1 Introduction

Lipases (E.C. 3.1.1.3) belong to a class of hydrolases and water-soluble enzymes that catalyze insoluble triacylglycerol hydrolysis to produce free fatty acids, diacylglycerols, monoacylglycerols, and even glycerol. They catalyze a whole range of reactions like hydrolysis, transesterification, and interesterification of other esters and ester synthesis, and display a range of regioselective, enantioselective, and stereoselective transformation properties [28, 71].

Lipases are present everywhere and from different sources but microbial lipases are the most commonly ones used in modern industrial processes such as detergent formulations, synthesis of biosurfactant, dairy formation, paper, cosmetics, and pharmaceuticals [33, 36, 83]. The microbial lipases are extracted industrially, much more than that of the plants and animals. Microbes have short generation cycle, pervasive nature, and great metabolic divergence so that the enzymes can be isolated easily [28]. Microbial lipases also have abilities to work at high temperatures, pH, and stable in organic solvents, chemoselective, regioselective, and enantioselective. In addition, they have higher output yields, ease of use genetic engineering, and rapid growth on cheap media, allowing more suitable production [77]. The most frequently utilized fungal genera in those applications are Penicillium, Mucor, Geotrichum, Rhizopus, and Aspergillus [3, 14, 29, 48]. Numerous species of Candida genus had also an extracellular ability to hydrolyze and synthesize various oils [5, 11, 35, 38, 68, 74], Alami et al. [4]. This ability gives them great prospects in lipase-producing industries [16].

Component of the medium composition greatly influences production of lipase especially carbon sources (lactose, glucose, and galactose), nitrogen (yeast extract, urea, ammonium nitrate, peptone, corn steep liquor, and amino acids), and surfactants as Tween 20 and Tween 80 [1, 19, 20]. In the published literatures, some investigators studied the effect of medium components and conditions on lipase activities of Candida spp. such as Tsuboi et al. [80], Jatta et al. [38], Ali et al. [5], Salihu et al. [68], Boonchaidung et al. [11], and Alami et al. [4], but none was recorded on C. catenulata.

Popular therapies for invasive candidiasis and mucocutaneous infections depend on some compounds such as vulvovaginal candidiasis (VVC) including polyene (nystatin, amphotericin B, and its formulations of lipids), azoles (fluconazole and voriconazole), and echinocandins (caspofungin) [59]. The azoles, as fluconazole, are one of the most popular antifungals, and also have a direct impact on Candida sp. morphology and hyphae development [34]. The impact of azole-mediated hyphal repression is important enough that hyphal branching is limited at subinhibitory azole concentrations and that the yeast-to-hyphal transformation is fully prevented at clinically significant doses [34]. It was announced that Candida albicans cells treated with azoles developed higher farnesol levels [37]. A fungal-produced quorum sensing agent is capable of preventing the development of filaments in C. albicans. Azoles target enzymes involved in sterol biosynthesis and azole treatment contributes to the formation of the sterol biosynthetic intermediate, farnesyl pyrophosphate, which indirectly promotes the overproduction of farnesol [7, 37].

Mutagenesis, either by chemical or physical agents, is one of the preferred methods for strain improvement to achieve maximum enzyme productivity in microbes. Several investigations mentioned to use this technique to improve lipase activity in mutants as Aspergillus fumigatus MTCC 9657 [64], Aspergillus japonicas [44], Aspergillus niger [79], Candida rugosa [60], and Rhizopus sp. BTS-24 [8]. Similarly, others reported mutagenic methods to enhance protease activity in mutants as Aspergillus niger [62] and Trichoderma harzianum T334 [75].

From previous study [35], we found that Candida catenulata produce high lipase activity (20.31 U/ml) in presence of fluconazole (than other antifungal drugs), so the current study aimed to optimize the fluconazole-treated C. catenulata in presence of variable factors to maximize the extracellular lipase production then to impact some mutagens with/without fluconazole on the lipase activity and on the cellular structures of C. catenulata.

2 Materials and methods

2.1 Test organism

Candida catenulata was kindly provided by Micro Analytical Center, Microbiology Lab, Cairo University. This species was isolated from blood sample in Abu Al-Rish Japanese Children’s Hospital.

2.2 Chemicals

Flucoral (SEDICO) containing 150 mg fluconazole was used as systemic antifungal agents. Sodium azide (SA) and ethidium bromide (EtBr) were purchased from ICN Biomedicals Inc., while ethanol (EtOH) was HPLC grade and was used as mutagens.

2.3 Culturing and maintenance

Candida catenulata was maintained in medium composed of (%): peptone (1), glucose (2), and agar (3) and incubated at 37 °C for 48 h. It was subcultured according to Bramono et al. [12] and kept in refrigerator until use.

2.4 Lipase extraction and assay

For lipase induction, C. catenulata was grown for 3 days in a liquid medium containing 0.7% yeast extract (modified from yeast nitrogen base) and 2.5% Tween 80 [80]. Fifty milliliters of the culture medium contained in a 200-ml flask was kept at 37 °C for 20 min shaking at 150 rpm and then filtered. The yeast cells were dried and weighted, while the filtrate was used for lipase assay. Culture filtrate (0.3 ml) was added to 1.0 ml of the substrate 1.12 mM of sorbitan monopalmitate (modified from alpha-naphthylpalmitate), 14 mM of citric acid, and 74 mM of Tris buffer (pH 5.5) [for 1 h] at 37 °C. The reaction was stopped by adding 0.5 ml of 0.5 N NaOH solution. Sorbitol released was reacted with 0.3 ml fast violet B (1 mg/10 ml H2O), and the optical density was measured at 520 nm on JENWAY 6300 spectrophotometer. Reaction mixture without culture filtrate was used as a blank. The enzyme activity was expressed in the international unit; one activity unit corresponds to the amount of enzyme that catalyzes the hydrolysis of 1 µmol of substrate (sorbitan monopalmitate in the present study) per minute [80].

2.5 Specific activity

It is the activity of an enzyme per milligram of total protein (expressed in μmol min−1 mg−1) or (U/mg). Specific activity measures the enzyme purity in mixture. It is the micromoles of product formed by an enzyme in a certain time (minutes) under specific conditions per milligram of total proteins. The precipitates of organism were used to estimate the dry biomass in each experiment [35].

2.6 Biomass

Dry biomass (g/l) was determined after drying the C. catenulata cells at 60 °C until constant weight.

2.7 Determination of protein concentration

The concentration of proteins was determined according to the method mentioned by Lowry et al. [50]. One milliliter of the enzyme preparation was added to a mixture which consisted of 0.1 ml of 4% NaOH, 0.5 ml of 2% Na2CO3, and 0.5 ml of freshly prepared solution [1% CuSO4 and 2% potassium sodium tartrate (1:1 v/v)]. They were well mixed and left for 10 min at room temperature. Thereafter, 0.1 ml of Folin-Ciocalteu reagent was added to the mixture and shaken well. After 30 min., the optical density was measured spectrophotometrically at 750 nm. Bovine serum albumin standard curve was used to estimate the protein concentrations (mg/ml).

2.8 Optimization of growth factors

Eight factors were studied to elucidate the optimum medium for maximizing lipase production and activity by C. catenulata. The effect of different media was carried out using Czapek Dox’s medium, Sabouraud medium, and Tsuboi et al. [80] medium (0.7% yeast extract + 2.5% Tween 80). Carbon sources of 2.5% sucrose, maltose, glucose, and Tween 80 were tested. Nitrogen sources (yeast extract, peptone, sodium nitrate, and ammonium sulfate) at 0.7% were used. The impact of metal ions, at concentration of 10 mM, of ZnSO4.7H2O, (NH4)2SO4, CuSO4.5H2O, and MnSO4.7H2O were assessed. Before medium sterilization, various pH values (4, 4.5, 5, 5.5, and 6) were adjusted for investigation. Different agitation were assayed in different incubator conditions as light static, dark static, light shaking, and dark shaking.

Different temperatures (25, 30, 35, 37, and 40 °C) and different incubation periods (1, 2, 3, and 4 days of incubation) were also tested. In all cases, the optimum factor for lipase production from each experiment was used in the next one. Lipase assay was estimated in culture filtrate after growth of C. catenulata disks in each experiment.

2.9 Cytological changes exerted by the most potent treatment on C. catenulata cells

The transmission electron microscopy (TEM) technique was performed at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Both untreated (control) and treated C. catenulata cells were collected separately by centrifugation (at 4000 rpm for 10 min) from 72-h-old cultures grown on liquid medium containing 0.7% yeast extract (modified from yeast nitrogen base) and 2.5% Tween 80 for lipase induction [80]. After washing with distilled water, the samples were prepared for examination by TEM using the procedures described below:

Specimens were fixed by immersing in freshly prepared aqueous potassium permanganate solution (1% w/v) for 5 min at room temperature. Fixed specimens were then washed three times by distilled water for 15 min, and then they were dehydrated in ethanol graded series each for 15 min. The absolute alcohol was replaced with acetone via a stepwise series of ethanol:acetone (2:1, 1:1, and 1:2 v/v), and then finally maintained in water-free acetone for an hour. Dehydrated specimens were infiltrated with the resin mixture through a graded series of resin:acetone and then in pure resin at 60 °C for 72 h then allowed to attain room temperature.

The dehydrated specimens fixed with potassium permanganate were embedded in blocks filled with epoxy resin mixture overnight in an oven at 60 °C then return to room temperature. Using a stereomicroscope (Leica), the mounted blocks were trimmed with razor blades to give a trapezoid-shaped face of less than 1 mm width and height. Ultra-thin sections were cut with a Leica ultracut (UCT) ultramicrotome and the sections (straight ribbon) were manipulated to picking up on grids.

The grids of copper hexagonal (2.05 mm mesh) were degreased and the sections were fastened to clean glass slide adhesive prior to staining. Sections were stained with double stain (uranyl acetate followed by lead citrate) according to Reynolds [67]. Stained sections were examined with transmission electron microscope [JEOL (JEM 1010)] at different magnifications. The images were captured by a built-in camera.

2.10 Mutagenesis of Candida catenulata and its effect on lipase enzyme activity

In the first experiment, the mutagens used to induce mutation on Candida catenulata were ethidium bromide (EtBr), sodium azide (SA), and ethanol (EtOH), and they were added singly. Their concentrations were 0.1, 0.3, and 0.5 µg/ml of ethidium bromide, 0.5, 1, and 2 mM of sodium azide, and 10, 30, and 70% of ethanol.

Four disks of C. catenulata were transferred to 20 ml of each mutagen (type and conc), shake carefully, and incubated for 1 h at room temperature. After incubation, the samples were centrifuged at 4000 rpm. The precipitates were transferred to 20 ml subculture liquid medium (1% peptone + 2% glucose) to make suspensions. Two milliliters of suspension was added to triplicate flasks, and each contains 60 ml of lipase medium (0.7% yeast extract + 2.5% Tween 80) and then incubated for 3 days at 37 °C. After incubation, the samples were centrifuged at 4000 rpm. Untreated C. catenulata was represented as control and was prepared similarly but without prior incubation.

The best concentration (with high lipase activity) from each mutagen treatment (of the previous experiment) was taken in combination, representing 0.1 µg/ml EtBr, 0.5 mM SA, and 10% EtOH singly or in combination with flucoral (150 mg fluconazole). Similarly, the previous experimental steps were carried out using optimized lipase medium.

In all cases, the filtrates were collected for lipase activities, protein concentrations, and specific activities. The cells were collected for TEM studies. All mutagen work was carried out under aseptic conditions in a laminar airflow cabinet.

2.11 Statistical analysis

Results were shown as mean ± standard deviations (mean ± SD) of triplicate samples. Data were analyzed by one-way analysis of variance (ANOVAs) using SPSS statistical tools. With Duncan’s test at P ≤ 0.05, the differences between mean values were also evaluated.

3 Results and discussion

3.1 Effect of different media

Table 1 indicates that lipase produced from C. catenulata achieved its maximum activity in medium composed of 0.7% yeast extract + 2.5% Tween 80 with enzyme activity of 20.498 U/ml, protein content of 2.48 mg/ml, and specific activity of 8.26 U/mg. In this medium, the dry biomass recorded the highest gain with 0.146 g/l. In agreement with this previous report, the investigated medium 0.7% yeast extract + 2.5% Tween 80 induced high Candida sp. lipase [80]. Minimum lipase activity was attained in a glucose yeast extract medium [81]. However, in Pseudomonas sp., glucose yeast extract peptone (GYP) was the optimum medium for lipase production [70].

Table 1 Effect of different media on lipase activity, protein concentration, specific activity, and dry biomass gain of C. catenulata
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3.2 Effect of carbon source

Table 2 reveals that maximum lipase production occurred in presence of Tween 80 with enzyme activity of 20.90 U/ml and specific activity of 8.42 U/mg. Sucrose also enhanced lipase production with activity and specific activity of 4.69 U/ml and 1.39 U/mg, respectively. Meanwhile, other carbon sources inhibited lipase activity. Tween substrates have a good inductive effect on the production of fungal lipase [46]. A linear increase in lipolytic activity was shown in presence of Tween 80 on growth medium of Mortierella vinace [30]. Cihangir and Sarikaya [17] suggested that Aspergillus niger grown on carbohydrate-supported media achieved lower lipolytic activity compared to oil as the sole substrate. Considering the dry biomass, Tween 80 recorded the highest value (0.146 g) compared to control and other sugars. Similarly, the main influencing factor for the growth of fungal lipase was oil as a carbon source [10].

Table 2 Effect of carbon
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3.3 Effect of nitrogen source

Maximum lipase production was obtained in presence of yeast extract as nitrogen source with activity of 21.0 U/ml and specific activity of 8.50 U/mg. This was followed by peptone and NaNO3, and minimum activity was recorded with (NH4)2SO4. Higher dry biomass gain of 0.148 g/l was achieved also in presence of yeast extract as nitrogen source in C. catenulata medium (Table 3). Yeast extract was very efficient in the production of lipase by the yeast Cryptococcus, spS-2 with activity of 26.0 U/ml [42], with 25.0 U/ml in Aspergillus niger [18, 26], and with 1.134 U/ml in Candida sp. KKU-PH2-15 [11]. Among the various nitrogen sources added to the modified glucose-yeast-peptone (GYP) medium, only NH4NO3, KNO3 enhanced lipase production, while the other inorganic nitrogen sources had an inhibitory effect on the production of lipase by Pseudomonas sp. [70].

Table 3 Effect of nitrogen
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3.4 Effect of metal ions on lipase production

It was observed that Cu+2 promoted the activity of lipase followed in descending order by Zn+2 and Mn+2 (Table 4). The highest lipase activity, specific activity, and biomass gain of 22.95 U/ml, 9.14 U/mg, and 0.154 g/l, respectively, were attained in presence of Cu+2 in the growth medium of C. catenulata. Maximum lipase activity in Lactobacillus delbrueckii subsp. bulgaricus was achieved with the addition of 0.01 M of CaCl2 [27]. The addition of 2% Ca+2 in production medium resulted in maximum lipase production, whereas the other metal ions decreased lipase production. The transition metals, especially Ni+2, led to decrease in activity of up to 88%, with somewhat lesser reductions related with Mg+2, Na+, K+, and Cu+2 [69]. This negative effect of metal ions was reviewed by Joseph et al. [40].

Table 4 Effect of metal ions on lipase activity, protein concentration, specific activity, and dry biomass gain of C. catenulata
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3.5 Effect of pH

pH has an impact on ionization of active sites involved in substrate binding and catalysis [21]. In this study, it was found that the highest lipase activity and specific activity were obtained at pH 5.5 with 23.1 U/ml and 9.31 U/mg, respectively (Fig. 1). Also, at pH 5.5, the highest dry biomass of 0.156 g/l was recorded. Any change in this pH value led to decrease in enzyme production. In previous reports, maximum lipase development of Cryptococcus spS-2 at pH 5.6 has been reported, with activity of 65.7 U/ml [42]. The peak of lipase activity was observed when the initial medium pH of Pseudomonas sp. was adjusted at 5.5 [70]. Penicillium roqueforti has achieved optimum lipase yield at pH 5.5 [52]. Lin et al. [49] and Rehman et al. [66] stated that pH 5.5 in Penicillium notatum and Antrodia cinnamomea was the optimal for lipase production. In contrary to ours, Jatta et al. [38] found that optimum pH for the lipase activity of C. albicans was 7 when grown on both Sabouraud dextrose broth (1600 U/mg) and Lee synthetic medium (200 U/mg).

Fig. 1
figure 1

Effect of pH on lipase activity, specific activity, and dry biomass from C. catenulata

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3.6 Effect of temperature

Optimum temperature was found to be 37 °C with lipase activity and specific activity of 23.3 U/ml and 9.39 U/mg, respectively (Fig. 2). The gradual increase in temperature led to concomitant increase in lipase activity while higher temperature above the optimum caused a significant reduction in enzyme activity. At 37 °C, the highest dry biomass of Candida catenulata had been recorded with gain of 0.157 g/l. Jatta et al. [38] found that optimum temperature for the lipase activity of C. albicans was 37 °C when it is grown on both Sabouraud dextrose broth and Lee synthetic media. Maximum yeast lipase development from Cryptococcus spS-2 was achieved at temperature 25 °C [42]. Lipase activity in Pseudomonas sp. was maximum at 37 °C [70]. The Rhodotorula glutinis [22] and Penicillium citrinium [53] have optimum temperature at 30 °C and 22 °C, respectively, whereas maximum lipolytic activity of Trichoderma harzianum occurred at temperature 40 °C [81].

Fig. 2
figure 2

Effect of temperature on lipase activity, specific activity, and dry biomass from C. catenulata

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3.7 Effect of light and agitation

Agitation is a significant variable that enhances the growth and lipase development by fungi. It enhances the rate of oxygen transfer and thus increasing the dispersal of oil micelles for greater interaction with fungal cells, and enhances the absorption of oil that induces lipase secretion [76]. In the present study, maximum lipase production and biomass gain by C. catenulata were obtained at dark agitated condition with 23.9 U/ml and 0.160 g/l, respectively (Table 5) followed by light static with activity of 17.34 U/ml and 0.157 g/l, respectively. Bacillus sudeticus showed that the activity of lipase on agitated culture was fourfold higher than non-agitated culture [85]. Activity of lipase in B. ribis also reported a 98% increase in agitated culture relative to static ones [55]. Similarly, Aspergillus sp. detected significant enhancement in lipase secretion in agitated condition [17]. A considerable lipase activity in the modified GYP medium of Pseudomonas sp. was obtained in shaking condition [70]. Also, Velmurugan et al. [82] reported that maximum fungal biomass development was obtained in darkness condition, and minimal production was obtained under the yellow light in five filamentous fungi (Monascus purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides, and Penicillium purpurogenum).

Table 5 Effect of different light and agitation states on lipase activity, protein concentration, specific activity, and dry biomass gain of C. catenulata
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3.8 Effect of incubation period

It was observed that lipase activity and biomass yield were increased to record the highest value at the 3rd day of incubation with activity of 24.1 U/ml and weight of 0.11 g/l. Further increase in incubation period above the optimum led to decrease in both parameters (Fig. 3). Similarly, Candida sp. developed the highest lipase activity on the 3rd day of incubation [80]. In the same context, Pseudomonas sp., incubation time for peak lipase activity was 3 days [70]. Pseudomonas flourescens reported optimum lipase activity after 24 h [43], while maximum lipase activity (25 U/ml) was observed after 72 h of incubation time in A. niger [26].

Fig. 3
figure 3

Effect of incubation period on lipase activity, specific activity, and dry biomass from C. catenulata

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3.9 Mutagenesis of Candida catenulata and its effect on lipase enzyme activity

The effect of mutagens was assayed on the lipase activity from C. catenulata (Table 6). All mutagen types and concentrations decreased the lipase activity, its specific activity, protein concentrations, and dry biomass gradually reaching their maximum decline at mutagen higher concentrations. Lipase activity, specific activity, and protein concentrations of control (no mutagen) still recorded the highest values 13.01 U/ml, 6.67 U/mg, and 1.95 mg, respectively. EtBr declined the lipase activity by about 50% (at 0.1 and 0.3 µg/ml) to more than 60% (at 0.5 µg/ml) compared to control. Lipase specific activity showed similar declined trend. However, the protein concentrations were significantly unchanged by increasing EtBr concentrations, but still lower than the control by about 0.1 folds. SA also decreased the lipase activity but not as sharp as EtBr. Lipase activity declined by 15% (at 0.5 mM SA), then 25% (at 1 mM SA), and finally by 50% (at 2 mM SA). Lipase specific activities were inversely proportional to SA concentrations. Protein concentrations showed similar declined trend. EtOH exhibited less harmful effect on lipase activity (as well as its specific activity) as compared to both EtBr and SA. EtOH decreased lipase activity by 28% and its specific activity by 15% of the value of control at higher concentration (70%). Protein concentrations were insignificantly decreased at higher EtOH concentrations (30 and 70%), while dry biomass decreased by all treatments over the control.

Table 6 Assay of lipase activity, protein concentration, specific activity, and dry biomass of untreated and treated Candida catenulata with different concentrations of either ethidium bromide, sodium azide, or ethanol
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Mutagenesis, either by chemical or physical agents, is one of the methods used for strain improvement to achieve maximum enzyme productivity in microbes. Several investigations mentioned to use this technique to improve lipase activity in mutants as Aspergillus fumigatus MTCC 9657 [64], Aspergillus japonicas [44], Aspergillus niger [79], Candida rugosa [60], and Rhizopus sp. BTS-24 [8]. Those results were in contrary with ours as the mutant-derived strains from either mutagen treatment reduced the lipase and specific activity as compared with the wild strain. It is interesting to mention that random point mutations may cause silent, nonsense, or missense mutations. Thus, those reduced activities may be due to missense point mutations that alter the gene sequence, change the amino acids of the enzyme, and so reduce (non-conservative) its activity [63]. It is also important to have a glimpse on the mode of action of the used mutagens: The mutagenicity of SA is accomplished through the production of an organic metabolite of SA [58] which first enters in the cell nucleus, interacts with DNA, and induces point mutation in the host genome [45]. The azide ion alters the structure of cytosine such that it forms hydrogen bonds with adenine, rather than guanine. This produces a cytosine to thymine transition [39]. However, EtBr is intercalating dye that form complexes with double-strand ds-DNA and single-strand DNA by forming close van der Walls contacts with the adenine–thymine base pairs. It also binds to the hydrophobic interior of the DNA molecule causing an increase in the distance between adjacent base pairs and so mutation of DNA [13]. Ethanol is a hydrophilic polar solvent that can create homogenous systems with water, so strip off essential water molecules from the enzyme and easily penetrate its surface causing its denaturation and losing its activity [25]. It also acts as mutagen via its first metabolite, acetaldehyde which stimulates chromosomal aberrations, sister-chromatid exchanges, and cross-links between DNA strands. It also impedes RNA formation in cells and in cell-free transcriptional systems [57].

In addition, dry biomass and protein concentrations decreased by all mutagen treatments over the control. The reduction of growth exerted by SA may be attributed to the decrease in ATP accessibility of the cell which may retard the cell division of the microbe by (1) interacting with enzyme activities and DNA in the cell [54], (2) altering the mitochondrial membrane potential [86], and (3) decreasing the cellular Ca-binding protein, which inhibit the proton pump [23]. EtBr affect the membrane constructions, slow cell division, and hold ATP biosynthesis by (1) interrupting the metabolism and protein synthesis and (2) inhibiting the formation of mitochondrial macromolecules by reducing the number of mitochondrial DNA [47]. Ethanol as hydrophilic polar solvent strips off essential water molecules from hydrophilic proteins of membranes causing their denaturation and losing their activities [25] including their ability to divide. This declined phenomenon on protein concentrations and growth was also observed by some investigators studying mutant stains. Protein synthesis in yeast protoplast is completely inhibited 3 min after SA addition to a final concentration of 10−3 M [6]. Survival rate of Saccharomyces boulardii was decreased by increasing concentration of EtBr [2]. Growth of Escherichia coli was partially inhibited by 1.2 × l0−4 M EtBr, a phenanthridinium trypanocide. Bacillus cereus was extremely sensitive to the growth-inhibitory action of ethidium (10−5 M) and morphological changes were observed [78]. Treating Candida albicans with 2% EtOH caused a 50% inhibition of germ tube induction, while doubling the EtOH concentration at all times triggered 80% inhibition of germ tube induction [15].

The data in Table 7 revealed that combination of lower concentrations of each mutagen (0.1 µg/ml EtBr + 0.5 mM SA + 10% EtOH, Comb-Fl) decreased lipase activity by exerting synergistic inhibition action. This action reduces C. catenulata virulence over their single impact on lipase activity, protein concentration, specific activity, and dry weight recording 6.61 U/ml, 1.63 mg, 4.05 U/mg, and 0.054 g/l.

Table 7 Assay of lipase activity, protein content, specific activity, and dry biomass of treated Candida catenulata with combination of ethidium bromide, sodium azide, and ethanol and/or fluconazole
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When 150 mg fluconazole was added to the mutagen combination (Comb + Fl), an increase in the production of the virulence factor lipase by C. catenulata (15.68 U/ml) was recorded with specific activity of 8.25 U/mg. This stimulation action indicated the antagonistic effect of fluconazole over the action of mutagens as well as the effectiveness of this new combination on increasing fungal pathogenicity.

The combination of mutagens with fluconazole decreased sharply the dry biomass over the Comb-Fl and the control (Table 7). This may be due to the cumulative stress produced by fluconazole over those produced on mutant derived by mutagen combinations. It was previously mentioned that fluconazole damages cell divisions and destabilizes the fungal cell membrane [72] causing cell content leakage, lysis, and eventual death [31].

The impact of fluconazole and mutagen combinations on lipase, specific activity, and protein concentrations is illustrated in Fig. 4. Results demonstrated that the highest lipase activity was recorded on optimum medium with fluconazole (Opt + Fl) followed by optimum medium without fluconazole (Opt-Fl, control) = optimum medium with both mutagen combination and fluconazole (Opt + Comb + Fl). The least value was estimated on the optimum medium with mutagen combination alone (Opt + Comb-Fl). Similar trend was observed with the specific activity and protein concentrations.

Fig. 4
figure 4

Effect of optimum medium (Opt) alone or in addition to mutagen combination (Comb) and/or fluconazole (Fl) on lipase, specific activity, and protein concentrations

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3.10 Examination of the cytological changes exerted by fluconazole treatment on C. catenulata cells using transmission electron microscope

The TEM photomicrograph of the control cells (untreated) of C. catenulata is shown in Fig. 5. It showed a typical morphology of Candida with a uniform central density, a typically structured nucleus, and a cytoplasm with several elements of endomembrane system and enveloped by a regular, intact cell wall as seen in Fig. 5a and b. Control culture showed yeast form and budding cells where cell and chromatin constrict and the cells were spherical to elongate with smooth surface (Fig. 5c-e). Untreated cells of C. catenulata presented a normal cell ultrastructure, such as compact and electron-dense cell wall, cytoplasmic membrane, homogeneous and electron-dense cytoplasm, nucleus, and mitochondria.

Fig. 5
figure 5

TEM micrograph of control C. catenulata. C, cytoplasm; CW, cell wall; CM, cytoplasmic membrane; N, nucleus; M, mitochondria. Magnifications a 120,000 × , b, d 100,000 × , and c, e 60,000 × 

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The typical ultrastructure of C. catenulata was not described in the available literatures. In this context, it may be mentioned that some other Candida spp. were ultrastructurally descripted as C. tropicalis [24], C. krusei [41], and C. albicans [9, 56].

The TEM photomicrograph of the treated cells of C. catenulata with 150 mg fluconazole is illustrated in Fig. 6. It was shown that the ultrastructures of treated C. catenulata displayed noticeable morphological changes.

Fig. 6
figure 6

TEM micrograph of fluconazole-treated cells of C. catenulata. Magnifications a-d 80,000 × , e, f, h 60,000 × , and g 50,000 × 

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Fluconazole treatments caused deformation in cell shape, disintegration of protoplasm, and disruption in the cell wall as well as the cytoplasmic membrane of the cells which led to the protoplasm leakage along with vacuolations as seen in Fig. 6a, d, e, g, h. Most cells were irregular and even hollow to form ghost cells where fluconazole induces formation of pores in yeast cells to evacuate their cytoplasmic contents (Fig. 6b, c, f). Many cell walls became incomplete or detached and some were broken. Their contents were either disappeared or were denatured (Fig. 6e, b). The cell shapes became irregular, and the organelles atrophied (Fig. 6g) and the organelles were completely disappeared (Fig. 6e, g).

In this study, fluconazole showed positive result in anticandidal activity test against C. catenulata, including a damaged cell wall. The results indicated that fluconazole interrupted the Candida cell membrane and increased the cell membrane permeability. The control Candida cells had uniform central density and intact cell wall (Fig. 6a, b). However, treatment with fluconazole exhibited notable alterations in the cells especially in the cell membrane and the cell wall (Fig. 6a-h). The treatment also caused the disposition of membranous bodies within the cell in addition to the decrease in the cytoplasmic volume (Fig. 6a, d). Moreover, certain number of cells apparently underwent drastic shape changes, some lysed and others collapsed (Fig. 6e). It seems that fluconazole induced dysfunctions of the cell membrane and the cell lost its metabolic functions.

In the present study, fluconazole concentrations expressed destructive effects on the ultrastructure of C. catenulata. These deformations may be attributed to its antifungal activity. Fluconazole exerts their fungistatic effect on Candida spp. by inhibiting their cell growths through the disruption of sterol biosynthesis and increased cell size due to an apparently high porosity level of the cell wall. In consequence, these cells disintegrated, perhaps due to increased turgor pressure [65]. Fluconazole contains a free nitrogen atom which binds to the heme group at the active site of Erg11 enzyme that is encoded by the ERG11 gene in Candida. Thus, it inhibits the heme protein, which in turn affects the fungal cytochrome P450 enzyme 14α-demethylase of lanosterol. This inhibition prevents the conversion of lanosterol to ergosterol [84]. Ergosterol is an essential component of the fungal cytoplasmic membrane, and subsequent accumulation of 14α-methyl sterols [61]. It also serves as a bioregulator of membrane fluidity, asymmetry, and consequently of membrane integrity in fungal cells [51], so the inhibition of 14 α-demethylase prevents the demethylation of lanosterol causing its accumulation and blocking the ergosterol production. This action causes the formation of a plasma membrane with altered structure and function [72].

In this connection, few investigators also detected those changes in the ultrastructure of some Candida spp. after their exposure to fluconazole concentrations. Giordani et al. [32] studied the impact of the antifungal agent fluconazole on the ultrastructure of C. albicans. They realized that its low concentrations (2 µg/ml) modified the cell wall structure and accumulated polysaccharides between cell wall and plasmalemma, while doubling its concentrations thickened the cell wall with no appearance of multilayer structure and formation of electron-dense lamella and increased the accumulation of the polysaccharides. In addition, Sohnle et al. [73] mentioned that exposing the cells of C. albicans to 1 µg/ml fluconazole has direct fungicidal activity on non-proliferating yeast cells by damaging the cell wall, cell membrane complex, and the interior contents of the cells.

4 Conclusion and future perspective

Maximum lipase production and biomass gain of C. catenulata could be obtained in medium composed of 150 mg fluconazole, 2.5% Tween 80, 0.7% yeast extract, and 10 mM CuSO4.5H2O, pH 5.5 at 37 °C with dark agitation, after 3-day incubation. This study concluded that the cultural condition plays an important role in lipase production and activity of C. catenulata, so it must be optimized before any experiment.

Lipase, specific activity, and protein concentrations were slightly enhanced on optimum medium enriched with both fluconazole and combined mutagen [0.1 µg/ml EtBr + 0.5 mM SA + 10% EtOH], so we can conclude that fluconazole can be the key factor for increasing pathogenicity and the lipase activity for industrial purposes but if the target was to reduce the fungal pathogenicity, only the combined mutagen 0.1 µg/ml EtBr + 0.5 mM SA + 10% EtOH will be effective.