fungal chitinase genes that express to chitinase enzymes (Deng et al. 2007). In nature, the generation of some nitrogen and carbon may be due to degradation of chitin via chitinase (Rana and Farhana 2019).
The rigidity and construction matrix of the fungal cell wall also depend on glucans as well as chitins. There are two forms of glucans, namely β-glucans, which join through β-(1,3)- or β-(1,6) bonds, and α-glucans, which join through α-(1,3)- and/or α-(1,4) bonds (Webster and Weber 2007). As mentioned previously (El-Tarabily et al. 2000), the action of chitinases and glucanase on the microbial cell wall lead to deacylated oligomer chitosan, disaccharide chitobiose, and the monomer N-acetylglucosamine. These changes cause disruption of microbial structure, cellular function, subsequent lysis and death. Biocontrol using microbes depends not only on the mycoparasitic nature but also on the hydrolytic enzymes of the biocontrol agent. Decline in mycelial biomass of pathogens was explained on the basis of lysis of the cell wall via lytic enzymes (Chang et al. 2007; Dukare et al. 2020). Starke et al. (2020) studied the attacking dead fungal biomass chitin by Ewingella americana pseudomonas isolates and reported the activity of enzyme on the glucans and chitin backbone. Utilization of fungal cell walls as the substrate of chitinase production by Myrothecium verrucaria (Vyas and Deshpande 1989) and Streptomyces viridificans (Gupta et al. 1995) was reported.
From previous indications, antibiosis of microorganisms may depend on the lytic enzymes as well as secretion of antimicrobial compounds; therefore, these enzymes have a significant role as antimicrobial compounds (Bowman and Free 2006; Shaikh and Sayyed 2015). Several secondary metabolites were detected in the fungal cultures and showed antimicrobial activities (Abdelghany 2014). The interaction among fungi, particularly in dual culture, was studied previously to stimulate the yielding of many array of metabolites (Chatterjee et al. 2016).
Thermal stability of enzymes is a critical factor of industrial applications of enzymes; therefore, thermodynamic studies have been carried out to support these phenomenon (Karam et al. 2017). Different parameters, such as enthalpy (∆H), Gibbs free energy (∆G), and entropy (∆S), were applied to judge the stability and tolerance of enzymes to increased temperature (Mostafa et al. 2018). Stimulation of chitinase production and antibiosis by live dual cultures of fungi was reported in many studies, but there has been a lack of reported work concerned with growing live fungus on dead fungal biomass. Therefore, the objective of the current study was achieved via dead biomass with thermodynamic studies of chitinase production.
EXPERIMENTAL
Chemical Used
All used chemicals and growth media contents, including colloidal chitin (crab shell chitin), potato dextrose agar (PDA) medium, Czapek Dox agar medium, malt extract medium, solvents, and buffers, were provided from Sigma-Aldrich, St. Louis, MO, USA.
Selection of Fungi Used in the Experiment
One fungus was isolated from an Oriental hornet (Vespa orientalis) trap growing as saprobe on dead Oriental hornets. The isolate was purified and cultivated on different media including PDA, Czapek Dox agar, and malt extract media for identification based on macro- and microscopic examination. The identification was completed according to previous keys (Raper and Fennell 1973; Domsch et al. 1980).
Preparation of Culture Filtrates
Dead fungal biomass of Aspergillus flavus was used to induce chitinase production. Five g of fungal fresh weight were washed 5 times with distilled water, then autoclaved at 121 °C for 30 min. The autoclaved biomass was added in Czapek Dox broth medium without sucrose, then inoculated with 5-mm diameter disc of active margins of fungus on a PDA plate. The inoculated glass bottle was then incubated at 30 °C, via 0.22-μM bacterial proof filter, and the medium broth (200 mL) was filtrated. The filtrate was used as a sources of crude chitinase and crude protein.
Colloidal Chitin Preparation and Assay of Chitinase Activity
A total of 10 g of chitin flakes were added slowly to 175 mL concentrated HCl and mixed gently for 3 h on a magnetic stirrer. This solution was then filtered to 500 mL of pre-chilled, distilled water with constant mixing and allowed to settle. A dense white precipitate formed that was then centrifuged at 10,000 rpm for 10 min at 4 °C. The precipitate was then washed in cold, distilled water repeatedly until the pH of the wash reached 5.5. The supernatant was discarded, and the colloidal chitin was then kept in a refrigerator for future use. Then, 1% of colloidal chitin was added to buffer solution (pH 5) of acetate, followed by sterilization in the autoclave. The enzyme substrate was used for evaluation of chitinase (Endochitinase) activity, which in the current method depended on the turbidity reduction of colloidal suspension containing chitin. The reaction mixture contained enzyme solution and colloidal chitin (1:1 ratio). One enzyme unit was defined as the quantity of enzyme necessary to reduce the turbidity at 510 nm of a chitin suspension by 5% (Tronsmo and Harman 1993). According to Bradford (1976), the protein level was assessed using bovine serum albumin.
Chitinase Activity at Different Conditions
Chitinase activity was studied using different concentrations of dead fungal biomass. The studied concentrations were 0.5, 1, 1.5, 2, 2.5, and 3 g/200 mL of Czapek Dox broth medium inoculated with chitnase producer fungus incubated at 30 °C for 8 days. Furthermore, the activity of chitinase at optimum biomass was evaluated at different interval times ranging from 1 to 7 h. Additionally, the filtrate containing enzyme was incubated for 1 h at different temperatures ranging from 10 to 70 °C to evaluate the activity of chitinase. For the effect of pH on chitinase activity, the Czapek Dox broth medium amended with the optimum dead fungal biomass was adjusted at different pH values ranging from 3 up to 9, followed by inoculation with chitnase producer fungus and incubated at optimum temperature for 8 d (Abdelghany et al. 2020).
Thermodynamic Parameters
Thermodynamic functions can be calculated from the effect of temperature as follows: ΔG, ΔH, and ΔS. The standard free energy change, which is a measure of the spontaneity of the chemical reaction, is expressed as,
(1)
where R is the gas constant (8.31432 J⋅K−1⋅mol−1), T is the absolute temperature (standard state = 298 K), and Ka is the equilibrium constant.
In a solid-liquid system Ka can be replaced by the distribution coefficient (Kd). In such a case ΔG is given as,
(2)
or:
(3)
The function ΔG provides a measure of the degree of complex formation. A higher negative ΔG results in a more complete formation of the complex. The free energy change is related to the changes in the enthalpy and entropy according to the following equation,
(4)
where ΔH is the enthalpy change (KJ mol-1) , ΔS is the entropy change (J K-1 mol-1), and T is the absolute temperature (°C).
Fungal Filtrate Extract Analysis via Gas Chromatography-Mass Spectrometry (GC-MS)
The used GC (THERMO Scientific Corp., Dani, Rome, Italy)-MS (ISQ Single Quadrupole Mass Spectrometer) for fungal filtrate extract analysis was under the following conditions : Injection of the extract in the capillary column TR-5MS (30 m × 0.32 mm × 0.25 μm) with different temperature cycles including 60 °C, followed by 240 °C, followed by gradually increase 30 °C/min up to 290 °C and held for 2 min. Helium was used as the carrier gas (One mL/min). An autosampler AS1300 (Thermo Fisher Scientific Inc., Dani, Rome, Italy) interconnected to the GC in the split style was used to inject 1 µL of the extract. Through mass spectra and retention time (RT), the detected compounds were identified and compared with identified compounds from the library mass spectra at the National Institute of Standards and Technology (Abdelghany et al. 2021).
Antimicrobial Activity of Fungal Extract
The filtrate extract of fungus grown with and without dead fungal biomass was tested against growth of various microorganisms including Mucor circinelloides, Aspergillus fumigatus, Candida albicans, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis using the Well agar diffusion protocol (Abdelghany et al. 2021). The medium growth was streaked with test microorganism, then a hole of agar was made by a 6-mm sterile cork borer. The dissolved extract in dimethyl sulfoxide (DMSO) (100 µL) was put into the well, followed by incubation of the plat at suitable temperature (37 °C and 25 °C for 24 h and 72 h for bacteria and fungi, respectively). The clear zone around each well was measured. Gentamycin as antibiotic and Ketoconazole as antifungal were used as control. The DMSO in the well was also used as another control (Qanash et al. 2022). Fold increase (FI, %) of the activity of the combination of extract with antibiotic or antifungal was also evaluated via the following Eq. 5):
(5)
Statistical Analysis
All experimental results were realized in triplicate. The standard deviation (SD) and variance were calculated using SPSS ver. 22.0 software (version 14, IBM Corp., Armonk, NY, USA).
RESULTS AND DISCUSSION
The chitinase producer fungus was isolated from dead Oriental hornets (Vespa orientalis) in a collected trap (Fig. 1A). It is known that the Oriental hornet is one of the main important insects that attack apiaries, particularly in Arab countries. The fungus was identified as A. fumigatus. As a result of fungus isolation from dead Oriental hornets that contain large content of chitin, this fungus was tested for the production of chitinase, but used dead fungal biomass of other fungus as the growth medium (Fig. 1A and AD).
Fig. 1. Oriental hornet trap (A), fungus growing without dead fungal biomass (B), and with dead fungal biomass (C)
The GC-MS analysis of fungal metabolites showed less detected compounds (13 compounds) in the supernatants of fungus culture cultivated without dead fungal biomass (Table 1 and Fig. 2) unlike the number detected of compounds (41 compounds) in the cultivated fungus with dead biomass (Table 2 and Fig. 3), indicating the stimulatory role of dead biomass in the biosynthesis of numerous compounds. Some compounds, including acetic acid ethyl ester, d-gala-l-ido-octonic amide, desulphosinigrin, hexadecanoic acid, and α-D-glucopyranose, 1-thio-, were detected in fungal metabolites cultivated with or without dead biomass, while other compounds were detected only in fungal metabolites cultivated with dead biomass, for example 6,8-di-C-α-glucosylluteolin; bistrimethylsilyl N-acetyl eicosasphinga-4,11-dienine; curan-17-oic acid, 19,20-dihydroxy-,methyl ester, (19S)-; spiro[5α-androstane-3,2′-thiazolidine; retinal; androsta-1,4-dien-3-one; panaxydol; costunolide; cyclo-(glycyl-L-tyrosyl); and 2-amino ethane thiolsulfuric acid.
Fig. 2. GC-MS chromatogram analysis of fungus metabolites growing without dead fungal biomass
Fig. 3. GC-MS chromatogram analysis of fungus metabolites growing with dead fungal biomass
Table 1. GC-MS Analysis of Fungus Metabolites Growing Without Dead Fungal Biomass
Table 2. GC-MS Analysis of Fungus Metabolites Growing with Dead Fungal Biomass
Brakhage and Schroeckh (2011) mentioned that the creation of new compounds can be stimulated by the co-culturing of microorganisms with other microbes. In another study (Rateb et al. 2013), synthesis of various metabolites by A. fumigatus was induced by the presence of dead cells of Streptomyces bullii in culture medium. Some of the detected compounds reflected antimicrobial activities. For example, Proteus mirabilis was inhibited by desulphosinigrin (Vinothkanna et al. 2014). However, the retinal and panaxydol were reported as secondary metabolites of some fungi (Prado-Cabrero et al. 2007; Schumacher et al. 2008; Collado and Viaud 2016), but it was recorded only when the fungus were grown in the presence of dead biomass (Table 2). Panaxydol and 3′,4′,7-trimethylquercetin displayed antifungal potential towards mucormycosis (Bharat et al. 2021). Antimicrobial activity was exhibited using costunolide against Staphylococcus aureus, Mycobacterium tuberculosis, Scopulariopsis sp., Candida albicans, Aspergillus niger, Curvulari lunata, and Magnaporthe grisea (Fischer et al. 1998; Luna-Herrera et al. 2007; Duraipandiyan et al. 2012). Many current detected metabolites were recognized as fungal metabolites in other studies with multiple biological functions. For example, curan-17-oic acid, 19,20-dihydroxy-,methyl ester,(19S)- was detected as one of the metabolites of the current tested fungus (Table 2) and of metabolites of Candida albicans in another study (Mohanad et al. 2016) and showed antifungal against Aspergillus fumigatus and antibacterial activity against Proteus mirabilis. Additionally, 7-hydroxy-6,9a-dimethyl-3-methylene-decahydro-A zuleno[4,5-B]furan-2,9-dione was identified as a metabolite of Pleurotus cornucopiae and appeared to exhibit antimicrobial activity (Renu and Dinesh 2015).
The isolate fungus was tested for chitinase activity using fungal dead biomass (Fig. 4). The obtained results indicated that the dead biomass stimulated the fungus to produce chitinase (42.9 Units/mL) compared with its productivity (10.3 Units/mL) without dead biomass. The presence of chitin in the cell wall of dead fungal mycelia was the main inducer for chitinase production.
Fig. 4. Chitinase activity of fungus growing without (A) and with dead fungal biomass (AD). Error bars ± indicated standard deviation
Dead fungal biomass was described previously by Baldrian et al. (2013), who observed that the polysaccharides represent the main structure (80 to 90%) of the fungal cell wall. From polysaccharides, chitin was detected in a high content of fungal cell walls (Zeglin and Myrold 2013). Ueno et al. (1990) mentioned fungal chitinase was able to lyse the dead and live walls of fungi. Different dead fungal biomasses, including C. lunata, A. flavus, A. niger, F. udum, and F. oxysporum, were used as substrates of chitinase production by Rhizobium sp. instead of chitin (Sridevi and Mallaiah 2008), with different activities of enzyme depending on the type of fungal mycelia. The highest activity of chitinase was observed in soils enriched with biomass of fungi subsequently with rapid decomposition of enzyme substrate (Zhao et al. 2013).
Increment of chitinase activity was observed with increment of dead fungal biomass up to 1.5 g, then it was gradually decreased (Fig. 5).
Fig. 5. Chitinase activity at different concentrations of dead fungal biomass. Error bars ± indicated standard deviation
Fig. 6. Chitinase activity at different incubation times. Error bars ± indicated standard deviation
The inhibitory action of high substrate was probably due to the presence of some compounds in dead fungal biomass that interfere with chitinase activity. Chitinase activity was studied by Sridevi and Mallaiah (2008) at different concentrations 0.5 to 5% of chitin. They showed that 2 to 3% was the optimum concentration of enzyme activity, beyond which no increment of chitinase activity was observed with further increase of chitin concentration. Activity of chitinase was tested at different times. Maximum activity was observed at 4 h, after which further increase in time did not show any an increase in enzyme activity (Fig. 6). At the 1st h, the activity (48.1 Unit/mL) was more than the activity (42.8 Unit/mL) at 7 h.
Increasing chitinase activity was observed with increasing incubation temperature up to 50 °C. High temperature was more effective than low temperature for enzyme activity (Fig. 7). Increasing temperature may increase the enzyme activity via decreasing viscosity resulting from the substrate. The current findings were similar to results observed in another reports associated with chitinase activity.
The stability of Pseudomonas aeruginosa chitinase (Thompson et al. 2001) and Penicillium oxalicum chitinase (Rodríguez et al. 1995) was recorded at 50 °C and at less than 45 °C, respectively. As mentioned in a recent study (Emruzi et al. 2020), the optimum temperature for chitinase activity of Serratia marcescens was 50 °C. Also, the stability of enzyme was observed at 90 °C for 60 min.
Thermodynamic studies on chitinase activity at different temperatures ranging from 10 to 60 °C evaluated the behavior of the enzyme. As shown in Fig. 8, thermodynamic properties indicated that ∆H° and ∆S° were 126.1 (KJ mol-1) and 431.5 (J mol-1 K-1), respectively (Table 3). This means that this reaction is an endothermic reaction up to 60 °C with an increase in disorder after 60 °C. Variation in ∆G° values indicated that the reaction at 10 to 20 °C is a nonspontaneous reaction, and at 30 to 60 °C the reaction has a spontaneous nature.
Fig. 7. Chitinase activity at different temperatures. Error bars ± indicated standard deviation
Table 3. Thermodynamic Parameters for Chitinase Activity at Different Temperatures
Fig. 8. Thermodynamic parameters of chitinase activity
The effect of different pH on chitinase productivity is visualized in Fig. 9. The optimum pH was 6, where the chitinase activity was 65.3 units/mL followed by pH 5. The obtained findings indicated that acidic pH was better than alkaline pH.
Fig. 9. Chitinase activity at different temperatures. Error bars ± indicated standard deviation
Activity of chitinase (48.5 units/mL) was more than its activity (20.8) at pH 9. The current results may be due to the fact that acidic conditions are more favorable for most fungi and their activities. A parallel behavior has been detected in other studies (Patel et al. 2019; Al-Rajhi et al. 2022a).
Antibacterial and antifungal activities of medium extract of fungus grown on medium with or without dead fungal biomass as inducer of antibiosis were documented (Table 4 and Fig. 10).
Table 4. Antimicrobial Activity of Culture Filtrate Extract of Fungus Growing With and Without Dead Fungal Biomass
