Table of Contents  
Year : 2019  |  Volume : 18  |  Issue : 4  |  Page : 403-410

Genetic improvement of fungal β-mannanase and its molecular differentiation

1 Department of Microbial Genetics, Division of Genetic Engineering and Biotechnology
2 Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, Egypt
3 Department of Plant and Microbiology, Science Collage, Helwan University, Helwan, Egypt

Date of Submission09-Jul-2019
Date of Acceptance19-Aug-2019
Date of Web Publication28-Jan-2020

Correspondence Address:
Professor Doctor Amal M Hashem
Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, El Buhouth Street, Dokki, Giza
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/epj.epj_32_19

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Background and objective β-Mannanase is an enzyme that has great potential in many industrial application including feed, food, pharmaceutical, cosmetics, production of mannan and manooligosaccharides, pulp and paper, bioethanol and biodiesel productions, and oil and textile industries. The aim of this study was to describe the potential of gamma and ultraviolet (UV) rays to optimize the production of industrially important β-mannanase enzyme by subjecting Penicillium citrinium Egy5LC368457 to these rays.
Materials and methods Various doses and times of UV and gamma irradiation were used. Genetic diversity was resolved by mistreatment with the Random Amplified Polymorphic Polymer (RAPD-PCR) technique. Ten RAPD oligonucleotide primers amplifying DNA of β-mannanase showed reproducible banding patterns.
Results and conclusion The results of this study revealed the highest β-mannanase activity was produced by gamma ray 150 Gy (37.42 IU/ml) with 2.27-fold higher than the wild type. A total of 64 bands were obtained from nine of these markers with 44% polymorphic bands. The size of the amplified bands ranged between ∼75 and 3000 bp. The genetic polymorphism value of each primer was determined, which ranged between 2 and 9 bands. The primer efficiency of amplification ranged between 3.13 and 23.44%, and the discriminatory power ranged from 4.5 to 25. In conclusion, UV and gamma ray irradiation can induct mutations, which can be carefully acclimatized and commercially propagated under suitable condition. RAPD technique could be successfully applied to the newly β-mannanase and can differentiate mutants.

Keywords: Penicillium citrinium, random amplified polymorphic polymer, ultraviolet and gamma rays

How to cite this article:
Abosereh NA, Ismail SA, Khattab OK, Nour SA, Abo-Elnasr AA, Hashem AM. Genetic improvement of fungal β-mannanase and its molecular differentiation. Egypt Pharmaceut J 2019;18:403-10

How to cite this URL:
Abosereh NA, Ismail SA, Khattab OK, Nour SA, Abo-Elnasr AA, Hashem AM. Genetic improvement of fungal β-mannanase and its molecular differentiation. Egypt Pharmaceut J [serial online] 2019 [cited 2023 Jan 30];18:403-10. Available from:

  Introduction Top

Microbes are the major source of most enzymes. The demand for these enzymes had occurred owing to growth of sustainable solutions [1],[2].

Various fungi can produce β-mannanase, such as Aspergillus awamori [3], Aspergillus oryzae [4], Penicillium humicola [5], Penicillium oxalicum [6], Trichoderma harzianum [7], and Rhodothermus marinus [8]. Many microbic β-mannanase production studies have targeted on the use of pure mannans like locust bean gum and konjac gum as inducers [9],[10]. Many wastes have low-value mannan-rich substrates, like coconut meal, palm nut cake, apple pomace and occasional extracts [11], and alternative hemicellulosic biomass like wheat bean and wheat straw, which may also be utilized in bioprocesses [12],[13].

Endo-β-1,4 mannanases (E.C. are enzymes that hydrolyze the (1,4)-β-D-mannosidic linkages within the main chain of mannans and heteropolysaccharides, consisting mainly of mannose, such as galactomannans and glucomannans, producing manno-oligosaccharides [14].

In fact, mannanases may well be additionally accustomed to cut back the body of occasional extracts [15],[16], biobleaching of pulp and detergent trade [5],[13],[17],[18],[19],[20], bioconversion of biomass wastes to fermentable sugars [21],[22], and upgrading of animal feed products [17],[23]. Furthermore, they are employed in the preparation of manno-oligosaccharides which used as prebiotics (non-nutritional food additives) for selective growth of human beneficial intestinal microflora (Bifido bacterium and Lactobacilli spp.) [24],[25].

For high production of enzyme, microorganisms can be improved by different mutagenesis as a successful method [26].

Ultraviolet (UV) and gamma (γ) irradiations can also be used to obtain mutants yielding higher enzymes production from conidia of thermophilic fungi [27]. The improvement of the mutants for the production of the enzymes occurred by re-constitution of the damaged genes of wild-type strains, with improvement of these properties.

For strain mutation, UV rays are very important inducers. The main effect of these rays is to modify the structure of pyrimidine (cytosine and thymine), causing the formation of thymine dimers, which distort the structure of DNA heliex and block further replication process [28].

Omear et al. [29] and Soliman et al. [30] have developed the haphazardly amplified polymorphic polymer (RAPD) markers, which have become one-in-all the foremost wide PCR-based DNA markers that can be used to detect the relation between species.

In this work, genetic improvement of fungal β-mannanase by using physical mutagen (UV and gamma) and the differentiation between wild-type strain and their mutant were done.

  Materials and methods Top

Fungal isolate and growth media

The fungal strain Penicillium citrinium Egy5 LC368457 was obtained from the Pharoesmomes of Ancient Egyptian Museum, Cairo, Egypt. There is No animal or human experimental. The culture was maintained on potato dextrose agar and incubated at 30°C for 7 days before storage at 4°C with monthly subculturing.


Two methods of mutations were employed for strain improvement:

Mutagenesis with gamma rays treatment

In this way, mutagenesis to the spore suspension of P. citrinium was carried out using gamma rays produced from Cobalt-60 (Co60) as a source of gamma radiation (by Egyptian Atomic Energy Authority, Nasser City, Egypt).

Five different doses of gamma radiation were employed: 0, 50, 100, 150, 200, and 250 Gy [27].

Mutagenesis with ultraviolet treatment

UV mutagenesis to the spore suspension of P. citrinium was treated with UV (power, 30 W, and wavelength, 254 nm), each 15 min intervals, with a distance of 18 cm from the UV light source. Then, the plates were stored in the dark for 2 h. After treatment, mutagenic of each treatment was diluted and plated on potato dextrose agar as complete media and Cazpek media as minimal media at 30°C for 5 days. A single colony from each treatment was subcultured for enzyme production [31].

Culture media

The basal medium for inoculum included the following (g/l): peptone (2); ammonium sulfate (1.5); urea (0.3); MgSO4.7H2O (0.5); K2HPO4 (10); and locust bean gum (10) [5]. The pH of the medium was adjusted at 5.3 before autoclaving. Each 250 ml Erlenmeyer flask contained 50 ml of the medium and was autoclaved for16 min at 121°C.

Screening of mutated isolates (production medium)

The production medium included the following (g/l): peptone (2.27); ammonium sulfate (1.7); urea (0.34); MgSO4 · 0.7H2O (0.6); K2HPO4 (7.5); and Coffee waste(3 g/flask). The pH of the medium was adjusted at 4.5 before autoclaving. Each 250 ml Erlenmeyer flask contained 50 ml of the medium and was autoclaved for 16 min at 121°C [5].

An inoculum culture was obtained by culturing the fungal strains in the above medium at 30°C for 48 h with shaking at 120 rpm. The culture flasks were inoculated by 8% of the inoculum and incubated at 30°C in a shaking incubator at 120 rpm for 12 days. Thereafter, the fermented medium was centrifuged, and the filtrate was used as the crude enzyme solution.

Analytical methods

Enzyme assay

An assay was performed by incubating 0.5 ml of appropriately diluted culture filtrate with 1 ml of 1% (w/v) locust bean gum (0.05 mmol/l) sodium citrate buffer at pH 5.0 for 10 min at 50°C [4]. The reducing sugars produced were determined using the Nelson–Somogyi technique [32]. One unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of mannose/ml/min.

Protein determination

To determine the specific enzymatic activity, the quantification of total amount of soluble protein was measured using the Lowry method [33].

Genomic DNA extraction

Genomic DNA was extracted from the most efficient productive strains and their best resulting mutants using Easy Quick DNA extraction kit (Qiagene, Netherlands and Germany) following the manufacturer’s instructions.

Random amplified polymorphic DNA (RAPD-PCR)

The RAPD-PCR technique was done using 10 oligonucleotide primers. The PCR was performed according to Plengvidhya et al. [34] in a 25 µl reaction volume, and amplification was programmed to 40 cycles after an initial denaturation cycle for 2 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 25°C for 1 min and an extension step at 72°C for 2 min, followed by extension for 10 min at 72°C in the final cycle.

Band analysis

The gels for control and exposed DNA were run for each of the 10 primers ([Table 1]). A DNA ladder of 100 and 250 bp was also run in each gel. The bands for PCR products were analyzed by Labimage version 5.21 programs. The banding patterns on the gels were transformed into tables of binary characters, where the appearance of a band was given the number one while the absence of the band was denoted by zero. [Table 6] was used to first, determine the total number of bands together with their molecular weights that were produced by a primer across all isolates starting from the lowest weight (bottom of the gel) to the highest weight (top of the gel) [35]; second, to determine the monomorphic or common bands (appearing in all isolates) and polymorphic bands (appearing in some isolates only) amplified by a primer; third, to determine the % efficiency of a primer. This was estimated as a percentage of the total number of bands amplified by the primer out of the total number of bands amplified by all primers across all species. This represents the availability of sequences complementary to the primer in the genome; and fourth, to determine the discriminatory power of the primer [36]. This is a percentage of the polymorphic bands amplified by a primer out of the total number of polymorphic bands given by all primers in all isolates.
Table 1 Numbers and sequences of the random amplified polymorphic polymer primers used

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  Results and discussion Top

Screening of β-mannanase activity by gamma-irradiated mutants

β-Mannanase is the most significant catalyst for hemicelluloses digestion, one of abundant groups of chemical compound in nature. This enzyme hydrolyzes mannan yielding mannotriose and mannobiose [37]. The present study is an attempt to improve the yield of enzymes production from Penicillium citrinum by gamma rays and/ or UV mutation. A range of doses (50–250 Gy) of gamma radiations were applied to induce mutation in cells of wild strain of Penicillium citrinum ([Figure 1]). It was found that the survival percentages decreased by increasing exposing dose of gamma rays. The survival percentages were 96, 86, 60, 40, and 10 at doses 50, 100, 150, 200 and 250 Gy, respectively. A total number of 42 mutant strains were subjected to different exposure time of gamma radiation. Approximately 26% of the mutants using 50, 100, 150, 200, and 250 doses of gamma radiation showed β-mannanase activities higher than the parent strain ([Table 2]). Similar results were revealed by Iftikhar et al. [38] who found that with the dosage 140 Gy, MBL-5 showed maximum extracellular lipase production. However, Shahbaziet al. [27] reported that gamma causes improvement of the activity of Ccase, CMCase, Avicellase, and Fpase for Trichoderma mutagenesis.
Figure 1 The effect of gamma dose at different dose interval on the survival mutation percentage of Penicillium citrinium.

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Table 2 Screening of β-mannanases activity in gamma-irradiated mutants

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Of all the mutants obtained, mutant designated 150-A had the highest increase in the β-mannanases activity (37.42 IU/ml). This is ∼2.27 folds of the parent strain, with specific activity above wild type of 2.18 folds. The mutants coded 200 J, 200 F, and 250 G gave ∼0.4 IU/ml. Similarly, an 81% increase in lipase activity has also been reported by Penicillium expansum after exposure with gamma irradiation [39].

The protein content in all the mutants ranged from 6.01 to 8.92 mg/ml. The mutant coded 50-A, 150-A,150-B, 250-A, and 250-D that gave higher β-mannanases (30.65, 37.42, 23.2, 26.13, and 25.03 IU/ml, respectively) were selected and identified as stable mutants that showed higher stability with five generations ([Table 3]).
Table 3 Stability of gamma mutants

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Screening of β-mannanase activity in ultravoilet-irradiated mutants

A total of 22 mutant strains of wild-type strain were developed at intervals of 60 min of exposure to UV irradiation ([Table 4]). Approximately, 27% of the mutant strains of P. citrinium generated from 15, 30, and 45 min of exposure to UV irradiation showed higher increase in β-mannanase activities compared with the wild-type strain. Of all the mutants generated, mutant designated UV30-B had the highest increase in mannanase activity, with ∼3-fold higher than the parent strain, whereas the mutant coded UV15-H gave 0.79 IU/ml. The mutant strains 30-B, 45-B, and 45-A showed low stability within three generations ([Table 5]). It was clear that UV rays were very harmful on the microorganism as only 3.57% germination was reported after 15 min of exposure, and lethal doses were found to be after 60 min ([Figure 2]). Our results agree with EL-Bondkly et al. [31] who showed that after exposure of Penicillium roquefortii strains to UV rays, the lethality percentage sharply increased with the increase in the periods of exposure to UV. Arotupin et al. [40] showed also UV irradiation produced higher increase in β-mannanase activities from Aspergillus glaucaus and Rhizopus japonicas. Prabakaran et al. [41] noticed that the highest production of cellulases was observed by Penicillium chrysogenum with UV exposure time of 5 min. Moreover, Bapiraju et al. [26] observed that UV-induced mutant of Rhizopus spp. showed higher lipase activity than the parent strain. Meanwhile, Irfan et al. [42] revealed that the UV radiation has increased the CMCase activity up to two times, whereas FPase activity exaggerated to a few times as compared with the parental strain. However, Burlacu et al. [43] reported that UV mutagenesis was a successful method for enhancing the xylanase activity compared with chemical mutagenesis.All of these results indicated that the survival of the parent strain depends on both of the type and period of mutagen treatment and on the microorganism’s nature.
Table 4 Screening of β-mannanases activity in ultraviolet-irradiated muatnts

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Table 5 Stability of ultraviolet mutants

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Figure 2 The effect of ultraviolet dose at different time interval on the survival mutation percentage of Penicillium citrinium.

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Molecular study

The molecular genetic variability among the wild-type and gamma mutants that showed higher productivity and stability (150-A, 150-B, 50-A, 250-Aand 250-D) was evaluated using ten random primers. Nine of ten primers (10-mer random primers: A04, A05, A07, A08, B12, C01, C02, C04, and C05, [Table 6]) gave positive and detectable bands ([Figure 3]). They generated a total of sixty-four RAPD different bands ranging approximately from 75 to 3000 bp size. Band numbers are ranging from two bands for primer OPC-04 to fifteen bands for primer OPA-07 ([Figure 3]). However, 13 (23%) bands were monomorphic, and two primers OPC-04 and OPA-04 did not generate monomorphic band.
Figure 3 Random amplified polymorphic DNA pattern of isolated DNA from Penicillium citrinium and their resulting mutants by using nine different random amplified polymorphic polymer primers (OPA-04; OPA-05; OPA-08; OPB-12; OPC-01; OPC-04; OPC-02; OPC-05; OPA-07): W:wild type, m1: 150-A, m2: 150-B, m3: 50-A, m4: 250-A, and m5: 250-D.

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Table 6 Fragments amplified by the nine primers in the wild-type and gamma mutant species and the % efficiency of amplification and discriminatory power of each primer

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Additionally, the primer efficiency of amplification ranged between 3.13% (OPC-04) and 23.44% (OPA-07) ([Table 6]).

On the contrary, the discriminatory power ranged from 4.5 (OPA-08 and OPA-02) to 25 (OPA-07). Nine of ten primers gave amplified bands (representative of these PCR products are shown in [Figure 3]). All primers succeeded in giving polymorphic and monomorphic bands, except primers OPC-04 and OPA-04. The high potency of the primer is indicative of an oversized space of the order that enhances and permits base pairing between primer and genomic deoxyribonucleic acid [44]. Moreover, discriminatory value of a primer depends only on the number of polymorphic bands produced by the primer relative to the total number of polymorphic bands produced by all primers.

Discriminatory power of a primer is more important than the primer efficiency in determining its DNA fingerprint. In this study, the two primers (OPA-08 and OPB-12) have similar efficiency (10.94), and the same total number of bands (7) gave different discriminatory power of 11.36 and 6.82, respectively.

Dendrogram was produced after numerical analysis of the DNA RAPD profiles using the Nei and Lis correlation coefficient, and unweighted pair group method with arithmetic averages algorithm (UPGMA) is shown in [Figure 4]. Numerical analysis revealed clearly two distinct clusters. The cluster 1 includes m4 and m5. The cluster 2 is divided into two subclusters; the first includes m3 and the second includes w1, m1, and m2. The highest similarity was scored between m1 and m2 (0.7) and the lowest similarity between m5 and m1 (0.415) ([Table 7]).
Figure 4 Patterns and dendrogram based on unweight pair group method with arithmetic average algorithm (UPGMA) of the DNA Genome patterns of the whole-cells of Penicillium citrinium and their mutants. [W:wild type, m1: 150-A, m2: 150-B, m3: 50-A,m4: 250- A ,m5: 250-D].

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Table 7 Similarity matrix computed with dice coefficient

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  Conclusion Top

UV and gamma ray irradiation can induce mutations, which can be carefully acclimatized and commercially propagated under suitable condition. RAPD technique could be successfully applied to the newly β-mannanases and can differentiate mutants. All of these results indicated that the survival of the parent strain depends on both of the type and period of mutagen treatment and on the microorganism’s nature.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]


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