Screening of asporogenic mutants of phytase-producing Aspergillusniger CFR 335 strain

Abstract

Objective:Aspergillus niger is one of the fungi exploited industrially for the production of various metabolites including the enzyme phytase. Intensive sporulation is a serious crisis in using this fungus, hence, in the present study an attempt was made to obtain an asporulating strain. Materials and methods:A. niger CFR 335, a local phytase-producing isolate, was induced to physical mutation by ultraviolet (UV) radiation and chemical mutation using different concentrations of ethyl methane sulphonate (EMS) and N-methyl N′-nitro N-nitrosoguanidine (NTG). Results: UV exposure for 30 min, 10 mM EMS and 25 µg ml⁻¹ of NTG were found to be optimum for maximum killing effect of 26.3%, 95% and 80% of the spores, respectively. In this experiment, a stable auxotrophic mutant (ala⁻) of A. niger CFR 335 with suppressed sporulation was obtained. The auxotrophic mutant M-1 (A. niger CFR 335 ala⁻) showed an improved specific activity of 1.26 U mg⁻¹ as compared with the parent strain (0.85 U mg⁻¹). Conclusion: Sporulation was drastically reduced in the mutant M-1 without affecting phytase yield, whereas the production rate was proportional to the rate of sporulation in the parent strain (A. niger CFR 335).

• asporulation
• Aspergillus niger CFR 335
• phytase
• ethyl methane sulphonate
• N- methyl N′-nitro N-nitrosoguanidine
• ultraviolet radiation

Introduction

Phytic acid (myo-inositol hexakisphosphate) is a storage form of phosphorus in plants [1], legumes [2] and oil seeds [3], [4]. The most striking chemical impact of phytic acid is its strong chelating ability with multivalent cations, to form cation–phytic acid complexes [5]. Minerals of concern in this regard include phosphorus, calcium, zinc, iron, copper and magnesium. In the context of human and animal nutrition, the following two aspects of phytic acid are critically important [6], [7]. (i) Monogastric animals have only low levels of phytate-degrading enzymes in their digestive tracts, and since phytic acid itself is not resorbed, feed for pigs and poultry is commonly supplemented with inorganic phosphate to meet the phosphorus requirements of these animals. (ii) Phytic acid is an antinutrient factor, since it complexes with proteins and a variety of metal ions and therefore decreases the dietary availability of these nutrients. The antinutritive properties, its value as a phosphorus source and its role in causing eutrophication have prompted researchers to develop a method to remove phytic acid completely from the feed or to hydrolyse it economically using phytase enzyme.

The phytases (myo-inositol hexakisphosphate 3- and 6-phosphohydrolases; EC 3.1.3.8 and 3.1.3.26) are a subfamily of the histidine acid phosphatases [8] and are found naturally in plants and microorganisms, particularly fungi. With the growth of the biotechnology industries, filamentous fungi, a principal source of enzymes and metabolites, have been widely employed in traditional food fermentation processes, such as the koji process [9]. Features such as low cost and high productivity have attracted many research efforts in both molecular genetic techniques and bioprocess improvements [10]. Exploitation of aspergilli began early with the use of Aspergillus niger and Aspergillus oryzae for the production of citric acid and taka-amylase through fermentation. Although several other fungi are currently in use, A. niger remains a favourite for industries. This may be attributed to its GRAS status, great secretory potential and the wealth of information accrued on its fermentation. Of all the organisms surveyed, A. niger was found to produce the most active extracellular phytase [11]. Various strains of A. niger have been shown to produce three extracellular acid phosphatases, a meso-inositol hexaphosphate phosphohydrolase (E.C. 3.1.3.8) (Phyt A phytase) with two pH optima (2.5 and 5.0) and a non-specific phosphomonoesterase (E.C.3.1.3.2) (Phyt B) with a pH optimum of 2.0–2.5 that hydrolyses phytin [12–15].

Apart from good yield of phytase enzyme, the fungus poses a serious threat to the researchers in rendering potential risk for the handlers. The fungus produces large amounts of dark conidiospores that hamper the extraction of enzyme and cause health risks such as allergic bronchopulmonary aspergillosis if not handled properly [16], [17]. A strain with phytase overproduction and lower sporulation rate is of high value from an industrial point of view. With this aim, the present study focused on developing a strain of A. niger CFR 335 with asporogenicity or delayed sporulation through induced mutations without any adverse effect on phytase production.

Materials and methods

Strain

A. niger CFR 335, a native isolate from soil samples obtained from poultry livestock areas, was used in the present study (Figure 1). About 50 different soil samples were screened for the isolate. The culture was grown for 4–5 days at 30°C on complete medium (CM) slants in triplicate. Completely grown slants were maintained in the refrigerator until further use.

Figure 1.  A. niger CFR 335; Czapek Dox Agar plate (left), Phytase Screening Medium plate (right) with clearing zone indicating hydrolysis of calcium phytate seeded in the agar medium.

Media

Complete medium (CM) comprised (g/l): yeast extract 2.5, malt extract 5.0, glucose 10.0 and agar 20.0; pH 5.5±0.2. Minimal medium (MM) comprised (g/l): yeast nitrogen base without amino acid and ammonium sulphate 1.7, succinic acid 10.0, sodium hydroxide 6.0, glucose 2.0 and agar 20.0; pH 5.8±0.2. Phytase screening medium (PSM) was as described by Howson and Davis [18].

A CAMAG UV chamber was used for physical mutation. Ethyl methane sulphonate (EMS) and N-methyl N-nitro N-nitrosoguanidine (NTG) and all the amino acids were obtained from Sigma Co., USA. The chemicals used for the phytase assay were obtained from Sigma and from Qualigen Chemicals, India Ltd.

Mutagenesis by UV radiation

Young spores, 16–18 h old, were harvested using 0.1% Tween-80. Then 1 ml samples of the spore suspension containing 2.4×10⁶ cells ml⁻¹ were subjected to UV irradiation for different time exposures (10, 20 and 30 min) in a sterile petri dish using a CAMAG UV chamber, which emits radiations of 254 nm at a distance of 25 cm [19]. The experiment was carried out in triplicate. The spore suspensions were intermittently agitated during the course of irradiation. The treated spore suspensions were then incubated at 4°C in the dark overnight for any DNA repair to take place. Suspensions were suitably diluted and appropriate dilutions were plated on CM and incubated for 4–5 days at 30°C. Spore-killing percentage was plotted against UV exposure.

Mutagenesis by EMS

Aliquots of 1 ml of the spore suspensions harvested as described above were treated with different concentrations of EMS (2, 4, 6, 8 and 10 mM) in sterile distilled water [19]. The suspensions were incubated for 1 h at 30°C with a brief agitation. This experiment was carried out in triplicate. After incubation, the mutagen was completely removed by centrifugation at 2000 rpm. The spore suspensions were thoroughly washed twice with sterile distilled water, resuspended in 0.01 M sterile buffered saline (pH 7.0) and then refrigerated overnight in the dark before plating for any DNA repair to take place. Suitably diluted spore suspensions were plated on CM and incubated for 4–5 days at 30°C. The percentage of spore killing was plotted against the concentration of EMS.

Mutagenesis by NTG

Aliquots of 1 ml of the spore suspensions were treated with different concentrations of NTG (5, 10, 15, 20 and 25 µg ml⁻¹). The suspensions were incubated for 1 h at 30°C with a brief agitation. The experiment was carried out in triplicate. After incubation, the mutagen was completely removed by centrifugation at 2000 rpm. The spore suspensions were thoroughly washed twice with sterile distilled water, resuspended in 0.01 M sterile buffered saline (pH 7.0) and then refrigerated overnight in the dark before plating for any DNA repair to take place. Suitably diluted spore suspensions were plated on CM and incubated for 4–5 days at 30°C. The percentage of spore killing was plotted against the concentration of NTG.

Characterization of putative mutants for auxotrophy

The UV, NTG and EMS mutagenized colonies grown on CM were replica plated using sterile velvette cloth on minimal medium (MM) plates and incubated for 24–36 h at 30°C [20]. The colonies that did not grow on MM plates were picked up from the master plate (CM) and individually tested for their growth factor requirement by inoculating into a liquid minimal medium containing appropriate concentrations of individual amino acids [21]. About 2000 colonies were screened for auxotrophic strains with amino acid markers. Growth of the culture in tubes supplemented with a particular amino acid indicated its amino acid requirement and was marked as the respective auxotroph. The experiment was carried out in triplicate and repeated to confirm the growth and stability of the mutant.

Characterization of putative mutants for reduced sporulation

About 500 putative mutants with morphological variations were screened for spore suppression. Spores were harvested from all the mutants after cultivating on CM plates for up to 10 days. The spores were counted using a haemocytometer (Feinoptic Bad Blankenburg, Germany) and were expressed as number of spores ml⁻¹. This was compared with the parent strain (A. niger CFR 335) after confirmation by repeated experiments and the average consistent result of the percentage reduction in sporulation was reported.

Characterization of putative mutants for phytase production

About 62 putative mutants with reduced sporulation were qualitatively screened for phytase enzyme by plating them on PSM containing 0.5% calcium phytate as substrate. About 10 positive mutants were selected based on the size of the zone of hydrolysis around the colony and quantitatively screened for phytase enzyme production by cultivating them in a wheat bran medium. Fresh wheat bran (50 g) was placed in a 1 L Erlenmeyer flask and autoclaved for 40 min at 121°C. The total moisture was maintained at 60% level with sterile distilled water. The medium was inoculated with 2.4×10⁶ cells ml⁻¹ of each of the mutants and incubated at room temperature for 5 days in well-aerated conditions. The crude enzyme was extracted from each of the mutant flasks using 1:5 w/v sodium acetate (0.2 M) buffer, pH 4.5.

Phytase assay

The crude enzyme preparation was quantitatively assayed for phytase enzyme by the method of Heinonen and Lahti [22]. The assay was initiated by mixing 1 ml of diluted (1:10) crude enzyme with 0.5 ml of sodium acetate (0.2 M) buffer, pH 4.5, and 0.5 ml of sodium phytate (15 mM) (Sigma). The reaction mixture was incubated at 40°C in a water bath for 45 min. The reaction was terminated by adding 2 ml of 15% trichloroacetic acid. Assay mixture (0.5 ml) was then mixed with 4 ml of 2:1:1 v/v of acetone, 10 mM ammonium molybdate and 5 N sulphuric acid (AAM solution) and 0.4 ml of citric acid (1 M). The amount of free phosphate released was determined spectrophotometrically at 355 nm. A standard graph was plotted using potassium dihydrogen phosphate with working concentration ranging from 30 to 360 µM. Protein quantifications were made by the method of Bradford [23] and compared with the standard prepared using bovine serum albumin.

The phytase production by different mutants obtained through induced mutation of A. niger CFR 335 was presented as the standard error means (±SEM). The linear and quadratic contrasts were determined by SAS [24] for different concentrations of EMS and NTG used for spore-killing percentage.

Results and discussion

Non-ionizing radiations such as UV at the wavelength of 254 nm is absorbed by DNA leading to thymine dimers that cause mispairing during DNA replication, which leads to permanent mutation if not repaired. Thus mutation is frequently employed in the field of microbiology for improving industrially important microorganisms for product development. Young spores of A. niger CFR 335 are often sensitive to induced mutation by UV radiation, as observed in the present study.

The results obtained in the present study indicated the highest spore-killing rate of 26.3% when the spores were exposed to UV for 30 min, which was 16% higher than the minimum killing rate of 10.2% when exposed for 10 min. With increase in the exposure time, killing rate increased and an inhibitory effect on growth was observed in CM. An improved catalytic efficiency and thermostability of Escherichia coli, pH 2.5, acid phosphatase/phytase expressed in Pichia pastoris was achieved by site-directed mutagenesis [25]. A similar kind of expression of gene of A. fumigatus wild-type phytase and the mutant genes were expressed in P. pastoris GS115 by site-directed mutagenesis with PCR [26], and the results revealed an increased specific activity of the Q23L from 0.51 U mg⁻¹ of the wild type to 1.09 U mg⁻¹ at pH 5.5. Similarly, in the present study an improvement in the specific activity of the parent strain from 0.85 U mg⁻¹ to 1.26 U mg⁻¹ of the mutant M-1 was observed (Table I). However, the exact mechanism and site of mutation are not known. An increase in phytase activity of A. niger NRRL 3135 phytase A (phy A) has been shown at intermediate pH levels (3.0–3.5) by site-directed mutagenesis of its gene at amino acid residue 300 [27]. It was shown that a single mutation, K300E, improved phytic acid hydrolysis at 37°C by 56% and 19% at pH 4.0 and 5.0, respectively.

Table I.  Phytase production of A. niger CFR 335 and its 10 mutants.

Strain	Total protein(mg ml^−1)	Specific activity (U mg^−1)
A. niger CFR 335 (parent)	1.75±0.02	0.85±0.002
M-1	0.94±0.01	1.26±0.002*
M-2	0.92±0.01	1.35±0.002*
M-3	1.28±0.02	0.76±0.001
M-4	1.17±0.01	0.76±0.001
M-5	1.23±0.02	0.81±0.002
M-6	1.31±0.02	0.99±0.002*
M-7	1.46±0.02	0.75±0.002
M-8	1.39±0.02	0.95±0.002*
M-9	1.53±0.01	0.84±0.002
M-10	1.19±0.01	0.91±0.001*

The results showed a highest spore killing rate of 95% when 10 mM ml⁻¹ of EMS was used and a lowest killing rate of 32% when treated with 2 mM ml⁻¹ of EMS. The killing percentage increased with the increase in the concentration of EMS (Figure 2). A maximum killing rate of 80% was observed when the spores were treated with 25 µg ml⁻¹ of NTG, while a minimum killing rate of ≥15% was observed with 5 µg ml⁻¹ of NTG. Beyond 25 µg ml⁻¹ concentration of NTG there was 100% killing of the spores (Figure 3). Thus, from the results it was observed that the killing percentage of the spores was proportional to the concentration of both EMS and NTG.

Figure 2.  Effect of ethyl methane sulphonate (EMS) on A. niger CFR 335 spores. Linear effect of EMS on spore killing was significant (p < 0.001).

Figure 3.  Effect of N-methyl N-nitro N-nitrosoguanidine (NTG) on A. niger CFR 335 spores. Linear effect of NTG on spore killing was significant (p < 0.001).

The parent strain (A. niger CFR 335), auxotrophic mutant (A. niger CFR 335 ala⁻) and other unstable auxotrophic mutants are shown in Figure 4. Even though the relationships between amino acid auxotrophy and colony morphology variations were concisely proved, the exact mechanism of occurrence of such a phenotype is not known. Amino acid auxotrophy might play a vital role in suppressing the spore formation, which is supported by the present observation. Of the 2000 colonies screened for auxotrophy, 2 colonies were found to be auxotrophic for alanine, valine and isoleucine. The mutant that had val⁻ and isoleu⁻ markers reverted back to its original form after repeated subculturing, whereas the ala⁻ auxotroph remained stable even after several subcultures.

Figure 4.  Centre plate shows parent strain (A. niger CFR 335) and the surrounding plates show its mutants.

A. niger CFR 335 ala⁻ auxotrophic mutant (Figure 5) showed about 88% reduction in the sporulation compared with the wild strain counterpart, which was confirmed by repeated cultivation in nutrient medium. The sporulation rate of the parent strain was found to be 3.2×10⁶ ml⁻¹ and the auxotrophic mutant had a rate of 3.84×10⁵ ml⁻¹. This mutant also showed morphological variations with white vegetative mycelium, unlike its parent counterpart, which is pale yellow in colour with black conidiospores (Figure 5). Quantitatively, the production of phytase by the auxotroph did not differ much with respect to the parent strain. A similar kind of experiment was carried out and an improved strain of A. niger showing 3.2-fold higher extracellular phytase activity was developed where hygromycin resistance was used as the mutant selection marker [28]. It was shown that the mutation of phiA, a gene from A. nidulans, resulted in reduced growth and severely reduced sporulation. The abnormality is traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. The possibility that changed expression of phiA is correlated with growth arrest, which is caused by inhibited V-ATPases, was suggested [29]. It was also revealed that the mutation of PkcA allele of calC2 strain of A. nidulans showed an alteration in hyphal growth and conidiogenesis [30].

Figure 5.  P, A. niger CFR 335 (parent); M, A. niger CFR 335 ala⁻ (auxotrophic mutant).

In conclusion, the results showed that 30 min of UV exposure, 10 mM EMS and 25 µg ml⁻¹ of NTG were effective in producing maximum killing effect of A. niger CFR 335 spores. An auxotrophic mutant of A. niger CFR 335 with a trait of delayed sporulation was obtained in the present study that may reduce the risk of allergic bronchopulmonary aspergillosis caused by long-term handling of the spores. The mutant also retained its characteristic of phytase yield, which is beneficial from the industrial point of view. Asporulation is an important genotypic change that bears relationship with the alteration of a particular gene responsible for the phialide and conidia formation. The possibility of the involvement of membrane diffusion caused by mutagens cannot be ruled out for delayed sporulation. Studies of this sort of investigation on fungal asporulation, particularly A. niger, are very sparse. Thus, such studies could open a new area in the field of microbiology and biotechnology, wherein numerous health risks related to fungal spores could be evaded. A study investigating the exact site of mutation leading to delayed sporulation is in progress.

Acknowledgements

The authors are grateful to the University of Mysore for a Research Fellowship and they are also thankful to the Director, Central Food Technological Research Institute, Mysore, India for permitting them to work at the Food Microbiology Department of the Institute. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.