Table of Contents  
Year : 2020  |  Volume : 19  |  Issue : 1  |  Page : 29-46

Optimization and comparative studies on activities of β-mannanase from newly isolated fungal and its mutant

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

Date of Submission25-Aug-2019
Date of Decision02-Oct-2019
Date of Acceptance08-Oct-2019
Date of Web Publication24-Mar-2020

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

DOI: 10.4103/epj.epj_48_19

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Background and objective β-Mannanase has potential industrial applications in pharmaceutical field, bioethanol production, coffee extraction, food and feed technology, etc. So finding a new and promising enzyme source is a very important issue. The aim of this study was to improve the biosynthesis of β-mannanase by different techniques, such as mutation and optimization of the culture parameters.
Materials and methods Five fungal isolates were tested for the production of β-mannanase. Enzyme activity, protein content, and specific activity were determined. The most potent isolated microorganism and its mutant were identified by using Transmission Electron Microscopy and 18SrDNA sequencing and phylogenetic analysis. Ultraviolet and gamma rays were used. Optimization studies were done to maximize enzyme production from the most potent microorganism and its highly productive and stable mutant, including culture conditions and medium compositions, and statistical optimization was also carried out. Primary characterization of β-mannanase was studied.
Results and conclusion In our research, we found a stable mutant strain obtained by using gamma radiation at 150 GY. The first step of the fermentation, optimized by one-factor-at-a-time technique, increased the biosynthesis of β-mannanase for Penicillium citrinium 150 GY from 65.9 to 219 IU/ml compared with the wild strain, which increased from 16.82 to 26.5 IU/ml. Statistical optimization improved P. citrinium 150 GY β-mannanase from 219 to 296 IU/ml by applying Plackett–Burman design and increased the level of β-mannanase biosynthesis to 351 IU/ml. Primary characterization of β-mannanase produced by P. citrinium and P. citrinium 150 GY proved that they are almost the same, except in a little shift to higher value (5°C) in optimum temperature.

Keywords: gamma radiation, Penicillium citrinium 150 GY, Penicillium citrinium, β-mannanase

How to cite this article:
Khattab OK, Ismail SA, Abosereh NA, Abo-Elnasr AA, Nour SA, Hashem AM. Optimization and comparative studies on activities of β-mannanase from newly isolated fungal and its mutant. Egypt Pharmaceut J 2020;19:29-46

How to cite this URL:
Khattab OK, Ismail SA, Abosereh NA, Abo-Elnasr AA, Nour SA, Hashem AM. Optimization and comparative studies on activities of β-mannanase from newly isolated fungal and its mutant. Egypt Pharmaceut J [serial online] 2020 [cited 2023 Feb 9];19:29-46. Available from:

  Introduction Top

β-Mannanase is the enzyme that cleaves the β-1,4-mannosidic linkages in mannans, galactomannans, glucomannans, and galactoglucomannans, which are found in many natural products ([Figure 1]) [2],[3].
Figure 1 Structural chemistry of mannan, galactomannan, glucomannan, and galactoglucomannan, taken from Srivastava et al. [1].

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β-Mannanase is produced by different microorganisms [4],[5]. The fungal strains are preferred owing to high yields and extracellular release of the enzymes and higher enzymatic activity [6],[7].

Owing to extensive use of β-mannanase in different industries, the interest in achieving microbial strains with high β-mannanase activity has increased. Therefore, several methods were used to increase β-mannanase biosynthesis, such as optimization of enzyme production, cloning of β-mannanase gene, random mutagenesis, and site-specific mutagenesis [8].

Random mutagenesis is often used as a rapid and feasible method. This technique relies on the exposure of the microorganism to a physical or a chemical mutagen. Physical mutagenesis can be performed with electromagnetic radiation, such as ultraviolet (UV) light, radiographies, gamma rays, or a particle radiation with beta and alpha particles or fast and thermal neutrons [7],[9].

Chemical mutagenesis can be achieved with alkylating agents (N-methyl-N’-nitro-nitrosoguanidine, ethyl methane sulfonate, N-methyl-N-nitrosourea, dimethyl nitrosamine, etc.), intercalating agents (ethidium bromide), or azides (sodium azide) [10],[11].

The current research was carried to find out new fungal isolates that can produce a significant level of β-mannanase using a cheap substrate, coffee waste.

Morphological characterization and phylogenic analysis were applied to identify the chosen fungal isolate. Mutagenesis of the most potent fungal strain was achieved by gamma radiation to enhance the production of β-mannanase.

Furthermore, optimization of the production of β-mannanase was performed by one-factor-at-a-time technique for both wild and mutant strains. The statistical methodology was applied to maximize the enzyme production of the most potent strain (wild or mutant). Primary characterization of the produced β-mannanase was also determined.

  Materials and methods Top


Locust bean gum was obtained from Sigma Chemicals (St Louis, USA) (a galactomannan from the seeds of Ceratonia siliqua, which consists of a straight-chain polymer of mannose with galactose branches on every fourth mannose). There is No animal or human experimental. All other chemicals used were of analytical grade.

Coffee residue

Green coffee beans were bought from a local market, Giza, Egypt. They were roasted, and the spent coffee ground was then milled by thermal water extraction. The residue of coffee was collected and used in the microbial culture after determination of the carbohydrate contents [12].


In this study, five fungal strains, primary identified as Aspergillus niger.1, Aspergillus niger.2, Penicillium spp. (isolated from Pharaoh mummies of Ancient Egyptian Museum, Cairo, Egypt), Fusarium spp., and Rhizoctinia spp. (isolated from plant), were screened for β-mannanase production.

All the fungal strains were maintained on potato dextrose agar (PDA) and incubated at 30°C for 7 days and stored at 4°C with monthly subculturing.

Mutagenesis with ultraviolet

UV mutagenesis to the spore suspension of Penicillium citrinium was done by treating with UV (power: 30 W and wavelength: 254 nm) each 15-min interval, with a distance of 18 cm from the UV light source, and then plates were stored in the dark for 2 h. Mutagenic suspension of each treatment was diluted and plated on PDA as complete media and Cazpek media as minimal media at 30°C for 5 days. Single colony with different morphology from each treatment was subcultured and tested for enzyme production [13].

Mutagenesis with gamma rays

In this way, mutagenesis of the spore of the most potent isolate was carried out using five different doses of gamma radiation (0, 50, 100, 150, 200, and 250 Gray) produced from Cobalt-60 (Co60) (Egyptian Atomic Energy Authority, Nasser, Egypt) [14].

Strain identification, 18s rDNA sequencing, and phylogenetic analysis

The most potent isolated microorganism and its mutant were identified by using Transmission Electron Microscopy (TEM, JEM-2100; JEOL, USA) after growing on PDA plate for 7 days. Molecular identification was carried out by using Easy Quick DNA extraction kit (Quiagene, Netherlands and Germany) following the manufacturer’s instructions.

The PCR was performed according to Plengvidhya et al. [15], in a total of 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.

Culture media

  1. Inoculum medium: this 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 [16]. 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 for 16 min at 121°C.
  2. Fermentation medium: the fermentation medium contains 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 [16].
  3. Cultivation conditions: an inoculum culture was obtained by culturing the fungal strains in the aforementioned 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 static condition and in a shaking incubator at 120 rpm for shaking of all the tested fungal species. Thereafter, the fermented medium was centrifuged, and the filtrate was used as the crude enzyme solution.

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 in sodium citrate buffer (50 mmol/l) at pH 5.5 for 10 min at 50°C [17]. The reducing sugars produced were determined using the Nelson–Somogyi technique [18]. 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 et al. [19] method.

Optimization of β-mannanase production

One-factor-at-a-time technique

The ability of the wild and mutant strains to utilize coffee waste in the fermentation medium was tested at different incubation periods (3, 5, 7, 10, 12, 14, and 18 days), while testing with static and shaking (120 rpm) culture techniques. Effects of different nitrogen sources were investigated by replacing different equivalent amount of nitrogen base from different tested organic or inorganic nitrogen sources. Different organic nitrogen sources such as peptone, yeast extract, Baker’s yeast, casein, corn steep liquor, and urea, whereas inorganic nitrogen sources, including sodium nitrate and ammonium sulfates, were individually tested as a sole nitrogen source.

The optimum concentration of the coffee waste and nitrogen source were evaluated in relation to enzyme yield. The strain that produced the highest β-mannanase was subjected to further optimization. The experiments were conducted in triplicate, and the results are the average of these three independent values.

Statistical optimization

  1. Plackett–Burman experimental design (PBD): to evaluate the relative important factors in the production of β-mannanase by the chosen isolate in substrate fermentation, PBD was applied [20]. Matrix contains seven dependent variables in two levels [(+1) and (−1)] with nine trials being selected, as described in [Table 1]. PBD was based on the first-order linear model:
    Table 1 Survey of some fungal strains for the production of extracellular β-mannanase in shaken cultures

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  2. where Y is the response (β-mannanase production), B0 is the model intercept, and Bi is the linear coefficient and Xi is the level of the independent variable. The main effect of each variable was determined by following equation:
    • where E(Xi) was the effect of the tested variable Mi+ and Mi− represented β-mannanase production from the trials where the independent variable (Xi) measured was present at high and low levels, respectively and N is the number of trials. The significance level (P value) of each tested variable was determined using Student’s t-test.
  3. Optimization of factors using response surface methodology (RSM): RSM was employed to investigate the accurate optimal levels of the main affecting factors obtained from PBD using central composite design (Box and Behnken) [21].

A study of four variables at five different levels (−1, +1, 0, −2, and +2) was carried out in a set of 30 trials ([Table 5]).

A second-order polynomial equation was used for the interpretation of correlation between variables and the response.

The equation is presented as follows:

where Y represents response or β-mannanase yield; β0, is model interception; βi, is linear coefficient; βii, is squared coefficient; βij, is interaction coefficient; and Xi and Xj are the coded levels of independent variables.

Primary characteristics of the crude β-mannanase produced by wild and mutant strains

Effect of pH

The optimum pH of the crude β-mannanase for wild and mutant strain was determined by carrying out the enzyme assay at different pHs (4.0–7.0) using sodium citrate buffer (50 mmol/l) for 10 min at 50°C.

Effect of temperature

The optimum temperature of the crude β-mannanase of wild and mutant strains was investigated by incubating the reaction mixture at different temperature ranged from 40 to 65°C for 10 min under standard assay condition.

Determination of thermal stability

Thermal stability of the crude β-mannanase for wild and mutant was investigated by incubating the enzyme in sodium citrate buffer (50 mmol/l) solution at pH 5.5 (without substrate) at different temperatures (40–65°C) for different time intervals (15, 30, 45, 60, 90, and 120 min). The residual enzyme activity was measured under the optimum conditions.

All the experiments were done in triplicate, and the recorded data were the mean values.

  Results and discussion Top

The production of extracellular β-mannanase from the fungal isolates

The shaking cultures of the five fungal isolates possessed different levels of β-mannanase, ranged from 0.0 to 16.3 IU/ml ([Table 1]). Rhizocotinia spp. failed to produce β-mannanase in all tested incubation periods, whereas Penicillium spp. recorded the highest β-mannanase activity (16.35 IU/ml) after 12 days and was chosen for further studies.

Microorganism Identification

The 18S rDNA gene sequence analysis indicated that the isolate is closely related to P. citrinium ([Figure 2]), which was known as β-mannanase producer [22]. The data of 18 Sr DNA partial sequence have been submitted to Gen Bank databases under the name of P. citrinium with accession no. of Egy5 LC368457.
Figure 2 Molecular phylogenetic tree of Penicillium citrinium strain Egy5 with related strains. Phylogenetic analysis was conducted in MEGA6 software (Tamura et al., 2013) using Maximum Likelihood method and basing on genes of ribosomal RNA (rRNA) and internal transcribed spacer. Accession number is indicated adjacent to each strain between parentheses. The tree is drawn to scale with branch lengths measured in the number of substitutions per site. Bootstrap values are indicated at nodes.

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Mutation by using ultraviolet rays

Of all the mutants generated by this method, mutant designated UV30-B increased mannanase biosynthesis with ∼threefold (48.34 IU/ml) higher than the wild type (16.48 IU/ml), whereas the lowest amount (0.79 IU/ml) was produced by mutant coded UV15-H. The mutant strains 30-B showed low stability within the three generation (unpublished data).

Mutation by using gamma rays

In an attempt to increase the production of β-mannanase, P. citrinium was mutagenized by exposing to different doses (50, 100, 150, 200, and 250 Gy).

The production of extracellular P. citrinium β-mannanase increased about 1.87 fold with mutant at dose 50 Gy (30.58 IU/ml), whereas at dose of 150 Gy, the resulted mutant increased the production (37 IU/ml) by about 2.27-fold more than the wild-type strain. To our knowledge, no one improved β-mannanase production by exposing the fungal strain to gamma rays. [Figure 3] shows high morphological difference between the wild strain (A&B) and the mutant (C&D) by using transmission electron microscopy.
Figure 3 Colonial and morphological characteristic of wild (a, b) and mutant (c, d) for most potent fungal isolate.

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Increasing enzyme production by exposure to the gamma radiation was recorded by other researches. Iftikhar et al. [23] found that the dosage 140 Gy in MBL-5 showed maximum extracellular lipases production. Moreover, Shahbazi et al. [14] recorded that gamma radiation at 250 Gy had accrued the activity of Ccase, CMCase, Avicellase, and Fpase for Trichoderma spp.

Optimization the production of Penicillium citrinium β-mannanase

In this study, we optimized the production of β-mannanase by P. citrinium and its 150-Gy mutant.

Effect of incubation period and culture technique on biosynthesis β-mannanase

The results in [Figure 4]a, b illustrate that in shaking cultivation the highest amount of β-mannanase (65.9 IU/ml) was produced by 150 Gy strain followed by 28.16 IU/ml obtained by 50 Gy mutant strain after 18 days. The highest value obtained by the wild strain (16.82 IU/ml) was produced after 14 days in shaking culture. By using static culture technique, the produced β-mannanase was reduced to half of that produced by shaking cultivation for both 150 Gy mutant strain and 50 Gy mutant strain (34.5 and 16.28 IU/ml, respectively). The wild-type strain produced 5.4 IU/ml only with the static cultivation. Therefore, 150 Gy mutant strain was chosen with the wild-type strain (for comparison) for further experiments with shaking culture technique.
Figure 4 (a) Biosynthesis of extracellular β-mannanase using shaken culture medium at different incubation periods for wild and mutant strains; (b) biosynthesis of β-mannanase using in static culture medium at different incubation periods for wild and mutant strains.

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Effect of different concentration of coffee waste

Agroindustrial byproducts are available in large amounts, and they represent a cheap source for the production of several enzymes [16],[24].

Since coffee waste is the byproduct produced in huge amount in the coffee industries, its uses as a carbon source in the fermentation medium can reduce the cost of the enzymatic synthesis and also solve the pollution problems.

The effect of different concentrations of coffee waste ([Figure 5]) indicated that increase in concentration to 18% increased the β-mannanase production by P. citrinium 150 Gy to 129 IU/ml, whereas the wild-type strain produced 16 IU/ml at the same concentration at optimum incubation periods using shaking technique.
Figure 5 Effect of different concentration of carbon source on the production of wild and mutant extracellular β-mannanase.

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Youssef et al. [25] revealed that the optimum coconut concentration was 5 g per 50 ml, yielding the highest mannanase activity (8.6 U/ml) and protein content.

Effect of different nitrogen sources on mannanase production

The nitrogen source had a great influence of the biosynthetic pathways of the bioactive metabolites of the produced strain [26]. Different nitrogen sources were examined for the production of β-mannanase. On equivalent nitrogen base, ammonium sulfate, peptone, and urea of the basal medium were substituted individually by different nitrogen sources. These included organic sources, namely, baker’s yeast, casein, corn steep liquor, peptone, urea, and yeast extract, and inorganic sources, namely, ammonium sulfate and sodium nitrate.

It was quite clear from the results in [Figure 6] that the mixed nitrogen source produced the highest level of β-mannanase for both P. citrinium and P. citrinium 150 Gy strains, which were 16.3 and 129.92 IU/ml, respectively.
Figure 6 Effect of different nitrogen source on the production of wild and mutant extracellular β-mannanase.

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El-Refai et al. [16] also recorded that the mixed nitrogen source was the best nitrogen source for production of Penicillium humicola β-mannanase.

Saad et al. [27] reported that sodium nitrate was the best inorganic source for production of Aspergillus tamarii NRC3 β-mannanase.

Effect of different concentration of nitrogen

The production of β-mannanase in P. citrinium and P. citrinium 150 Gy mutant was increased up to 26.5 and 219 IU/ml as the concentration of nitrogen source of the culture medium increased to1.2 and 1.4%, respectively.

The β-mannanase production was almost stable by increasing the nitrogen concentration of the culture medium to 1.8% ([Figure 7]).
Figure 7 Effect of different concentration of nitrogen source on the production of wild and mutant extracellular β-mannanase.

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Youssef et al. [25] indicate that the increase in β-mannanase production was observed with raising the NH4Cl concentration to 0.25% and decreased gradually at higher concentrations.

The aforementioned optimization producers increased the biosynthesis of β-mannanase by P. citrinium 150 Gy mutant strain to 219 IU/ml, which was higher by 8.26-fold from β-mannanase produced by the wild-type P. citrinium (26.5 IU/ml) after optimization. Therefore, P. citrinium 150 Gy mutant strain was used for statistical optimization.

Statistical optimization for the production of the mutant strain β-mannanase

  1. PBD
    • PBD was used to investigate the relative interaction and the variable of different parameters for the culture processing. Eleven trials for seven variables ([Table 2]) clarify the wide variation in the production of β-mannanase from 12.9 to 296 IU/ml, which implied the great influence of different factors in the fermentation process. The highest value of the β-mannanase 296 IU/ml was produced in trial 8, which contained the following: coffee waste, 10; nitrogen mixture, 1.8%; KH2PO4, 8.5; and inoculum, 5 ml, at 140 rpm, at pH 5 after 21 days. On the contrary, the lowest value of β-mannanase 296 IU/ml was produced in trial 6, which contained coffee waste, 10; nitrogen mixture, 1%; KH2PO4, 5.5; and inoculums, 5 ml at 100 rpm at pH 4 after 21 days.
      Table 2 Results for Plackett−Burman experiment

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    • The main effects of the tested parameters on the production of β-mannanase were calculated and graphically represented in [Figure 8]. All the examined factors except inoculum possessed positive effect.
      Figure 8 Main effects of independent variables on β-mannanase production by Penicillium citrinium according to the results of the Plackett–Burman design.

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    • Confidence level, P-effect and t-test of the statistical analysis of the PBD are indicated in [Table 3]. The variables showed high confidence level above 99% for nitrogen complex, KH2PO4, initial pH, and rpm, and they were selected for further optimization.
      Table 3 Statistical analysis of Plackett–Burman design showing coefficient values, effect, t values, and P values for each variable on β-mannanase analysis

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    • The first-order model describing the correlation of the seven factors and the β-mannanase activity could be presented as follows:
  2. RSM

The optimal levels of the most effective variables (nitrogen complex, KH2PO4, initial pH, and rpm) arising from PBD were determined by applying RSM involving CCD in 30 trials ([Table 4]).
Table 4 Central composite design and response β-mannanase activity by Penicillium citrinium

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Culture medium containing in g/l, peptone (25), ammonium sulfate (19.15), urea (3.83), MgSO4 · 0.7H2O (0.6), K2HPO4 (8.5), and coffee waste(10 g/flask), adjusted at pH 5 (before autoclaving) inoculated by 8% of the inoculums and incubated at 30°C in a shaking incubator at 140 rpm for 21 days was used as the central point of the CCD.

The independent variables with their coded matrix and responses are listed in [Table 4]. In this table, experimental and predicted values for β-mannanase activity are presented. Variation in the enzyme yield from 65 to 351 IU/ml was observed during the 30 runs of the experiments. The highest level of the produced β-mannanase was 351 IU/ml obtained in run 5, which indicated that the optimal levels of the tested variable were as follows: KH2PO4, 8.5; rpm, 120; and nitrogen complex, 1.8% at pH5.0.

The determination coefficient (R2) shows the accuracy of the model. The R2 value is 0.9562, indicating that the 95.62% variability in the response is explained by the independent variables. Therefore, the present value of R2 confirms the reliability of the current model for the production of β-mannanase and also exhibited a good correlation between the experiment and the theoretical values ([Table 5]).
Table 5 Analysis of Fractional Factorial design for β- mannanase activity by Penicillium citrinium

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A second-order polynomial equation used for the interpretation of correlation between variables and the response is presented as follows:

Y, represents response or β-mannanase yield and X1, X2, X3, and X4 are KH2PO4, RPM, nitrogen complex, and pH, respectively.

The mathematical optimal point of the equation was 403.8 IU/ml at 120 rpm and 1.6975% of mixed nitrogen with 8.5 g/l of KH2PO4 at pH, 5. Three-dimensional graphs of the regression equation ([Figure 9]) explained main and interaction effects of KH2PO4 and rpm; KH2PO4 and mixed nitrogen; KH2PO4 and pH; rpm and mixed nitrogen; rpm and pH; mixed nitrogen and pH on β-mannanase production by P. citrinium 150 GY mutant strain respectively.
Figure 9 Explained main and interaction effects of (a) KH2PO4 and rpm, (b) KH2PO4 and mixed nitrogen, (c) KH2PO4 and PH, (d) rpm and mixed nitrogen, (e) rpm and PH, and (f) mixed nitrogen and pH on β-mannanase production by Penicillium citrinium 150 Gy mutant strain correspondingly.

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The P value was used as a tool to check the significance of each coefficient, which in turn indicates the pattern of the interaction between the variables [28]. The statistical analysis of data ([Table 5]) indicated high significant effect of terms with smaller P values (P<0.05) on β-mannanase production.

The results obtained by analysis of variance analysis ([Table 4] and [Table 5]) showed a significant F value (23.39547) which implied the model to be significant. Model terms having values of prob ˃ F (1.17E−07) less than 0.05 were considered significant.

It was reported by many researchers that the statistical optimization model for fermentation process could overcome the limitation of classic empirical methods and was proved to be more significance for the optimization production of β-mannanase [5],[29],[30],[31].

Validation of the model

The validity of the proposed model was estimated by prediction of P. citrinium β-mannanase production for each trial of the matrix. The experimental results in [Table 3] show that the maximum observed β-mannanase production (351 IU/ml) was very close to the predicted value (352 IU/ml).

Verification of the optimization models

As shown in [Table 6] under the optimization condition for P. citrinium 150-GY, β-mannanase was reached to 351 IU/ml with specific activity of 21.57. The results indicated that the optimized condition accelerated about 9.5-fold times than basal medium.
Table 6 A verification experiment demonstrating the optimization steps targeted β-mannanase biosynthesis by wild and mutant Penicillium citrinium

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Primary characteristics of the crude wild and mutant Penicillium citrinium β-mannanase

Effect of pH of the reaction

The results in [Figure 10] show that P. citrinium and its mutant β-mannanase was optimally active at pH 5.5; below or above this pH, the activity decreased.
Figure 10 Effect of pH of the reaction mixture on the crude Penicillium citrinium β-mannanase. Control: at pH 5.0 represent 100%.

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In general, optimum pH for the activity of most fungal mannanases was in the acidic range [16],[32].

Effect of temperature of the reaction mixture

It was quite clear that β-mannanase produced from the mutant strain had optimum temperature at 60°C than that produced by the wild strain at 55°C; on the contrary, the gradual decrease in the temperatures around the optimum was slowly decreased for mutant one than the wild, as indicated in [Figure 11].
Figure 11 Effect of temperature of the reaction on the crude Penicillium citrinium β-mannanase. Control: at pH 5.5 and temperature 50°C represent100%. (a)Thermal stability of crude enzyme (wild); (b)Thermal stability of crude enzyme (mutant)

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Chantorn et al. [33] showed that maximum activity of bacterial mannanase was at 50°C. There are several commercial advantages in carrying out enzymatic reactions at a higher temperature [34]. Enzymatic digestion at a high temperature (60–65°C) may reduce microbial contamination of the material being processed. In addition, higher temperatures increase the rate of substrate digestion and increase the solubility of the polymeric substrates such as carbohydrates, rendering them more amenable to enzymatic attack [35].

Thermal stability

The crude enzyme solution of wild strain and its mutant strain was incubated in the absence of its substrate at 40, 45, 50, and 55°C in a water bath, and the residual activities at different periods up to 2 h were determined under the optimum conditions (pH 5.5, 55°C) for wild and (pH 5.5, 60°C) for mutant.

After 2 hr, the crude enzyme in the wild type retained 97.1% of its activity at 40°C and 94.35% at 45°C, but in the mutant, the crude enzyme kept its activity, and there is no any loss at 40°C and retained 95.4% after 2 h ([Figure 12]).
Figure 12 (a, b) Thermal stability of the crude Penicillium citrinium β-mannanase.

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After heating the crude enzyme of wild and mutant at 50°C up to 15 min, most of the activity was retained (96.86 and 98.45%, respectively), and still retained 71.35 and 86.21%, respectively, of its activity after 60 min At 50°C, the P. citrinium and P. citrinium 150 GY mutant strain retained 71.35 and 86.21%, respectively, of its activity after 60 min

At 55°C, the thermostability of both enzymes was almost equal after 1 h (74%).

The produced β-mannanase from both wild and mutant strains was quite stable than that recorded by El-Refai et al. [16], which recorded that Penicillium humicola β-mannanase lost ∼20% of its activity after one hour at 50°C.

  Conclusion Top

In our work, we found that gamma mutant was stable at 150 Gy. The first step of the fermentation optimized by one-factor-at-a-time technique increased the biosynthesis of β-mannanase for P. citrinium 150 Gy from 65.9 to 219 IU/ml comparing with the wild strain, which increased from 16.82 to 26.5 IU/ml.

Statistical optimization improved P. citrinium 150 Gy β-mannanase level from 219 to 296 IU/ml by applying PBD and increased the level of β-mannanase biosynthesis to 351 IU/ml. Primary characterization of β-mannanase produced by P. citrinium and P. citrinium 150 Gy proved that they are almost the same except in a little shift to higher value (5°C) in optimum temperature.

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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], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12]

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

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