Harnessing Mucor spp. for Xylanase Production: Statistical Optimization in Submerged Fermentation Using Agro-Industrial Wastes

Xylan is the primary hemicellulosic polymer found in lignocellulosic agricultural wastes and can be degraded by xylanase. In the current research, Mucor circinelloides and M. hiemalis were tested for their ability to produce xylanase from tangerine peel by submerged fermentation. Experiments on five variables were designed with Box–Behnken design and response surface methodology. Analysis of variance was exercised, the xylanase output was demonstrated with a mathematical equation as a function of the five factors, and the quixotic states for xylanase biosynthesis was secured. In addition, xylanase was partially purified, characterized, and immobilized on calcium alginate beads. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis were consisted of incubation temperature (30 and 20°C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. M. circinelloides and M. hiemalis demonstrated the highest xylanase activities after RSM optimization, with 42.23 and 35.88 U/mL, respectively. The influence of single, interchange, and quadratic factors on xylanase output was investigated using nonlinear regression equations with significant R2 and p values. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69- and 1.97-fold purification, and 30.74 and 31.34% recovery with 292.08 and 240.15 U/mg specific activity, respectively. Partially purified xylanase from M. circinelloides and M. hiemalis demonstrated the highest activity at neutral pH and 60 and 50°C, respectively. The immobilized M. circinelloides and M. hiemalis xylanase retained 84.02 and 79.43% activity, respectively. The production of xylanase from M. circinelloides and M. hiemalis utilizing RSM is deemed profitable for the decomposition of the agro-industrial wastes.


Introduction
Biotechnological processes on filamentous fungi have enabled the industrial utilization of their ability to produce valuable enzymes due to their ease of propagation and increased production of extracellular enzymes with specific characteristics such as stability over a wide range of pH and temperature [1][2][3].
The class Zygomycetes, order Mucorales, including the genus Mucor, are mainly saprobic fungi that can grow well on various agro-industrial wastes with the potential to produce extracellular hydrolytic enzymes that can be utilized in a variety of industrial applications [4].
The amount of fungal enzyme manufactured is determined by the conditions of the fermentation process and the need to optimize these conditions for low enzyme output cost [11]. The fungal enzyme manufacturing is prevalently implemented by submerged or solid-state fermentation [12]. Submerged fermentation, utilized in 90% enzyme industry, occurs in the presence of water excess, providing a soft handling and best control [13,14]. Immobilization of enzymes and entrapment of enzymes in polymers such as alginate eliminate the need to isolate the enzyme from the outcome solution, facilitate enzyme recycling, enable repetitive and continuous use, and control enzyme activity [15][16][17][18].
This study aimed to evaluate the production of highvalue xylanase by Mucor spp. using agro-industrial byproducts as a cheap substrate in submerged fermentation conditions. The optimization of xylanase production, as well as characterization and immobilization of partially purified xylanase, was investigated.

Materials and Methods
2.1. Tested Fungi. Mucor circinelloides AUMC 6696.A (Accession no. MT509983) and M. hiemalis AUMC 6031 (Accession no. MT365791) [19] were utilized in the current search for xylanase production. Pure cultures were kept in potato dextrose agar (PDA) tubes and preserved at 4°C for further use.

Enzymes Preliminary Screening
2.2.1. Screening on Agar Plates. Czapekʼs agar medium (g/L: KH2PO 4 , 1; NaNO 3 , 2; MgSO 4 .7H 2 O, 0.5 and CaCl 2 .2H 2 O, 0.5) was supplemented with 10 g/L of xylan powder as a carbon source for xylanase production. The pH was set to 7, and the impregnated plates were preserved at 28 ± 2°C for five days and then screened for enzymes production [20]. After immersing the cultured agar plates in iodine solution for 15 minutes, they were examined for the presence of a clear zone.

Screening by Submerged Fermentation (SmF).
Pomegranate peel, tangerine peel, and wheat straw were desiccated at 65°C for 24 h, then squelched to grist, and used as substrates (10 g/L) in Czapekʼs mineral salts broth. The broth containing flasks was adjusted to pH 7 and autoclave sterilized. The strains' spore suspensions (10 7 spores/mL) were utilized to inoculate 250-mL flasks holding 100 mL submerged broth. The impregnated flasks were preserved at 28 ± 2°C for seven days on both shaking and static conditions. Subsequently, broth media were centrifuged, and supernatants were maintained for further enzymatic analysis.

Quantitative
Screening of Xylanase. Xylanase efficiency was measured according to Miller [21]. In addition, 0.5 mL enzyme supernatant was added to 0.5 mL xylan (1% w/v) in acetate buffer, and the mixture was incubated at 50°C for 30 min. Afterward, the interaction was intercepted by appending 1 mL of 3,5-dinitrosalicylic acid reagent and incubated at 100°C for 10 min. After cold dishing, the absorbance was measured at 570 nm using a spectrophotometer (Jenway 7315, UK). The amount of reducing sugars was determined using xylose as a standard for plotting the calibration curve. All the tests were carried out three times, and the outputs were expressed as an average value. A unit of the enzyme was acquainted as the quantity of the enzyme per one mL required to release one μmol of reducing sugar from a substrate per 60 seconds under the optimum trial conditions [22].  The data were given as averages of three replicates (mean ± SD). Values followed by the different letters are significantly different at p < 0:05. . Response surface methodology (RSM) tactic using Box-Behnken design (BBD) was exercised to determine the optimum factors for boosted xylanase production, including A, temperature; B, pH; C, incubation period; D, inoculum size; and E, substrate concentration (Table 1). Forty-six experiments with the central points were employed to satisfy the polynomial pattern established on a Box-Behnken design (BBD, 5 variables) attained by Minitab 19® software (Version19.1.1.0. LLC). At three-level and five factors, experimental BBD was examined, and the number of the tests (N) was determined corresponding to the subsidiary equation:

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where k is the digit of factors and C0 is the digit of central points equal to 6. The impact of variables on the simulation (Y) was evaluated by utilizing a second-order polynomial equation to determine the quixotic states of the xylanase biosynthesis.

Partial Purification of Xylanase from Mucor Strains.
After the incubation period under optimum conditions, the contents of the broth culture were centrifuged, and the supernatant was utilized for enzyme assay. Xylanase activity and protein concentration were measured in the supernatant according to Miller [21] and Lowry et al. [23], respectively, utilizing the standard of bovine serum albumin to generate the calibration curve spectrophotometrically at 750 nm. Crude enzyme solution was partially purified by precipitation using cold acetone. Precooled acetone (-20°C) was subjoined to the enzyme solution until the volume ratio between enzyme solution and acetone reached 1 : 1; 1 : 2; 1 : 3; 1 : 4, and 1 : 5 (v/v). The solution was left at -20°C overnight to allow protein precipitation. The precipitates were gathered by centrifugation at 10000 rpm for 15 min and resuspended in a small volume of (sodium citrate buffer, pH 4.8, 0.05 M). These samples were used for determining the activity of xylanase, purification factor, and enzyme recovery yield [24]. Protein was estimated, and suitable precipitants for characterization were selected. The following equations were used to calculate the partially purified xylanase's specific activity, yield, and purification fold.
2.5. Characterization of Partially Purified Xylanase. The optimum temperature for partially purified xylanase activity was determined to be in the range of (30-90°C), and the thermal stability was determined after premaintaining the    [27], and the assay of free and immobilized xylanase was performed. The immobilization yield was assayed according to the equation [28].
where Ai is the immobilized enzyme activity and Af is the free enzyme activity.

Response Surface Methodology for Optimization of
Xylanase Production. Table 1 shows the independent factors with their competent levels employed in optimization of xylanase output, while BBD of the independent factors along with predicted as well as experimental values are depicted in Table 3. The production of xylanase by M. circinelloides was predicted using the following equation: The highest xylanase activity of both M. circinelloides    (9), the incubation period (9 days), inoculum size (3 mL), and substrate concentration (3 g/100 mL). In comparison, the run No. 46 for M. hiemalis consisted of incubation temperature (20°C), pH value (7), the incubation period (9 days), inoculum size (3 mL), and substrate concentration (3 g/100 mL).
Analysis  Contour plots explained the relationship between parameters and defined each factor's optimum scale for xylanase efficiency by M. circinelloides (Figures 1(a)-1(j)) and M. hiemalis (Figures 2(a)-2(j)). The response surface plot constructed any two variables, while other variables were maintained at their optimal level. Contour plots of xylanase activity by M. circinelloides revealed significant interactions between incubation temperature with pH, incubation period, and substrate concentration; pH with incubation period and substrate concentration; and incubation period with inoculum size and substrate concentration. In contrast, contour plots of the interactions between incubation temperature with incubation period, inoculum size, and substrate concentration; pH with substrate concentration; incubation period with inoculum size and substrate concentration; and inoculum size with substrate concentration significantly influenced xylanase production by M. hiemalis. The remaining interactions insignificantly influenced xylanase production.
3.3. Partial Purification of Xylanase. Extracellular xylanase from M. circinelloides and M. hiemalis was partially purified from broth cultures by using different acetone concentra-tions. The highest xylanase activity (11.89 and 10.73 U/mL from M. circinelloides and M. hiemalis, respectively) was obtained via crude filtrate precipitation with acetone at ratio 1 : 4 (Table 6). Xylanase purification resulted in 1.69-fold purification and 30.74% xylanase recovery with a specific activity of 292.08 U/mg from M. circinelloides, while in 1.97-fold purification, 31.34% recovery with a specific activity of 240.15 U/mg was obtained from M. hiemalis xylanase ( Table 7).

Characterization of Partially Purified
Xylanase. M. circinelloides partially purified xylanase was highly active at 60°C (total activity 100%) and decreased gradually at 50-30°C and 70-90°C, while xylanase activity of M. hiemalis was highly active at 50°C (total activity 100%) and decreased gradually at 40-30°C and 60-90°C. The relative xylanase stability from both M. circinelloides and M. hiemalis was high at 30°C and decreased in the range of 40-90°C (Figures 3(a) and 3(b)). Furthermore, partially purified xylanase from both strains had the highest activity and stability (100%) at pH 7.0, and then, at higher or lower pH values, activity and stability of xylanase were reduced (Figures 3(c) and 3(d)).
After incubation with 10 mM K + , Mg 2+ , Ba 2+ , and Ni 2+ , xylanase activity of both strains decreased with the exception of K + , which increased M. hiemalis xylanase activity by 40.34% compared to the control, while stability increased except for Ba 2+ with M. circinelloides enzyme and K + and Mg 2+ with M. hiemalis enzyme. Detergents including Tween 80, Tween 20, urea, and Na 2 CO 3 at low concentrations of 1%, 1%, 1%, and 50 mM, respectively, reduced M. circinelloides and M. hiemalis partially purified xylanase activity. In contrast, there was a significant increase in activity and decreased stability at high concentrations of the tested detergents, except urea, which decreased M. hiemalis partially purified xylanase activity at its applied high concentration (5%) ( Table 8). The data were given as averages of three replicates (mean ± SD). The data were given as averages of three replicates (mean ± SD).

Xylanase Immobilization and Scanning Electron
Microscopy. M. circinelloides enzyme had an immobilization yield of 84.02%, while M. hiemalis enzyme had a yield of 79.43% (Table 9). A scanning electron microscope was utilized to investigate the surface superficial of calcium alginate beads together and without entrapped xylanase (Figure 4). The vents of calcium alginate beads were examined in the micrographs of calcium alginate beads without entrapped xylanase, whereas together with xylanase, the vents were coated with intense molecule xylanase.

Discussion
Fungi play an excellent source for the production of various beneficial enzymes. Xylan represents the principal hemicellulosic xylopolymer found in plant cell walls. The biodegradation of xylan functions as an essential role of plant materials natural [29]. In this research, M. circinelloides and M. hiemalis were investigated as potential sources for producing xylanase.
Many fungi were proven to have multiple xylanase forms and can efficiently breakdown xylan. In addition, the  14 BioMed Research International substrate type influenced the concentration and number of various expressed forms of xylanase [30,31]. On the contrary, the substrate installation and growth factors affect the fungal growth and the biosynthesis of enzymes. Therefore, there is an urgent need to modify the biosynthesis of the enzymes by optimizing fungal production.
In the current research, the low p values, which are attained by the F test, and high R 2 values indicated that the employed model attained a high significance, and its sufficiency was confirmed [32]. For xylanase production on wheat bran by solidstate fermentation by M. indicus and M. hiemalis using RSM, the optimum temperatures were 40.0 and 43.4°C, respectively, and 51.3 and 53.2 h for incubation time, respectively, while the highest xylanase activities were 43.1 and 43.8 U/g for M. indicus and M. hiemalis, respectively [33]. Atalla et al. [34] reported a maximum xylanase activity from A. oryzae (0.37 U/mL) by utilizing rice straw waste, while orange peel exhibited low xylanase production (0.17 U/mL). Cui and Zhao [35] highlighted the magnitude of the substrate concentration for xylanase biosynthesis, where the enzyme activity by Penicillium sp. WX-Z1 demonstrated a gradual rise in wheat bran concentration. Statistical analysis of xylanase production from Penicilliumoxalicum ZH-30 by RSM showed that the linear, quadric terms and initial pH and temperature interaction significantly impacted the optimal conditions for raised xylanase biosynthesis at pH 7.38 and temperature 31.1°C. Under optimal conditions, the portended and experimented xylanase activities were 14.33 and 14.50 U/mL, respectively [36].
Box-Behnken design statistical analysis by Cao et al. [37] demonstrated that the linear and quadric terms of cultivation time, pH, and substrate concentration variables significantly affected xylanase production by Aspergillus nigerAN-13 using wheat bran under submerged fermentation. In submerged fermentation with corncob, Aspergillus niger KIBGE-IB36 was found to be a high producer of xylanase [38] and different spectra of xylanase synthesis using wheat bran, rice husk, orange peel, and pomegranate peel [5]. RSM method by Azzouz et al. [39] reported a 65.01% increase in xylanase output by Aspergillus niger strain BG on 84% humidified wheat bran with a pH of 2.5 at 37°C and incubation for 66 h. Statistical analysis of xylanase output by Aspergillus foetidus in submerged fermentation with soybean scraps revealed that variables pH and the interaction of pH and temperature influenced xylanase biosynthesis, with good xylanase activity (13.98 U/mL) at pH 7.0, 28°C, and 120 rpm for 168 hours [40]. Response surface methodology by Ramanjaneyulu and Rajasekhar [41] for maximal production of xylanase (4560 U/mL) by Fusarium sp. BVKT R2 demonstrated that the optimal conditions were sorbitol 1.5%, yeast extract 1.5%, pH of 5.0, temperature of 32.5°C, and shaking of 175 rpm.
High temperature increases the solubility of reactants and products by decreasing viscosities, resulting in faster hydrolysis [42], and prolonged energetic existence would fabricate enzymes convenient for promoted and active biomass diversion. Therefore, thermostability is the most substantial property for the enzyme used under extreme bioprocessing conditions to be efficient [8]. RSM optimization of xylanase yield by Aspergillus niger 3-fold and 1.41-fold purification was attained with about 6.2% yield, and the highest activity of the purified xylanase was observed at pH 6 and 50°C. The produced xylanase exhibited high thermal and pH stability, with more than 90% residual activity between 30 and 40°C and pH 3-9 after incubation of 24 h, with half-lives of 30 min at 50 and 60°C [43]. Liet al. [36] reported a temperature range of 50-60°C suitable for the industrial application of xylanase from P. oxalicum ZH-30. Supplementation of Tween 80 as additional surfactant improved (72:4 ± 1:42 U/g) the titer of xylanase on sugarcane bagasse by T. viride-IR05 [44]. Regarding xylanase immobilization, the formation of an ionic bond of Ca 2+ with carboxylate groups of sodium alginate helm to formulate mechanically firm grids of alginate gel that fulfill the demands for efficient hydrogel systems acting as carriers of bioactive molecules in several industrial applications [45]. Immobilized Talaromyces thermophilus xylanase covalently bound by glutaraldehyde to chitosan, chitin, amberlite, duolite, florisil, and gelatin gave immobilization yield of 89.0, 87.8, 89.3, 81.1, 96.2, and 98.8%, respectively [28].

Conclusion
The present study utilized the response surface methodology via the Box-Behnken design to improve xylanase production by M. circinelloides and M. hiemalis. The experimental results are consistent with predicted responses. The produced enzyme was partially purified, characterized, and immobilized. The optimum parameters for xylanase production by M. circinelloides and M. hiemalis consisted of incubation temperature (30 and 20°C), pH value (9 and 7) incubation period (9 and 9 days), inoculum size (3 and 3 mL), and substrate concentration (3 and 3 g/100 mL), respectively. The partial purification of M. circinelloides and M. hiemalis xylanase yielded 1.69-and 1.97-fold purification, and the immobilized xylanase retained 84.02 and 79.43% activity, respectively. Response surface methodology was effective and satisfactory and investigated many factors simultaneously. More research is needed to scale up enzyme production for a wide range of applications.